bluuebunny/crossref_metadata_2025 huggingface dataset:
Apple Pomace Supplementation Favorably Alters Hepatic Lipid Metabolism in Young Female Sprague‐Dawley Rats fed a Western Diet
Authors: Skinner Roy Christopher , Warren Derek C. , Lateef Soofia N. , Benedito Vagner A. , Bryner Randall W. , Tou Janet C.
Published: June 2021

Apple pomace is a waste byproduct of apple processing that is high in nutrients including polyphenols, pectin, insoluble fibers, and fructose that may contribute to alleviation of some metabolic diseases. Non‐alcoholic fatty liver disease is the most common chronic liver disease in Western countries with the cause attributed to a high fat, high sucrose (Western diet) consumption. The objective of this study was to determine the effects of apple pomace supplementation on hepatic lipid metabolism. Growing (age 22–29 days) female Sprague‐Dawley rats were randomly assigned (n=8 rats/group) to consume a standard purified rodent diet (AIN‐93G), AIN‐93G +10% kcal apple pomace, Western diet (45% fat, 33% sucrose), or Western + 10% kcal apple pomace diets for 8 weeks. Histological evaluation showed rats consuming Western diets had the highest hepatic steatosis although apple pomace supplementation diet led to a lower degree of fat infiltration. Gene expression of hepatic lipogenesis by RT‐qPCR showed no significant differences of transcription factors, carbohydrate response element binding protein (ChREBP) and sterol element binding protein‐1c (SREBP‐1c), or the enzymes, fatty acid synthase (FAS) and stearoyl‐CoA desasturase‐1 (SCD‐1) among diet treatments. Rats fed Western diet had higher (p<0.01) gene expression of diacylglycerol O‐acyltransferase 2 (DGAT2) than Western + 10% apple pomace. The results suggest lower triacylglycerol synthesis reduced hepatic fat infiltration in rats fed Western diet + 10% apple pomace. In a hypercaloric state, apple pomace supplementation, attenuated hepatic steatosis induced by the consumption of a Western diet, despite the high levels of fructose in this supplement.

Support or Funding Information

This project was supported by Dean Daniel J. Robison, West Virginia University Davis College of Agriculture, Natural Resources, and Design and the West Virginia University Office of the Provost Fellowship.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of butyrate in ethanol‐induced intestinal immune responses and associated gut‐liver injury
Authors: Cresci Gail A.M. , Glueck Bryan
Published: June 2021

Purpose

Following a physiologic insult, an immune response needs to be adequately mounted and resolved for proper organ function homeostasis. The immune response to ethanol exposure differs based on multiple factors including the duration of ethanol exposure. While chronic ethanol exposure is proinflammatory, acute ethanol can dampen immune responses. Butyrate, a fermentation byproduct of the gut microbiota, influences gut inflammation and immunity. Our prior work reveals butyrate protects against EtOH‐induced intestinal and hepatic injury. This study aimed to determine butyrate's impact on intestinal innate immune responses following chronic‐binge EtOH exposure.

Methods

Female C57BL/6J age‐matched mice were adapted to a control liquid diet for 5 days, then randomly assigned to a 5% v/v (27% total kcal) ethanol‐containing diet or an isocalorically‐substituted maltose dextrin containing diet for EtOH for 10 days. Ethanol‐fed groups were allowed free access to the diet and control mice were pair‐fed. On day 11, mice were orally gavaged a 5 g/kg dose of EtOH or isocaloric maltose and mice were euthanized 9 hours later. Mice were provided equimolar glycerol or tributyrin (5mM) as part of the liquid diet over the 10 days of EtOH exposure as well as added into the EtOH or maltose gavage at a dose of 2.5 mM. Proximal colon was dissected and mRNA and protein expression of markers of immune and inflammatory response and neovasculature were evaluated.

Results

In response to chronic‐binge EtOH exposure, genes and proteins for immune and inflammatory mediators had decreased expression in proximal colon including markers for leukocytes, macrophages, neutrophils, cytokines and chemokines. Interestingly, EtOH exposure was also associated with decreased mediators of angiogenesis and markers of vasculature. However, mice co‐treated with tributyrin during EtOH exposure had comparable mRNA and protein expression of several key immune and inflammatory parameters and enhanced makers of angiogenesis as pair‐fed control mice. These preserved responses were associated with protection of intestinal tight junction proteins and liver injury.

Conclusion

These findings show tributyrin protected against reduced vasculature and blunted immune responses in the proximal colon caused by chronic‐binge ethanol exposure. These data suggest butyrate could be a potential protective supplement to ethanol consumption and future studies investigating role in human models is warranted.

