bluuebunny/crossref_metadata_2025 huggingface dataset:
β1Pix Stabilizes Nedd4‐2 and Plays a Critical Role in ENaC Regulation by AMPK in Kidney Epithelial Cells
Authors: Ho Pei‐Yin , Li Hui , Pavlov Tengis S. , Staruschenko Alexander , Hallows Kenneth R.
Published: June 2021

The epithelial Na+ channel (ENaC) is expressed in many salt‐reabsorbing epithelia at the apical membrane, including in the aldosterone‐sensitive distal nephron of the kidney where it is the final regulator of Na+ balance, blood volume, and blood pressure. Our previous work has established that the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits ENaC by promoting the binding of the ubiquitin ligase Nedd4‐2 to ENaC. Here, we have further demonstrated that functional β1Pix, a Rho‐GEF signaling protein, is required for AMPK‐dependent ENaC inhibition in CHO cells. Epithelial volt‐ohmmeter measurements were performed to examine the role of β1Pix in regulating ENaC‐dependent currents in polarized kidney cortical collecting duct (CCD) cells. Lentiviral shRNA‐mediated knockdown of β1Pix expression in mpkCCDcl1 cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization‐deficient mutant unable to bind 14‐3‐3 proteins (Δ602‐611) increased ENaC currents in mpkCCDc14 cells, whereas overexpression of wild‐type β1Pix had an opposite effect. Through additional immunoblotting and co‐immunoprecipitation studies, we explored the role of β1Pix in the regulation of ENaC by AMPK and the interplay of β1Pix, 14‐3‐3 proteins and Nedd4‐2. Treatment with AMPK activators promoted the binding of β1Pix to 14‐3‐3 proteins in CCD cells. However, the association between Nedd4‐2 and 14‐3‐3 proteins was not significantly altered by AMPK activation, β1Pix knockdown, or overexpression of wild‐type or β1Pix‐Δ602–611 mutant in cells. Moreover, β1Pix knockdown or overexpression of β1Pix‐Δ602–611 inhibited Nedd4‐2 protein expression in CCD cells. This inhibition was associated with a parallel reduction in Nedd4‐2 phosphorylation at Ser‐328, a site that is phosphorylated by several kinases including AMPK which is critical for cellular Nedd4‐2 protein stability. Decreased Nedd4‐2 protein stability in the setting of β1Pix knockdown was confirmed by cycloheximide chase studies. Together, these results suggest that functional β1Pix is important for Nedd4‐2 Ser‐328 phosphorylation and thus Nedd4‐2 stability. Overall, our findings suggest that AMPK activation promotes, potentially via AMPK‐dependent β1Pix phosphorylation, the association of β1Pix, 14‐3‐3 proteins, and Nedd4‐2 into a complex that inhibits ENaC by enhancing Nedd4‐2 binding to ENaC followed by ENaC ubiquitination and targeting for degradation. This regulation of ENaC and other epithelial transport proteins by AMPK may have a pro‐survival role by inhibiting energy‐consuming ion transport processes during conditions of ischemia and other cellular stresses that occur with acute kidney injury.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Piezo1 Regulation of Lung Endothelial Barrier Function in Left Heart Failure
Authors: Friedrich Emily , Hong Zhigang , Zhong Ming , Rehman Jalees , Komarova Yulia , Malik Asrar B.
Published: June 2021

Pulmonary edema is a potentially life‐threatening condition in which interstitial and alveolar spaces fill with fluid. This can be the result of left heart failure and a rise in lung capillary pressure. Piezo1 is a mechanosensitive ion channel expressed in endothelium that is activated by changes in plasma membrane curvature due to high pressure. Here, we address the role of Piezo1 in sensing increased lung capillary pressure that leads to endothelial barrier disruption through disassembly of Vascular Endothelial (VE)‐cadherin (VE‐cad) adhesion at adherens junctions (AJ). We showed that pulmonary capillary pressure rise of 16 cmH2O elicited increased permeability to liquid and albumin in lungs of Piezo1+/+ mice and these responses were prevented in mice deleted of Piezo1 gene in endothelial cells (Piezo1iΔEC). Piezo1iΔEC mice demonstrated no significant change in lung vascular albumin permeability at 24h after aorta constriction, which causes capillary pressure and left heart failure, in contrast to Piezo1+/+ mice, which demonstrated 4‐fold increase in permeability. Consistent with increased lung capillary permeability, activation of Piezo1‐mediated signaling increased VE‐cadherin phosphorylation at both Y685 and Y731 that coincided with VE‐cad internalization and AJ disassembly. Thus, Piezo1‐activated signaling in endothelial cells causes endothelial barrier disruption. These results uncover endothelial cell expressed Piezo1 as a new drug target for treatment pulmonary edema associated with left heart failure.

