bluuebunny/crossref_metadata_2025 huggingface dataset:
Empagliflozin effects on ischemic contracture and I/R injury in isolated mouse hearts perfused with or without insulin
Authors: Uthman Laween , Nederlof Rianne , Eerbeek Otto , Baartscheer Antionius , Buchholtz Ninée , Coronel Ruben , Hollmann Markus W. , Weber Nina C. , Zuurbier Coert J.
Published: June 2021

Introduction

The kidney‐targeted Empagliflozin (EMPA) has shown large unexpected cardiovascular benefits, which cannot be explained by general cardiovascular risk factors. We recently reported that EMPA has sodium hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes. Classic NHE inhibitors such as Cariporide (CARI) delay ischemic contracture onset and reduce ischemia reperfusion (I/R) injury in the heart. Here we examined whether EMPA delays contracture onset and reduces I/R injury. Both normal‐ and low‐glycogen hearts were used, because the rate of glycogen depletion during ischemia affects contracture onset and IR injury.

Methods/Results

Isolated mouse hearts were perfused with and without 50 mU/L insulin, to create normal‐ and low‐glycogen hearts respectively, and subjected to 25 min I and 120 min R. 1μM EMPA, 10μM cariporide (CARI; positive control) or vehicle was administered prior to ischemia until 10 minutes post‐reperfusion. In normal‐glycogen hearts, EMPA and CARI did not change ischemic contracture (in seconds, vehicle 1056±56, EMPA 900±44, CARI 880±84) and IR injury (infarct size (%): vehicle 59±6, Empa 62±10, Cari 68±4), however in low‐glycogen hearts, both EMPA and CARI delayed ischemic contracture onset (vehicle 459±25, EMPA 559±22, CARI 588±31). Higher EMPA concentration further delayed contracture onset. Only CARI reduced I/R injury (from 51±6 to 34±5). The presence of insulin (same concentration used) did not alter NHE activity, nor the ability of CARI or EMPA to inhibit NHE, in isolated cardiomyocytes. The increased IR injury in normal‐glycogen hearts was associated with increased end‐ischemic lactate and glucose‐6‐phosphate accumulation, and consequently decreased mitochondrial hexokinase ll.

Conclusion

EMPA does not protect against I/R injury in isolated hearts, yet EMPA delayed ischemic contracture onset in low‐glycogen hearts, suggesting a direct cardiac, ATP preserving, effect of EMPA during ischemia. Cardiac glycogen content interferes with the effects of NHE inhibitors on I/R injury in isolated hearts. These data indicate that empagliflozin and cariporide may inhibit NHE in different manners.

Support or Funding Information

None.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PVN Orexin Receptor 1 Knockdown Effect on Metabolism and Fluid Homeostasis
Authors: Bigalke Jeremy Andrew , Jiang Enshe Jasen , Hahka Taija Marie , Chen Qing Hui , Shan Zhiying Jenny
Published: June 2021

Previous research has shown the importance of orexin, a neuropeptide, in the regulation of appetite, sleep, and arousal. However, limited research has been done on the impact of orexin in the hypothalamic paraventricular nucleus (PVN) on the control of metabolism and body fluid homeostasis. In this study, we investigated the role of PVN orexin signaling on metabolism through chronic knockdown of orexin 1 receptor (OX1R) using AAV1‐OX1R‐shRNA. Seven‐week‐old male Dahl Salt sensitive rats were divided into two groups and were microinjected with either AAV2‐scembled‐shRNA (n=6) or AAV2‐OX1R‐shRNA (n=5) into the PVN. After one week recovery from the surgery, all rat baseline body weights were measured. There was no significant difference found between the groups. The rats were then fed with a high salt diet (HS, 4% NaCl) for 5 consecutive weeks, after which body weights were once again measured and the rats were then transferred into individual metabolic cages. Rats were given 24 hours to get accustomed to the new environment and their food intake, water intake, urine excretion, and fecal output were measured for 24 hours. The results showed that PVN OX1R knockdown rats had a lower body weight when compared to control rats after the 5 weeks of high salt intake (Control: 336.6±9.10, vs. OX1R‐shRNA: 299.8±14.62 g P<0.05). In addition, chronic knockdown of PVN OX1R showed a significant decrease in water intake (Control: 43.67±3.72, vs. OX1R‐shRNA: 24.96±5.16 mL P<0.05), urine output (Control: 27.0±3.32, vs. OX1R‐shRNA: 14.04±2.75 mL P<0.05), and food intake (Control: 20.85±2.16, vs. OX1R‐shRNA: 13.68±1.84 g P≤0.05), but there was no fecal output alteration observed between PVN OX1R knockdown rats and control rats. At the end of this protocol, rats were euthanized and their brains were removed and subjected to either real time PCR or immunostaining to test mRNA or protein expression of the genes of interest in the PVN. Real time PCR showed that AAV2‐OX1R‐shRNA injection decreased OX1R mRNA level by 41%, and melanocortin‐4 receptor (MC4R) mRNA by 68% compared to control vector injection group. Immunostaining showed that both vasopressin immunoreactivity and positive vasopressin expressing cells are dramatically decreased in the PVN of AAV2‐OX1R‐shRNA rats as well. These observations coupled with the importance of MC4R in controlling satiety, and vasopressin in controlling urine water excretion, suggest that orexin plays a large and complex regulatory role in fluid homeostasis within the body as well as in appetite, through direct mediation by the PVN.