Support or Funding Information

NIH NIAAA R00 ‐ 4R00AA023266‐03

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ursolic acid potentiates paclitaxel induced antitumor effects on esophageal squamous cell carcinoma cell through the Akt signaling pathway
Authors: Meng Ruo Yu , Kim Soo Mi
Published: June 2021

Development of chemoresistance, poor prognosis, and metastasis often renders the current treatments for esophageal cancer ineffective. The present research is designed to investigate ursolic acid (UA), a component of numerous medicinal plants, either alone or in combination with paclitaxel can inhibit the growth of esophageal squamous cell carcinoma (ESCC) through the Akt/FOXM1 signaling pathway. UA plus paclitaxel treatment inhibited the proliferation of TE‐12 and TE‐8 cells in a dose‐dependent manner when compared to treatment with UA or paclitaxel alone. In colony formation assay, UA potentiated the inhibition of colony formation by paclitaxel when compared to treatment with a single agent. Combination treatment induced apoptosis by increased levels of cleaved poly ADP‐ribose polymerase (PARP) and cleaved caspase‐9 protein and inhibited the invasion and metastasis of ESCC cells. In cell cycle analysis, UA plus paclitaxel induced the G1 cell cycle arrest when compared to treatment with UA or paclitaxel alone. In addition, UA potentiated paclitaxel‐induced inhibition of Akt function and increased phosphorylation form of Akt. These results suggest that UA effectively potentiates the efficacy of chemotherapeutic agents such as paclitaxel via inhibition of proliferation and increased apoptosis by inactivation of Akt in ESCC cells. Taken together, UA enhances the therapeutic efficacy of paclitaxel in esophageal cancer and is a potential clinical anticancer agent for the prevention and/or treatment of ESCC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exposure to a reduced ambient temperature induces browning of bone marrow derived adipocytes in vitro
Authors: Symonds Michael , Velickovic K , Lugo‐Leija H A. , Bloor Ian , Law James , Sacks H , Sottile Virginie
Published: June 2021

Brown and beige adipocytes are characterised as expressing the unique mitochondrial uncoupling protein (UCP) 1 for which the primary stimulus in vivo is cold exposure. The ability to promote the abundance of UCP1 can have a beneficial effect on energy balance as well as lipid and carbohydrate metabolism and as such is a potential anti‐diabesity target. This process has not been studied widely in vitro, for which the impact of modulation of the thermal environment is not well established. The aim of our study was therefore to examine whether cold‐induced UCP1 activation can be achieved in vitro.

We thus report an in vitro model to induce adipocyte browning using bone marrow (BM) derived mesenchymal stem cells (MSC) of both human and mouse origin. It relies on differentiation at a cool ambient temperature of 32°C instead of the standard temperature of 37°C. The low temperature promoted browning in MSC‐derived adipogenic cultures, with increased adipocyte number (including multilocular cells), and upregulation of adipogenic and thermogenic factors, especially UCP1. Lipid droplets were smaller and more numerous. The bioenergetic status of cells was then investigated using a Seahorse XF96 flux analyser, and the overall respiratory responses calculated for the six key regulators of mitochondrial function. Cells differentiated at 32°C exhibited enhanced uncoupled respiration and metabolic adaptation, with intensified oxidative and glycolytic pathways. Cold‐exposed differentiated cells showed a marked translocation of regulatory hormones to adipocyte nuclei, suggesting a previously unknown role in the browning process. To further determine whether this adipogenic phenotype acquired at 32°C may be reversible as found in vivo, cells differentiated at 32°C were subsequently transferred to standard culture conditions (37°C). This showed that cells transferred from 32°C to 37°C showed a pattern of UCP1 expression similar to that of cells differentiated at 32°C i.e. they retained their beige‐like characteristics.

Taken together these results indicate that BM‐MSC can be driven to forming beige‐like adipocytes in vitro by exposure to a reduced temperature. This in vitro model will now provide a powerful tool to further elucidate the regulation of UCP1 function in order to provide a better understanding of the endocrine regulation of the browning process in human adipocytes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimized Induction of Neurospheres from Porcine Mesenchymal Stem Cells
Authors: Schomberg Dominic T. , Wood Aleksandar , Singh Harpreet , Meudt Jennifer J. , Shanmuganayagam Dhanansayan
Published: June 2021

Neurospheres are free‐floating clusters of cells that provide researchers a unique method to investigate neural stem cells. Stem cells typically grow as an adherent monolayer on the culture surface, however, when introduced to proper media and growth factors without adherent substrates, these stem cells will form non‐adherent three‐dimensional aggregates called neurospheres. Induction of neurospheres increases multipotency and regenerative capacity of some stem cells, such as adipose‐derived mesenchymal stem cells (adMSCs). Currently, there are no standardized methods for neurosphere induction and evaluation which limits their utility. Studies into cancer, neural development, and regenerative medicine would all benefit from an optimized neurosphere induction protocol. We aimed to establish an optimized protocol for inducing neurospheres from adMSCs in an efficient and reproducible manner.

Primary swine adMSCs were induced to neurospheres with media comprised of varying concentrations of neurobasal medium, B27, and glutamine, epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF) and morphometric parameters of growth (total neurospheres, total cells, cell density and neurosphere diameter) were evaluated daily for eight days. After the eight‐day induction period, the multipotency of neurospheres was determined by assessing presence of neuronal markers nestin, Sox2 and Oct4. We also evaluated for ideal conditions under which neurospheres formed without any signs necrosis at the core. Our optimized neurosphere culture methods produce neural stem cell that best maintain neural‐multipotency and provide a standardized and reproducible protocol for neuronal stem cell propagation.

