bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dietary docosahexaenoic acid (DHA) halts progression of nonalcoholic steatohepatitis (NASH), but does not promote fibrosis remission in Ldlr−/− mice
Authors: Lytle Kelli A. , Wong Carmen , Jump Donald 
Published: November 2023

Nonalcoholic fatty liver disease (NAFLD) is a major public health concern. NASH, the progressive form of NAFLD, is characterized by hepatosteatosis, inflammation, oxidative stress and fibrosis. NASH is a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. We previously established that DHA attenuated the onset of western diet (WD)‐induced NASH in Ldlr−/− mice. Herein, we evaluate the efficacy of DHA to slow progression and promote disease remission in mice with pre‐existing NASH. Mice maintained on the WD for 22 weeks developed mild NASH. These mice were randomized to 3 groups: (Group 1) WD‐Base [euthanized at 22 weeks]; (Group 2) WD + Olive oil; and (Group 3) WD + DHA. Olive oil was added to ensure isocaloric diets and DHA was at a therapeutic dose, i.e., 2% of total calories. A Control Group was maintained on laboratory chow for the duration of the study. Mice in Groups 2 and 3 were euthanized after 8 weeks on the new diets. DHA had no effect on WD‐induced changes in body weight, food intake, plasma glucose, TLR4 agonists or leptin. DHA, however, decreased blood lipids (triglycerides, cholesterol) and Toll‐like receptor 2 agonists. Analysis of hepatic histology, lipids and gene expression established that the NASH phenotype worsened in WD + Olive oil group, but not WD + DHA. Hepatic gene expression markers of inflammation, oxidative stress and fibrosis in the WD‐Base and WD + DHA groups were comparable. While DHA supplementation increased hepatic omega‐3 fatty acids and repressed further total hepatic fatty acid accumulation, it failed to fully eliminate fibrosis. Taken together, these studies establish that DHA supplementation, at physiologically relevant levels, attenuates disease progression in mice with pre‐established NASH, but does not promote full NASH remission.Support or Funding InformationSupported by NIH: DK094600
FTY720/Fingolimod Reduces Steatosis in a Murine Model of Non‐Alcoholic Fatty Liver Disease
Authors: Rohrbach Timothy D , Asgharpour Amon , Avni Dorit , Cazanave Sophie C , Bedossa Pierre , Sanyal Arun J , Spiegel Sarah D 
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a metabolic disease characterized by a spectrum of related liver disorders. Liver steatosis is an early initiator of NAFLD followed by the development of nonalcoholic steatohepatitis. Approximately 30% of adults in the United States are affected by NAFLD and although obesity and insulin resistance are risk factors for NAFLD, there is currently no approved therapy for this disease. To make matters worse, NAFLD can progress to liver cirrhosis and ultimately hepatocellular carcinoma. Sphingolipid metabolites, particularly ceramide and sphingosine‐1‐phosphate (S1P), have recently received attention for their potential roles in insulin resistance and hepatic steatosis. FTY720/Fingolimod, an FDA approved pro‐drug for treatment of multiple sclerosis, is phosphorylated in vivo to its active phosphorylated form by sphingosine kinase 2 and has been shown to interfere with the actions of S1P and inhibits several sphingolipid metabolic enzymes. Therefore, in this study we investigated its effect in the C57BL/6 x 129S murine model of NAFLD. Mice were fed a high fat diet (42% kcal from fat) with sugar water (HFD+SW) for 16 weeks with concurrent oral administration of a clinically relevant dose of FTY720 (0.3 mg/kg). FTY720 significantly decreased liver lipid accumulation and steatosis. Mass spectrometry analysis confirmed that FTY720 was phosphorylated to its active form, FTY720‐phosphate. Although levels of S1P and monohexosylceramide were significantly decreased in blood by administration of FTY720, no major changes in ceramide were detected. Moreover, liver sphingolipid levels including ceramide, monohexosylceramide, sphingomyelin, dihydroceramide, dihydromonohexosylceramide, and dihydrosphingomyelin were all decreased (p < 0.05) by FTY720 treatment. Additionally, FTY720 treatment caused a significant decrease in activation of Akt known to play a critical role in steatosis. Taken together, these findings support the notion that targeting sphingolipid metabolism could lead to reduced hepatic steatosis.Support or Funding InformationSupported by NIH T32 Training Grant 5T32DK007150‐39.