Support or Funding Information

NIHR15 HL129213 (Shan) and Michigan Tech and Portage Health Foundation Research Excellent Fund (Shan).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reciprocal Interaction between Hypothalamic Orexin‐A and Corticotropin‐releasing Factor under Stressed Condition
Authors: Sinen Osman , Bülbül Mehmet
Published: June 2021

In response to stressful stimuli, expression of orexin‐A (OXA) is increased within central nervous system. The orexinergic cells reside in lateral hypothalamic area (LHA) have dense projections on hypothalamic paraventricular nucleus (PVN), furthermore, corticotropin‐releasing factor (CRF) receptors are expressed in orexin‐producing neurons within LHA. However, the interaction between these neuropeptides is understood incompletely. Using acute restraint stress (ARS) model in rats, the purpose of the present study was designed to investigate (i) the interaction between hypothalamic OXA and CRF, and (ii) the contribution hypothalamic OXA in CRF‐mediated glucocorticoid release. Adult male Wistar rats underwent stereotaxic cannulation for intracerebroventricular (icv) drug injection and microdialysis 7 days prior to the experiments. Under basal and stressed conditions, microdialysis samples were collected from LHA and PVN to monitor the alterations in OXA and CRF, respectively, whereas, their antagonists were administered centrally prior to the stress loading. The immunoreactivities for both neuropeptides were detected in hypothalamic sections by immunohistochemistry. The corticosterone (CORT) concentrations in plasma samples were assayed using enzyme immunoassay. Compared to their basal levels, OXA and CRF concentrations in microdialysates were increased in ARS‐loaded rats, while they were attenuated significantly by the receptor antagonists. ARS increased the immunoreactivities for OXA and CRF in hypothalamus remarkably, while increasing the plasma CORT. Central preadministration of the antagonists reduced the ARS‐induced increased hypothalamic immunoreactivites and plasma CORT. Under stressed condition, OXA and CRF seem to play in a synergistic signaling pathway through their specific receptors. The endogenous hypothalamic OXA appears to contribute to the CRF‐mediated neuroendocrine stress response. The interaction between OXA and CRF may lead to better understanding of the etiopathogenesis of stress‐related disorders.

Support or Funding Information

The present study was supported by the Research Foundation of Akdeniz University (Project number: TSA‐2017‐2418)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of the Nuclear PKA Holoenzyme
Authors: Clister Terri , Sample Vedangi , Zhang Jin
Published: June 2021

cAMP and PKA activity is regulated in time and space to ensure proper cellular responses. Recently a novel pool of activatable PKA holoenzyme was discovered to reside in the nucleus, expanding the traditional model of PKA signaling. Based on live‐cell imaging we hypothesize that the regulation of the nuclear PKA holoenzyme is dependent on phosphodiesterases (PDEs) anchored to A‐kinase Anchoring Proteins (AKAPs) which create a local microdomain with a lower concentration of cAMP, thus controlling the activation of anchored nuclear PKA. Using superresolution (pcSOFI) and FRAP techniques we've characterized AKAP95 in the nucleus. Co‐IP experiments using nuclear extracts revealed that both the RIIα subunit of the PKA holoenzyme, and multiple PDE4D isoforms interact with AKAP95, supporting our hypothesis and providing specific candidates to further explore this regulation. Our ICUE3 cAMP biosensor was localized to AKAP95 to measure the levels of cAMP directly around AKAP95 in response to differently localized sources of cAMP. We are able to generate cAMP in different cellular compartments in a dose‐dependent manner by targeting genetically encoded soluble adenylyl cyclase (sAC) and stimulating transfected cells with sodium bicarbonate. When a low concentration of cAMP is generated, only cAMP produced in the nucleus can be detected in the vicinity of AKAP95, whereas cAMP produced at the plasma membrane was not detected within this microdomain. These results support the hypothesis that AKAP95 and PDE4Ds create a microdomain that prevents the activation of scaffolded nuclear PKA holoenzyme except by local cAMP signals. We are still exploring the endogenous source of nuclear PKA activation, but hypothesize that cAMP generated by internalized GPCR signaling at the endosome could activate AKAP95‐tethered PKA.

Support or Funding Information

This work was funded by R01 DK073368.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Driving Forces and Metabolic Resilience
Authors: Muoio Deborah Marie
Published: June 2021

