bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating Phosphorylation Differences in FFA4 Short and Long Isoforms
Authors: Senatorov Ilya S. , Cheshmehkani Ameneh , Moniri Nader H. 
Published: June 2021

The G protein‐coupled receptor Free Fatty Acid Receptor 4 (FFA4) is an attractive drug target due to its profound influence on a variety of endocrine, inflammatory, and metabolic processes. Humans contain a unique long splice variant of FFA4, which contains an additional 16 amino acid insert within the 3rd intracellular loop, and has not been found to be expressed in any other mammals. Although the shorter FFA4 isoform (FFA4‐S) is ubiquitously expressed, human FFA4‐L has only been found in colorectal tissue. More interestingly, although both isoforms are agonized by the same ligands, upon agonism, FFA4‐L has been demonstrated to possess an intrinsic bias towards β‐arrestin signaling rather than coupling to G proteins. Since it is known that receptor phosphorylation is required for interaction with β‐arrestin, the purpose of the current study was to determine whether any differences exist between the long and short isoform of FFA4 in terms of basal, homologous, or heterologous phosphorylation.MethodsWhole cell phosphorylation assays using 32P‐labeled orthophosphate were performed in HEK293 cells transiently expressing FFA4 isoforms or mutants. Inquiries into G‐protein receptor kinase (GRK) activity were performed using small interfering RNA (siRNA) followed by whole cell phosphorylation assay. Inquiries into protein kinase A (PKA) or protein kinase C (PKC) activity utilized the adenylyl cyclase activator forskolin to induce PKA activity, PKA inhibitor H‐89, PKC activator phorbol‐myristol acetate (PMA), and PKC inhibitor BIMII. Arrestin recruitment assays were conducted using yellow fluorescence protein (YFP) tagged arrestin‐3 and visualized via confocal microscopy. Truncated mutants were generated by sequential deletions via overlap primers and PCR mutagenesis.ResultsSimilar to our previously reported results with FFA4‐S, homologous phosphorylation of FFA4‐L is mediated by GRK6, whereas heterologous phosphorylation is mediated by PKC. FFA4‐L has lower basal phosphorylation compared to FFA4‐S. Truncation of FFA4‐L at S356 fully inhibits receptor phosphorylation and abrogates arrestin recruitment, matching reported observations for FFA4‐S at corresponding S340.ConclusionThe same kinases are responsible for homologous and heterologous phosphorylation of FFA4‐S and FFA4‐L, while basal phosphorylation of FFA4‐L remains lower compared to FFA4‐S. The arrestin sensor for both isoforms is located on the C‐terminal tail of the receptor following S340 (FFA4‐S) or S356 (FFA4‐L).Support or Funding InformationNIH/NIDDK DK098730 to NHM and Diabetes Action Research and Education Foundation grants to NHMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Signaling Bias and Antagonism of Pilocarpine for M3 Muscarinic Acetylcholine Receptor
Authors: Pronin Alexey N. , Wang Kelly , Slepak Vladlen Z. 
Published: June 2021

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth since the 19th century and classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with its main therapeutic target ‐ muscarinic M3 receptor (M3R). We found that pilocarpine was 1,000 times less potent in stimulating mouse eye pupil constriction than muscarinic agonists oxotremorin‐M (Oxo‐M) or carbachol (CCh), even though all three ligands have similar Kd values for M3R. In contrast to CCh or Oxo‐M, pilocarpine does not induce Ca2+ mobilization via endogenous M3R in HEK293T or mouse insulinoma MIN6 cells. Pilocarpine also fails to stimulate insulin secretion, and instead, antagonizes insulinotropic effect of Oxo‐M and CCh‐induced Ca2+ upregulation. However, in HEK293T or CHO‐K1 cells overexpressing M3R, pilocarpine induces Ca2+ transients like those recorded with another Gq‐coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in PIP2. In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R, but not M3R. Moreover, pilocarpine blocked CCh‐stimulated PIP2 hydrolysis in M3R‐overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates ERK1/2 in MIN6 cells. The stimulatory effect on ERK1/2 was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward b‐arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level and signaling pathway downstream of this receptor.Support or Funding InformationThis work was supported by the National Institutes of Health Grants RO1DK105427 and RO1DK111538This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The soluble epoxide hydrolase attenuates pathological vascularization by preventing astrocyte loss in a retinopathy of prematurity model in mice
Authors: Hu Jiong , Bibli Sofia Iris , Wittig Janina , Lin Jihong , Popp Rüdiger , Hammes Hans‐Peter , Fleming Ingrid 
Published: June 2021