Receptor for advanced glycation endproducts modulates leukotriene B4 receptor 1 signaling
Authors: Ichiki Takako , Koga Tomoaki , Okuno Toshiaki , Saeki Kazuko , Yamamoto Yasuhiko , Sakaguchi Masakiyo , Yokomizo Takehiko 
Published: November 2023

Leukotriene B4 (LTB4) is a potent lipid chemoattractant for leukocytes. Leukotriene B4 receptor 1 (BLT1), a high‐affinity G protein‐coupled receptor (GPCR) for LTB4, is expressed in phagocytes and immune cells, and plays important roles in acute and chronic inflammatory responses. Although the LTB4‐BLT1 axis is known to promote inflammation, no studies have defined the binding proteins that modulate LTB4‐BLT1 signaling. Recent studies showed that receptor for advanced glycation end products (RAGE) modulates GPCR signaling via direct interaction with the receptors or downstream adapter molecules. RAGE is a member of the immunoglobulin superfamily, and is involved in several inflammatory disorders like BLT1. In this study, we investigated the functional interplay between BLT1 and RAGE. We found that RAGE and BLT1 were co‐immunoprecipitated and RAGE enhanced LTB4‐BLT1‐dependent ERK phosphorylation. RAGE inhibited LTB4‐BLT1‐dependent activation of nuclear factor‐κ B (NF‐κ B) and up‐regulation of pro‐inflammatory cytokines and chemokines. RAGE‐dependent inhibition of NF‐κ B was blunted by treatment with a MEK inhibitor, suggesting that RAGE suppresses LTB4‐BLT1‐dependent NF‐κ B signaling by enhancing MEK‐ERK pathway. Meanwhile, the velocity of LTB4‐dependent neutrophil migration was attenuated by soluble RAGE, which acts as an inhibitory decoy protein for RAGE signaling, or by RAGE deficiency. Furthermore, both LTB4‐dependent ERK phosphorylation in neutrophils and LTB4‐dependent chemotaxis in a murine peritonitis model were attenuated in RAGE‐deficient mice compared with wild‐type mice. These results indicate that RAGE potentiates LTB4‐dependent neutrophil migration by enhancing ERK phosphorylation. Collectively, our findings reveal that RAGE interacts with BLT1 and modulates LTB4‐BLT1 signaling through the potentiation of the MEK‐ERK pathway.Support or Funding InformationT. Ichiki is a Research Fellow of the Japan Society for the Promotion of Science. This work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Nos. 22116001, 22116002, 15H05901, 15H05904, 15H04708, 25860223, 24590386, 15K08316, 24102522 and 25460374).
