bluuebunny/crossref_metadata_2025_split huggingface dataset:
Altered Lipid and Glucose Homeostasis in Mice with Liver‐Specific Deletion of Protein Kinase Cbeta
Authors: Mehta Kamal D , Hassan Faizule , Coppola Vincenzo , Shu Yaoling 
Published: June 2021

It is widely recognized that a Western‐style diet increases the risk of non‐alcoholic fatty liver disease (NAFLD) development and its subsequent progression to NASH. Several mechanisms have been proposed to explain the dramatic inducing effect of dietary cholesterol on the progression of inflammation and apoptosis/necrosis of hepatocytes and nonparenchymal cells. Some of these mechanisms are related to dysregulational lipid homeostasis, mitochondrial dysfunction, and oxidative stress. However, the diet‐induced signaling events integrating with the mechanistic processes driving lipid accumulation, mitochondrial dysfunction, and inflammation in non‐alcoholic steatohepatitis (NASH) are not well understood. We recently reported that PKCβ can coordinate multiple sequential and parallel pathways that cooperatively trigger dysregulation of triglyceride, cholesterol and glucose homeostasis, mitochondrial signaling and autophagy, thus can drive critical events in the genesis and progression of NAFLD/NASH. Furthermore, PKCβ has also been reported to be involved in the activation of two main downstream pathways, nuclear factor‐κB and C‐Jun N‐terminal kinase. Consistent with the role of PKCβ in metabolism, systemic PKCβ deficiency protected mice from diet‐induced adiposity, hepatic steatosis and insulin‐resistance. The goal of this study is to define the individual contribution of hepatic PKCβ in the control of hepatic metabolism. To accomplish this, we generated mice specifically lacking the expression of PKCβ in the liver, which represents the first tissue‐specific deletion of this kinase. We compared body compositions, hepatic lipids, insulin sensitivity, glucose tolerance, and insulin signaling responses between high‐fat/high‐cholesterol‐fed control PKCβflox/flox and PKCβLiv−/− mice. We found that hepatocyte‐specific PKCβ deficiency protects from diet‐induced adiposity, with modest alterations in glucose homeostasis. Our data establish that hepatocyte‐restricted PKCβ deletion is sufficient to protect from steatosis, emphasizing this kinase's relevance as a drug target in NAFLD. Hence, hepatocyte PKCβ is a critical hub for integrating nutritional inputs with downstream metabolic events.Support or Funding InformationNIH R01 HL138198This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genistein, daidzein, and resveratrols stimulate PGC‐1β‐mediated gene expression
Authors: Kamei Yasutomi , Uchitomi Ran , Nakai Shiho , Matsuda Rintaro , Onishi Takumi , Hatazawa Yukino 
Published: June 2021

PGC‐1β is a transcriptional co‐activator of nuclear receptors such as the estrogen receptor‐related receptor (ERR). Transgenic overexpression of PGC‐1β in mice increases energy expenditure and suppresses high‐fat diet‐induced obesity. In this study, we screened various food‐derived and natural compounds using a reporter assay system to measure the transcriptional activity of PGC‐1β. Soy‐derived isoflavones, genistein and daidzein, and several resveratrols activated PGC‐1β. Genistein, daidzein, and trans‐oxyresveratrol activated ERR‐responsive element‐mediated reporter activity in the presence of PGC‐1β. Stable overexpression of PGC‐1β in C2C12 myoblasts increased the expression of medium‐chain acyl‐CoA dehydrogenase (MCAD), an important enzyme in fatty acid β‐oxidation. Genistein and daidzein increased MCAD mRNA levels and mitochondrial content in PGC‐1β‐expressing C2C12 cells. These compounds activated ERR/PGC‐1β complex‐mediated gene expression, and our findings may be a practical foundation for developing functional foods targeting obesity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Characterization of Leucine‐Rich Repeat Containing 8 A (LRRC8A) Homomeric Channel
Authors: Yamada Toshiki , Denton Jerod S. , Strange Kevin 
Published: June 2021