Support or Funding Information

This work was supported by National Institutes of Health Grants T32 grant and HL45638

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 Sulfide inhibits store‐operated calcium influx by selectively targeting STIM1‐Orai3 interactions.
Authors: Fresquez Adriana Maria , White Carl
Published: June 2021

Hydrogen sulfide (H2S) is an endogenous signaling molecule that posttranslationally modifies cysteine residues on target proteins involved in regulating the physiology and pathophysiology of neurodegeneration, blood pressure, metabolism, and inflammation. We recently determined that H2S impinges on Ca2+ signaling‐regulated processes by inhibiting store operated Ca2+ entry (SOCE). The SOCE machinery consists of the plasma membrane‐localized Orai channels and ER‐localized STIM proteins. Interestingly, H2S inhibits SOCE mediated by STIM1‐Orai3 and not STIM1‐Orai1 or Orai2 interactions. The current study tested the hypothesis that H2S achieves selectively and disrupts STIM1‐Orai3 interactions by targeting cysteines unique to Orai3. The approach was to transiently transfect either CFP‐ or mCherry‐tagged Orai3 into HEK293 cells stably expressing STIM1‐YFP and measure SOCE by Ca2+ imaging. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2 sequences. Mutation of either of these cysteines to serine, alone or in combination, abolished SOCE inhibition by the H2S donor GYY4137. Residues C226 and C232 in Orai3 are predicted to form an extracellular intermolecular disulfide. To determine the requirement for disulfide formation, cells were pretreated with DTT. When performed prior to GYY4137 treatment, disulfide reduction also abolished inhibition of SOCE by GYY4137. SOCE is activated upon ER Ca2+ store depletion when STIM interacts with and gates open Orai channels. We postulated that H2S inhibited SOCE by inhibiting STIM1‐Orai3 interactions. To test this, cells were treated with GYY4137 both before and after store depletion. GYY4137 treatment decreased SOCE only when cells were pretreated with GYY4137. These data suggest that H2S modification of Orai3 inhibits the initiation of STIM1‐Orai3 interactions but cannot disrupt them once formed. We next visualized the interaction between STIM1‐YFP and Orai3‐mCherry using confocal microscopy and measured the rate of YFP‐mCherry puncta formation upon store depletion. Pre‐treatment with GYY4137 caused a decrease in puncta formation rate. Taken together, these data are consistent with a model in which H2S impairs SOCE by modifying a disulfide bridge on Orai3 resulting in decreased STIM1‐Orai3 interactions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic L‐type CaV1.2 channel trafficking facilitates CaV1.2 clustering and cooperative gating
Authors: Ghosh Debapriya , Nieves‐Cintrón Madeline , Tajada Sendoa , Brust‐Mascher Ingrid , Horne Mary C. , Santana Luis F. , Navedo Manuel F.
Published: June 2021

The L‐type CaV1.2 channel plays an essential role in gene expression, cell excitability and muscle contraction. These channels clustered throughout the plasma membrane of excitable and non‐excitable cells. This has been suggested to contribute to regional variations in channel activity and the concerted opening and closing of adjacent CaV1.2 channels (e.g. cooperative gating), but whether dynamic intracellular and near‐membrane trafficking of the channel modulates these features is unknown. Here we show dynamic transport of CaV1.2 in vesicles of circular and tubular shape with diverse intracellular and submembrane trafficking patterns. Microtubules and actin filaments are required for dynamic movement of CaV1.2 vesicles, which undergo constitutive homotypic fusion and fission events to sustain CaV1.2 clustering, channel activity and cooperative gating. In conclusion, our study reveals very diverse and unique intracellular and near‐membrane dynamics of the CaV1.2 channel in which homotypic vesicle fusion may be a key event promoting CaV1.2 membrane organization and clustering to facilitate its physical proximity for cooperative gating and fine‐tune control of Ca2+ signals.

Support or Funding Information

NIHRO1HL098200, NIHRO1HL121059

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 regulation of muscle growth by miR‐23a
Authors: Beach Cassandra Lynn , Petrocelli Jonathan , Stott Nicole , Marino Joseph
Published: June 2021

Background

Micro‐RNAs (miRNAs) are a class of small, non‐protein coding RNAs that posttranscriptionally regulate gene expression through the degradation or translational inhibition of their target complimentary messenger RNAs (mRNAs). Developing evidence indicates the importance of miRNAs in the myogenic program and their influence on disorders affecting muscle growth and metabolism.

Purpose

The purpose of this study was to determine whether miR23a regulates myotube formation in skeletal muscle under lipid‐induced atrophic conditions.

Methods

C2C12 myoblasts were transfected with a mimic complex to increase levels of miR23a or vehicle at seeding, then induced to differentiate into myotubes. Cells were treated with palmitic acid (0.1mM) at day 0 and day 2 of differentiation. Cells were collected at day 4 of differentiation. Real‐time PCR was used to investigate changes in miRNA expression and myosin heavy chain staining was performed to determine differences in myotube formation.

Results

Cells that received mimic had increases in miR‐23a levels. Palmitic acid‐induced reductions in myotube formation were abrogated with overexpression of miR‐23a.

Conclusions

These data suggest that miR23a may exist in a regulatory pathway that alters muscle growth in the presences of saturated fatty acid. Future work is ongoing to understand the mechanisms by which miR23a may regulate skeletal muscle growth under lipid‐induced atrophic conditions.

Support or Funding Information

Faculty Research Grant

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 Effects of Palmitic Acid on MicroRNA‐26a Expression and Skeletal Muscle Insulin Signaling
Authors: Petrocelli Jonathan Joseph , Beach Cassandra , Stott Nicole , Marino Joseph
Published: June 2021

Background

MicroRNAs (miRs) are non‐protein coding RNAs that interact with messenger RNAs (mRNAs) to post transcriptionally regulate protein translation. The miRs important in regulating skeletal muscle insulin resistance are not fully identified.

Purpose

The purpose of this study was to determine the role of miR‐26a on lipid induced insulin resistance in skeletal muscle cells.