Evidence supporting a causal link between mitochondrial performance and disease risk has been bolstered by the advent and application of state‐of‐the‐art multi‐omics molecular profiling technologies, such as genomics, proteomics and metabolomics. Although these are powerful tools that can produce provocative molecular signatures of disease, they still do not tell us how well (or not) mitochondria resident in specific tissues are able to maintain the free energy of ATP hydrolysis (ΔGATP, i.e. ATP:ADP ratio)–the energetic currency that powers normal cell and organ function. Thus, we came to recognize that progress toward deciphering the precise role of mitochondria in the disease process requires sophisticated approaches that can assess changes in carbon flux and bioenergetics in intact mitochondria working to regenerate ATP, and maintain ΔGATP, in the context of physiologically relevant energy demands and thermodynamic constraints (i.e. backpressure). To this end, we developed a new multiplexed assay platform for interrogating respiratory kinetics and thermodynamics under multiple substrate and energetic conditions. Unlike conventional respirometry methods, the new platform permits comprehensive assessment of respiratory fluxes (JO2) and energy transduction, which refers to the transfer of energy in carbon fuels–to electron potential energy (NAD(P)H/NAD+ redox)–to membrane potential–to ΔGATP. Importantly, these assays are performed in the context of dynamic extramitochondrial ATP:ADP concentrations (i.e. ΔGATP) that are precisely controlled within a (near) physiologic range. In simple terms, our diagnostic assays mimic an exercise “stress test” by evaluating how well a given population of mitochondria, fueled by specific mixtures of carbon substrates, responds to a graded energetic challenge. We have been combining this platform with mass spectrometry‐based metabolomics and proteomics to evaluate mitochondrial remodeling, and corresponding changes in respiratory power and efficiency, in response to a variety of nutritional and genetic maneuvers. This seminar will discuss new insights pertaining to the interplay between mitochondrial driving forces and metabolic resilience.

Support or Funding Information

Supported by grants from NIDDK and NHLBI.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The actin‐binding protein cortactin controls transendothelial migration and organ infiltration of acute lymphoblastic leukemia T‐cells
Authors: Castellanos Ramón , Vadillo Eduardo , Schnoor Michael
Published: June 2021

T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive neoplasm due to high rates of organ infiltration and relapse, which are major obstacles for curing the disease. Infiltration of T‐ALL cells requires extravasation, a migratory process highly dependent on actin cytoskeleton dynamics. Cortactin is an actin‐binding protein that regulates actin remodeling and thus cell migration. High expression of cortactin is associated with invasiveness in several types of solid tumors, and recently, expression of cortactin in adult B‐cell chronic lymphocytic leukemia was correlated with negative prognostic factors. However, it is unknown whether cortactin plays a role in T‐ALL biology. Here, we analyzed cortactin expression in the murine and human T‐ALL cell lines 6645/4 and Jurkat, and cortactin functions using different in vitro and in vivo methods such as transendothelial migration, bone marrow (BM) colonization and xenotransplantation assays. Compared to normal T cells, leukemic T cells expressed significantly higher levels not only of cortactin but also of the integrin VLA‐4 and CXCR4, the receptor for the T cell chemokine CXCL12, which are both required for T cell extravasation. Leukemic T cells transmigrated significantly more, and cortactin levels were significantly higher in cells capable of transmigrating across endothelial monolayers towards CXCL12. Of note, only leukemic T cells with highest cortactin levels were capable of invading BM‐like organoids. Preliminary in vivo assays showed that leukemic T cells had different levels of cortactin depending on the infiltrated organ. Surprisingly, once leukemic T cells completed infiltration they diminished cortactin expression suggesting that cortactin is needed for initial organ invasion, but not for subsequent functions. Taken together, our results highlight an important role of cortactin in T‐ALL infiltration; and contribute to a better understanding of T‐ALL aggressiveness. Thus, cortactin may be a potential pharmacological target to prevent T‐ALL organ infiltration.

Support or Funding Information

CONACyT

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Effects of Cancer Mutations on Mre11 Function and the DNA Damage Response
Authors: Harris Caitlin , Savas Jessica , Shanle Erin Karina
Published: June 2021

When DNA is damaged, a DNA damage response (DDR) pathway is initiated to prevent mutations from being passed on to daughter cells. The MRN complex in humans (MRX in yeast) functions to start the DDR pathway by detecting DNA damage and is comprised of Mre11, Rad50, and Nbs1 in humans. Although mutations in members of the MRN complex pre‐dispose individuals to cancer, the functional effects of Mre11 cancer mutations have not been well characterized. The goal of this project was to investigate the effects of Mre11 cancer mutations on the DDR pathway using yeast‐based assays because the complex is highly conserved in eukaryotes. Using a database of human tumor mutations, we identified 135 cancer mutations in human Mre11, 46 of which occurred at residues that are conserved between yeast and human. A subset of these mutations were screened for functional effects in yeast by creating the cancer mutations at the conserved residues in yeast Mre11. Yeast expressing wild‐type or mutant Mre11 were then assessed for DNA damage sensitivity using ultraviolet light and methyl methanesulfonate. Results indicate that N‐terminal point mutations at in yeast Mre11 do not confer sensitivity to DNA damage in yeast cells suggesting that these regions may have conserved sequence without conserved function. Using yeast‐based assays, cancer mutations can rapidly be screened for functional effects on DDR pathways.

Support or Funding Information

Longwood University Faculty Research and Development Grants

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for the Accumulation of 3‐Hydroxypropionate and β‐Alanine in Arabidopsis thaliana Seedlings via Isoleucine Degradation
Authors: Goldfarb Margo H , Rouhier Kerry A
Published: June 2021

