bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR Cas9‐mediated deletion of Biliverdin reductase A (BVRA) in mouse liver cells models a broad spectrum of NAFLD Phenotype
Authors: Adeosun Samuel O. , Nwaneri Assumpta C. , Hinds Terry D. , Stec David E. 
Published: November 2023

Obesity is the leading cause of Non Alcoholic Fatty Liver Disease (NAFLD) which is also associated with insulin resistance and diabetes. NAFLD include a spectrum of pathologies which starts with fat accumulation (steatosis). The presence of a ‘second hit’ drives the continuum from steatosis to steatohepatitis, fibrosis, cirrhosis and liver failure/cancer. We previously showed that bilirubin treatment protects mice from hepatic steatosis. Biliverdin reductase (BVRA) ‐the key enzyme for bilirubin synthesis, also signals through MAPK/AKT which is an important insulin‐regulated pathway.The aim of this study was to test the hypothesis that BVRA protects against steatosis via regulation of intracellular fat processing through a combination of direct signaling and bilirubin generation.We used the CRISPR‐Cas9 system to delete/disrupt a 16.5kbp fragment between Exon1 and Exon 5 of the BVRA gene in a mouse liver cell line –Hepa 1c1c7 and studied NAFLD‐related phenotypes. Using a novel protein UnaG which fluoresces only when bound to bilirubin, we show that both intracellular bilirubin and conversion of biliverdin to bilirubin (BVRA activity) by live cells and by cell lysates were markedly reduced in these BVRA‐KO cells. With no treatment, these cells recapitulated the transcriptomic signature of drug‐induced steatosis as FOXA1, HHEX and SREB1F –genes associated with fatty acid processing, were changed in a manner that favors fat accumulation. Consequently, when exposed to free‐fatty acids, Nile‐Red staining coupled with flow cytometry showed that BVRA‐KO cells accumulate 42% more fat (p=0.007) and 100% more triglycerides (p=0.002) compared to wildtype cells. They also showed a blunting of insulin‐stimulated AKT phosphorylation (up to 41% less) compared to wildtype cells. Lastly, untreated BVRA‐KO cells express significantly higher levels (1.7–3.2 folds) of markers of fibrosis versus wildtype cells.In conclusion, our data shows that BVRA plays a critical role in protecting against lipid accumulation and in the pathophysiology of NAFLD. Studies are on‐going to determine the independent contributions of the signaling and reductase activities of BVRA and thus to identify the best BVRA‐directed therapeutic target. Also, given that the deletion of this important gene required no [additional] second‐hit to precipitate phenotypes like fibrosis which represents a later stage in the NAFLD continuum, these cells may be a novel in‐vitro model for studying a broader spectrum of NAFLD phenotypes.Support or Funding InformationNIH grant PO1HL 51971 and P20GM‐104357
Western diet feeding downregulates hepatic RECK expression and induces NASH with fibrosis
Authors: Cunningham Rory , Sheldon Ryan , Meers Grace , Kandikattu Hemanth Kumar , Chandrasekar Bysani , Rector Randy Scott 
Published: November 2023

Nonalcoholic steatohepatitis (NASH) is characterized by increased accumulation of fat in liver, along with inflammation and fibrosis. During liver fibrosis development, there is a shift in the intrahepatic balance where extracellular matrix accumulation exceeds its degradation, resulting in excessive collagen deposition. Reversion‐inducing‐cysteine‐rich protein with Kazal motifs (RECK) is a novel membrane‐anchored protease inhibitor. While its expression and protective role in myocardial fibrosis are reported, its expression and role in the development and progression of hepatic fibrosis are not known. Here we examined the effects of short‐term (6 weeks) and long‐term (18 weeks) western diet (WD; 44.9% fat; 17% sucrose; 1% cholesterol) feeding on hepatic RECK expression and fibrosis development in male C57/BL6 wild‐type (WT) mice. Six weeks of WD feeding resulted in significant hepatic steatosis and mild inflammatory cell infiltration, but not hepatic fibrosis. This histological presentation occurred in conjunction with significantly elevated hepatic RECK mRNA expression and protein content (p<0.05). However, following 18‐weeks of WD feeding (versus low‐fat control diet; 10% fat), RECK mRNA and protein expression was dramatically reduced by ~50% in the liver, despite no significant changes in hepatic SP‐1, SHP, or FXR mRNA expression. Importantly, reduced RECK expression was associated with (i) significant hepatic steatosis, (ii) histological increases in hepatic immune cell infiltration and hepatic fibrosis, and (iii) elevated expression of key genes involved in hepatic fibrogenesis (Tgfb1 and Col1α1), total macrophage content (Emr1), M1 macrophage markers (Tnfα, Cd11c, Nos2), cellular adhesion (Selectin E, Icam, Ccl2, Ccr2), and extracellular matrix regulators (TIMP‐1, MMP‐13, and MMP‐2). These results demonstrate that the progression of western diet‐induced NASH with fibrosis is associated with suppressed RECK and elevated pro‐fibrotic mediators in the liver. These results suggest that overexpression or induction of RECK may have therapeutic potential in hepatic fibrogenesis. Our future studies will determine this possibility using a transgenic mouse model with liver‐specific overexpression of RECK.Support or Funding InformationThis work was supported by a Veterans Affairs VHA‐CDA2 grant (IK2BX001299).
Palmitate Increases Protein Palmitoylation in Liver Cells
Authors: Estrada Andrea L , Stewart Claire M , Kim Paul Y , Wang Dong , Wei Yuren , Pagliassotti Michael 
Published: November 2023