Methods

C2C12 mouse skeletal muscle cells were treated to increase levels of miR‐26a (mimic) using commercially available kits (Qiagen). C2C12 mouse myoblasts were grown to full confluence, serum starved for 3 hours then treated with 0.5 mM palmitic acid (PA) to induce insulin resistance. Cells received PA for 24 hours followed by a 100 nM insulin treatment for 15 minutes. Real‐time PCR was used to examine levels of miR‐26a, and Western blotting was utilized to investigate insulin sensitive protein expression.

Results

Cells receiving mimic had increases in miR‐26a levels and cells receiving palmitic acid had decreased responsiveness to insulin. Overexpressing miR‐26a levels in C2C12 muscle cells improved responsiveness to insulin in the presence of palmitic acid.

Conclusions

Here we show that increasing miR‐26a levels in vitro can improve insulin responsiveness in mouse skeletal muscle cells with lipid induced insulin resistance. The mechanisms of how miR‐26a is operating in skeletal muscle need to be further explored.

Support or Funding Information

Faculty Research Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of 17β‐Estradiol on Endothelial Cell Expression of Inflammation‐Related miRNAs
Authors: Levy Ma’ ayan V. , Hijmans Jamie G. , Stockelman Kelly A. , Brewster Lillian M. , Reiakvam Whitney R. , Goldthwaite Zoe A. , Greiner Jared J. , DeSouza Christopher A.
Published: June 2021

MicroRNAs (miRs) are short single stranded noncoding RNAs that are involved in the regulation of a number of physiological and pathological processes. miRs down regulate target gene expression post‐transcriptionally by degrading messenger RNA and/or by blocking translation. It is now recognized that miRs play a key role in regulating inflammatory processes underlying cardiovascular disease (CVD). For example, altered expression of specific miRs, such as miR‐126, miR‐146a, miR‐150 and miR‐181b have been linked with heightened vascular inflammation, atherosclerosis and CVD risk. It is well established that 17β‐estradiol is anti‐inflammatory and vasculo‐protective. Indeed, 17β‐estradiol‐mediated reductions in vascular inflammation have been linked with reduced pathogenesis of CVD. However, the mechanisms by which 17β‐estradiol influences vascular inflammation are not fully understood. Accordingly, the aim of this study was to determine the effects of 17β‐estradiol on endothelial cell expression of miR‐126, miR‐146a, miR‐150 and miR‐181b. Cultured human umbilical vein endothelial cells (HUVECs) were plated at a density of 5.0×105 cells/condition. Cells were treated with media alone or media containing 17β‐estradiol (100 nM) for 48 h. To determine miR expression, RNA was extracted from 1.0×105 cells and quantified using RT‐PCR. Cellular expression was normalized to RNU6 and calculated as fold change in ΔΔCt from control (N=4, experimental units). Cellular expression of miR‐126 (2.40±0.56 fold), miR‐146a (2.40±0.58 fold), miR‐150 (2.01±0.36 fold) and miR‐181b (2.47±0.56 fold) was significantly increased (240%, 240%, 200% and 250% respectively) in response to 17β‐estradiol treatment compared with control. These data indicate that 17β‐estradiol beneficially affects cellular expression of inflammation‐associated miRs. Higher cellular expression of miR‐126, miR‐146a, miR‐150 and miR‐181b is associated with reduced vascular inflammation by targeting nuclear factor κB (NFκB) activity. The alteration of the cellular expression of these miRs may play a role in the anti‐inflammatory and vasculo‐protective effects of 17β‐estradiol.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of miR‐101‐3p Prevents Human Aortic Valve Interstitial Cells Calcification through Regulation of CDH11/Sox9 Expression
Authors: Chen Jianglei , Lin Yi , Sun Zhongjie
Published: June 2021

Background

Calcific aortic valve disease (CAVD) is one of the most prevalent forms of cardiovascular disease in the elderly population. There is no effective treatment for CAVD due to unknown mechanism,. The purpose of this study is to investigate whether miR‐101‐3p plays a role in human aortic valve interstitial cell (HAVIC) calcification and elucidate the underlying mechanism.

Methods and Results

Small RNA deep sequencing and qPCR showed a significant increase of miR‐101‐3p in HAVICs isolated from calcified human aortic valves compared with normal valves. In vitro study using cultured primary HAVICs showed that mimic of miR‐101‐3p promoted calcification and upregulated osteogenesis pathway, while inhibition of miR‐101‐3p in the osteogenic conditioned medium prevented calcification and inhibited the osteogenic differentiation in the HAVICs. We found that miR‐101‐3p directly targeted CDH11 and Sox9 expression which were also downregulated in the calcified human aortic valves and HAVICs. Inhibition of miR‐101‐3p restored the CDH11 and Sox9 expression in calcific condition, downregulated asporin expression and prevented osteogenesis in the HAVICs.

Conclusions

Upregulation of miR‐101 plays a crucial role in the calcification process in the HAVICs by directly regulating the expression of CDH11 and Sox9. Inhibition of miR‐101 prevents the calcification in the HAVICs and downregulates asporin through restoring CDH11 and Sox9 expression. Thus, miR‐101 may be an effective therapeutic target for CAVD.