Amino acids serve several cellular processes outside of proteinogenesis that influence the growth and development of the plant, as well as the generation of metabolic energy. Here, we examine the contribution of branched‐chain amino acid degradation to the production of β‐alanine in the model organism, Arabidopsis thaliana. β‐alanine is a key intermediate in the synthesis of the essential molecules vitamin B5 and coenzyme A (CoA). Despite the importance, the current knowledge surrounding β‐alanine lacks strong evidence for the primary biosynthetic pathways in higher plants. Previous studies found that polyamines, uracil, and propionate can all serve as sources for β‐alanine. Given that the catabolism of branched‐chain amino acids (BCAAs) valine and isoleucine result in the production of propionyl‐CoA (a derivative of propionate), we hypothesized that these BCAAs could also serve as a source of β‐alanine. Propionyl‐CoA is typically converted to acetyl‐CoA via β‐oxidation, however, one of the intermediates, malonate semialdehyde, can react to form β‐alanine. To trace the production of β‐alanine from propionyl‐CoA and the BCAA isoleucine, we treated wild‐type and transgenic seedlings with isotopically‐labeled precursors and analyzed extracts using 13C‐NMR and GC‐MS. We observed the production of both β‐alanine and a key intermediate, 3‐hydroxypropionate (3HP) in samples treated with propionate and isoleucine. Our data shows that the fed 13C‐isotopes were efficiently incorporated into and recycled through propionyl‐CoA metabolism, resulting in the production of 13C‐labeled β‐alanine. These findings suggest that while there is evidence for the production of β‐alanine via polyamines, uracil, and propionate in other plants; isoleucine, and possibly valine, can also serve as a precursor to this metabolically important molecule.

Support or Funding Information

Clare Boothe Luce Undergraduate Scholars program funded by the Henry Luce Foundation (Grant #9601)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effects of Uncaria tomentosa on acetylsalicylic acid and paclitaxel
Authors: Contreras Jessica Irene , Larios Kelsy N. , Nogaj Luiza A.
Published: June 2021

For thousands of years, the indigenous women of Peru have relied on traditional medicine as a method for treating illnesses. While conventional medicine exists in Peru today, women continuously turn to the remedies they know and trust when modern medicine is inaccessible. The vast amounts of ethnopharmacological plants are utilized for a large span of conditions such as digestion, headaches, and inflammation. In particular, Uncaria tomentosa commonly known as Cat's claw, a bark like plant native to the Amazonian regions of Peru has been investigated for its historical anti‐inflammatory uses and was found to contain many antioxidant properties. However, not much is known about the effects of U. tomentosa on the molecular mechanism of drugs such as paclitaxel (taxol) and acetylsalicylic acid (aspirin). To address this question, a randomized survey was conducted on women and health care providers within the region of Cusco, Peru. The survey revealed that U. tomentosa remains a prominent healing aid within the Peruvian culture. In addition the survey showed that women overwhelmingly chose natural remedies over professional medical care. The following investigation was conducted to test the in vitro effects of U. tomentosa on the molecular mechanism of drugs paclitaxel and acetylsalicylic acid on MCF‐7 cells. An optimal concentration of paclitaxel and acetylsalicylic acid was established for further use in viability assays, MTT and Cytotoxicity. Preliminary data suggests that a 0.6 uM concentration of paclitaxel decreases cell viability by 55% and a 0.4 mM concentration of aspirin decreases cell viability by 20%. In addition, the preliminary data from molecular viability assay shows that the addition of U. tomentosa to acetylsalicylic acid and paclitaxel has an additive effect. Further analysis is necessary to determine the molecular mechanism of U. tomentosa on the activity of acetylsalicylic acid and paclitaxel.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Factors contained in the expansion medium regulate the expression stem cell markers CD146 and SUSD2 on human placenta‐derived mesenchymal stromal cells and modulate their differentiation capacity in vitro
Authors: Abruzzese Tanja , Aicher Wilhelm K
Published: June 2021

Human mesenchymal stromal cells (MSCs) are investigated in vitro and in vivo manifold to serve as cellular therapy in different clinical situations including the regeneration of muscular tissues eventually. For production of MSCs for experimental purposes expansion media often include fetal bovine serum (FBS) as source of growth factors. In clinical situations xenogenic components such as FBS must be avoided in many countries. Therefore, different xeno‐free MSC expansion media were developed and FBS was replaced e.g. by human serum (hS), platelet lysate (hPL) or hPL plus human plasma (hPLP). However, differences in these medium complements could influence the phenotype and characteristics of MSCs. We therefore compared the expression of cell surface markers, the differentiation capacity and the expression of key cytokines involved in muscular regeneration in human placenta‐derived MSC‐like cells (pMSCs) expanded in different xeno‐free and GMP‐compliant expansion media. We report that addition of 5% hPL to either commercial MSC expansion medium (medium with FBS + 5% hPL) or to GMP‐compliant MSC expansion medium (DMEM + 5% hS + 5% hPL) significantly increased the MSC proliferation (p<0.05), and reduced the expression of CD146 (p<0.05), whereas CD73 and CD90 and other markers remained unchanged, on both transcript and protein levels. Expression of stem cell marker SUSD2 was not significantly regulated by hPL. The osteogenic differentiation capacity was reduced in pMSCs expanded in medium complemented with 5% hP + 5% hPL when compared to pMSCs incubated in medium containing FBS without hPL, but not in DMEM + hS compared to DMEM+ hS + hPL. Significant changes in the expression of neuro‐ or myo‐regenerative cytokines including BDNF, bFGF, GDNF, HGF, IGF‐1, IGF‐2, TGFβ, or VEGF in MSCs was not influenced by the composition of the GMP‐compliant expansion media. However, some interindividual differences between pMSCs from individual donors and between batches of hPL were noted. In contrast, the composition of expansion media did not influence osteogenesis or expression of stem cell markers CD146 and SUSDA on human bone marrow‐derived MSCs. We conclude that the expression of CD146 and the osteogenic differentiation potential of human pMSCs is influenced by the expansion medium. The expression of the regenerative cytokines investigated was not biased by hPL in the expansion medium. Expansion of pMSCs in GMP‐compliant medium enriched by hP + hPL could therefore be an advantage when bone formation by the MSCs applied should be avoided in clinical situations.

