bluuebunny/crossref_metadata_2025_split huggingface dataset:
AMPK Enhances Enterocyte Migration and in vivo Intestinal Barrier Function
Authors: Sun Xiaofei , Xue Yansong , Du Min , Zhu Meijun 
Published: November 2023

Impairment in gut epithelial integrity and barrier function is associated with many diseases, such as inflammatory bowel disease, obesity and diabetes. Inadequate mucosal wound healing is associated with defective enterocyte migration along the crypt‐villus axis. AMP‐activated protein kinase (AMPK), an energy sensor, has been suggested to mediate epithelial barrier function and enhances wound healing processes in cultured cells. However, the causal relationship between AMPK and intestinal barrier function remains underdetermined. The objective of this study was to provide direct evidence of the regulatory role of AMPK on intestinal barrier function and enterocyte migration in vivo. To test, mice with AMPKα1‐floxed gene were cross‐bred with VilCre mice to specifically knock out AMPKα1 gene in intestinal epithelial cells where villin is expressed. The migration of enterocyte and intestinal barrier function were compared between AMPK VilCre knockout (KO) and wild type (WT) male mice at the age of 2.5 months. AMPK VilCre KO mice had increased intestinal epithelial paracellular permeability to FITC‐dextran compared with WT mice, indicating a leaking gut. In addition, mucosal thickness and the migration of BrdU‐labeled enterocytes were decreased in AMPK VilCre KO mice. CDX2 is the key transcription factor committing cells to the intestinal epithelial lineage. Immunofluorescence staining further showed an abnormal expression pattern of CDX2 in AMPK VilCre mice, where CDX2 expression was extended from crypt to the middle of villi, while CDX2 expression resided along the epithelial cells in WT mice. Taken together, these data clearly show that AMPK is required for proper intestinal barrier function and enterocyte migration in vivo possibly through regulating cdx2 expression.Support or Funding InformationNIH R15HD073864
AMPK Improves Intestinal Epithelial Differentiation via Regulating CDX2
Authors: Sun Xiaofei , Yang Qiyuan , Du Min , Zhu Meijun 
Published: November 2023

BackgroundGut epithelium forms the largest and critical barrier against the external environment, which is constantly renewed. The homeostasis of intestinal barrier is based on a delicate regulation of epithelial proliferation and differentiation. AMP‐activated protein kinase (AMPK) regulates gene expression and cellular differentiation, and its inactivation is as a major etiological factor for many diseases. However, the role of AMPK in intestinal epithelial barrier function has been only sparsely studied.ObjectiveThe objective of this study was to evaluate the regulatory role of AMPK on intestinal epithelial differentiation and uncover its underlying mechanism.ResultsAICAR, an AMPK activator, improved the intestinal epithelial barrier function of Caco‐2 cells as indicated by increased transepithelial electrical resistance (TEER) and reduced paracellular FITC‐dextran permeability. Consistently, AICAR treatment enhanced the expression of epithelial differentiation markers, E‐cadherin and Villin, Alkaline Phosphatase activity, and the formation of tight junction based on Calcium switching assay and ZO‐1 immunostaining. Transfection of Caco‐2 cells with AMPK wild type (WT) plasmid, which enhances AMPK activity, improved epithelial barrier function and epithelial differentiation, while K45R, a dominant negative mutant, impaired. In addition, AMPK enhanced the expression of CDX2, the key transcription factor committing stem cells to intestinal epithelial lineage, which was associated with enrichment of H3K4me3, a permissive histone modification in the cdx2 promoter, while reducing inhibitory histone modification, H3K27me3. Immunofluorescence staining revealed a lower level of CDX2 expression in Caco‐2 cells with AMPK dominant negative mutant compared to that with AMPK WT.ConclusionAMPK enhances intestinal barrier function and epithelial differentiation through inducing the expression of CDX2.Support or Funding InformationNIH R15HD073864
Activation Of The Constitutive Androstane Receptor Enhances Wound Healing – A New Link Between Xenobiotic Sensing And Intestinal Mucosal Homeostasis?
Authors: Hudson Grace , Zamponi Alexandra , Alston Laurie , Chang Thomas , Hirota Simon Andrew 
Published: November 2023