Support or Funding Information

The research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF North Central, NF Network, and NF Team Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the function of the histone methyltransferase PRMT1 during post‐embryonic development in Xenopus tropicalis.
Authors: Shibata Yuki , Shi Yun‐Bo
Published: June 2021

Anuran amphibians such as Xenopus tropicalis offer a unique opportunity to study how the adult organs and their organ‐specific stem cells are formed in vertebrates during the so‐called postembryonic development. The postembryonic events such as the maturation/remodeling of many organs are totally regulated by thyroid hormone (TH). Of particularly interest among TH target genes is protein arginine methyltransferase1 (PRMT1), which can function as a TR co‐activator. We have shown that PRMT1 mRNA and protein expression peaks at the climax of metamorphosis in the intestine when adult stem cells are forming de novo and proliferating, and PRMT1 overexpression enhanced the expression of TH target genes and increased the number of adult intestinal stem cells during metamorphosis, suggesting a critical role during the formation of adult stem cells in the intestine. To determine the function of the endogenous PRMT1 gene during post‐embryonic development and intestinal stem cell development, we adapted the gene editing technology to knockout PRMT1 gene in X. tropicalis.

We used Transcriptional activator–like effector nuclease (TALEN) technology to generate PRMT1 knockdown frogs (F0). After generating PRMT1 heterozygous frogs (F1) with out of frame mutations from the F0 animals, we tried to produce PRMT1 homozygous knockout frogs by intercross the F1 frogs. We measured the survival rate, animal size, and the developmental stage of the resulting tadpoles, including wild type, heterozygous and homozygous animals.

As the result, we successfully generated PRMT1 heterozygous (F1) frogs including out frame mutation (8 base deletion), and PRMT1 homozygous knockout (F2) animals by intercross with F1 frogs. Surprisingly, all PRMT1 homozygous tadpoles died within 14 days after fertilization although they completed embryogenesis to form apparently normal tadpoles. In addition, morphological analysis showed that PRMT1 knockout tadpoles delayed animal development and growth.

PRMT1 knockout tadpoles completed embryogenesis normally but had delayed development and tadpole lethality. The availability of knockout tadpoles will allow us to study the role of PRMT1 during T3 ‐induced metamorphosis, thus overcoming the difficulty to study PRMT1 during postembryonic development in mammals due to the lethal phenotype in PRMT1 knockout mice.

Support or Funding Information

JSPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle specific MnSOD deficiency leads to complex II‐specific inactivity of ETC and contractile dysfunction, but increases muscle mass
Authors: Ahn Bumsoo , Ranjit Rojina , Pharaoh Gavin , Piekarz Katarzyna , Matsuzaki Satoshi , Premkumar Pavithra , Riddle Kaitlyn , Humphries Kenneth , Richardson Arlan , Van Remmen Holly
Published: June 2021

Multiple pathological conditions exhibit excess mitochondrial ROS and muscle weakness in parallel, including aging, denervation and cancer cachexia. To directly test the role of mitochondrial ROS in skeletal muscle weakness and atrophy we generated a skeletal muscle specific knockout mouse model in which MnSOD (Sod2), the primary superoxide anion scavenger in the mitochondrial matrix, was deleted using a Cre‐Lox approach driven by a constitutively active human skeletal actin promoter (HSA‐Cre). Female wild type and MnSOD deficient (mSod2 KO) mice were studied at 6–8 months of age. In mSod2 KO mice, the observed rate of superoxide generation from isolated skeletal muscle mitochondria was significantly elevated, and hydrogen peroxide generation using permeabilized fibers increased by ~5 fold. ATP generation rate and electron transport chain (ETC) activity were severely reduced in response to Complex II linked substrate, suggesting superoxide‐induced complex II inactivity. Reduced ATP generation rate and ETC activity were consistent with severe exercise intolerance in mSod2 KO mice. To determine the effects of mitochondrial ROS on skeletal muscle weakness, we first tested in situ contractile function using gastrocnemius. Maximum isometric specific force was decreased by ~30 % in mSod2 KO mice with direct stimulation on muscle, but the reduction in force was further diminished (~50%) after stimulation of the sciatic nerve suggesting neuromuscular disruption. Morphological assessment exhibited increases in NMJ size and fragmentation in mSod2 KO mice supporting that mitochondrial dysfunction can feed back to initiate NMJ disruption. Surprisingly, gastrocnemius muscle mass was increased in mSod2 KO mice, which was associated with increased numbers of central nuclei and hyperplasia via fiber branching. In summary, our data suggest that excess mitochondrial superoxide caused a selective loss of complex II activity and mitochondrial defects, leading to severe exercise intolerance. Increased cytoplasmic hydrogen peroxide induced necrosis‐mediated regeneration, which increased muscle mass by fiber branching.