Regulation of cell volume in response to osmotic perturbations is an essential cellular process. The volume‐regulated anion channel (VRAC) is ubiquitously expressed in vertebrate cells and mediates the swelling‐induced release of chloride and organic solutes. VRAC is encoded by LRRC8 gene family members, LRRC8A‐E. VRAC is a heteromeric channel comprising an essential LRRC8A subunit plus at least one other LRRC8 member. Several high‐resolution cryo‐EM structures of the LRRC8A homomeric channel have recently been published. However, until now, the detailed functional and regulatory properties of the LRRC8A homomeric channel have not yet been characterized. Here, we show that HCT116 cells in which all five Lrrc8 genes have been knocked out produce a small whole‐cell current (~20 pA/pF at +100 mV) when transfected with a plasmid encoding human LRRC8A. In striking contrast to the heteromeric VRAC current, the LRRC8A current is not activated by cell swelling or inhibited by cell shrinkage. However, when cells are patch clamped with an intracellular solution of low ionic strength, the current exhibits slow and modest activation, and is dramatically activated by cell swelling. The degree of swelling‐induced LRRC8A current activation is directly and dose‐dependently related to intracellular ionic strength. Consistent with the heteromeric VRAC current, swelling‐induced activation of LRRC8A current is prevented by removing intracellular ATP. We are currently evaluating the anion selectivity and pharmacological properties of LRRC8A. Our studies provide important new insights into the functional and regulatory properties of the LRRC8A homomeric channels.Support or Funding InformationThis work was funded by National Institutes of Health grant R01DK051610 to K.S.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of exogenous ceramide on barrier function of human airway epithelial cells
Authors: Kalsi Kameljit Kaur , Jackson Sonya , Baines Deborah Louise 
Published: June 2021

Maintaining the integrity of the airway epithelial barrier is essential in preventing increased permeability to harmful molecules and pathogens, which may lead to increased risk of developing respiratory infection and disease. We have previously shown that in human airway epithelial cells metformin increases tight junction (TJ) assembly after pathogen disruption by promoting the abundance and assembly of full length occludin, and that this process involves phosphorylation of occludin via an AMPK‐PKCζ pathway. During bacterial infection alpha toxin activates the acid sphingomyelinase/ceramide system which can cleave membrane proteins releasing ceramide to form ceramide rich domains, this signals the reorganization of receptors and activation of protein kinase C isoforms such as PKCζ. This study aimed to investigate the effect of N‐Palmitoyl‐d‐erythro‐sphingosine (C16:0 ceramide) on epithelial integrity of human airway epithelial cells (H441 cells). TJ assembly was measured after calcium switch assay by measuring the transepithelial electrical resistance (TEER) and paracellular transport of sodium‐fluorescein which was used as an index of permeability (Papp) in the presence of 0.1 μM or 1 μM ceramide. We found that after 24h there was 11% change in TEER (138 ± 15 vs 125 ± 14 Ω/cm2, P = 0.34; n=8) and 10% change in Papp (16 ± 6 vs 18 ± 7 cm/s x 10−6, P = 0.19; n=3) with 0.1 μM ceramide compared to control. No changes were observed with 1 μM ceramide. Immunofluorescence staining for occludin, claudin‐1 or claudin‐3 did not show a difference in localisation with ceramide treatment compared to control untreated cells. These results indicate so far that in airway epithelial cells activation of PKCζ by ceramide only evokes a minor effect on tight junction reassembly or permeability.Support or Funding InformationAstraZeneca R&DThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced Exosome Release and Inhibited TRPML1 Channel Activity in Podocytes from Mice with Podocyte‐Restricted Deletion of Asah1 Gene
Authors: Li Guangbi , Zhang Qinghua , Yuan Xinxu , Li Pin‐Lan 
Published: June 2021