BackgroundCompounds released from the intestinal microbiota may play a role in maintaining mucosal homeostasis, but little is known about the receptors that sense and respond to these compounds. Recently a cytosolic xenobiotic sensor, called the pregnane X receptor (PXR), was identified as a receptor for microbial metabolites, and shown to play a key role in regulating intestinal epithelial barrier and wound healing following injury. In the current study, we sought to determine whether the constitutive androstane receptor (CAR), which shares structural, functional and pharmacological features with the PXR, plays a role in regulating wound healing and mucosal repair.MethodsCaco‐2 intestinal epithelial cells were used to model wound healing. Wounds formed using Ibidi culture inserts were treated with CITCO, a selective human CAR agonist, and imaged for 24 hours. Wound closure was quantified and cell migration tracks assessed. To interrogate the intracellular events involved in the CITCO‐induced responses, we assessed the role of p38 MAPK and ERK1/2 signaling in our assays, along with proliferation and migration (xCELLigence impedance‐based migration assay). To determine the role of the CAR in vivo, we assessed recovery from dextran sulphate sodium (DSS)‐induced colitis (2.5% W/V in drinking water; 5 days exposure, followed by 9 days of recovery) in CAR‐deficient mice and WT mice treated with TCPOBOP (3 mg/kg; IP; daily during recovery phase), a selective rodent CAR agonist.ResultsActivation of the CAR enhanced wound closure in Caco‐2 monolayers, which was associated with increased cell migration, but not proliferation. The enhancement of cell migration was dependent on p38 MAPK signaling. Treating WT mice with a CAR agonist accelerated their recovery from DSS‐induced colitis. In contrast, CAR‐deficient mice exhibited delayed recovery.ConclusionsOur data suggest that the CAR regulates the cellular events that contribute to wound healing and mucosal restitution following injury. While the microbe‐derived compounds that activate the CAR have yet to be identified, its close relative, the PXR, has been shown to respond to such ligands. Taken together, we posit that xenobiotic receptors play a key role in regulating the response to injury and may provide new therapeutic targets to enhance mucosal repair.Support or Funding InformationCrohn's & Colitis Canada; Canadian Foundation for Innovation; The Dr. Lloyd Sutherland Investigatorship in IBD/GI Research
Chronic Alcohol Consumption Alters Rhythms in Key Enzymes and Transcription Factors Involved in Lipid Metabolism: Potential Role of the Circadian Clock
Authors: Valcin Jennifer Allison , Udoh Uduak Sunday , Swain Telisha , Bailey Shannon Marie 
Published: November 2023

Chronic alcohol consumption disrupts hepatic lipid metabolism causing fatty liver disease or steatosis. The molecular and cellular mechanisms responsible for alcohol‐induced fat accumulation in hepatocytes remain unclear. New studies indicate that many metabolic processes, including lipid metabolism, are influenced by the molecular circadian clock. At the core of this molecular oscillator are the two transcription factors BMAL1 and CLOCK that drive rhythmic expression of other clock and clock‐regulated metabolic genes. We previously reported that chronic alcohol consumption disrupts the function of the molecular circadian clock in the liver. However, the impact this has on downstream clock‐regulated pathways, like lipid metabolism, is unknown. The purpose of this study was to determine whether chronic alcohol disrupts clock‐controlled rhythmic expression of triglyceride metabolism genes and transcription factors that regulate lipid metabolism. For these studies, male hepatocyte‐specific BMAL1 knockout (HBK) mice and their wild‐type (WT) control littermates were kept under a 12:12 hour light‐dark cycle and pair‐fed control or alcohol‐containing diets for 5 weeks. Livers were collected at 4‐hour intervals for a 24 hour period and gene expression was measured by qPCR. In WT alcohol‐fed mice, we observed a dampening in the diurnal rhythms in several triglyceride metabolism genes. Alcohol significantly decreased the diurnal gene expression rhythm of patatin‐like phospholipase domain‐containing 3 (Pnpla3), an enzyme that mediates triglyceride breakdown. This result suggests that the alcohol‐mediated decrease in Pnpla3 may contribute, in part, to triglyceride accumulation in liver. Rhythmic expression of Pnpla3 was also completely abolished in liver of both control and alcohol‐fed HBK mice, suggesting a role of the clock in Pnpla3 regulation. The diurnal rhythm in acetyl CoA carboxylase 2 (Acc2) was also decreased in livers of WT alcohol‐fed mice with further dampening observed in livers of HBK alcohol‐fed mice. Interestingly, the diurnal rhythm of Acc2 was phase shifted in the HBK control‐fed compared to WT controls. In addition, diurnal rhythms in sterol regulatory element binding protein 1c (Srebp1c) and Acc1, both of which are involved in fatty acid synthesis, were also dampened in livers of WT and HBK alcohol‐fed mice. Overall, our findings indicate that chronic alcohol consumption disrupts normal diurnal rhythms of lipid metabolism processes, suggesting that alterations in the balance of these tightly regulated oscillations likely contribute to the alcoholic steatosis.Support or Funding InformationThis work is supported by NIH grant R01 AA020199
TRPV4 Modulates CYP2E1 Function in Nonalcoholic Steatohepatitis Progression
Authors: Seth Ratanesh Kumar , Das Suvarthi , Dattaroy Diptadip , Alhasson Firas A , Michelotti Gregory A , Nagarkatti Mitzi , Nagarkatti Prakash , Bell Phillip D , Liedtke Wolfgang B , Diehl Anna Mae , Chatterjee Saurabh 
Published: November 2023