Support or Funding Information

NIH/NIA to Dr. Van Remmen (R01 AG050676) and a VA Merit grant to HVR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling Calcium‐Induced Inhibition of Oxidative Phosphorylation
Authors: Malyala Sathyavani , Zhang Yizhu , Bazil Jason N.
Published: June 2021

Ischemic myocardium is characterized by a chronic elevation in cellular calcium concentration. In this setting, mitochondria accumulate calcium to levels that compromise their ability to synthesize ATP. Although total mitochondrial calcium can reach up to 1 M, the vast majority is presumed to be present as insoluble calcium phosphate granules. We recently reported calcium levels below thresholds that trigger mitochondrial permeability transition significantly lowers the rate of ADP‐stimulated respiration. To explain these data, we tested the following hypotheses using a novel mathematical model of cardiac mitochondrial energetics and calcium handling: i) depressed membrane potential caused by sodium/calcium cycling lowering the driving force for ATP production; ii) direct inhibition of calcium on mitochondrial processes critical for ATP production; iii) calpain activation leading to a decrease in ATP synthesis capacity; iv) depletion/reduction of available ADP for phosphorylation caused by calcium biding, incorporation into calcium phosphate granules, or net loss of adenine nucleotide. The model includes mechanistic descriptions of mitochondrial metabolism and energetics in addition to calcium uptake, sequestration, and efflux pathways. Based on the model analysis, sodium/calcium cycling lowers mitochondrial membrane potential, but does not lead to lower rates of oxidative phosphorylation. However, direct inhibition of calcium on ATP production did agree with these data. We also found that calpain activation is a possible explanation, but we experimentally ruled this mechanism out. Finally, ADP complexation with calcium did not affect the rate of oxidative phosphorylation, but net loss of nucleotides via incorporation into calcium phosphate granules or efflux out of mitochondria can explain these data. The model will be used to design further experiments to determine the most likely mechanism capable of explaining the calcium‐induced inhibition of oxidative phosphorylation.

Support or Funding Information

R00‐HL121160

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Protective Effects of Atrial Natriuretic Peptide Infusion in Salt‐Sensitive Hypertension
Authors: Ilatovskaya Daria , Levchenko Vladislav , Zietara Adrian , Winsor Kristen , Spires Denisha R. , Staruschenko Alexander
Published: June 2021

Atrial Natriuretic Peptide (ANP) is a hormone secreted by cardiomyocytes in response to increased atrial volume; it stimulates water and sodium excretion by the kidneys and relieves pressure on the circulatory system. In our previous studies we compared wild type Dahl Salt Sensitive (SS) rats to SS rats lacking the Nppa gene which encodes ANP (SSNppa−/−). The data revealed that salt‐induced blood pressure development was significantly exacerbated in the SSNppa−/− rats. Furthermore, SSNppa−/− rats exhibited reduced urinary sodium excretion and diuresis, as well as aggravated kidney damage compared to wild type controls (FASEB J 2017 31:855.8.). These data led to a hypothesis that ANP infusion can have beneficial effects during the development of salt‐induced hypertension.

Dahl SS rats were fed a high salt diet (HS, 4% NaCl) for 21 days, with a continuous i.v. infusion of ANP (100 ng/kg/day) administered from day 0 or day 14 onward to test preventive and therapeutic effects of ANP, respectively. Blood pressure was monitored throughout the study, and urine samples were collected in metabolic cages on days 0 and 21. At the end of the protocol animals were sacrificed and kidney tissues were harvested and stained with Masson's trichrome to evaluate renal damage. Fibrosis, glomerular injury, and protein casts formation were assessed; urine and plasma samples were also analyzed to estimate electrolyte homeostasis.

ANP infusion had beneficial effects on BP and renal function compared to control animals. We observed a decrease in blood pressure in both groups infused with ANP during 0–21 days (21D) and 14–21 days (7D) of a HS challenge. Kidney to body weight ratio did not differ between the groups; however, we observed a trend for reduced renal hypertrophy in the 21D animals. Metabolic cage studies revealed no changes in diuresis; urinary electrolyte analysis showed a more effective sodium excretion in the 21D rats. Assessment of kidney damage demonstrated a decrease in protein casts in both 7D and 21D rats: 3.8% ± 0.47%, 2.7% ± 0.24%, and 2.0% ± 0.41% of the tissue surface in the control, 7D and 21D groups, respectively. Glomerular injury had a similar pattern: scores were 2.86 ± 0.09, 2.62 ± 0.07, and 2.41 ± 0.06. Renal fibrosis was alleviated in the 21D group (1.4% ± 0.18%, compared to 1.9% ± 0.49% and 3.1% ± 0.31% in the 7D and control groups).

Therefore, ANP infusion had both preventive and therapeutic effects. Our data revealed that ANP administration during the HS diet challenge resulted in decreased blood pressure, increased electrolyte excretion, and reduced tissue damage.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Hypotheses through Discovery of Relevant Models of Epithelial Transport
Authors: Sarwar Dewan , Yu Tommy , Atalag Koray , Carlson Brian , Gennari John , Hunter Peter J. , Neal Maxwell , Nickerson David P.
Published: June 2021

Scientists often utilize computational models to help understand experimental observations or to investigate hypotheses in ways that are not practically achievable experimentally. The first step in constructing such models is to discover existing models which can be reused to help in such an investigation. We are developing novel research tools to enable the discovery and exploration of computational models relevant to specific research questions or hypotheses. This can aid biologists and clinicians to test their clinical or experimental hypotheses from a given collection of disparate mechanisms and/or observations such as diseases, drug actions and clinical observations.