Support or Funding Information

NIH R01 HL118558, AG049780, DK093403

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 human immune response to RSV includes a loop between the tumor suppressor p53 and expression of APOBEC3 genes
Authors: Gladwell Wesley , Menendez Daniel , Li Heather , Yost Oriana , Resnick Michael A. , Kleeberger Steve
Published: June 2021

Background

Respiratory syncytial virus (RSV) infects nearly all children by theirsecond year of life with re‐infections occurring throughout adolescence andadulthood. RSV infection typicallyresults in mild host symptoms. However, susceptible individuals can develop severe lower respiratory tract infectionsand bronchiolitis, sometimes leading to hospitalization or death. Recently we established that in addition toits protective role against cancer development, the tumor suppressor p53 alsocontributes to host immune responses regulating the expression of several genesincluding the APOBEC3 family of DNA cytidine deaminases (A3), consideredsentinels in innate immune response against viral infections.

Objective

Usinghuman lung cancer cells, we tested the role of p53 in response to RSV infectionand its replication through the regulation of the A3 gene family as well asother components of the immune response.

Methods

A549 and H1299 cells were infected with RSV (Strain A2). Cells were thenharvested 72–120 hours post‐infection and qPCR and Nanostring arrays? were usedto evaluate mRNA expression of RSV‐g, P53, A3s and other immune targets. Expression of RSV was assessed? by ConfocalMicroscopy and cytofluorometry while Annexin V/PI assay was used to evaluateapoptosis.

Results

In response to acute and chronic RSV infection in human lung cancer cells, p53 was activated and had an impact on RSV replication, which involves theexpression in a p53 dependent manner of several members of the A3 gene familyas well as other innate immune genes. APOBEC3G (A3G) was highly induced in a dose‐ and time‐dependent mannerafter RSV only in lung cancer cells harboring WT p53. Accordingly, overexpression of A3G in lungcancer cells reduced RSV replication. Furthermore, a Nanotstring transcriptome analysis (immune code set) after RSV infectionrevealed a strong p53 dependent regulation of immune response genes, includingseveral components of the innate immune antiviral response. In addition, RSV infection induced p53phosphorylation at serine 15 and triggered p53‐dependent apoptosis in p53 functional cells while p53 deficiency correlated with increased viralreplication and increased syncytia phenotype.

Conclusions

Overall, our findings establish that the “guardian of the genome” role ascribed to p53 also extends to a unique component of the immune system – the A3 genes – revealing novel components in the antiviralimmune response against RSV infection.

Support or Funding Information

Supported by NIEHS Division of Intramural Research Program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Do Omega‐3 Fatty Acids Prevent the Development of Nonalcoholic Fatty Liver Disease?
Authors: Gagen Ashley N. , Schaller Megan L. , Maciejewski J. Claire , Sandoval Karin E. , Witt Ken A. , Wooten Joshua S. , Guilford Brianne L.
Published: June 2021

The prevalence of obesity in the United States is at an all‐time high. It is estimated that 80–90% of the obese population has nonalcoholic fatty liver disease (NAFLD), which is characterized by excess lipid accumulation in the liver (when lipid exceeds >7% of total liver mass). NAFLD can progress to nonalcoholic steatohepatitis (NASH) that includes development of hepatic inflammation, fibrosis, and cell death. Omega‐3 polyunsaturated fatty acids found in fish oils exhibit beneficial effects on regulating inflammation and may hold therapeutic potential as a preventative treatment of NAFLD.

PURPOSE

Assess the preventative effects of omega‐3 fatty acids on the development of NAFLD in high‐fat fed mice.

METHODS

Male C57BL/6 mice were randomized into four diet groups (n = 12/group) for 32 weeks: 10% low fat fish oil (LFFO), 41% high fat‐fish oil (HFFO), 10% low fat‐lard (LFL), or 41% high fat‐lard (HFL). At study completion, mice were euthanized and livers were frozen in O.C.T. compound in 2‐methylbutane while submerged in liquid nitrogen and stored at −80°C. Livers were cryosectioned at a thickness of 10 μm at −21°C. Fluorescence immunohistochemistry using Nile Red then allowed for the microscopic localization and quantification of neutral lipid droplets. The area of positive staining was calculated as a percentage of total section area, and an average neutral lipid droplet size was determined using Nikon Elements. A glucose tolerance test was performed at end study and the trapezoidal method was used to calculate total area under the curve (AUC).

RESULTS

Body weight in the HFL group was significantly greater than all other groups but glucose total AUC was only greater in HFL compared LFFO (p < 0.05). In addition, body weight (p < 0.05) and fasting glucose (p < 0.05) was greater in HFFO compared to LFFO. Based on this data, we expect a diet rich in omega‐3 fatty acids will mitigate hepatic lipid accumulation during prolonged feeding of a high fat diet. It is expected that HFL will have significantly greater liver lipid accumulation than HFFO, LFFO, and LFL.

CONCLUSIONS

These findings would suggest a protective effect of omega‐3 fatty acids on development of NAFLD.

Support or Funding Information

Supported by NIH R21NS090282‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects Of Dates (Phoenix Dactylifera L.) On Basal And Stimulated Gastric Acid Secretion Due To Histamine, Pentagastrin And Carbachol In Rats
Authors: Onwuchekwa Chinedu , Okon Bassey
Published: June 2021

Local folk claims that dates (Phoenix dactylifera L.) has a gastric beneficial role in humans. This study was carried out to examine the effects of dates on basal and stimulated gastric acid secretion (GAS) in the Wistar rats. Each group of rats consisting of 6 rats (180 – 210 g) were treated with aqueous undialyzed extract of dates (1.5 mg/kg, 2.5 mg/kg and 4 mg/kg) orally for 21 days. At the last day of the treatment, the rats were fasted for 24 hours, anaesthetised with ketamine. GAS ( mEq/l) induced by histamine, pentagastrin and carbachol administration were assessed by the continuous perfusion technique. Data were analysed using student's t‐test and ANOVA at p= 0.05.