Emerging evidence shows that oxidative stress via the activation of cytochrome p450 2E1 (CYP2E1) is key to the progression of inflammation in Nonalcoholic steatohepatitis (NASH). However, the molecular mediators and its pathways that regulate CYP2E1 mediated oxidative stress in NASH remain obscure. In the present study, the CYP2E1 substrate pyrazole or bromodichloromethane (BDCM) were used to induce CYP2E1 mediated oxidative stress, inflammation and NASH pathology in high‐fat diet fed mice. Results showed that the transient receptor potential vanilloid channel 4 (TRPV4) mRNA expression and protein levels were significantly elevated in parallel to increases in CYP2E1 and correlated well with increased lipid peroxidation, IL1β, MCP1, TNFα and HMGB1 levels in NASH livers and in Kupffer cells. TRPV4 knockout (KO) mice showed exacerbated inflammation, increased CYP2E1 protein (2 fold higher than wild type), lipid peroxidation (4‐Hydroxynonenal, 4‐HNE >4 fold), inflammatory cytokines (>25 fold), infiltration of leukocytes, sinusoidal endothelial dysfunction (SED) marker genes, HMGB1 levels, decreased phosphorylated endothelial nitric oxide synthase (pNOS3) and nitric oxide (NO) release. Mechanistically, diallyl sulfide (CYP2E1 inhibitor) or NO donor DETANONOate administration to TRPV4 KO mice completely abrogated enhanced inflammation and NASH pathophysiology. Use of NO donor significantly decreased CYP2E1‐mediated lipid peroxidation, proinflammatory genes and SED markers in TRPV4 KO mice suggesting that TRPV4 might regulate CYP2E1‐function via NOS3 phosphorylation and increased NO release. In vitro, CYP2E1‐mediated lipid peroxidation and levels of HMGB1, a damage‐associated molecular patterns (DAMPs) was increased in HepaRG cells in the presence of TRPV4 antagonist but had no significant effect on NO release. Since the CYP2E1‐mediated release of 4‐HNE and DAMPs from necrosed hepatocytes is known, the resident macrophages were primed with 4‐HNE. NOS‐3 phosphorylation, NO release and Phospho‐AKT levels from Kupffer cells were decreased with TRPV4 antagonist treatment while there was a concomitant increase in MCP‐1 and TNF‐α levels. Interestingly, Kupffer cells treated with TRPV4 agonist reversed decreased pNOS3 and pAKT levels. The results obtained shows that TRPV4 modulated CYP2E1‐mediated oxidative stress in hepatocytes, inflammation and endothelial injury by increasing NOS3‐mediated release of nitric oxide in the microenvironment via the AKT pathway. Based on the above, targeting TRPV4 or its downstream signaling cascade might be a promising therapeutic strategy in NASH.Support or Funding InformationThis study was supported by NIH pathway to independence award 7R00ES019875‐03 to Saurabh Chatterjee
Reduced Jejunal Basal Chloride Secretion In Diabetes Is Reversed By Genistein Diet
Authors: Al‐Nakkash Layla , Masood Faisal , Catmull Shawn 
Published: November 2023