Computational tools and standards have evolved over the years such as CellML to encode mathematical models of biological systems; SemGen to annotate models; and the Physiome Model Repository (PMR) to store these models and their comprehensive descriptions to ensure the deposited knowledge is persistent, identifiable, discoverable and sharable. Our long term goal is to develop a knowledge management system capable of constructing multi‐scale renal nephron models from information extracted from PMR to investigate transport phenomena in the kidney at the cellular and subcellular level and the impact on nephron function.

We have used the SemGen tool to comprehensively annotate a preliminary cohort of renal epithelial transport models available in PMR to provide the initial knowledgebase for model discovery and exploration, and in particular, to extract a model suitable for testing specific clinical or experimental hypotheses. One use of such exploration is the identification of missing resource(s), which could be addressed via more accurate description of existing resources or the addition of new resource to the repository.

Specifically, we present here a web‐based tool enabling scientists to discover and explore epithelial transport models that are relevant to their specific research questions. Biologists and clinicians can use this tool to discover models with specific transport proteins, solute species, genes, anatomical location, or other relevant information. The result of this discovery is a “recipe” describing the computational model that can be assembled to help the user investigate their hypothesis.

Support or Funding Information

Supported by the MedTech Centre of Research Excellence

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydrogen Peroxide Increases Intracellular Sodium Concentration in the Medullary Thick Ascending Limb Isolated from Sprague‐Dawley Rats
Authors: Zheleznova Nadezhda N. , Cowley Allen W.
Published: June 2021

Elevations of renal medullary hydrogen peroxide (H2O2) produce a salt‐sensitive form of hypertension as shown by chronic infusion of H2O2 into the renal medulla of a single remaining kidney of normotensive Sprague‐Dawley (SD) rats (Makino et al., Hypertension, 2003) and salt‐resistant consomic SS.13BN rats. Conversely, chronic medullary infusion of catalase (H2O2 scavenger) into the renal medulla of Dahl salt‐sensitive rats significantly reduced salt induced hypertension (Taylor and Cowley, Am J Physiol Regul Integr Comp Physiol, 2005). The goal of this study was to assess the effects of H2O2 upon intracellular sodium concentration [Na+]i and the role of mTORC1 and mTORC2 signaling in the medullary thick ascending limb (mTAL) isolated from the outer medulla (OM) of SD rats.

Methods

rats were anesthetized, the kidney flushed with saline, removed, and mTALs dissected from the inner stripe of the OM at 4°C under a stereomicroscope. The mTALs were attached to poly‐Lysine coated cover slips and incubated with HBSS containing 10 nM of the Na+‐sensitive fluorescence dye NaGreen and 0.05% Pluronic acid F‐127 for 30 min at room temperature. The mTALs were washed and then placed in a heated chamber (37°C) mounted on the stage of an inverted Nikon TE‐2000U microscope for recording.

Results

It was found that 100 uM H2O2 resulted in a significant increase in [Na+]i (p<0.05). The responses to pretreatment of mTAL with 10 uM PP242 (mTORC1/2 inhibitor), 200 nM rapamycin (mTORC1 specific inhibitor), or 1 mM furosemide (Na+/K+/2Cl− inhibitor) were then studied. We found that each of these inhibitors nearly abolished H2O2 induced increases in intracellular Na+. Western blot analysis of isolated mTAL from SD rats for each of the treatments found that 100 uM H2O2 increased the activation of AKT (pAKTSer473; a surrogate for mTORC1/2 activation) and S6 (pS6Ser235/236; a surrogate for mTORC1 activation) and were inhibited by their respective inhibitors.

Conclusion

Together these observations indicate that H2O2 acts through the mTOR signaling pathways to inhibit NKCC2 activity, which would in turn be expected to lead to sodium retention and hypertension.

Support or Funding Information

HL 122662

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α‐actinin 4 Knockdown Reduced Vasopressin‐Induced Aquaporin‐2 Expression in the Kidney Collecting Duct Cells
Authors: Ho Cheng Hsuan , Yu Ming‐Jiun
Published: June 2021

Vasopressin is a pituitary peptide hormone that regulates aquaporin‐2 (AQP2) gene expression in the kidney collecting duct principal cells. Dysregulation of vasopressin‐regulated AQP2 gene expression is associated with many water balance disorders. Previously in the collecting duct cell model (mpkCCD), we identified α actinin 4, a cytoskeletal actin‐binding protein that was reported to translocate into the nucleus and acts as a transcriptional co‐activator. Here, we found that short hairpin RNA (shRNA)‐mediated α actinin 4 knockdown in the mpkCCD cells reduced vasopressin‐induced AQP2 mRNA and protein levels, suggesting a role of α actinin 4 in vasopressin‐induced AQP2 gene expression. However, vasopressin did not induce nuclear translocation of α actinin 4, suggesting a different model of α actinin 4 action. α actinin 4 was reported as a transcriptional co‐activator of the glucocorticoid receptor whose agonist dexamethasone was shown to enhance vasopressin‐induced AQP2 gene expression in the mpkCCD cells in a dose‐ and time‐dependent manner. We thus tested whether α actinin 4 interacts with glucocorticoid receptor to enhance vasopressin‐induced AQP2 gene expression. In support of our hypothesis, shRNA‐mediated glucocorticoid receptor knockdown almost blunted vasopressin‐induced AQP2 mRNA and protein expression. Our results are consistent with a role of α actinin 4 in vasopressin‐induced AQP2 gene expression involving glucocorticoid receptor.