Support or Funding Information

This work was supported in part by grants from the DFG, BMBF and by institutional funding.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying Lysine Acetylation of Aminoacyl‐tRNA Synthetases in Escherichia coli
Authors: Fan Chenguang , Gan Qinglei
Published: June 2021

Aminoacyl‐tRNA synthetases (AARSs) generate aminoacylated tRNAs for ribosomal protein synthesis, and play key roles in maintaining high fidelity of translation. A series of acetylome studies have shown that the family of AARSs is one of the protein families which have both the most acetylation sites and the highest acetylation levels. However, those putative acetylation targets have been less studied, mostly because of the challenge to produce homogeneously acetylated proteins at specific positions which needs the knowledge of specific acetyltransferases for target lysine residues. To overcome this issue, we applied the genetic code expansion strategy to co‐translationally incorporate acetyllysine at the target positions directly. We totally generated 25 site‐specifically acetylated variants of four AARSs. Kinetic analyses were performed to biochemically characterize the impact of site‐specific acetylation on AARS functions, including both tRNA aminoacylation and editing activities. The results showed that impacts of acetylation were different for class I and class II AARSs, and also varied among the same class of AARSs. Combined with quantitative proteomic studies, we proposed that the impact of acetylation on AARS editing activities could be more significant than that on tRNA aminoacylation activities. To the best of our knowledge, this is the first work to show the association of protein acetylation with the AARS editing function. Furthermore, using threonyl‐tRNA synthetase (ThrRS) as a representative, both in vivo and in vitro studies were performed to characterize the acetylation and deacetylation processes of AARSs. Although nonenzymatic acetylation and CobB‐dependent deacetylation were concluded, our results also indicated existence of additional acetyltransferases and deacetylases for AARSs.

Support or Funding Information

This work was supported by Oak Ridge Associated Universities, Arkansas Biosciences Institute, and the University of Arkansas.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational determination of the binding kinetics of the pH‐Low Insertion Peptide
Authors: Freshwater Kasey , Flint Bethann , Mertz Blake
Published: June 2021

Membrane active peptides (MAPs) have the potential to be utilized in a number of biomedical applications due to their ability to alter the membrane through surface interactions. MAPs with pH‐dependent activity can be harnessed for use in medical imaging and targeted drug delivery to cells afflicted with diseases characterized by acidosis, such as cancer, arthritis, and heart disease. An example of this type of MAP is the pH‐Low Insertion Peptide (pHLIP), which interacts with the membrane in three states: in State I, pHLIP is randomly coiled in solution; upon addition of lipid vesicles, pHLIP binds to the membrane surface and gains partial helicity (State II), and upon acidification, pHLIP fully folds, inserting unidirectionally into the membrane (State III). Currently, the peptide‐membrane interactions leading to formation of stable complexes (i.e., state II) are poorly understood. To better understand this mechanism, we employed coarse‐grained molecular dynamics (CGMD) simulations of pHLIP interacting with either 1‐palmitoyl‐2‐oleoyl‐sn‐3‐phosphocholine (POPC) or mixed POPC:1‐palmitoyl‐2‐oleoyl‐3‐sn‐phosphoserine (POPS) lipid bilayers to determine the effect of headgroup charge on the ability of pHLIP to bind to a membrane surface. Our goal was to rigorously determine the kinetics of binding between pHLIP and the bilayer and to identify the key biophysical interactions that lead to a stable binding event, as well as characterizing the effect of headgroup composition on these behaviors. Our preliminary results show that the interaction of pHLIP with a POPC bilayer initiates at the N‐terminus and induces partial helicity. As the concentration of the anionic POPS lipids increases, the interactions between the anionic residues of pHLIP in the C‐terminus and the charged lipid head groups will be less favorable due electrostatic repulsions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Substrate Selectivity of a P4‐ATPase Flippase
Authors: Dudek David , Daleke David
Published: June 2021