The aqueous extract of undialyzed dates significantly decreased (p<0.05) GAS induced by the histamine, pentagastrin and carbachol in a dose‐ dependent manner over 80 minute's period in all the treated groups. The gastroprotective action of dates may be multifactorial and this may include an anti‐secretory action.

Support or Funding Information

Usmanu Danfodiyo University, Sokoto Nigeria

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Canonical and non‐canonical inflammasomes are differentially regulated in NAFLD, which may play important roles in sensing hyperlipidemic DAMPs and initiating the pathogenesis of the disease and liver inflammation
Authors: Drummer Charles Edward , Johnson Candice , Wang Hong , Yang Xiao‐Feng
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) is becoming increasingly common in countries with a Western‐style high fat diet. The caspase‐1‐dependent inflammasome pathway contributes to the pathogenesis of NAFLD and other chronic liver diseases. Current understandings are that caspase‐1 and caspase‐4 work synergistically in the canonical inflammasome pathway. However, recent research indicates that caspase‐4 can be activated independently and activate the non‐canonical inflammasome pathway. While the pathogenesis of the canonical inflammasome pathway in NAFLD has been studied, the non‐canonical inflammasome pathway is not well understood in this severe liver disease. We hypothesized that the non‐canonical pathway has its own up‐stream and down‐stream mediators, which may be independent of that associated with the canonical pathway. To test our hypothesis, we performed experimental data mining in various NAFLD NIH‐NCBI‐GEO microarray datasets. Our analysis revealed the following findings: 1) there is no overlap of the top ten upregulated pathways between the canonical and non‐canonical inflammasome pathways; 2) there is no overlap of the top ten downregulated pathways between the canonical and non‐canonical Inflammasome pathways; 3) the top pathways specifically mediating upregulation of non‐canonical pathways included two cell biology pathways apoptosis signaling and death receptor signaling; 4) the non‐canonical inflammasome pathways were metabolically related to adipose macrophage signaling (ATM) and docosahexaenoid acid (DHA) signaling; and 5) the non‐canonical inflammasome pathway was associated with down‐regulation of three retinoid × receptor alpha signaling pathways (FXR/RXR activation, LXR/RXR activation VDR/RXR activation). Of note, these nuclear receptors are metabolism master regulators, which were recently classified into our newly proposed anti‐inflammatory homeostasis‐associated molecular pattern (HAMP) receptors model (PMID: 29065888). Our results have demonstrated that canonical and non‐canonical inflammasome pathways are differentially regulated in NAFLD and that three retinoid × receptor alpha signaling pathways may specifically inhibit the upregulation of non‐canonical inflammasome pathways but not the canonical pathways. These findings have provided novel insights on differential regulatory mechanisms of canonical and non‐canonical inflammasomes in the liver and their potential roles in sensing hyperlipidemic DAMPs and initiating the pathogenesis of NAFLD.

Support or Funding Information

NIH‐NHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sub‐organ Fractionation of Hepatic Cells after Antisense Oligonucleotide Treatment in Mice
Authors: Murray Heather , Peralta Raechel , Damle Sagar , Lee Richard , Prakash Thazha , Seth Punit , Fu Wuxia , Murray Sue , Guo Shuling
Published: June 2021

Nonalcoholic Steatohepatitis (NASH) causes chronic liver disease and leads to advanced fibrosis and cirrhosis. Current research is unclear which liver cell population is key for treatment of the disease. Antisense oligonucleotide (ASO) drugs are a likely therapy for NASH due to their preferential distribution to the liver. Moreover, conjugates can be attached to the ASO and enable targeted cell specific drug delivery to the liver. Conjugations to the ASO also facilitate enhanced potency in targeted cell types by utilizing different uptake mechanisms. Unconjugated ASOs preferentially accumulate in the non‐parenchymal (NP) cells of the liver including liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) before reaching the hepatocytes. Conversely triantennary N‐acetyl galactosamine (GalNAc)‐conjugation of ASOs results in a shift of ASO to preferentially accumulate in the hepatocytes over the NP cells (Prakash et al. NAR, 42: 8796, 2014). A recent study was designed to investigate the ASO activity in hepatocytes, LSEC, KC, and HSC from mice fed a standard chow diet compared to mice fed a Western diet. The liver pathology of the mice fed a Western diet at the time of ASO treatment was indicative of a NASH diseased state (steatosis, steatohepatitis and fibrosis). Mice were injected subcutaneously, at the indicated doses, with unconjugated or GalNAc‐conjugated ASO for 6 weeks. Following treatment, mouse livers were perfused, the hepatocytes were isolated by Percoll gradient and the NP cells by Optiprep (Lodixanol) gradient. NP cells were further isolated into enriched populations by fluorescence‐activated cell sorting (FACS). Whole liver and isolated cell populations were evaluated for RNA reduction after treatment. Drug concentration in each cell type was also measured using an ELISA based assay. As expected, there was target mRNA reduction with unconjugated and conjugated ASOs in all hepatocyte cell fractions. There was also targeted mRNA reduction in all NP cell populations in both healthy and diseased mice. However, the HSC cell population contained the least amount of ASO resulting in the lowest level of targeted reduction. These results suggest that GalNAc‐conjugated ASO can decrease targeted RNA not only in hepatocyte cells, but also in LSEC, KC and HSC cells despite a lower dose and a sever diseased state. This finding illustrates the ability of GalNAc‐conjugated ASO to enhance drug potency yet still allow drug delivery to all liver cell types. Therefore, ASO treatment is an attractive therapy for liver diseases, such as NASH, that involve multiple cell types contributing to disease progression.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiorespiratory Fitness Stipulates Presence and Normalization of NAFLD In a Community‐Based Adults Enrolled in a Lifestyle Modification Program
Authors: Tomeleri Crisieli Maria , Kano Hugo Tadashi , Barros Layene Peixoto , Burini Roberto Carlos
Published: June 2021