Support or Funding Information

This work was supported by Ministry of Science and Technology, Taiwan to MJY (MOST104‐2320‐B002‐064‐MY3)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effectiveness of Total Water Intake Guidelines in Maintaining Lowered Urine Osmolality
Authors: Seal Adam , Johnson Evan , Pérronet Francois , Jansen Lisa , Summers LynnDee , Adams J.D. , Perrier Erica , Guelinckx Isabelle , Kavouras Stavros
Published: June 2021

The reference values for daily total water intake from fluids and foods (TWI) from the Institute of Medicine (IOM) are 2.7 and 3.7 liters for females and males, respectively. Recent studies suggest that for optimal water intake 24h urine osmolality should be maintained below 500 mmol·kg−1.

Purpose

To determine the total water intake requirement for healthy adults to maintain 24h urine osmolality below 500 mmol·kg−1.

Methods

Urinary concentration was assessed in 48 men and 50 women (age: 41±14 y, body mass index: 26.2±5.1 kg·m−2) via urine osmolality from 24h samples (UOsm). TWI was assessed via water turnover using the deuterium dilution (D2O) method throughout a 7‐day time period, corrected for metabolic water production. The diagnostic accuracy of TWI to identify UOsm ≤ 500 mmol·kg−1 was evaluated using receiver operating characteristic (ROC) analysis in men and women separately.

Results

UOsm was 511±232 and 402±203 mmol·kg−1 and TWI was 3.54±1.08 L·d−1 and 3.20±1.27 L·d−1 in men and women, respectively. 32% of men and 55% of women met IOM guidelines for TWI. ROC analysis for TWI detecting UOsm ≤ 500 mmol·kg−1 in men yielded an area under the curve (AUC) of 77.4% with sensitivity, specificity, and threshold values of 83.3%, 64.5%, and 3.39 L·d−1, respectively. In women, the AUC was 82.4% with sensitivity, specificity, and threshold values of 85.7%, 72.1%, and 2.61 L·d−1, respectively.

Conclusion

Considering threshold values in men and women of 3.39 L·d−1 and 2.61 L·d−1, respectively, maintaining TWI in line with IOM guidelines of 3.7 L·d−1 in men and 2.7 L·d−1 in women should be sufficient for most individuals to maintain 24h urine osmolality less than 500 mmol·kg−1.

Support or Funding Information

This research was supported by Danone Research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Farnesoid X receptor (FXR) activation induces up‐regulation of multidrug and toxin extrusion proteins (MATEs) in human renal proximal tubular cells
Authors: soodvilai sunnhapas , Wongwan Teerasak , Chatsudthipong Varanuj
Published: June 2021

Farnesoid X receptor (FXR) is a nuclear transcriptional factor which highly expressed in the liver, intestine, and kidney. Its plays an essential role in regulation of membrane transporters to eliminate endogenous and xenobiotic compounds. The previous study has shown that endogenous ligand, chenodeoxycholic acid (CDCA) induced by cholestasis down‐regulated hepatic organic cation transporter 1 (OCT1) which plays a critical role in the hepatic secretion of organic cations. It is interesting whether FXR activation regulates multidrug and toxin extrusion protein1 (MATE1) and kidney specific MATE2‐K that play an essential role in the renal cationic drugs efflux into the lumen. The present study showed that treatment with 20 μM CDCA, an endogenous ligand of FXR, for 24 hours stimulated MATE1‐ and MATE2‐K‐mediated 3H‐MPP+ transport in human renal proximal tubular cell line (RPTEC/TERT1 cells). The effect of CDCA was abolished by co‐treatment with a FXR antagonist, Z‐guggulsterone. These data indicated that the stimulatory effect of CDCA on MATEs required FXR‐dependent mechanism. Furthermore, treatment RPTEC/TERT1 cells with CDCA at both physiological level (20 mM) and pathophysiological level (cholestasis) (80 μM) increased mRNA expression of MATE1 and MATE2‐K. Taken together, FXR activation up‐regulates mRNA expression of MATE1 and MATE2‐K resulting in increased cation transports in the proximal tubular cells in both physiological and pathological conditions.