The ceramide hydrolysis by lysosomal acid ceramidase (AC) has been reported to regulate lysosome function, which determines the degradation of multivesicular bodies. In this regard, we found that urinary exosome excretion was remarkably increased in podocyte‐specific AC gene knockout (Asah1fl/fl/Podocre) mice. Given the importance of transient receptor potential mucolipin 1 (TRPML1) channel‐mediated Ca2+ release to lysosome function and its regulation by sphingolipids, the present study tested a hypothesis that podocyte‐specific Asah1 gene deletion inhibits TRPML1 channel activity, leading to lysosome dysfunction and exosome release from podocytes. It was found that exosomes released from Asah1fl/fl/Podocre podocytes were remarkably higher than WT/WT podocytes as detected by nanoparticle tracking analysis. Super‐resolution microscopy showed less fusion of multivesicular bodies and lysosomes in Asah1fl/fl/Podocre podocytes compared with WT/WT podocytes. Using GCaMP3 Ca2+ imaging, it was found that ML‐SA1 as a specific cell‐permeable TRPML1 inducer remarkably increased Ca2+ release from lysosomes by 7.35 folds in WT/WT podocytes. Pre‐treatment of podocytes with carmofur, a specific AC inhibitor almost totally blocked ML‐SA1‐induced Ca2+ release in WT/WT podocytes. In Asah1fl/fl/Podocre podocytes, however, ML‐SA1 was found to have no effects on TRPML1 channel‐mediated Ca2+ release, but sphingosine as an AC product of ceramide hydrolysis remarkably increased TRPML1 channel‐mediated Ca2+ release in both WT/WT podocytes (5.42 folds) and Asah1fl/fl/Podocre podocytes (4.52 folds). Using whole‐lysosome patch clamp recording, we detected ML‐SA1‐induced Ca2+ currents in a dose‐dependent manner through the membrane of lysosomes isolated from WT/WT podocytes. Compared to WT/WT podocytes, intact lysosomes isolated from Asah1fl/fl/Podocre podocytes had evidently blunted opening of TRPML1 channels in response to its agonist, ML‐SA1. AC associated sphingolipids, sphingomyelin, ceramide and sphingosine had different effects on TRPML1 channel activity in WT/WT podocytes, with inhibition by sphingomyelin, no effect from ceramide, but enhancement by sphingosine (2.25 folds). In Asah1fl/fl/Podocre podocytes, sphingosine remained to increase ML‐SA1‐induced TRPML1 channel‐mediated Ca2+ release (3.96 folds). These results suggest that the AC deficiency decreases TRPML1 channel activity due to reduction of sphingosine production, which may contribute to lysosome dysfunction and enhanced exosome release.Support or Funding InformationThis study was supported by NIH grants DK54927, DK120491, and HL057244.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Depolarization of mitochondrial membrane by H2O2 regulates Ca2+ signalling in Endothelial Cells in Intact Blood Vessels
Authors: Zhang Xun , Wilson Calum , Lee Matthew , McCarron John G 
Published: June 2021

Hydrogen peroxide (H2O2) is a key mitochondrial derived reactive oxygen species (ROS) that acts as a signalling hub in regulating signalling transduction, vasocontraction and vasodilation. Although the physiological roles of ROS in mechano‐stress signal transduction, vascular relaxation and permeability of endothelial cells is acknowledged, there is uncertainty on the mechanisms by which how H2O2 regulates endothelial signalling. In the present study, the effects of H2O2 on Ca2+ signalling and on vasocontraction and vasodilation were investigated in the endothelium of intact rat mesenteric arteries. Endothelium‐dependent vasodilation of intact mesenteric arteries to acetylcholine (ACh) was suppressed by H2O2 (100 μM) and a greater vasoconstriction to phenylephrine occurred. To investigate the underlying endothelium‐dependent mechanisms of H2O2 on vascular response, intracellular Ca2+ signalling in endothelial cells of intact arteries was visualized and recorded with high‐speed and spatial‐resolution. H2O2 (100 nM – 1 mM) alone did not significantly alter basal Ca2+ activity in endothelial cells. However, H2O2 inhibited acetylcholine‐evoked Ca2+ signalling as revealed by decrease in the amplitude of the Ca2+ signal in each activated cell and a reduced number of cells activated. H2O2‐evoked inhibition of Ca2+ signalling was reversed by catalase (1000 U/ml). To determine if the effects of H2O2 were explained by decreased IP3 production or decreased IP3‐evoked Ca2+ release, photoactivatable caged‐inositol 1,4,5‐trisphosphate (cIP3) was used to bypass the synthesis of IP3. Ca2+ release evoked by photolysis of cIP3 supressed by 100 μM H2O2. These results suggest that H2O2 may alter the activity of IP3 receptors. One mechanism by which this may occur is by H2O2 altering mitochondrial activity. Mitochondria exert a profound control of IP3‐evoked Ca2+ release. Acetylchonline‐evoked Ca2+ release was inhibited by proton uncoupler carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) and rotenone, a mitochondrial complex inhibitor. H2O2 (100 μM) depolarized mitochondria membrane potential as measured by the mitochondrial membrane potential sensitive dye, TMRE. The depolarization of mitochondria decreased the organelle's ability to produce ATP and sequester Ca2+ from the local microdomain near IP3 receptors. A higher local concentration of Ca2+ around IP3 receptors inhibited Ca2+ release from endoplasmic reticulum. In summary, H2O2 inhibit Ca2+ release from the internal store in endothelial cells by depolarizing mitochondrial membrane potential to suppress vasodilation in intact mesenteric arteries.Support or Funding InformationWellcome Trust and British Heart FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fecal metabolomic response of IL‐10‐deficient mice supplemented with Goji
Authors: Wang Hongbin , Zhang Shuming , Zhu Meijun 
Published: June 2021