Phospholipids, the primary structural components of cell membranes, are distributed unequally across the membrane. The sphingolipids, including sphingomyelin (SM) and glycosphingolipids, are enriched on the exofacial surface of the plasma membrane or the luminal surfaces of internal organelles, while the cytofacial leaflet is enriched in the aminophospholipids, phophatidylserine (PS) and phosphatidylethanolamine (PE). Contributing to maintenance of this asymmetry is a combination of slow passive transbilayer phospholipid movement, trapping by cytofacial proteins and the activity of a number of energy‐dependent transporters. The substrate specificity and directionality of these pumps defines the transbilayer asymmetry of phospholipids. Two transporter superfamilies, ABC transporters and P‐type ATPases, account for these activities and move lipids in either the exofacial (floppase) or cytofacial (flippase) directions, respectively. In previous studies we have characterized the biochemical requirements and substrate specificity for a PS specific flippase activity in human red blood cells. Transport is ATP‐dependent and sensitive to vanadate and sulfhydryl‐modification. This flippase is selective for the sn‐1,2‐isomer of PS. Transport is diminished by modification of the PS headgroup amine (with the exception of mono‐methylation), carboxyl and phosphate groups, while a variety of fatty acyl groups and the D‐serine analog are accommodated. Red blood cells express two members of the P4‐type ATPase subfamily, ATP8A1 and ATP11C, that are likely responsible for this activity. Here, we report on the heterologous expression, purification, and functional assaying of hexahistidine‐tagged constructs of Atp8a1 with and without its cognate accessory subunit, Cdc50a. Purified Atp8a1 is vanadate‐sensitive and dependent upon Cdc50a co‐expression and PS for robust ATPase activity, although lipid specificity is not dependent on the presence of Cdc50a. In addition to reconstituting the flippase activity of Atp8a1 and ATP11c in vitro, our efforts are focused on rational mutagenesis of Atp8a1 to identify the lipid transport path and the determinants of substrate specificity, directed by homology modeling and the lipid‐binding characteristics of the closest members of the P‐type superfamily. X‐ray crystal structures of the SERCA pump in conformation E2 (PDB 2AGV) display a PE glycerol backbone and head group tightly pinched by helices M2 and M4 with PE's acyl chains extending between helices M2 and M6. This PE‐binding site implies a mechanism for how the P4lipid‐binding site and transport pathway evolved from ancestor P‐types designed to move simple cations. With these clues, we aim to identify the PS binding site and deduce the origin of the sn‐1,2‐stereospecificity of Atp8a1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating regulation of the PI 4‐kinase Pik1 at the Saccharomyces cerevisiae trans‐golgi network
Authors: Diefenderfer Carolyn M , Fromme J. Christopher
Published: June 2021

Vesicle‐mediated protein transport among eukaryotic cellular compartments is dynamic and tightly‐regulated. At the Saccharomyces cerevisiae trans‐Golgi Network (TGN), the conserved small GTPases Ypt31 (Rab11 homologue) and Arf1 collaborate with an essential pool of phosphatidylinositol 4‐phosphate (PI4P) to recruit vesicle coats, vesicle transport motors, and tethering proteins to nascent outbound vesicles. The conserved lipid kinase Pik1 (PI4KIIIβ homologue) generates PI4P at the TGN, and work in several model systems suggests that it is regulated by close ties to the activities of Ypt31, Arf1, and the ArfGEF (guanine nucleotide exchange factor) Sec7 (BIG1/2 homologue). However, the mechanisms underlying this regulatory network are unclear. Using a combination of imaging and in vitro protein‐protein interaction studies, we have found that Pik1 relies on Sec7‐activated Arf1 for proper localization to the TGN and may, once at the TGN, influence the activity of Ypt31. Together, these data provide a clearer picture of how cooperation among Ypt31, Arf1, and Pik1/PI4P choreographs trafficking from the TGN.

Support or Funding Information

NSF GRFP DGE‐1650441; NIH/NIGMS

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Papuamine prevents the fusion between autophagosomes and lysosomes by inhibiting the maturation of autophagosomes
Authors: Fujimoto Keiko , Tadakuma Ryo , Tanaka Chiaki , Hirota Yuko , Miyauchi Yuu , Ishii Yuji , Tanaka Yoshitaka
Published: June 2021

Papuamine, an alkaloid isolated from a marine sponge Haliclona sp., has been shown to have antifungal and antimycobacterial activities. Recent studies have reported that papuamine exhibits cytotoxicity and induces apoptosis in MCF7 breast cancer cells, which are attributable to the induction of autophagy. However, the effect of papuamine on autophagic flux remains uncertain. Therefore, the aim of this study was to investigate the effect of papuamine on autophagy.

Western blot analyses showed that treatment of HeLa cells with papuamine causes a markedly increase in LC3‐II, an autophagosome‐specific marker. Likewise, confocal immunofluorescence microscopy revealed that not only the number of, but also the size of, LC3‐positive vesicles increased in papuamine‐treated cells. However, in cells treated with papuamine LC3‐positive vesicles were not significantly colocalized with a late endosome/lysosome marker LAMP‐1. In addition, papuamine did not affect lysosomal properties including acidic pH, activities and distribution of lysosomal enzymes. These results demonstrate that papuamine inhibits the fusion between autophagosomes and lysosomes, thereby leading to the accumulation of autophagosomes. Furthermore, our data suggest that papuamine‐induced impairment of both the autophagosomes‐lysosomes and the enlarged autophagosomes formation is due to a blockade of Rab5‐to‐Rab7 conversion.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MiR‐590‐3p Restores Autophagy in Cultured Human Glomerular Endothelial Cells Exposed to High Glucose
Authors: Liu Wenjie , Shi Yi
Published: June 2021

Background

Autophagy is an important mechanism for homeostasis. Previous data mining indicated that miR‐590‐3p was involved in diabetes‐induced endothelial dysfunction. The present study was designed to investigate the role of microRNA‐590‐3p (miR‐590‐3p) in the regulation of autophagy in cultured endothelial cells incubated with high glucose.

Methods

Cultured human aortic endothelial cells (HAEC) and glomerular endothelial cells (HGEC) were subjected to high glucose (44.4mM). To overexpress miRNA‐590‐3p, lipofectamine RNAiMAX was used for transfection. Cells were collected for quantitative real‐time polymerase chain reaction and western blotting.