Biochemical Characterization of HDL/LDL‐like Particles Secreted by Astrocytes
Authors: Nguyen Amanda , Cabrera Karina 
Published: November 2023

Lipids from the diet or those synthesized in the liver are packaged with proteins into HDL, LDL, and VLDL particles that circulate to places where they are needed. These particles are lipid core vesicles with protein wrapped around the external surface. However, these particles can not cross the blood brain barrier due to their size, so the bulk of neural lipid must be synthesized de novo in the brain. With their extensive processes of dendrites and axons, neurons have a plasma membrane surface several hundred times larger relative to a cell of the same soma size in another tissue and thus have much more lipid to synthesize, manage, and keep healthy. In order to address this biosynthetic need, the brain uses astrocytes to synthesize the bulk of the lipid and then transport it to neurons. Lipid and cholesterol synthesized in astrocytes are packaged into “HDL/LDL‐like” particles along with apolipoproteins and are released to be picked up by adjacent neurons with special receptors that recognize the apolipoproteins on the surface of these particles. Our initial proteomic studies identified ApoE and Apo1A as major components of the astrocyte secretome along with ApoC. Utilizing astrocytes isolated from mice at different ages from 4 to 28 months, we observed that ApoE protein levels secreted from the astrocytes decreased 3.5 fold in the oldest animals. We have hypothesized that the reduction in apolipoprotein production, ApoE in particular, is associated with a decrease in the maintenance of the extensive neuronal plasma membrane which results in enhanced susceptibility of the aged animal for damage to neurons and subsequent degeneration. We have developed an equilibrium sucrose centrifugation protocol to isolate these HDL/LDL‐like particles based on density as well as a more rapid isolation procedure using Cleanascite media to isolate all the lipid containing material and apolipoproteins from the astrocyte conditioned media. We are currently performing quantitative GC‐MS analysis to determine the lipid composition of these particles and if that composition changes with the age of the astrocytes. Western blot analysis is being utilized to determine if the apolipoprotein composition also changes with age of the astrocytes. These studies will clarify the role of lipid turnover/maintenance in the brain as it relates to the progression of neurodegenerative processes.Support or Funding InformationMcNair Scholars Program, Cowles Endowed Professorship Research Fund, Trinity University
Characterization of a Glycerophosphocholine Acyltransferase (GPCAT) from Saccharomyces cerevisiae
Authors: Anaokar Sanket , Lager Ida , Glab Bartosz , Banas Antoni , Stymne Sten , Patton‐Vogt Jana 
Published: November 2023

Glycerophosphopholipids (phospholipids) are essential components of biological membranes. The maintenance of membrane composition through regulated synthesis and turnover of phospholipids is crucial to cell functioning. Phosphatidylcholine (PC) is the major phospholipid found in eukaryotic membranes. The two main pathways by which PC is synthesized in yeast are the methylation pathway and the Kennedy pathway (CDP‐DAG pathway). However, a novel pathway has been identified in S. cerevisiae and plants. This pathway involves glycerophosphocholine (GPC), the product of PC deacylation, as the starting substrate. It is proposed that GPC is converted to lysophosphatidylcholine (lysoPC) by accepting an acyl chain. This conversion is catalyzed by an enzymatic activity termed glycerophosphocholine acyltransferase (GPCAT). The lysoPC is then acylated, primarily by a lysophosphatidylcholine acyltranferase (LPCAT) encoded by ALE1, to form PC. Here we report on the identification of the gene encoding GPCAT and the characterization of this pathway in S. cerevisiae. The yeast deletion set was screened to identify the GPCAT‐encoding gene. Biochemical studies utilizing microsomes derived from strains lacking or overexpressing GPCAT in wild type and ale1 delete strains confirm in vitro GPCAT activity. In vivo metabolic labeling studies employing radiolabelled GPC were performed in various strain backgrounds in which the established pathways for PC biosynthesis were compromised and the GPCAT gene was either missing or overexpressed. Overall, our results indicate that the GPCAT gene acts as an GPC acyltransferase and that this novel pathway contributes to PC biosynthesis in the cell.Support or Funding InformationNational Institute of Health Grant: R15 GM104876
Phosphorylation of yeast glycerol 3‐phosphate acyltransferase Gpt2 regulates the timing of TAG synthesis and lipid droplet morphology
Authors: Tavassoli Marjan , Nagler Bernadette , Shabits Brittney , Sipila Patrick , Athenstaedt Karin , Zaremberg Vanina 
Published: November 2023