PurposeDisruption of endoplasmic reticulum (ER) homeostasis, or ER stress, is a characteristic feature of non‐alcoholic fatty liver disease (NAFLD). Elevated fatty acids, in particular saturated fatty acids, induce ER stress in numerous cell types including hepatocytes. However, the specific mechanisms that link fatty acids to ER stress in the liver are poorly understood. The ER is responsible for numerous post‐translational modifications including palmitoylation. Protein palmitoylation plays a crucial role in protein stability, function, and subcellular location. The aim of this study was to assess whether palmitate selectively increases protein palmitoylation in liver cells.MethodsH4IIE liver cells (n=3) were treated with control media or control media supplemented with palmitate (250μM), a saturated fatty acid that induces ER stress. Treatments were administered in the absence or presence of bromopalmitate (200μM), a potent inhibitor of palmitoylation, for 6 hours. In order to characterize the palmitoylated proteome, we utilized an acyl‐biotin exchange technique to capture palmitoylated proteins. The protein lysate was separated via SDS‐PAGE, and probed for using a fluorophore‐conjugated streptavidin antibody.ResultsPalmitate increased palmitoylation by approximately 20% compared to control. Bromopalmitate restored protein palmitoylation in palmitate treated cells to levels observed in control. Preliminary results suggest that the presence of bromopalmitate reduced palmitate‐induced X‐box binding protein 1 splicing (a marker of ER stress). In conclusion, palmitate increased protein palmitoylation over a time course that coincides with ER stress.Support or Funding InformationNIH: DIK072017
miR‐195 and RNA‐binding protein CELF1 jointly regulate translation of IGF2 receptor in intestinal epithelium
Authors: Zhang Yuan , Chung Hee Kyoung , Xiao Lan , Wu Jing , Turner Douglas J. , Jaladanki Rao N. , Wang Jian‐Ying 
Published: November 2023

Insulin‐like growth factor‐2 receptor (IGF2R) specifically binds to the IGF‐2 and its activation is crucial for stimulation of mucosal regeneration after small bowel resection, but the exact mechanism underlying the control of cellular IGF2R abundance remains largely unknown. microRNAs and RNA‐binding proteins (RBP) regulate the stability and translation of target mRNAs and are involved in many aspects of cellular functions. This study tested the hypothesis that miR‐195 and RBP CELF1 (also named as CUG‐binding protein 1) modulate IGF2R expression posttranscriptionally.MethodsStudies were conducted in intestinal epithelial cells (IECs) and mice.ResultsmiR‐195 directly interacted with the Igf2R mRNA via its 3′‐untranslated region (3′‐UTR) rather than its 5′‐UTR and coding region, and ectopically expressed miR‐195 repressed IGF2R translation without effect on its mRNA stability in cultured IECs. In contrast, intestinal epithelial tissue‐specific miR‐195 knockout in mice increased levels of IGF2R expression in the small intestinal mucosa, which was associated with an increase in mucosal growth. Moreover, CELF1 also associated with the Igf2R 3′‐UTR and inhibited IGF2R translation. Further studies showed that miR‐195 and CELF1 repressed IGF2R translation synergistically. Functionally, decreased IGF2R by miR‐195 altered the structure and homeostasis of Golgi apparatus, since there were significant redistribution of syntaxin 6 (Golgi structural protein) at Golgi network and endosomal compartments following reduction in IGF2R by miR‐195.ConclusionmiR‐195 and CELF1 repress IGF2R translation synergistically, thus down‐regulating intestinal epithelial renewal by altering Golgi stability and functions.Support or Funding InformationDepartment of Veterans Affairs (RNJ, DJT, JYW) and NIH (JYW)
Expression and localization of the major receptor of vasoactive intestinal peptide (VPAC1) along the length of the intestine
Authors: Jayawardena Dulari , Guzman Grace , Gill Ravinder , Alrefai Waddah , Onyuksel Hayat , Dudeja Pradeep 
Published: November 2023

Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide with a broad array of physiological functions in the nervous system and peripheral organs including the intestine. This peptide exerts its action via binding to VPAC1, VPAC2 and PAC1, which are G‐protein coupled receptors (GPCR). VPAC1 is the major receptor constitutively expressed in many cell types and is responsible for majority of VIP's biological activity. However, the distribution of VPAC1 along the length of the gastrointestinal tract and its sub cellular localization in intestinal epithelial cells has not been fully defined. Therefore, the current studies were undertaken to determine VPAC1 distribution and localization so that VIP based therapies can be targeted to specific regions of the intestine.MethodsReal time PCR, immunoblotting and immuno‐fluorescence staining were used to determine the mRNA, protein and cellular localization of VPAC1 respectively. Intestinal tissues from male C57Bl/6 mice (6–8 weeks) and human tissues as well as colonic adenocarcinoma cell lines were used. The results indicated that the mRNA levels of VPAC1 in mouse intestine were highest in colon followed by jejunum and ileum. In parallel, the VPAC1 protein levels were higher in the mouse colon (proximal > distal) followed by the jejunum and ileum. Immuno‐fluorescence studies in mouse colonic tissue sections demonstrated that the receptor was specifically localized to the luminal surface as evident by co‐localization of the VPAC1 protein with the apical marker villin but not with the basolateral marker sodium potassium ATPase. In the human intestine, VPAC1 mRNA expression exhibited an expression pattern similar to mouse intestine (colon > jejunum> ileum). Furthermore, in human colon, VPAC1 predominantly showed apical localization as observed by immuno fluorescence staining. Western analysis demonstrated almost equal protein levels of VPAC1 in human adenocarcinoma cell lines e.g. Caco2, T84, HT‐29 and SKC015 cells. The luminal localization was further confirmed by relatively high apical staining for VPAC1 in differentiated Caco2 monolayers. Our current data showed differential expression of VPAC1 along the length of intestine and demonstrated its localization on the luminal membrane of colonocytes. The physiological relevance of this expression and apical localization of VPAC1 remains elusive. We speculate that apical localization of VPAC1 in intestinal epithelial cells has physiological relevance in recognizing secreted peptides in the intestinal lumen and supports the potential therapeutic use of VIP nanomedicine via oral route to target gut inflammation.Characters with spaces: 2901Support or Funding InformationSupported by NIDDK and VA Merit
Gene Expression of the GIP and GLP‐1 Receptors in the Horse
Authors: Kheder Murad H. , Dudley Kevin , Sillence Martin N. , de Laat Melody A. 
Published: November 2023

IntroductionThe Glucagon‐like peptide‐1 (GLP‐1R) and gastric insulinotropic polypeptide (GIPR) receptors are members of the secretin family of G‐protein coupled receptors that regulate insulin secretion from pancreatic beta cells. Both receptors are well studied in rodents and humans, but there are limited data available for horses. In horses, GIP and GLP‐1 contribute to hyperinsulinaemia, which causes a painful foot disease called laminitis.AimThis study aimed to determine whether the GIPR and GLP‐1R genes are expressed in equine tissues.MethodsArchived tissue samples (pancreas, skeletal muscle, heart, liver, kidney, digital lamellae (which is the organ affected by laminitis), tongue and small intestine) from healthy horses were subject to mRNA extraction and cDNA synthesis prior to primers design and PCR. Sequencing of the PCR product was undertaken to confirm amplification of the target gene.ResultsBoth the GIPR and GLP‐1R were expressed in the pancreas as expected, and this tissue was used as a positive control for tissue distribution studies. The GIPR was expressed in the heart, liver, kidney and small intestine. The GLP‐1R gene was expressed in all tissues except the tongue and small intestine. Sequence identity to the Equus caballus mRNA sequence available from GenBank was 98% for GIPR and 100% for GLP‐1R.DiscussionThese data indicate that GIPR and GLP‐Rs may be potential therapeutic targets for the treatment/prevention of hyperinsulinaemia and laminitis in horses. The expression of both receptors in tissues other than the pancreas suggests that they may have other physiological functions and this requires investigation.Support or Funding InformationAustralian Research Council (ARC)
FFA2‐mediated 5‐HT release increases HCO3− secretion, but induces mucosal injury in rat duodenum
Authors: Maruta Koji , Akiba Yasutada , Kaji Izumi , Kaunitz Jonathan D 
Published: November 2023