RATIONALE

Non‐alcoholic fatty‐liver disease(NAFLD) is the most frequent liver disease in western countries and has obesity as its main risk and cardiovascular mortality as worse outcome. NAFLD might be secondary to environmental factors such as energy dense food and sedentary lifestyle.

PURPOSE

To characterize the effects of physical activity and fitness on the presence of NAFLD in a community‐based adults submitted to a Lifestyle Modification Program(LiSM).

METHODS

Baseline data from 1010 individuals, selected for the LiSM, from 2005 to 2017, were cross‐sectionally analysed for medical, anthropometry, plasma biochemistry, physical activity (IPAQ version 8) and fitness(hand grip, flexibility and cardiorespiratory‐Balke's treadmill protocol). The algorithm based on BMI, waist circumference (WC), triglycerides and GGT was used to develop the “fatty liver index” (FLI), which varies between 0 and 100 with a FLI ≥ 60 ruling in the NAFLD. Longitudinal analysis were undertaken in data from 583 subjects submitted to a 10‐wk LiSM intervention with daily supervised mixed‐physical exercises(5x/wk, 80min/session/60–80% VO2max) and dietary counseling. FLI values were splitted into quartiles for comparison with co‐variables. SAS version 9.2 was used to complete ANOVA and evaluated FLI quartiles(Q1–Q4). Tukey's post hoc was applied with significance level set at p<0.05.

Results

FLI quartiles were Q1(<36.5), Q2(36.6–59.8), Q3(59.9–84.7) and Q4(>84.8). Q4 was predominantly male, lower schooling, lower income and in (self‐ perceptive) bad health. Q4 showed also lower physical activity and lower aerobic fitness without changes in flexibility and strength. FLI correlated negatively with VO2max(r=−0.49, p<0.00001)values. After LiSM, 95 subjects had reduced their FLI from ≥60(M0) to <60(M1). In a (%)muscle mass dependent effect, VO2max determined the FLI responses to LiSM,

Conclusion

Physical inactivity/aerobic fitness were risk factors for higher FLI, overcome by LiSM effects on aerobic conditioning.

Support or Funding Information

Suported by CNPq and CAPES

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing the Impact of Ethanol on Developing and Adult Zebrafish (Danio rerio)
Authors: Chiari John B. , Charles Jamie M. , McGinnis Courtney L.
Published: June 2021

Zebrafish have been used for many years in developmental and reproductive biology, yet little is known about the adult zebrafish liver. Additionally, many of the mammalian liver cell types have yet to be characterized in zebrafish, including Kupffer cells, oval cells, and stellate cells. There is minimal information available regarding zebrafish liver transporters and nuclear receptors. Therefore, it is important to understand the mechanism of xenobiotic metabolism in zebrafish to support their use as a model organism for studying the fate and biological effects of xenobiotics. The value of zebrafish as a model organism is based on a broad set of gene similarities (among fish species and humans) involved in metabolism and transportation of endogenous and exogenous chemicals. While there are molecular similarities between zebrafish and humans, there are also differences that may impact how compounds are metabolized. Therefore, it is absolutely necessary to investigate and validate the assumptions that zebrafish possess conserved genes, enzymes, and processes involved in all stages of metabolism. This study investigates the impact of ethanol exposure on liver toxicity markers in adult Danio rerio and the impact of ethanol treatment on developing zebrafish. For the adult study, liver tissue from sham and ethanol injected fish were collected, pooled and RNA was isolated for qPCR. Relative expression levels for each gene were determined and normalized to β‐actin. Of the seventeen genes that were assessed, our results demonstrate a significant up‐regulation of the following genes following ethanol treatment: abcb4 (mdr3), abcc1 (mrp1), abcc4 (mrp4), abcc9 (sur2), abcg5, mate1, oatp1 and slc4a2 (ae2). Alcohol metabolism increases acetate production, a precursor for cholesterol and fatty acid synthesis. Historically, this increase in acetate levels driven by alcohol, is a contributor of hepatic steatosis (fatty liver). This was demonstrated in a chronic ethanol exposure study of developing zebrafish from 10dpf to 90dpf, which displayed hallmark signs of hepatic steatosis. An upregulation in slc4a2, a sterol and cholesterol transporter aimed to eliminate dietary cholesterol, was found, consistent with the digestion of high levels of acetate. Furthermore, upregulation of abcc4, a prostaglandin transporter was found, indicating oxidative stress within the liver. Additionally, the upregulation of the other liver transporters is consistent with normal hepatic ion flow during xenobiotic metabolism.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Whole Milk and Full‐Fat Dairy Products on Vascular Function in Adults with Elevated Blood Pressure
Authors: Roy Stephen , Lapierre Stephanie , Baker Brett , Fico Brandon , Gourley Drew , Shah Jay , Delfausse Laura , Tanaka Hirofumi
Published: June 2021