BackgroundChronic gut inflammation is an important risk factor for colorectal cancer (CRC). Our recent study suggests that dietary Goji reduces gut inflammation and increases Bifidobacterium and butyrate‐producing bacteria in interleukin (IL)‐10‐deficient mice. We hypothesized that enhanced Bifidobacterium and other probiotics associated with Goji supplementation alter gut metabolites, which exert beneficial effects to host epithelial cells.MethodsThe GC‐MS analysis was used to profile the fecal metabolomic changes due to Goji supplementation to uncover the potential mechanisms underlying the preventive effects of Goji supplementation on inflammatory bowel diseases.ResultsThirty‐four compounds were altered due to Goji supplementation, including polyunsaturated long‐chain fatty acids (PUFAs), sugar and sugar alcohols, benzene derivatives, pyridines derivatives, dicarboxylic acids, short‐chain hydroxy acids. Pathway analysis showed that the α‐linolenic acid and linoleic acid metabolism were dramatically affected by dietary Goji intake. Four ω‐6 PUFAs including linoleic acid and arachidonic acid were reduced due to Goji supplementation. Fecal levels of most amino acids, positively correlated with gut inflammation and CRC risk, tended to decrease in the Goji supplemented group along with decreased urease activity.Conclusiondata provides valuable metabolic clues for unravelling the beneficial effects of Goji supplementation against gut inflammation and related diseases.Support or Funding InformationUSDA‐NIFA #2018‐67017‐27517 and WSU ERI competitive grantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Characterization of the Atlantic salmon, Salmo salar L., Di/Tripeptide Transporters PepT1a and PepT1b
Authors: Gomes Ana Silva , Rønnestad Ivar , Murashita Koji , Vacca Francesca , Cinquetti Raffaella , Barca Amilcare , Aksnes Anders , Bossi Elena , Verri Tiziano 
Published: June 2021

Amino acids and small peptides in the form of di/tripeptides are key nutrients in animal diets and essential contributors to growth. In mammals, di/tripeptides represent a highly significant fraction of dietary protein after digestion and are transported across the brush‐border membrane of the intestinal epithelium via a single transport protein, the Peptide Transporter 1 (PepT1), a member of the Solute Carrier 15 (Slc15) family. PepT1 is a low‐affinity/high‐capacity transporter that uses an inwardly‐directed proton electrochemical gradient to drive the uptake of di/tripeptides, and peptidomimetics, across the cell membrane. However, in teleost fishes two pept1 genes, namely pept1b and pept1a, have been identified. PepT1b has been studied in many teleost species, including the Atlantic (A.) salmon (Salmo salar L.), whereas PepT1a remain unexplored. The aim of our study was to characterize, the A. salmon PepT1a vs. PepT1b in terms of tissue/organ distribution and function, and possibly extrapolate their physiological role(s).Despite the extra (4th) whole genome duplication (WGD) in the Actinopterygian lineage, A. salmon has only two pept1 genes (namely pept1a and pept1b), like in other telosts that didn't undergo the extra WGD, e.g., the zebrafish (Danio rerio). At the amino acid level, the two paralogues share 77% similarity, 64% identity. Tissue distribution and relative abundance of A. salmon pept1a and pept1b revealed that both transcripts have a similar expression profile. A. salmon pept1a is highly expressed in the proximal intestine (pyloric caeca ≈ anterior midgut > midgut >> posterior midgut), but ~5‐fold less than pept1b, which is expressed in the same gut regions. No expression signals were detected in the anterior and posterior stomach or the anterior and posterior hindgut.As assessed by heterologous expression in Xenopus laevis oocytes and Two‐Electrode Voltage Clamp measurements, A. salmon PepT1a is, like PepT1b, a low‐affinity/high‐capacity system, able to sustain an electrogenic, sodium‐independent and pH‐dependent transport of di/tripeptides. Transport associated currents show that the protein exhibits a K0.5 value for the reference dipeptide Gly‐Gln of 0.5 mM at pH 7.6 and of 0.07 mM at pH 6.5.The pre‐steady state and steady‐state transport currents analysis indicate important functional differences between A. salmon PepT1a and PepT1b transporters. The difference in expression levels and a significantly dissimilar substrate specificity for certain di/tripeptides, such as lysine‐containing peptides, supports the idea of distinct roles in peptide recognition and transport for PepT1a and PepT1b.Support or Funding InformationFunded by RFFV; 247978 SalmoFeedPlus and CargillThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ezrin is a novel target in cyst initiation in ADPKD
Authors: Dixon Eryn E , Woodward Owen M 
Published: June 2021