Obesity and diabetes are often associated with disturbances in intestinal function, i.e. slowed gastrointestinal transit. The objective of this study was to characterize jejunal secretory function in a clinically relevant model of diabetes and obesity: the leptin‐deficient ob/ob mouse. We measured transepithelial short circuit current (Isc), across freshly isolated segments of jejunum from 11‐week old ob/ob and lean C57Bl/6J male and female mice. Separate segments of jejunum were frozen for western blot determination of key proteins involved in secretory transport. Basal Isc was significantly decreased in ob/ob female mice (82±7 μA/cm2, n=9, P<0.05) versus leans (107±14 μA/cm2, n=9), and genistein‐diet reversed this (112±12 μA/cm2, n=17, P< 0.05). Basal Isc was significantly decreased in ob/ob male mice (54±7 μA/cm2, n=11), versus leans (85±12, n=10, μA/cm2, P<0.05) and genistein reversed this (109±10 μA/cm2, n=7, P<0.05). Inhibition with clotrimazole and ouabain (both 100 μM, basolateral) was significantly reduced in all ob/ob mice compared to leans (P<0.05). Expression of total CFTR (normalized to GAPDH) was significantly decreased 2‐fold (P<0.05) in all ob/ob mice compared to leans. Our data suggests that the reduced basal jejunal Isc in all ob/ob mice is a consequence of reduced CFTR expression, decreased activity of the basolateral KCa channel and Na+/K+‐ATPase (males also exhibit reduced NKCC1 expression), each of which likely contributes towards this phenotype in jejunum of the ob/ob mouse. Genistein diet has beneficial effects on basal Isc: in males mediated by increased Na+/K+‐ATPase‐sensitive Isc and increased NKCC1expression, and, in females mediated by increased KCa‐sensitive Isc and increased adenosine expression. Improved understanding intestinal dysfunctions in the ob/ob jejunum, may allow for the development of novel drug targets to treat obesity and diabetes, and may also be of benefit in CF‐related diabetes.Support or Funding InformationSupported by: Soy Health Research Program, Diabetes Action Research and Education Foundation, and MWU intramural funds.
Disruption of Serotonin Signaling Results in Loss of Coordinated Tone Development and Subsequent Inhibition of Peristalsis in Guinea Pig Distal Colon
Authors: McGill Mahalia , Klinger‐Lawrence MaryBeth , Herrera Gerald 
Published: November 2023

This project was designed to examine the effects of the selective serotonin reuptake inhibitor, Fluoxetine, on colonic motility using the isolated guinea pig distal colon. The Gastrointestinal Motility Monitor system (“GIMM”, Catamount Research and Development Inc., St. Albans, VT) was used to analyze peristaltic motility during fecal pellet propulsion, as well as to conduct spatiotemporal analysis of motility patterns. Peristaltic motility in colonic segments was significantly slowed by Fluoxetine at concentrations of 20, 30 and 40 μM (p<0.05 vs. vehicle). Indeed, in colonic segments treated with 40 μM Fluoxetine, peristaltic motility was completely abolished (at least 1 of 3 trials with no peristaltic motility) in 4 out of 5 experimental preparations. This striking disruption in peristaltic motility by Fluoxetine was associated with a pronounced change in spatiotemporal motility patterns. In the presence of 40 μM Fluoxetine, average colonic diameter during the motility trial was substantially increased relative to pre‐treatment diameter (36.6 ± 21.5 % increase from baseline diameter, n=5). In vehicle‐treated colonic segments, average diameter remained relatively unchanged from pre‐treatment levels (0.5 ± 3.5 % change from baseline diameter, n=11). These findings suggest that serotonin signaling in the wall of the gut is required for normal peristaltic propulsive motility, and that disruption of serotonin signaling using Fluoxetine results in a loss of coordinated tone in the wall of the gut. Understanding the exact role of serotonin in normal gut motility is essential to gaining insights into novel therapeutic strategies for treating bowel dysfunction.Support or Funding InformationThis study was supported by Catamount Research and Development
Notch and mTOR Regulate Proliferation of Human Gastric Cancer Cells
Authors: Wong Gabriela , Demitrack Elise S , Samuelson Linda C 
Published: November 2023