Although serotonin (5‐HT) is an important mediator of ion secretion and propulsive motility, excessive 5‐HT release induces emesis and diarrhea, treatable with 5‐HT3 antagonists. The short‐chain fatty acid receptor FFA2 is expressed on 5‐HT synthesizing enterochromaffin (EC) cells. The selective FFA2 agonist phenylacetamide‐1 (PA1) increases the rate of duodenal HCO3− secretion (DBS) via 5‐HT4 receptor activation. We hypothesized that EC cell FFA2 activation increases 5‐HT release, affecting duodenal mucosal defenses under physiological and pathological conditions. We measured DBS in vivo in a PA1‐perfused duodenal loop, measuring 5‐HT released into the portal vein (PV) with or without the selective FFA2 antagonist GLPG0974 (0.1 – 10 μM). Intestinal injury was induced by indomethacin (IND) with or without PA1 (0.1 – 1 mg/kg, ig), GLPG0974, the 5‐HT3 receptor antagonist ondansetron (Ond), the 5‐HT4 receptor antagonist RS23597. Luminal perfusion with PA1 (1 μM and 0.1 mM) similarly augmented DBS, whereas 5‐HT release into the PV was 8‐fold higher in response to 0.1 mM PA1 compared with 1 μM. GLPG0974 dose‐dependently inhibited PA1‐induced DBS increase with corresponding reductions of 5‐HT release. IND alone induced small intestinal ulcers with duodenal sparing, whereas PA1 + IND dose‐dependently induced duodenal lesions. PA1 + IND‐induced duodenal lesions were inhibited by GLPG0974 and Ond, but not affected by RS23597. These results suggest that physiological 5‐HT concentrations released into the PV in response to FFA2 activation enhance duodenal mucosal defenses, whereas excessive 5‐HT release induced by FFA2 overactivation disrupts defenses via 5‐HT3 activation, injuring the duodenal mucosa during COX inhibition. FFA2 overactivation may be implicated in the genesis of symptoms such as emesis, bloating, and diarrhea.Support or Funding InformationSupported by VA Merit Review, NIH R01 DK54221
GLP‐2 enhances duodenal SGLT1‐mediated glucose absorption via EGF signaling
Authors: Kaji Izumi , Akiba Yasutada , Kaunitz Jonathan D. 
Published: November 2023

Glucagon‐like peptide 2 (GLP‐2) released from enteroendocrine cells has trophic, barrier strengthening, and anti‐inflammatory effects on the gut mucosa. Chronic administration of GLP‐2 accelerates the rate of jejunal glucose absorption through upregulation of sodium‐dependent glucose transporter (SGLT)1 expression. Nevertheless, glucose absorption by duodenal epithelial cells in response to the acute administration of GLP‐2 has not been investigated. We assessed SGLT1‐mediated electrogenic glucose absorption by measuring short‐circuit current (Isc) in Ussing chambers in mucosa‐submucosal preparations of rat distal duodenum and in full‐thickness preparations of mouse duodenum. GLP‐2 receptor (GLP‐2R) localization was confirmed by immunohistochemistry in frozen duodenal sections. Mucosal application of glucose (10 mM) increased Isc in muscle‐stripped mucosa‐submucosal preparations of duodena from overnight‐fasted rats, but had no effect after feeding. This glucose‐evoked Isc was completely reversed by the SGLT inhibitor phloridzin, consistent with electrogenic SGLT1‐mediated absorption. Full‐thickness duodenum from fed mice responded to mucosal glucose, accelerated by 4‐h fasting. Pretreatment with GLP‐2 (20 – 100 nM) for 15 min significantly enhanced the glucose‐induced increase in Isc. GLP‐2 did not alter electrogenic Cl− secretion evoked by serosal nicotine (0.1 mM), bethanechol (1 mM), or forskolin (1 μM) in rat and mouse duodenum. The enhancement of the glucose absorptive Isc was mimicked by mucosal incubation with epidermal growth factor (EGF, 50 ng/ml) for 15 min, abrogated by the tyrosine kinase inhibitor AG18 (0.1 mM). Since GLP‐2R is not expressed on duodenal epithelial cells, GLP‐2‐mediated EGF release from interstitial cells with consequent activation of epithelial ErbB (EGF receptor) may underlie the acute alteration of SGLT1 activity. GLP‐2R immunoreactivity was detected in vasoactive intestinal peptide (VIP)‐containing myenteric neurons in the duodenum, whereas a selective antagonist for VIP receptors (VPAC1) (1 μM) had no effect on GLP‐2‐induced enhancement of glucose absorptive Isc. In full‐thickness preparations with myenteric neurons from mouse duodenum, the nitric oxide synthase inhibitor L‐NAME (0.1 mM) or tetrodotoxin (1 μM) had no effect on GLP‐2‐enhanced glucose‐induced Isc, inconsistent with mediation of GLP‐2‐related acute acceleration of glucose absorption via SGLT1 by a neural pathway. These findings suggest that the GLP2R‐EGF pathway in duodenal epithelial cells acutely affects nutrient absorption, another important and novel effect of GLP‐2.Support or Funding InformationSupported by VA Merit Review, NIH R01 DK54221
Utilizing Shared Virtual Microscopy Slides to Create Innovative Anatomical Science Resources in an Integrated Curriculum
Authors: Goldman Haviva M. 
Published: November 2023