Glycerol‐3‐phosphate acyltransferases catalyze the acylation of glycerol‐3‐phosphate at the sn‐1 position yielding 1‐acyl glycerol‐3‐phosphate (lyso‐phosphatidic acid). This is the first and rate limiting reaction in de‐novo synthesis of phosphatidic acid (PA), the key intermediate for the formation of all glycerophospholipids and triacylglycerols. In all eukaryotic organisms glycerol‐3‐phosphate acyltransferases as well as acyltransferases catalyzing the subsequent acylation reaction converting lyso‐PA to PA occur in redundancy. This led to the hypothesis that isoenzymes are forming different PA pools which supply different pathways. In the model organism yeast Saccharomyces cerevisiae two glycerol‐3‐phosphate acyltransferases have been identified, Gpt2 (Gat1) and Sct1 (Gat2). Most recently we demonstrated the important role of Gpt2 (but not Sct1) in cells challenged by oleic acid as the only carbon source. Whereas growth in oleic acid containing medium induced massive lipid particle accumulation in wild‐type cells, cells lacking GPT2 failed to accumulate these storage compartments of triacylglycerols and other non‐polar lipids, exhibited a strong growth defect and cell viability was significantly reduced. We were intrigued by the fact that the intracellular distribution of Gpt2 and its phosphorylation status changed under these growth condition compared to growth on glucose containing medium. We have identified several phosphorylation sites in Gpt2 by mass spectrometry, which are localized to a serine rich region in the carboxy‐terminus of the protein. Serine to alanine or to aspartate mutations as well as deletion of the serine rich region containing these residues, were introduced in the yeast genome to investigate the role of phosphorylation in the regulation of Gpt2. Importantly, all phosphorylation mutants were able to support life of cells lacking Sct1, indicating these mutants are functional. We show that manipulations of Gpt2 phosphorylation status strongly and specifically affect TAG accumulation, but not metabolism of the most abundant glycerophospholipids during late log phase of growth. Importantly, alterations in the TAG content are accompanied by marked changes in lipid droplet morphology in the respective mutants. Altogether our results support a role for Gpt2 phosphorylation in regulating the timing of TAG synthesis and lipid droplet morphology.Support or Funding InformationThis work has been financially supported by the Natural Sciences and Engineering Research Council of Canada (to VZ and BS) and the Austrian Science Fund (FWF; Project P21251 to KA).
Molecular biology of calcium trigger vesicle fusion in situ
Authors: Cialowicz Katarzyna , Duncan Rory , Rickman Colin , Brown Euan , Euston Matthew 
Published: November 2023

Calcium ions in the human body serve as second messengers and are responsible for cell homeostasis. Muscle contractions and neuronal communication are supported by electrical activity. Voltage‐activated calcium channels are crucial for many cellular events. They play an important role in transducing the initial stimulus to the effector systems that modulate insulin secretion in pancreatic cells or neurotransmitter at nerve terminals. Dysfunction of these processes may cause many conditions, including diabetes or Alzheimer's disease. In recent years the amount of research on Ca2+ channels has markedly increased, but there are limitations related to physical restrictions in the spatial resolution of fluorescence microscopy. This phenomenon results in a loss of information with regard to the true location of a point source that is emitting light. Newly developed methods such as Stimulated Emission Depletion Microscopy (STED) or Photoactivated localization microscopy (PALM) allow imaging close to the molecular scale.The general aims of this project so far were focused on understanding the mechanism of action and distribution of N‐type calcium channels by using novel tools I have helped to develop for calcium channel imaging.To find out if Cav2.2 Ca2+ (Neuronal type) channels are in a clustered conformation or randomly distributed in the cellular plasma membrane, gated‐ (g)STED was used. After measuring the size of the signals in gSTED image data we conclude that Cav2.2 are patterned in clusters at the plasma membrane. To support that conclusion and characterize our imaging capability, more experiments using known size, sub‐diffraction fluorescent beads were conducted.To quantify the mobility and location of N‐type calcium channels PALM was used. The principle of PALM is activating several photoactivable fluorescent molecules and imaging their signals until irreversible photo‐bleaching occurs. This process of activation and inactivation is then repeated over thousands of frames to ensure all molecules have been imaged. Using a photo‐uncageable fluorescent dye mono‐conjugated with a peptide toxin from a marine snail, allows for imaging single endogenous channel nano‐scale locations. This experiment is significantly better than the current state‐of‐the‐art and revealed the patterning of large cohorts of single endogenous ion channels. To verify mobility of N‐type calcium channels series of images were analysed using bespoke particle tracking scripts.Exocytosis is a process in which secretory vesicles are fused to the plasma membrane. Vesicles proximal to the plasma membrane are triggered to fuse by a rise in local free Ca2+ ions. The probability of the release of synaptic vesicles is hypothesized to increase with the number of proximal calcium channels. We combined our novel approaches to generate calcium ‘activity maps’ of the plasma membrane to investigate the relationship between calcium channels and exocytosis.