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. Several signalling pathways have been implicated in the process of ROP but a novel drugable target is the cytochrome P450 (CYP)/soluble epoxide hydrolase (sEH) pathway. The latter enzymes play a crucial role in biological processes by generating bioactive epoxides and diols from endogenous polyunsaturated fatty acids (PUFAs). This study aimed to characterize the effects of the newly identified sEH‐derived w‐3 PUFA diol; 19,20‐dihydroxydocosapentaenoic acid (DHDP) in a mouse ROP model.ROP in mice was achieved by placing pups in 75% oxygen for 5 days from postnatal day (P) 7 until P12, before moving animals back to room air for an additional 5 days. This procedure resulted in the development of avascular areas that were significantly larger in retinas from sEH−/− mice than in their wild‐type (WT) littermates at P17. Avascularization was accompanied by angiogenesis in the form of characteristic vascular “tufts” which were more evident in retinas from sEH−/− mice. Although there was no difference in vascular obliteration, a marked decrease in the astrocyte network was observed in sEH−/− mice after exposure to high oxygen. Mechanistically, astrocyte loss in hyperoxic conditions was a consequence of elevated mitochondrial (mt) DNA damage mediated by increased interaction of the PARP‐1 DNA binding domain with mtDNA, as revealed by chromatin immunoprecipitation studies. Exposure of astrocytes to 75% O2 in vitro resulted in elevated PARP‐1 cleavage/activation; a phenomenon reversed in the presence of 19,20‐DHDP. Administration of 19,20‐DHDP during hyperoxia preserved the complex of presenilin 1 with mitochondrial carrier homolog 1 (MTCH‐1) in the outer mitochondria membrane, inhibiting the translocation of the MTCH‐1 to the inner mitochondria membrane and subsequently inhibiting PARP‐1 cleavage. The intravitreal injection of 19,20‐DHDP at day 7 significantly prevented astrocyte loss caused by high O2 levels at day 8, and reduced both the avascular area as well as vascular tuft formation at P17 in the sEH−/− mice.Our date indicate that the sEH dependent generation of 19,20‐DHDP from w‐3PUFA is required for retinal astrocyte survival and preservation of DNA integrity under hyperoxic conditions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lodoxamide protects against LXR‐mediated steatosis through G protein‐coupled receptor 35 in Hep3B cells and primary mouse hepatocytes
Authors: Nam So‐Yeon , Im Dong‐soon 
Published: June 2021

BackgroundG protein‐coupled receptor 35 has been an orphan receptor, although synthetic agonists and antagonists have been developed. Cromolyn and lodoxamide, mast cell stabilizers, were reported as agonists. Recently, cromolyn exhibited antifibrotic effects through actions on hepatocytes and stellate cells. However, the function of GPR35 in hepatocytes has not been investigated.METHODSUsing an in vitro model of liver X receptor (LXR)‐mediated hepatocellular steatosis, the roles of lodoxamide and GPR35 were investigated in human and mouse hepatocytes.RESULTST0901317, a specific LXR activator, induced lipid accumulation in Hep3B human hepatoma cells. This lipid accumulation was inhibited by lodoxamide, the most potent agonist of GPR35. The protective effects of lodoxamide were inhibited by CID2745687, a specific GPR35 antagonist or by siRNA trnasfection. T0901317 treatment induced expression of SREBP‐1c, a key transcription factor for lipid synthesis. Lodoxamide inhibited the induction of SREBP‐1c proteins in a GPR35‐dependent manner. Using specific inhibitors of cellular signaling components, lodoxamide‐induced inhibition of lipid accumulation was found to be mediated through p38 MAPKs and JNK, but not through Gi/o proteins, PI3K, and ERKs. Furthermore, the protective effect of lodoxamide was confirmed in mouse primary hepatocytes.CONCLUSIONTherefore, the present data suggest that GPR35 may function to protect against lipid accumulation in the liver.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 Genetic Polymorphisms on the Sulfation of 17α‐Ethinylestradiol (EE2) by Human Cytosolic Sulfotransferase SULT1E1
Authors: El Daibani Amal A. , Alherz Fatemah A. , Abunnaja Maryam S. , Liu Ming 
Published: June 2021