We have previously demonstrated that the solitary addition of nonfat dairy products to the normal routine diet was capable of improving overall vascular function. However, it is unknown whether whole fat dairy products mediate similar vascular responses as most of the available cohort studies do not support the association between full‐fat dairy products and arterial blood pressure. Therefore, the purpose of the study was to determine the efficacy of conventional full‐fat dairy products as part of the normal routine diet to improve vascular function in adults with elevated blood pressure. A total of 60 otherwise healthy men and women (mean age of 58 ± 2 years) with pre‐ or stage I hypertension (systolic and diastolic blood pressure: 120–159/80–99 mmHg) were recruited. They underwent a randomized controlled cross‐over dietary intervention encompassing 4 weeks of 4 daily servings of dairy (whole milk, cheese, or yogurt) and 4 weeks of a daily isocaloric non‐dairy control (coconut milk, orange juice, peanuts, and applesauce) provided by investigators. A 2‐week washout period was incorporated between the two dietary conditions to remove any residual effects. Body weight did not change throughout the experimental period. Brachial artery flow‐mediated dilation, an index of endothelium‐dependent vasodilation, did not change significantly in either dairy (5.7 ± 0.5 vs. 5.4 ± 0.6%) or control (6.5 ± 0.5 vs. 5.6 ± 0.6%) conditions. Central arterial stiffness, as measured by carotid‐femoral pulse wave velocity, did not change significantly following either dairy (11.3 ± 0.3 vs. 10.9 ± 0.3 m/sec) or control (11.2 ± 0.3 vs. 11.0 ± 0.3 m/sec) conditions. When arterial stiffness was assessed using ultrasound‐derived distention measures, including carotid artery compliance, arterial distensibility, and elastic modulus (preliminary samples of n=37), the results were the same as no significant changes were recorded. Central arterial systolic blood pressure did not change significantly for both dairy (125 ± 1 vs. 122 ± 2 mmHg) and control (125 ± 1 vs. 123 ± 2 mmHg) conditions. These results suggest that unlike non‐fat dairy products, addition of wholefat dairy products to the normal diet do not improve vascular function in adults with elevated blood pressure.

Support or Funding Information

Supported by the Dairy Research Institute.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Sympathetic and RAAS Responses to a Low Sodium Diet
Authors: Babcock Matthew C. , Robinson Austin T. , Watso Joseph C. , Migdal Kamila U. , Wenner Megan M. , Stocker Sean D. , Farquhar William B.
Published: June 2021

A common sodium recommendation intake target is 2300 mg/d; however the AHA has stated that no more than 1500 mg per day is ideal. Concerns have been expressed that reducing sodium intake too low could lead to maladaptive sympathetic and RAAS responses and some of this concern comes from studies where sodium/volume depletion diets were used (as low as 230 mg/d sodium).

PURPOSE

To assess the sympathetic and RAAS responses of reducing sodium intake from the widely recommended 2300 mg/d to <1500 mg/d. We hypothesized that this level of sodium restriction would stimulate RAAS but not increase sympathetic activity, since this level of sodium intake should not cause severe sodium/volume depletion.

METHODS

Eight subjects (age: 25±1 yrs, BMI: 23.4±3.9 kg/m2, 4 men) consumed 2300 mg Na+/day and 1000 mg Na+/day sodium diets (order randomized) for 10 days each. Subjects collected their urine for the final 24 hours preceding in‐lab testing. Testing was performed on the last day of each diet. Blood samples were collected for assessment of catecholamines and RAAS hormones. Angiotensin II and aldosterone concentrations were assessed in six of eight subjects. Subjects then lay quietly for 10 minutes while undergoing recording of beat‐to‐beat blood pressure (via photoplethysmography), R‐R interval (via 3‐lead ECG), and muscle sympathetic nerve activity (MSNA) (via microneurography).

RESULTS

Twenty‐four hour urinary sodium excretion was significantly lower when dietary sodium was reduced to 1000 mg/d (93±8 vs. 45±12 mmol/24 hrs, P<0.01). Resting mean arterial pressure was not different following either diet (81±2 vs. 81±2 mmHg, P>0.20). Reducing sodium intake to 1000 mg/d increased plasma renin activity (1.5±0.3 vs. 2.2±0.4ng/mL/hr, P<0.05), angiotensin II concentration (12.5±1.5 vs. 19.1±3.2 pg/mL, P<0.05), and aldosterone (11.7±1.3 vs. 20.4±4.6 ng/mL, P=0.052). However, reducing sodium intake to 1000 mg/d did not alter MSNA burst frequency (13.8±0.9 vs. 13.0±1.2 bursts/min, P>0.20), burst incidence (23.9±2.3 vs. 21.8±3.0 bursts/100 heart beats, P>0.20), or plasma norepinephrine concentrations (237±46 vs. 173±57 pg/mL, P>0.20).