Results

After six hours stimulation with high glucose, expressions of miR‐590‐3p were significantly reduced in HGEC, but not in HAEC, while expressions of miR‐590‐5p were not changed in both cells. After 48 hours, high glucose treatment blunted autophagy in HGEC, which was shown as reduced protein expressions of microtubule‐associated proteins 1A/1B light chain 3B‐II (LC3B‐II), autophagy‐related genes (ATG) 5, and beclin‐1, as well as increased protein expressions of SQSTM1/p62. Overexpressing miR‐590‐3p restored the impairment of autophagy in HGEC. Incubation of HGEC with high glucose reduced phosphorylation of 5′ AMP‐activated protein kinase (AMPK) and increased phosphorylation of mammalian target of rapamycin (mTOR), two upstream signaling of autophagy. Overexpressing miR‐590‐3p increased the phosphorylation of AMPK and reduced the phosphorylation of mTOR, suggesting that miR‐590‐3p restored autophagy in HGEC through AMPK/mTOR pathway. High glucose stimulation did not alter apoptosic protein expressions in HGEC or HAEC, including bcl‐2, pBAD and caspase‐3.

Conclusion

High glucose incubation reduces autophagy in HGEC which is prevented by overexpressing MiR‐590‐3p through AMPK/mTOR signaling pathways.

Support or Funding Information

The present study was supported by the National Nature Science Foundation of China (81573418) and ZSYXRC‐014

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA expression profiles in a human induced pluripotent stem cell‐derived model of diabetic cardiomyopathy
Authors: Mishra Manoj K. , Horikoshi Yuichi , Pan Xiaoqing , Liu Pengyuan , Bai Xiaowen , Bosnjak Zeljko J. , Kriegel Alison J. , Liang Mingyu
Published: June 2021

Diabetic cardiomyopathy (DCM) is a common and important health concern in the rapidly growing population of diabetic patients. At present, no specific drug treatment exists to prevent diabetes‐induced cardiac dysfunction, a complex multifactorial condition that is distinguished by a unique set of metabolic, structural, and molecular changes within the DCM. The advances in disease modeling using human induced pluripotent stem cells (iPSCs) have opened novel paths to study human physiology and disease at the cellular level. Here, we characterized an in vitro cell based model for DCM developed by exposing human induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) to a diabetic‐like environment (10 mM glucose, 10 nM human‐endothelin‐1, and 1 mM cortisol). The treated cells exhibited metabolic shift (demonstrated by reduced glucose oxidation and increased fatty acid oxidation) and hypertrophy (demonstrated by hypertrophic morphology and increased expression of hypertrophic markers such as BNP, ANP, ACTA1 and MYH7. MicroRNAs (miRNAs) are key factors in controlling complex regulatory networks in cellular functions and disease processes. We performed a small RNA deep sequencing analysis to identify miRNAs associated with this in vitro DCM model. We identified 57 differentially regulated (p<0.05) miRNAs (26 up‐ and 31 down‐regulated) in diabetic CMs compared with control iPSC‐CMs (10 mM mannitol). The differential expression of several miRNAs was verified by real‐time PCR. These findings suggest miRNAs might be involved in this model of DCM. Further investigation will be required to examine functional roles for these miRNAs and identify the mechanisms by which these miRNAs might contribute to the development of DCM.

Support or Funding Information

Supported by NIH Grant P01GM066730.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Angiopoietin‐1 Protects 3T3‐L1 Pre‐Adipocytes from Saturated Fatty Acid‐Induced Cell Death
Authors: Son Yura , Paton Chad Michael
Published: June 2021

Crosstalk between endothelial cells and adipocytes is vital to adipocyte functions, but little is known about the mechanisms or factors controlling the process. Angiogenesis is a critical component linking the endothelium to healthy adipogenesis, yet it is not known if or how it is involved in adipocyte physiology. Therefore, the purpose of this study was to determine the effect of Angiopoietin‐1 (Ang‐1) and ‐2 (Ang‐2) as well as their receptor, Tie‐2, on adipocyte physiology. 3T3‐L1 pre‐ and mature adipocytes were found to express Ang‐1, Ang‐2, and Tie‐2 which decrease upon PUFA‐treatment. Furthermore, 3T3‐L1 cells treated with recombinant Ang‐1 or Ang‐2 increased expression of the anti‐apoptotic gene Bcl‐x and decreased expression of the pro‐apoptotic gene Casp‐8. Next, pre‐adipocytes were treated with saturated fatty acids (SFA) to induce cell stress. SFA‐mediated splicing of X‐box‐binding protein‐1 (Xbp‐1s) was reduced by co‐treatment with Ang‐1 and cell viability was improved in the presence of SFA + Ang‐1. Taken together, these results indicate that Ang‐1 may protect pre‐adipocytes from SFA‐induced apoptosis and ER stress.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intestinal Axin1 Regulation of Epithelial Functions and Host‐Microbial Interactions in Health and Inflammation
Authors: Garrett Shari , Zhang Yong‐guo , Sun Jun
Published: June 2021

Background

Axin1 is a scaffold protein in the β‐catenin destruction complex which if disrupted can lead to inflammatory bowel disease or colorectal cancer. Our previous study has demonstrated that Axin1 expression was reduced by pathogenic Salmonella colonization. The physical binding between beta‐catenin and Axin1 was also decreased during bacterial infection. However, the role of Axin1 in modulating intestinal tissue architecture and microbial homeostasis is unknown.