Support or Funding Information

The study was supported by the grant from Mahidol University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transepithelial Na and Urea Transport Pathways in Rat Inner Medullary Thin Limbs of Henle's Loop
Authors: Evans Kristen K. , Yu Alan S. L. , Pannabecker Thomas
Published: June 2021

Identification of key transport pathways for Na and urea in thin limbs of Henle's loops will further explain how solute recycling and countercurrent mechanisms are integrated to produce the corticomedullary osmotic gradient. We studied transepithelial Na and urea fluxes by in vivo microperfusion of papillary descending and ascending thin limbs (DTLs and ATLs, respectively) and by in vitro microperfusion of isolated DTLs and ATLs from the upper and lower 50% of the inner medulla (IM). In vitro studies have shown that transepithelial water permeability (Pf) is high in DTLupper and low in DTLlower, ATLupper and ATLlower. In vivo microperfusion studies confirmed the low Pf in papillary DTLs and ATLs. Mean transepithelial Na and urea permeabilities (PNa, Purea) with in vitro lumen perfusion rates of ~30 nl/min in DTLupper were ~60 E‐5 cm/sec and in DTLlower and ATL were ~350 to 450 E‐5 cm/sec; Na and urea fluxes in DTLlower are directly proportional to lumen perfusion rates between 5 to 30 nl/min. In vivo microperfusion studies confirmed the high PNa and Purea in papillary DTLs and ATLs. With in vitro microperfusion, Purea in DTLupper and DTLlower was not inhibited by peritubular phloretin (0.25 mM) indicating that urea fluxes occur independently of known facilitated urea transporters such as UT‐A1, UT‐A2 or UT‐A3. Furthermore, Purea in DTLlower was not inhibited by ouabain (10 mM) or by a lumen‐to‐bath Na concentration gradient (143 mM/lumen and 0 mM/bath) indicating the absence of Na‐dependent urea transport. On the other hand, Purea in DTLupper and DTLlower were almost completely inhibited by peritubular lanthanum (5 mM), with Purea recovering in both segments following 20 min lanthanum washout. Lanthanum (5 mM) also completely inhibited PNa in DTLlower; however, PNa remained completely inhibited following 20 min washout. Activation energy for transepithelial Na and urea transport determined by in vitro microperfusion during 37°‐ to −16° C temperature transitions was found to be low in DTLupper (~1–2 kJ/mol) compared to DTLlower and ATL (~13 kJ/mol). Urea transport in DTLupper and Na and urea transport in DTLlower and ATL likely occur in large part, by way of a plasma membrane or paracellular channel‐like pathway. The distinctive transport characteristics suggest that there are different mechanisms involved with PNa and Purea in upper and lower DTLs, respectively.

Support or Funding Information

NIDDK DK083338 (TP)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Is regulation of microglial functions by T3 dependent on the micro‐environment?: Insights from the brainstem respiratory control network of newborn mice
Authors: Rousseau Jean‐Philippe , Kitazono Kota , Yoshioka Yusaku , Noda Mami , Kinkead Richard
Published: June 2021

We previously showed that thyroid hormones (THs) facilitate respiratory control development in newborn rodents. To understand the underlying mechanisms, we tested the hypotheses that TH, more precisely T3, 1) influences microglial functions in the brainstem of newborn mice and 2) this effect is region dependent. Measurements were performed on primary cultured mouse microglia isolated from the mixed glial cultures of postnatal day 3 C57BL/6J mice. Primary cultured neurons from cortex and brainstem were obtained from embryonic day 14–16 C57BL/6J mice. Microglia from cortex and brainstem were either cultured alone (DMEM) or co‐cultured with respective neurons (DMEM + neurobasal medium) under standard conditions (37°C, 5% CO2 / 90% humidity). Microglial motility (μm traveled) was monitored using a time‐lapse video‐microscopy system over 3h with or without T3 (1μM). Phagocytosis was quantified and observed in brainstem microglial cells pre‐incubated with 54 μg/ml Dextran, Rhodamine B (10 000 MW, neutral) in DMEM for 1h followed by incubation in DMEM with or without T3 (1 μM) for 3h. The cells were then fixed by 4% PFA. In the presence of neurons, but not in single culture, exposure to T3 increased brainstem microglial motility by 350% relative to control over the 3 h period (13.0 ± 4.5 vs 45.2 ± 7.8 μm; P=0.004). Exposing cortex cells to T3 also increased microglial motility; however, the effect was less important (156%; 46.0 ± 4.9 vs 72.1 ± 8.8 μm; P=0.009). The region of origin could also influence microglial functions in co‐culture as brainstem microglia showed decreased motility compared to cortex microglia (with and without T3). By comparison with controls, brainstem microglial cells cultured alone responded to T3 exposure by a ~4‐fold increase in phagocytosis as indicated by the fluorescence intensity of Dextran Rhodamine B (average arbitrary unit/area; 5.4 ± 1.1 vs 1.3 ± 0.2 AU/μm2; P=0.001). We conclude that 1) T3 facilitates multiple functional responses of microglia including motility and phagocytosis; both functions are important in regulation of synaptic function during development. 2) Microglial activation is specific to the surrounding environment, such as presence of thyroid hormones and brain regions. Microglia therefore appear as important effectors of TH action in the development of the respiratory network. Understanding the mechanisms by which these cells influence the generation and regulation of respiratory activity offers new approaches to alleviate respiratory disorders in newborn, especially those born pre‐term.