Analysis of Large Conductance Ca2+‐Activated K+ (BK) Channels Dynamics in Living Cells
Authors: Miguelez Allende , Duncan Rory , Brown Euan 
Published: November 2023

Large conductance Ca2+‐activated K+ (BK) channels are central in diverse cellular processes including neurotransmitter release or smooth muscle contraction. BK channel activation is driven by both intracellular Ca2+ and voltage, and they are essential for rapid membrane repolarisation. Moreover, BK channels are expressed in human β‐cells regulating insulin secretion. Indeed, in diabetes mellitus BK channels activity is disrupted.Previous electrophysiological studies suggested that N‐type (Cav2.2) calcium‐ and BK‐channel molecules must be in close proximity in the plasma membrane. Therefore, we developed novel methods to analyze the dynamics and distribution of BK channels at the level of single molecules in cell membranes using super‐resolution microscopy, molecular manipulation and electrophysiology. We expressed a cDNA construct that encodes the BK‐channel α‐subunit (BKα) fused to a photoactivatable fluorescent protein mutant of mCherry (PAmCherry) and localized single channel molecules. We also analyzed BK channel localization with gated stimulated emission depletion (g‐STED) microscopy using a BKα‐EGFP construct. Furthermore, we integrated both total internal reflection fluorescence (TIRF) microscopy and patch‐clamp for the study of BK channels function and localization.In order to study Ca2+ channel proximity with BK channels and organelles like secretory vesicles we transfected secretory cells with our BKα‐PAmCherry construct or use the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system to create genetically modified cells, labelling endogenous Cav2.2 channels with fluorescent‐Conotoxin (specific blocker for Cav2.2) that can be photoactivated. With this experiment we could analyse both channel membrane localization and proximity to secretory vesicles and exocytosis sites, using caged Ca2+ to trigger secretory vesicle fusion with the cell membrane. By co‐transfecting cells with both a GCaMP construct localized in the inner cell membrane layer and BKα‐PAmCherry constructs we can track the intracellular Ca2+ rise due to GCaMP fluorescence emission and how depolarization affects BK channels dynamics.These experiments revealed the dynamics, proximity and patterning of large cohorts of single ion channel protein molecules and their functional relationship with the secretory machinery.
Identification of the Eletriptan Binding Site on P‐glycoprotein Determined from Nuclear Magnetic Resonance Spectroscopy
Authors: Wilt Laura , Roberts Arthur 
Published: November 2023

P‐glycoprotein (Pgp) serves as the gatekeeper across the blood‐brain barrier and protects the brain from chemical insults; however, this protective role reduces brain penetration of neurotherapeutics. The drug class triptans are used in the treatment of migraines and have a range of Pgp transport rates. We have chosen to work with eletriptan (ETT) because this triptan drug has a high transport rate and represents the best opportunity to decipher the mechanism of transport. The affinity and the number of binding sites to Pgp were estimated using fluorescence quenching. ATPase activity of reconstituted Pgp revealed ETT to have a monophasic curve and was fit to Michaelis‐Menten equation. Using paramagnetic relaxation enhancement (PRE) NMR of cysteine‐labeled Pgp, the ETT binding site was identified to lie in the central binding cavity. ETT molecules bound to Pgp were also found to be in an extended conformation by exchange‐transferred NOE (etNOE) spectroscopy. The position of ETT was validated using saturation transfer difference (STD) NMR.Support or Funding InformationNIH ‐ 1021RR571390, AHA ‐ 1021RR571388, Pfizer, Inc., University of Georgia, Bioexpression and Fermentation Facility at UGA, Complex Carbohydrate Research Center at UGA