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Hepatocyte lipotoxicity is characterized by aberrant mitochondrial metabolism, which predisposes cells to oxidative stress and apoptosis. Previously, we reported that translocation of calcium from the endoplasmic reticulum to mitochondria of palmitate-treated hepatocytes activates anaplerotic flux from glutamine to α-ketoglutarate (αKG), which subsequently enters the citric acid cycle (CAC) for oxidation. We hypothesized that increased glutamine anaplerosis fuels elevations in CAC flux and oxidative stress following palmitate treatment. To test this hypothesis, primary rat hepatocytes or immortalized H4IIEC3 rat hepatoma cells were treated with lipotoxic levels of palmitate while modulating anaplerotic pathways leading to αKG. We found that culture media supplemented with glutamine, glutamate, or dimethyl-αKG increased palmitate lipotoxicity compared with media that lacked these anaplerotic substrates. Knockdown of glutamate-oxaloacetate transaminase activity significantly reduced the lipotoxic effects of palmitate, whereas knockdown of glutamate dehydrogenase (Glud1) had no effect on palmitate lipotoxicity. C flux analysis of H4IIEC3 cells co-treated with palmitate and the pan-transaminase inhibitor aminooxyacetic acid confirmed that reductions in lipotoxic markers were associated with decreases in anaplerosis, CAC flux, and oxygen consumption. Taken together, these results demonstrate that lipotoxic palmitate treatments enhance anaplerosis in cultured rat hepatocytes, causing a shift to aberrant transaminase metabolism that fuels CAC dysregulation and oxidative stress.
© 2019 Egnatchik et al.
BACKGROUND - DNA and RNA fractions from whole blood, serum and plasma are increasingly popular analytes that are currently under investigation for their utility in the diagnosis and staging of disease. Small non-coding ribonucleic acids (sRNAs), specifically microRNAs (miRNAs) and their variant isoforms (isomiRs), and transfer RNA (tRNA)-derived small RNAs (tDRs) comprise a repertoire of molecules particularly promising in this regard.
RESULTS - In this designed study, we compared the performance of various methods and kits for isolating circulating extracellular sRNAs (ex-sRNAs). ex-sRNAs from one healthy individual were isolated using five different isolation kits: Qiagen Circulating Nucleic Acid Kit, ThermoFisher Scientific Ambion TRIzol LS Reagent, Qiagen miRNEasy, QiaSymphony RNA extraction kit and the Exiqon MiRCURY RNA Isolation Kit. Each isolation method was repeated four times. A total of 20 small RNA sequencing (sRNAseq) libraries were constructed, sequenced and compared using a rigorous bioinformatics approach. The Circulating Nucleic Acid Kit had the greatest miRNA isolation variability, but had the lowest isolation variability for other RNA classes (isomiRs, tDRs, and other miscellaneous sRNAs (osRNA). However, the Circulating Nucleic Acid Kit consistently generated the fewest number of reads mapped to the genome, as compared to the best-performing method, Ambion TRIzol, which mapped 10% of the miRNAs, 7.2% of the tDRs and 23.1% of the osRNAs. The other methods performed intermediary, with QiaSymphony mapping 14% of the osRNAs, and miRNEasy mapping 4.6% of the tDRs and 2.9% of the miRNAs, achieving the second best kit performance rating overall.
CONCLUSIONS - In summary, each isolation kit displayed different performance characteristics that could be construed as biased or advantageous, depending upon the downstream application and number of samples that require processing.
Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound carriers with complex cargoes including proteins, lipids, and nucleic acids. While the release of EVs was previously thought to be only a mechanism to discard nonfunctional cellular components, increasing evidence implicates EVs as key players in intercellular and even interorganismal communication. EVs confer stability and can direct their cargoes to specific cell types. EV cargoes also appear to act in a combinatorial manner to communicate directives to other cells. This review focuses on recent findings and knowledge gaps in the area of EV biogenesis, release, and uptake. In addition, we highlight examples whereby EV cargoes control basic cellular functions, including motility and polarization, immune responses, and development, and contribute to diseases such as cancer and neurodegeneration.