In human, previous pharmacokinetic studies have shown the metabolism of 17α‐ethinylestradiol (EE2) through sulfation, with estrogen sulfotransferase (SULT1E1). Since SULT1E1 is a major responsible enzyme that mediates the biotransformation of endogenous estrogens and estrogen‐like drugs, genetic polymorphisms of SULT1E1 may significantly impact the 17α‐ethinylestradiol‐sulfating activity by SULT1E1. Subsequently, this study was performed to investigate the implications of SULT1E1 genetic polymorphisms in the sulfation of 17α‐ethinylestradiol. By using the site directed mutagenesis technique, five selected recombinant SULT1E1 allozymes were generated, expressed and purified. Compared with the wild‐type SULT1E1, purified allozymes of SULT1E1 were shown to exhibit significantly distinct sulfating activity with 17α‐Ethinylestradiol as a substrate. In summary, the obtained results provide evidence related to differential metabolism of 17α‐ethinylestradiol in individuals with different SULT1E1 genotypes, which may be useful for further in‐vivo study contributed to the therapeutic‐ as well as adverse effects of 17α‐ethinylestradiol through sulfation.Support or Funding InformationThe College of Pharmacy, University of ToledoThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergistic neuroprotective effect by combination treatment with DAPK1 and Pin1 inhibitor on ischemic stroke
Authors: Won Jinyoung , Kang Hyunbon , Hong Yonggeun 
Published: June 2021

DAPK1 (death‐associated protein kinase 1) and Pin1 (peptidyl‐prolyl isomerase 1) are positive mediators of the neuronal death signaling pathway in cerebral ischemia. Pin1 induces the cis‐trans conversion on a phosphorylated serine/threonine (pS/T) amino acid residue of target protein that is regulated by the calcium/calmodulim (CaM)‐dependent kinase termed DAPK1. Whereas DAPK1 and Pin1 activation is required for neuronal death induced by ischemia/reperfusion (I/R) injury, this mechanism has not been elucidated. Thus, the purpose of this study was to determine the effects of DAPK1/Pin1 on pathogenesis of cerebral ischemia. Ischemic stroke model was induced by middle cerebral artery occlusion (MCAo). Experimental animals were randomly divided into three groups; vehicle (control), single inhibitor (DAPK1 inhibitor or Pin1 inhibitor), inhibitor combination (DAPK1 inhibitor and Pin1 inhibitor). Inhibitor groups including single injection and combined injection groups showed the decrease of neurological deficit and the altered body weight compared to control. Also, the low mortality rates were shown in inhibitor groups. In addition, the expansion of ischemic core from the striatum to the cerebral cortex was inhibited in inhibitor groups. Particularly, combination group showed significantly decreased ischemic lesions compared to control group. These results indicated that suppression of DAPK1 and Pin1 catalytic activity could enhance the neuroprotective effect against ischemic insult.Support or Funding InformationFunding: NRF‐2013R1A2A2A01067169, NRF‐2017R1A2A2A01067169, KRIBB‐KGM4611714This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microarray Analysis of Long Non‐coding RNA and mRNA Expression Profiles in Diabetic Cardiomyopathy Using Human iPSCs‐Derived Cardiomyocytes.
Authors: Pant Tarun , Mishra Manoj , Bai Xiaowen , Bosnjak Zeljko J. , Ge Zhi‐Dong , Dhanasekaran Anuradha 
Published: June 2021