BackgroundThe Notch signaling pathway regulates proliferation and differentiation of stem cells in the stomach. We have recently shown in the mouse that constitutive Notch activation stimulates proliferation of LGR5‐expressing antral stem cells and that inhibition of mTORC1 attenuates the Notch‐mediated increase in proliferation. The goal of this study was to test whether Notch and mTOR regulate proliferation of human gastric cancer (GC) cells.MethodsAGS, MKN45 and NCI‐N87 human GC cell lines were treated with vehicle (DMSO), DAPT (Notch inhibition), rapamycin (mTORC1 inhibition), or both for 5 consecutive days. Proliferation was measured each day using the CellTiter 96 Proliferation Assay. Gene expression analysis of Notch pathway components, and stem and differentiated cell markers was performed using qRT‐PCR.ResultsNotch inhibition did not significantly affect growth of AGS cells, but significantly inhibited growth of MKN45 and NCI‐N87 cells after 2 days of DAPT treatment. Rapamycin treatment significantly reduced proliferation of all 3 cell lines. Proliferation was further reduced in all 3 cell lines with combined Notch and mTOR inhibition, suggesting the necessity of both pathways to sustain GC cell growth. Gene expression analysis revealed that AGS and MKN45 GC cells express Notch1 and Notch2 receptors. In AGS cells, Notch inhibition reduced mRNA abundance of the stem cell marker and Notch target gene olfactomedin 4 (OLFM4). Furthermore, Notch inhibition in AGS cells significantly increased expression of the deep mucous cell markers TFF2 and SPDEF. These findings are in accordance with the role of Notch to stimulate proliferation and repress cellular differentiation in the mouse stomach.ConclusionNotch and mTOR are required to sustain growth of human GC cells in vitro. Further research to understand these mechanisms might benefit GC therapies by identifying specific pathways that directly impact cancer cell growth.Support or Funding InformationAPS Undergraduate Summer Research Fellowship; NIH F32‐DK093349; NIH P01‐DK062041 and NCI P50‐CA130810
The Impact of Dietary Soy on Gut Microbiome
Authors: Gill India R , Uno Jennifer K 
Published: November 2023

The metabolism of daidzein, a major compound of soy, produces equol in the presence of intestinal bacteria. Equol is more biologically active and its role in human health remains controversial. It has been shown to have links to tumor regression, tumor progression, cardiovascular health and neurologic function. Soymeal is a common product used in the mouse chow of research labs. Given its controversial role in host health, mouse facilities at the University of North Carolina at Chapel Hill switched to a non soy‐based diet. The purpose of this study is to examine the impact that the removal of soy has on bacterial communities. We expect to see a decrease in phyla such as bifidobacteria that play a role in the metabolism of isoflavones like daidzein. Mice were fed a soy‐based diet (Prolab Isopro RMH 3000) and a non soy‐based diet (Harlan Laboratories). Bacterial DNA was extracted from stool and phyla were examined and quantified by qPCR. In soy fed mice, we saw a slight decrease in universal gut bacteria as well as increases in bifidobacteria and lactobacillus. Bifidobacteria was the only phyla that changed significantly, with a 13.63 fold increase (n=7, p=0.05). In order to determine if soy is directly responsible for the microbial changes, we employed the use of zebrafish. Zebrafish were first treated with amoxicillin to clear bacterial flora and bacteria were allowed to recolonize in the presence of or absence of soy. We saw 12.5% and 13.5% decrease in lactobacillus and bifidobacteria, respectively (n=5, p=0.046 and p= 0.05). There was no significant change in total bacteria. This indicates that dietary soy can cause a significant change in the composition of the gut microbiome. This study warrants further research into the role soy plays in regulating microbial communities and emphasizes the need for diet regulation in research facilities.Support or Funding InformationElon University