Copyright Â© 2016 Elsevier Ltd. All rights reserved.
BACKGROUND & AIMS - Penetration of the esophageal epithelium by food antigens is an early event in the pathogenesis of eosinophilic esophagitis (EoE), but the precise relationship among eosinophilia, dilated intercellular spaces (DIS), and decreased barrier function is unclear. We investigated the correlation between site-specific mucosal impedance (MI) measurements of ion flux and esophageal histology, and whether MI measurements can be used to distinguish between patients with active and inactive EoE.
METHODS - MI was measured (in Ω) in 10 patients with active EoE (>15 eosinophils [eos]/high-power field [HPF]) and in 10 with inactive EoE (<15 eos/HPF, as a result of treatment), and mucosal biopsy specimens were collected from 4 esophageal sites (2, 5, 10, and 15 cm above the Z-line). MI also was measured in 10 individuals without esophageal symptoms (controls). MI measurements, eos/HPF, and DIS grade were compared among patients with EoE and controls.
RESULTS - The esophageal MI values were significantly lower in patients with active EoE (1909 Ω) compared with inactive EoE (4349 Ω) or controls (5530 Ω) (P < .001). Biopsy specimens from 4 patients with active EoE contained fewer than 15 eos/HPF and lower-grade DIS than in patients with active disease. There were significant inverse correlations between MI and eos/HPF (rs = -.584), as well as between MI and DIS (rs = -.531; P < .001). The MI cut-off value of 2300 Ω identified patients with active EoE with 90% sensitivity and 91% specificity, and high-grade DIS with 89% sensitivity and 82% specificity.
CONCLUSIONS - In patients with EoE, eosinophilia and DIS correlate with MI measurements of ion flux. Endoscopic MI measurement in the esophagus is safe and easy to perform, and can be used to assess activity of diseases such as EoE.
Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.
Quantitative dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) provides estimates of physiologically relevant parameters related to tissue blood flow, vascular permeability, and tissue volume fractions which can then be used for prognostic and diagnostic reasons. However, standard techniques for DCE-MRI analysis ignore intra-voxel diffusion, which may play an important role in contrast agent distribution and voxel signal intensity and, thus, will affect quantification of the aforementioned parameters. To investigate the effect of intra-voxel diffusion on quantitative DCE-MRI, we developed a finite element model of contrast enhancement at the voxel level. For diffusion in the range of that expected for gadolinium chelates in tissue (i.e., 1 × 10(-4) to 4 × 10(-4) mm(2)/s), parameterization errors range from -58% to 12% for K(trans), -9% to 8% for ve, and -60% to 213% for vp over the range of K(trans), v(e), v(p), and temporal resolutions investigated. Thus the results show that diffusion has a significant effect on parameterization using standard techniques.
Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na(+)-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na(+)/leucine transporter LeuT, our results suggest that Na(+) binding at the conserved second Na(+) binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.
Astrocytes provide metabolic, structural, and synaptic support to neurons in normal physiology and also contribute widely to pathogenic processes in response to stress or injury. Reactive astrocytes can undergo cytoskeletal reorganization and increase migration through changes in intracellular Ca(2+) mediated by a variety of potential modulators. Here we tested whether migration of isolated retinal astrocytes following mechanical injury (scratch wound) involves the transient receptor potential vanilloid-1 channel (TRPV1), which contributes to Ca(2+)-mediated cytoskeletal rearrangement and migration in other systems. Application of the TRPV1-specific antagonists, capsazepine (CPZ) or 5'-iodoresiniferatoxin (IRTX), slowed migration by as much as 44%, depending on concentration. In contrast, treatment with the TRPV1-specific agonists, capsaicin (CAP) or resiniferatoxin (RTX) produced only a slight acceleration over a range of concentrations. Chelation of extracellular Ca(2+) with EGTA (1 mM) slowed astrocyte migration by 35%. Ratiometric imaging indicated that scratch wound induced a sharp 20% rise in astrocyte Ca(2+) that dissipated with distance from the wound. Treatment with IRTX both slowed and dramatically reduced the scratch-induced Ca(2+) increase. Both CPZ and IRTX influenced astrocyte cytoskeletal organization, especially near the wound edge. Taken together, our results indicate that astrocyte mobilization in response to mechanical stress involves influx of extracellular Ca(2+) and cytoskeletal changes in part mediated by TRPV1 activation.