Long non‐coding RNAs (lncRNAs) are 200 nucleotides long and represent the non–protein‐coding transcripts of our genome capable of regulating vital cell processes like transcription, translation and chromosome remodeling. Studies have reported the association of these deregulated non‐Coding RNAs (ncRNAs) with the development of diabetes. However, the potential role of these lncRNAs and their changing expression profiles in Diabetic Cardiomyopathy (DCM) has not been explored. Advancing field of stem cell technology offers us the opportunity to use human induced pluripotent stem cell‐derived cardiomyocytes ( hiPSC‐CMs) for disease modeling. We hypothesized that disease phenotype can alter the expression profile of these lncRNAs. In the present study utilizing the previously established in vitro DCM model, we investigated the deregulated genome profile. We performed (lncRNA and mRNA) microarray analysis on the disease modeled hiPSC‐CMs.ResultsThe array data revealed around 2046 lncRNAs to be differentially regulated between diseased and control group. Genome profiling of mRNA found 1582 probes with significant differential expression. In addition, Pathway and Gene ontology (G.O) analysis for differentially expressed mRNAs indicated that both Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) to be upregulated in the entire DCM group, distinct pathways which are associated with DCM were also investigated.ConclusionThese results provide a better understanding about deregulation of lncRNAs that are associated with disease pathogenesis.In the future, these lncRNAs can not only serve as early diagnostic biomarkers but also as potential therapeutic targets.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Pathway for Fatty Acid: Palmitate Transport from Plasma into Fat Vacuoles and Mitochondria in the Heart
Authors: Bassingthwaighte James B. , Barr Nathan 
Published: June 2021

As a part of a multi‐university, multi‐investigator program on integrated function of the heart, we are targeting the metabolism and regulation of the endothelial cell (EC) and the cardiomyocyte (CM) and the interactions between these two cell types. Models for blood flow, glucose, fatty acid and for oxygen are essential steps. Initial models are “eternal” in that there is no protein anabolism or catabolism, thus all enzymes remain at fixed concentrations. The emphasis is on the physico‐chemical balances (charge, ionic regulation, mass, volume, energy, reducing equivalents, carbon, oxygen, phosphorus and so on), on the limitations to the kinetics imposed by the energetics, and the responses to transients rather than steady state analysis. Fatty acid, the prime substrate for cardiac metabolism, is tightly bound to as many as 12 sites on plasma albumin, yet is about 1/3 extracted during a single capillary passage. The modeling of palmitate (a 16:0 fatty acid) flux starts with the release from these sites, the rebinding at multiple sites, diffusion in the plasma, and uptake across the capillary endothelial plasmalemma by a fatty acid transporter, e.g. CD36, facilitated diffusion by fatty acid binding protein (FABP) across the 1 micron thick EC, then facilitated or diffusive transport into the interstitial fluid space to the CM surface and transmembrane passage. The next stage, acylCoA formation, leads 2 ways. One is to di‐ and tri‐acyl glycerol (TAG) and lipid vacuole formation and the reverse, a storage and buffering system. The other is for acyl‐CoA to cross the mitochondrial membrane, undergo beta‐oxidation and insert the two‐carbon products into the tricarboxylic acid (TCA) cycle to provide energy for ATP generation through oxidative phosphorylation, thereby setting the balance between oxygen delivery by flow and ATP generation. The models serve for data analysis and hypothesis testing, account for the kinetics of enzyme binding and the resultant concentration‐dependent substrate capacitance (cellular volume of distribution), and provide mass balance. They serve as working hypotheses until disproven, and are “open source” on our website (www.physiome.org), so that they can be tested and improved by the scientific community. Such models will serve as tools to link overall cellular function to the underlying physiology and its regulation at the proteomic and genetic levels.Support or Funding InformationThese efforts are a part of the Physiome Project sponsored by NIH/NHLBI U01‐HL122199 Cardiac Energy Grid and our Simulation Resource (http://www.physiome.org).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomarkers and components of the interactive Physical and Cognitive Exercise System (iPACES™ v2.0) for Mild Cognitive Impairment (MCI): Cortisol, Dehydroepiandrosterone (DHEA‐S), and Insulin‐like Growth Factor (IGF1)
Authors: Wall Kathryn Mary , Stark Jessica , Karla‐Lall Anvit , Schillaci Alexa , Christian Hannah , Doty Carolyn , McLaren Elizabeth , Saulnier Tobi , Cohen Brian D. , Anderson‐Hanley Cay 
Published: June 2021