Autosomal dominant polycystic kidney disease (ADPKD), characterized by progressive renal cystogenesis and other extrarenal manifestations, is one of the most common monogenetic diseases, and is caused by loss of function mutations in PKD1 and PKD2, which encode transmembrane proteins polycystin 1 and 2 (PC1/2). The polycystin proteins have been hypothesized to function as a complex that has been localized to the plasma membrane, cilia, endoplasmic reticulum, and junctions of epithelial cells. The downstream effects of either PC1 or PC2 function loss have not been elucidated. Previously, an investigation of smaller cysts from nephrectomized human ADPKD kidneys revealed a pattern of increased cell width and an altered apical compartment. Subsequently, we observed a decreased abundance and disrupted localization of ezrin, a master scaffold and apical compartment regulator, in similarly characterized cysts. Following this observation, we explored the role of ezrin disruption in a three‐dimensional in vitro model of cystogenesis. Acute inactivation of Pkd2 lead to significant loss of ezrin protein. We confirmed the regulatory relationship between PC2 and ezrin with immunohistochemistry in a genetically inducible in vivo short induction model of Pkd2 inactivation (Pkd2fl/fl Pax8 rtTA TetOCre) that revealed a kidney tubule wide decrease in ezrin at the apical membrane. Lipid protein overlay assays and immunoprecipitation experiments demonstrate both PC2 and ezrin interact with similar phosphoinositides, and possibly with each other, suggesting a role for PC2 in apical compartment and junctional assembly. Functionally, we found that PC2 is critical in junctional integrity, with acute inactivation of Pkd2 causing decreases in transepithelial electrical resistance in inducible primary renal epithelial cells (Pkd2fl/fl Pax8 rtTA TetOCre). Ezrin, expressed in all segments of the kidney, is a regulator of many components of the junctions and apical compartment of renal epithelial cells. The disruption of ezrin following inactivation of Pkd2 in both in vitro and in vivo models, and shared interacting lipids, suggests that PC2 may play a role in the regulation of ezrin in renal epithelial cells and that ezrin may be involved in the initiation of cystogenesis following loss of PC2.Support or Funding InformationNIDDK F31 NRSA, PKD Foundation, NIDDK Baltimore Polycystic Kidney Disease Research and Clinical Core CenterThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional and Structural Insights into RSC‐mediated Nucleosome Remodeling via In‐Vivo Crosslinking
Authors: Evans Brian , Wilkins Bryan 
Published: June 2021

In eukaryotes, genetic information is stored as chromatin. Within the chromatin, DNA is wound around octameric units of histone proteins. The histone‐DNA complex forms the nucleosome and represents the most basic level of DNA compaction. A class of proteins known as chromatin remodelers physically reposition nucleosomes along the chromatin fiber. This can directly affect and mediate many important processes such as DNA replication, repair, and transcription. Albeit, due to their large sizes and numerous auxiliary subunits, chromatin remodeler interactions at the nucleosome have yet to be fully characterized and remain relatively obscure. The RSC complex is the most abundant remodeling complex of budding yeast, with homologs in both animals and plants. In this work, we use synthetic biology to trap histone‐RSC remodeler interactions in living cells using the unnatural amino acid p‐benzoylphenylalanine (pBPA). We site‐specifically insert pBPA into histones and identify remodeler binding via the addition of short peptide fusion tags to our interaction target protein. This system has the potential to illuminate chromatin remodeler complex interactions which can then be used to identify and assign biologically important structures. By properly implementing the system, we exemplify the potential of this technology for creating crosslinking maps for RSC. This conclusion is supported by Western blot analysis and a clearly identified crosslink between a myc‐tagged subunit of RSC (Sth1 protein) with histone H2A and histone H3. In addition, we show that binding to H3 (but not H2A) is dependent upon PTMs and a defined acetylation event at lysine 14 on histone H3. In cells that lack H3 K14ac, Sth1 can no longer be sequestered to the nucleosome providing in vivo mechanistic details about RSC‐nucleosomal function/structure relationships.Support or Funding InformationNIH R15 Academic Research Enhancement Award (AREA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.