Methods

To explore the novel mechanism of Axin1 in regulating the functions of intestinal epithelia in health and inflammation, we generated a novel Axin1 conditional knockout model in the intestinal epithelial cells (Axin1ΔIEC).

Results

We found that Axin1ΔIEC mice had less PCNA and β‐catenin compared to the control Axin1 loxp mice, suggesting the reduction of cell proliferation in intestinal epithelial cells without Axin1. Axin1ΔIEC mice exhibit altered goblet cell spatial distribution as well as disordered Paneth cells in the small intestine. Furthermore, in a Salmonella‐colitis model in vivo and cell cultures in vitro, we found bacterial protein AvrA expression modulates the β‐catenin/Axin1 interaction.

Conclusion

Lacking intestinal Axin‐1 led to reduced proliferation and abnormal goblet cells and Paneth cells, suggesting that intestinal epithelial development and differentiation is regulated in an Axin1 dependent manner. Axin1 is involved in regulating the host‐microbial interactions in health and inflammation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CREB‐Regulated miR‐27b Is Linked to Hepatic Insulin Resistance by Targeting Insulin/Akt Signaling
Authors: Hsu Shu‐hao , Chen Yen‐Ju
Published: June 2021

Insulin resistance (IR) is a predisposed condition of type 2 diabetes and commonly leads to fatty liver, in which hepatocytes exhibit excessive lipogenesis and gluconeogenesis. The role of microRNAs (miR) in liver‐associated IR remains largely unknown. Among microRNAs that participate in metabolic signaling, miR‐27b is a critical regulator of lipogenesis in both liver and adipose tissues; however, it is not clear if dysregulation of miR‐27b contributes to IR in liver. To address this question, we established several IR experimental models, including palmitate‐treated mouse AML12 hepatocytes, and dietary‐induced mouse fatty liver; Interestingly, miR‐27b was upregulated in all of these IR models. Furthermore, bioinformatic analysis predicted that miR‐27b targets several genes involved in Akt phosphorylation, including Pdpk1, Pik3ca (P110), Pik3r1 (P85), and Pik3cd. Based on these data, we hypothesize that miR‐27b over‐expression results in defective Akt signaling upon insulin stimulation. Indeed, over‐expression of miR‐27 in human HepaRG liver cells and mouse primary hepatocytes significantly suppressed Akt phosphorylation at both T308 and S473 residues and down‐regulated Pdpk1, Pik3r1 (P85), and Pik3ca (P110) at protein level upon insulin stimulation. Our luciferase reporter assay using 3T3 cells further demonstrated that miR‐27 directly targeted Pdpk1 and Pik3r1 3′UTR. Next, we investigated the mechanism by which miR‐27 is regulated in insulin signaling. Interestingly, we found that insulin significantly reduced miR‐27 expression below 50% of control in both HepaRG and AML12 cells. Further study on the detail mechanisms revealed that miR‐27b expression is positively regulated by cAMP response element‐binding protein (CREB), which is an IR promoter and regulated by insulin. Using HepaRG and AML12 cells, we demonstrated that miR‐27 is significantly down‐regulated by siRNA‐ or metformin‐mediated knockdown of CREB, but up‐regulated by forksolin‐induced activation of CREB. Also, miR‐27b is significantly increased when CREB is up‐regulated in mouse livers by either overnight fasting or high‐fat‐diet feeding, whereas decreased when CREB is down‐regulated in livers of mice treated with metformin. Taken together, our in vitro and in vivo data demonstrated that insulin may negatively regulate miR‐27 by repressing CREB transcriptional activity and the abnormal activation of hepatic CREB in IR animals may cause the aberrant elevation of miR‐27b to further decrease insulin sensitivity.

Support or Funding Information

This project is funded by Ministry of Science and Technology in Taiwan (Project#105‐2628‐B‐002 ‐017‐MY3)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution of histone H3 variants in eukaryotes
Authors: Waterborg Jakob Harm
Published: June 2021

Phylogenetic and Tree‐Of‐Life analysis of histone H3 proteins was performed for more than 600 eukaryotic species across all major phyla using available sequence database information, examining and curating more than 5000 sequences. A major aim of this study was to determine whether Replication‐Independent constitutively expressed and Replication‐Coupled S phase expressed histone H3 variants identified in animal and plant phyla arose from a single gene duplication event. Alternatively, did independent gene duplication events with analogous functional differentiation give rise to functionally similar RI and RC H3 histones in animals and plants? Multiple histone H3 gene duplication events were identified and some were correlated with functional histone H3 diversification. Experimental assessment of RI‐ and RC‐functions in coexisting H3 variants in selected species will be required. Uncertainty in the evolutionary relationship between various protist phyla and the animal‐plant‐fungal crown phyla as yet prevents a definitive answer to the major question posed. Conservation and variability of all 135 canonical histone H3 protein residues was determined, revealing almost absolute conservation of all residues for which post‐synthetic modifications have been observed in any crown phylum species: lysine acetylation and methylation, serine and threonine phosphorylation, and arginine methylation.