© 2014 Wiley Periodicals, Inc.
Fluorescent dyes have been designed for internal cellular component specificity and have been used extensively in the scientific community as a means to monitor cell growth, location, morphology, and viability. However, it is possible that the introduction of these dyes influences the basal function of the cell and, in turn, the results of these studies. Electrochemistry provides a noninvasive method for probing the unintended cellular affects of these dyes. The multianalyte microphysiometer (MAMP) is capable of simultaneous electrochemical measurement of extracellular metabolites in real-time. In this study, analytes central to cellular metabolism, glucose, lactate, oxygen, as well as extracellular acidification were monitored to determine the immediate metabolic effects of nuclear stains, including SYTO, DAPI dilactate, Hoechst 33342, and FITC dyes upon the pheochromocytoma PC-12 cells and RAW 264.7 macrophages. The experimental results revealed that the SYTO dye 13 significantly decreased glucose and oxygen consumption and increased extracellular acidification and lactate production in both cell lines, indicating a shift to anaerobic respiration. No other dyes caused significantly definitive changes in cellular metabolism upon exposure. This study shows that fluorescent dyes can have unintended effects on cellular metabolism and care should be taken when using these probes to investigate cellular function and morphology.
Cells communicate with neighboring cells and condition their local environment by secreting soluble factors into the extracellular space. These well-studied facets of cell biology are essential for the establishment and maintenance of physiological homeostasis. However, accumulating evidence has revealed that specific ligands, enzymes, and macromolecules are distributed into the extracellular space by virtue of their association with small vesicles, which are released by a variety of cell types. Although the biological significance of such vesicles was initially debated, purification and subsequent functional studies have shown that these extracellular vesicles are bioactive organelles carrying a wide range of protein and nucleic acid cargoes. In many cases these vesicles are laden with molecules that are involved in cell signaling, although other diverse functions are being revealed at a rapid pace. In this Perspective, we discuss recent developments in the understanding of the major pathways of extracellular vesicle biogenesis and how these vesicles contribute to the maintenance of physiological homeostasis.
Accurate quantification of cell specific rates and their uncertainties is of critical importance for assessing metabolic phenotypes of cultured cells. We applied two different methods of regression and error analysis to estimate specific metabolic rates from time-course measurements obtained in exponentially growing cell cultures. Using simulated data sets to compute specific rates of growth, glucose uptake, and lactate excretion, we found that Gaussian error propagation from prime variables to the final calculated rates was the most accurate method for estimating parameter uncertainty. We incorporated this method into a MATLAB-based software package called Extracellular Time-Course Analysis (ETA), which automates the analysis workflow required to (i) compute cell specific metabolic rates and their uncertainties; (ii) test the goodness-of-fit of the experimental data to the regression model; and (iii) rapidly compare the results across multiple experiments. ETA was used to estimate the uptake or excretion rate of glucose, lactate, and 18 different amino acids in a B-cell model of c-Myc-driven cancer. We found that P493-6 cells with High Myc expression increased their specific uptake of glutamine, arginine, serine, lysine, and branched-chain amino acids by two- to threefold in comparison to low Myc cells, but exhibited only modest increases in glucose uptake and lactate excretion. By making the ETA software package freely available to the scientific community, we expect that it will become an important tool for rigorous estimation of specific rates required for metabolic flux analysis and other quantitative metabolic studies.
Copyright © 2013 Wiley Periodicals, Inc.