Support or Funding Information

Supported by NSERC, Fonds de Recherche en Santé du Québec and Research Support Center, Graduate School of Medical Sciences, Kyushu University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Maternal Breathing Dysfunction During Pregnancy Alters Microglial Activities and Increases Risk for Psychiatric Disorders in Her Offspring
Authors: Cahill Michael E. , Vanderplow Amanda , Kiernan Elizabeth , Johnson Stephen , Baker Tracy , Watters Jyoti
Published: June 2021

Each year, over half a million women develop sleep disordered breathing (SDB) by the third trimester, the prevalence of which is increasing in parallel with the obesity epidemic. While negative consequences of SDB during pregnancy on the health of the mother and newborn are becoming better appreciated, nothing is known about the impact of SDB on the neural function of her offspring. SDB is a potent inflammatory stimulus, inducing chronic inflammation that contributes to many of the morbidities associated with SDB. Since epidemiological studies suggest that maternal inflammation is associated with the development of neuropsychiatric disorders in her offspring, we tested the hypothesis that in utero intermittent hypoxia impairs cognitive behavior in the offspring. Pregnant rat dams were exposed to chronic intermittent hypoxia (8 hrs/day, 2 min 10.5% O2 separated by 2 min of 21% O2) or normoxia from gestation days 10–21 (GIH and GNX, respectively). Juvenile (3 week) and adult (8 week) offspring were tested in a Y‐maze spontaneous alternation task, a well‐validated index of spatial working memory. We found that spontaneous alternation performance was severely impaired in juvenile and adult male GIH offspring relative to GNX offspring, suggesting GIH results in early developmental emergence of working memory impairments. In contrast, female GIH offspring did not exhibit impairments in spatial working memory at either age. Microglia isolated from the forebrain of male GIH offspring exhibited significant reductions in the expression of the phagocytic receptor gene complement receptor 3 (CD11b), suggesting that GIH impairs microglial synaptic pruning activities. In the same samples, expression levels of DNA methyltransferase enzymes (DNMTs), that are responsible for repressing gene expression, were increased. Since hypoxia enriches the transcriptional activation mark H3K4me3 in microglial cultures, we hypothesize that GIH upregulates DNMT gene expression via epigenetic histone alterations to fetal microglia, which represses microglial phagocytic gene expression. Together, these data suggest that GIH: 1) sex‐dependently alters cognitive phenotypes in GIH offspring, and 2) may induce epigenetic suppression of microglial phagocytic activities that impair microglial synaptic pruning during development, altering forebrain synaptic connectivity in male offspring.

Support or Funding Information

Supported by R01 NS085226 (JJW) and HL105511 (TLB)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“It's Complex”: Student Preconceptions about the Difficulty of Physiology Courses
Authors: Walton Kristen LW.
Published: June 2021

Students' pre‐conceived ideas about the difficulty of specific classes can affect their experience in these courses. Anxiety, uncertain motivation, and potential reticence to engage with topics regarded as difficult to learn can impact student success in these courses. In our student population, informal student feedback suggests that our physiology courses are widely regarded as difficult and challenging. Faculty who teach physiology at other institutions have noted this student perception as well, and suggest that students find this discipline difficult because of its complexity and the requirement for mechanistic reasoning (J. Michael, Adv Physiol Edu 2006). This study aimed to evaluate students' perceptions about physiology, including its relevance to other aspects of biology, levels of biological organization that it encompasses, and overall difficulty. The study population was drawn from students enrolled in two different biology majors courses at Missouri Western State University, a regional, comprehensive, primarily undergraduate institution with a large proportion of first‐generation college students. An anonymous survey, using 10 questions scored on a Likert‐type scale plus an open‐ended question, was administered on the first day of class. 40% of the respondents had previously taken a physiology course (anatomy & physiology, animal physiology, or plant physiology). Ninety‐seven percent of respondents agreed or strongly agreed that it is important to understand how cells communicate with each other to understand organismal function. However, interestingly, fewer students (71% of respondents) agreed or strongly agreed that “it is important to understand the molecules and ions that are involved in specific organ functions.” Survey responses to the statement “numbers and equations are rarely used when describing physiological events” were neutral on the Likert scale, suggesting that students may not anticipate using math in physiology courses. Open‐ended responses to the question “Do you view physiology as a difficult subject to learn?” frequently included comments about “complexity of the organism” and “a lot of information to learn”. Data from this survey can be used to help address specific student concerns about course content and may be useful to inform teaching strategies.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Drosophila based teaching laboratory used to investigate potential obesogens
Authors: Daggett Melissa A. F.
Published: June 2021

Drosophila is an emerging model organism for studying the effects of genetics, diet, and suspected environmental obesogens on the regulation of metabolism and physiological function. Obesogens are chemicals that are suspected endocrine disruptors thought to interfere with mechanisms involved in lipid metabolism and may be partly responsible for the increase in obesity. This teaching laboratory uses a modified version of the floating larvae or density assay originally developed as a genetic screen in Drosophila that identified genes that influence the metabolism and accumulation of lipids. This is a simple, inexpensive assay that can be used by undergraduate students to screen potential environmental obesogens by adding them to a standard fly diet and recording results that a highly reproducible and easily observed. The assay is suitable for incorporation into a course based undergraduate research project (CURE).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.