CONCLUSION

These preliminary data suggest that reducing sodium from 2300 mg/d to 1000 mg/d stimulates RAAS but does not alter resting MSNA or catecholamines in healthy young adults. These findings suggest that diets as low as 1000 mg Na+/day can be consumed without stimulating the sympathetic nervous system.

Support or Funding Information

Supported by NIH Grant 1R01HL128388

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 Impact of Antibiotics on the Intestinal Microbiome and the Gut‐Brain Axis in Zebrafish
Authors: Bell Emily A. , Ball Alex G. , Deprey Kirsten L. , Uno Jennifer K.
Published: June 2021

Anxiety disorders affect over 40 million adults in the United States. Current research indicates a correlation between antibiotic exposure and the onset of anxiety. Recent findings illustrate the microbiome's role in this relationship through the bidirectional communication pathway between the gut and the brain, known as the gut‐brain axis. This study utilizes zebrafish as a model organism to examine the impact of antibiotics on the composition of the microbiome, and subsequently stress‐related behaviors. It is hypothesized that antibiotic treatment will lead to sustained changes in the microbiome and an increase in anxiety‐like behavior. Adult zebrafish were treated with antibiotics for one week then put through a net‐stress and death‐induced odors, after which, swimming behaviors were tracked. Intestinal tissue was collected and bacterial DNA was isolated to quantify total intestinal bacterial using qPCR. In order to assess stress hormone production, intestinal RNA was isolated and corticotropin‐releasing hormone binding protein (CRHbp) was quantified via RT‐PCR. Results confirmed antibiotic treatment induced a 20% decrease in universal gut bacteria in the treated fish (n = 12 p < 0.05) versus control fish. Antibiotic treatment also correlated with decrease in exploratory behavior, illustrated by a significant decrease in top tank entries (n = 12, p < 0.01) indicating treated fish were stressed. Moreover there was a corresponding 2‐fold increase in CRHbp RNA (n= 5, p < 0.05) signifying an increase in cortisol production. Interestingly, following reestablishment of the gut microbiome, fish demonstrated exploratory behavior as seen by an increase in top tank entries (p < 0.01), demonstrating a reduction in stress correlated with reestablishment of intestinal bacteria. There were no significant differences in CRHbp expression in reestablished fish. Collectively, these results signify a decrease in the diversity of the gut microbiome may elicit anxiety in zebrafish. This data warrants further investigation into the mechanism of the gut‐brain axis and how antibiotics influence microbial‐induced changes in behavior.

Support or Funding Information

This study was funded by Elon 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.
Coordination of Homeostatic Functions by Intrascapular Brown Adipose Tissue‐ and Pancreas‐related Command Neurons
Authors: Butcher Sierra Morgan , Hamling Brooke Victoria , Desmoulins Lucie Delphine , Zsombok Andrea
Published: June 2021

Preautonomic neurons located in the brainstem and hypothalamus influence glucose metabolism, food intake, and energy homeostasis through outputs to visceral organs. The existence of command neurons (neurons with connections to multiple organs) suggests that coordinated autonomic output modulates homeostasis. In this study, we identified command neurons related to the pancreas and the intrascapular brown adipose tissue (iBAT) using isogenic strains of pseudorabies virus (PRV), PRV‐614 and PRV‐152, respectively. Co‐localization of the two PRVs identified iBAT‐pancreas command neurons in the brainstem and hypothalamus in male C57bl/6J mice. Command neurons were also identified in the parasubthalamic nucleus (PSTN), a subnucleus of the lateral hypothalamus that serves as a conduit between the brainstem and hypothalamus and has previously been implicated in food intake and cardiovascular function. Since the pancreas and the iBAT are important for glucose and energy homeostasis, we hypothesized that stimulation of the PSTN modulates food consumption and glucose tolerance. A viral construct (stimulatory designer receptor exclusively activated by designer drugs) was injected to the PSTN. Following a fasting period, mice received an i.p. injection of clozapine‐N‐oxide (CNO, 1mg/kg) to activate PSTN neurons 30 minutes prior to the reintroduction of food or a glucose challenge (2 g/kg). Stimulation of the PSTN increased food intake during the first 30 minutes of feeding. The average food intake in mice that received CNO was 0.052 ± 0.0076 g of food ingested/g of body weight, while the average in those that received vehicle was 0.0346 ± 0.0036 g of food ingested/g of body weight (p=0.0468, n=8). The average cumulative food intake after 6 hours was significantly increased in mice treated with CNO compared to vehicle injected mice (0.1386 ± 0.0177 g of food ingested/g of body weight vs. 0.0984 ± 0.005 g of food ingested/g of body weight) (p<0.05, n=8). On the other hand, stimulation of the PSTN did not alter glucose tolerance. The average area under the curve (AUC) was not significantly different among the groups (CNO and glucose: 1362.98 ± 60.52 AUC, vehicle and glucose: 915.37 ± 27.71 AUC, CNO and saline: 1156.51 ± 53.31 AUC) (p>0.05, n=8). These findings revealed that stimulation of the PSTN increased cumulative food intake but had no effect on glucose tolerance. In summary, our study demonstrates the existence of iBAT‐pancreas command neurons in brain areas including the PSTN and suggests that the PSTN is involved in the control of food intake.

Support or Funding Information

National Institutes of Health R01 DK099598 for AZ

Board of Regents for BH

Core Facilities (Tulane School of Medicine and Louisiana State University Health Sciences Center)

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