Dementia cases are on the rise among our aging global population, and thus there is increasing urgency to identify efficacious interventions for preserving or ameliorating cognitive decline. Physical exercise, cognitive training and combined physical and cognitive interventions have been found to slow the decline of cognitive abilities in those with mild cognitive impairment (MCI) but the biological mechanisms underlying these changes need further clarification. This quasi‐experimental within‐subjects design compared changes in biomarkers during two‐week exposures to components of the interactive physical and cognitive exercise system (iPACES™). Participants were evaluated over eight weeks at two‐week intervals. Biomarker levels of Cortisol, IGF1 and DHEAS were assessed at each evaluation through saliva (passive drool collection). Each saliva sample was analyzed using enzyme‐linked immunosorbent assays (ELISAs). Additionally, each sample was analyzed using bicinchoninic acid (BCA) protein assay to normalize protein concentration. Cognitive outcomes were also assessed, focusing on executive function via Stroop, Trails, and Flanker tests. This pilot study enrolled 14 older adults, 13 of which met criteria for MCI (MoCA<26), with ten study completers. Paired t‐tests revealed significant (ps<.05) cognitive improvements in Stroop and Flanker from baseline during all active component conditions (e.g., pedaler, game, and iPACES™), but not during placebo. Biomarkers did not change significantly during placebo or pedaler conditions, but during the game intervention, DHEAS declined significantly (p=.04), and during iPACES™ intervention, DHEAS declined further and IGF1 declined significantly (p=.05), consistent with prior research (McTiernan, et al., 2005; Yuichiro, et al., 2010). Furthermore, the changes (from baseline to the end of the 8‐week trial) for cognition were moderately correlated with biomarker changes (r's ranging from −.22 to −.48), suggesting a link between neurobiological mechanisms and cognitive outcomes. Further research is needed to replicate and extend this pilot research; in particular, it would be useful to compare such interventions in a randomized controlled trial.Support or Funding InformationNational Institute on Aging STTR Grant #R41AG053120 and Union College Undergraduate Research GrantThis 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 chronic lactate administration prior to endurance training on mitochondrial adaptations in mouse skeletal muscle
Authors: Takahashi Kenya , Kitaoka Yu , Matsunaga Yutaka , Hatta Hideo 
Published: June 2021

Lactate is considered as not only an oxidizable fuel, but also a signal molecule inducing mitochondrial biogenesis. In this study, we hypothesized that lactate administration prior to training augments mitochondrial adaptations. ICR male mice were randomly assigned to Con (n=9), Tr (n=7) or La+Tr (n=8) group. Mice in Tr and La+Tr were performed treadmill running (20 m/min, 60 min) immediately after administration of PBS (same volume as lactate) or sodium lactate (1 mg/g BW) via i.p. injection for 21 consecutive days. Mice in Con were administrated same volume of PBS on the same days. All the mice were performed lactate tolerance test (LTT: i.p. injection of 1 mg/g BW of sodium lactate) 24h after final training. Blood samples were collected pre, 5, 15, 30, 60 and 180 minutes after the lactate administration. Soleus and plantaris muscle were taken for the biochemical analysis 24h after LTT. As a result, some mitochondrial enzyme activities in soleus and plantaris muscle were significantly higher in La+Tr than Tr (CS: P<0.05, β‐HAD: P=0.05, COX: P<0.01 in soleus, CS: n.s., β‐HAD: n.s., COX: P<0.05 in plantaris). However, mitochondrial OXPHOS protein content was not different between Tr and La+Tr. MCT1 protein content was significantly higher in La+Tr than Con (+18%; P<0.01) in plantaris muscle, but not MCT4. As a result of LTT, blood lactate concentrations after lactate administration were significantly lower in Tr (60 min: P<0.05 vs. Con) and La+Tr (30 min: p<0.01, 60 min: P<0.01 vs. Con) than Con. These data indicate that lactate upregulates mitochondria functionally, and that lactate administration increased MCT1 protein content leading to lower blood lactate concentration after lactate administration.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.