The shift towards integrated medical education curricula and digital imaging has transformed the teaching of histology, leading to new challenges and opportunities for the discipline (Drake et al, Anat Sci Educ 7:321–5, 2014). Educators face limited curricular time and support for histology laboratories, and a demand for integrated, interactive and clinically applied learning activities. Virtual microscope technology has already transformed the histology laboratory, providing a high quality and flexible alternative to microscopes. A shared virtual microscopy database provides a resource for educators that can be used to create customized content that can readily be adapted towards an integrated and applied curriculum.Drexel University College of Medicine is moving towards an integrated curriculum where much of the course content is delivered electronically. We utilize our University's virtual microscopy collection within our customized online independent‐learning modules. Slides have been annotated to demonstrate important structures so that students can learn on their own using independent learning modules. These are then supplemented with images from shared virtual microscope collections (for example University of Michigan and University of Iowa collections, and now numerous others via the Virtual Microscopy Database) as mystery slides in live sessions. Several examples of teaching modules and in‐class exercises that utilize shared microscopy resources will be demonstrated during this presentation. These include tutorials, review exercises and clinical application exercises integrating histology with gross anatomy, radiology, neuroscience and embryology, as well as pathology. Through the creation and use of these innovative resources we are able to ensure that histology remains the important bridge it always has been, providing a linkage between gross morphology, physiological function and the biochemical and molecular basis of disease.
Intestinal Dysbiosis During Neonatal Development Alters the Microbiota‐Gut‐Brain Axis in Adulthood
Authors: Hennessey Carly H , Sladek Jessica , Miller Elaine , Kim Janis , Kaur Manvir , Gareau Melanie G 
Published: November 2023

The neonatal period of life is critical for bacterial colonization of the gastrointestinal (GI) tract, formation of GI mucosal barrier function, and neural development. Previous studies from our group have shown that infection of adult mice with a bacterial pathogen causes changes in the microbiota‐gut‐brain (MGB) axis, including intestinal dysbiosis and behavioral deficits. However, the relationship between intestinal dysbiosis during the early stages of development and cognitive and behavioral function in adulthood have not been investigated. We hypothesized that dysbiosis due to bacterial infection in early life would lead to a dysregulated MGB axis with lasting deficits into adulthood. A mouse model (C57BL/6) of neonatal bacterial infection with enteropathogenic Escherichia coli (EPEC; strain e2348/69) was used to study effects of intestinal dysbiosis on the MGB axis. Behavior was assessed using the light/dark box (anxiety‐like behavior) and the novel object recognition (NOR) task (cognitive function). The microbiota was characterized by qPCR, and Ussing chambers were used to study intestinal physiology. Mice infected with EPEC as neonates showed impaired memory as adults (p<0.05), without evidence of anxiety‐like behavior compared to sham‐infected controls. This was accompanied with down‐regulation of innate immune pattern recognition receptors (Nod1 and Nod2) and regulatory cytokines (IL10), coupled with up‐regulation of pro‐inflammatory cytokines (TNF‐α) and the serotonin biosynthesis pathway (Tph) in the hippocampus (p<0.05). Neonatal infection resulted in long‐lasting intestinal dysbiosis (decreased Lactobacillus, increased Enterobacteriaceae) and intestinal pathophysiology characterized by increased secretory state (short circuit current; Isc) and permeability (conductance; G) (p<0.05). Transcriptional analysis of the colon showed down‐regulation of protective gut factors (RegIIIγ) and increased NF‐κB activation in adulthood (p<0.05). In conclusion, our data demonstrates that neonatal bacterial infection leads to alterations of the development of the MGB axis that extend through to adulthood. These findings may have important clinical implications for pediatric patients exposed to bacterial enteric pathogens during early development.Support or Funding InformationThis work was funded by an APS undergraduate summer research fellowship (JK); NIH 5R21MH108154‐01 (MGG), U54 HD079125 (MGG).