rat hepatoma cell line h4iiec3  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Glutamate–oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux

doi: 10.1074/jbc.RA118.004869

Figure Lengend Snippet: Removal of extracellular glutamine attenuates lipotoxicity. Primary rat hepatocytes and H4IIEC3 cells were treated with 400 μm PA, either in the presence (2 mm) or absence of Gln. A, cell toxicity assessed by PI fluorescence after 24 h of treatment. B, caspase activity in H4IIEC3 cells after 12 h of treatment. In both panels, measurements are normalized to BSA (vehicle)-treated cells cultured with 2 mm glutamine. Data represent mean ± S.E., n = 4; *, p < 0.05, different from vehicle; †, p < 0.05, different from each other (comparison with cells of the same type).

Article Snippet: H4IIEC3 cell culture The H4IIEC3 rat hepatoma cell line was purchased from the ATCC.

Techniques: Fluorescence, Activity Assay, Cell Culture, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Glutamate–oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux

doi: 10.1074/jbc.RA118.004869

Figure Lengend Snippet: Effects of replacing medium glutamine with downstream products of glutamine metabolism. A, primary rat hepatocytes or H4IIEC3 cells were treated with 400 μm PA and cultured with 2 mm Gln, Glu, or αKG. Cell death was assessed by PI fluorescence at 24 h. B, relative cell death after 24 h of treatment with palmitate in the presence of 2 mm glutamine or a mixture of 1 mm α-ketoglutarate and 1 mm aspartate (αKG/Asp). In both panels, PI fluorescence of palmitate-treated cells is normalized to BSA (vehicle)-treated cells cultured with 2 mm glutamine. Data represent mean ± S.E., n = 4; *, p < 0.05, different from vehicle; †, p < 0.05, different from each other (comparison with cells of the same type).

Article Snippet: H4IIEC3 cell culture The H4IIEC3 rat hepatoma cell line was purchased from the ATCC.

Techniques: Cell Culture, Fluorescence, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Glutamate–oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux

doi: 10.1074/jbc.RA118.004869

Figure Lengend Snippet: GOT activity promotes glutamine-dependent palmitate lipotoxicity. A–C, H4IIEC3 cells were transfected with control siRNA (NC1) or siRNA specific for Glud1 (A), GOT1 (B), or GOT2 (C) and assayed for markers of apoptosis after 12 h of treatment with 400 μm PA. D, primary rat hepatocytes were transfected with control siRNA (NC1) or GOT2 siRNA and assayed for markers of apoptosis after 12 h of treatment with 400 μm PA. All palmitate treatment conditions are normalized to BSA-treated (vehicle) cells transfected with control siRNA. Data represent the mean ± S.E., n = 4; *, p < 0.05, different from vehicle; †, p < 0.05, different from each other.

Article Snippet: H4IIEC3 cell culture The H4IIEC3 rat hepatoma cell line was purchased from the ATCC.

Techniques: Activity Assay, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Glutamate–oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux

doi: 10.1074/jbc.RA118.004869

Figure Lengend Snippet: AOA reduces palmitate-induced cell death and activation of oxidative metabolism. H4IIEC3 cells were treated with 400 μm palmitate in combination with 500 μm of the transaminase inhibitor AOA (PA+AOA) and compared with palmitate-treated (PA) cells. A, cell toxicity was assessed after 24 h of treatment and normalized to BSA (vehicle)-treated conditions. B, oxygen consumption rates of H4IIEC3 cells treated with vehicle, PA, or PA+AOA were measured after 6 h of treatment. Data represent mean ± S.E., n = 4 for toxicity, n = 3 for oxygen uptake; *, p < 0.05, different from vehicle; †, p < 0.05, different from each other.

Article Snippet: H4IIEC3 cell culture The H4IIEC3 rat hepatoma cell line was purchased from the ATCC.

Techniques: Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Glutamate–oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux

doi: 10.1074/jbc.RA118.004869

Figure Lengend Snippet: Isotopic enrichments of intracellular metabolites indicate flux rerouting in response to AOA treatment. Unlabeled medium glutamine was replaced with [U-13C5]glutamine and used to isotopically enrich H4IIEC3 cells treated with vehicle (BSA), 400 μm PA, or a combination of 400 μm palmitate and 500 μm AOA (PA+AOA). After extraction and GC-MS analysis of intracellular metabolites, mass isotopomer distributions were corrected for natural isotope abundance using the method of Fernandez et al. (34). APE of selected metabolites was calculated using the formula APE = 100% × Σi = 0N (Mi × i)/N, where N is the number of carbon atoms in the metabolite and Mi is the fractional abundance of the ith mass isotopomer of the metabolite. APE provides a measure of the fractional synthesis of a metabolite from the isotope tracer (i.e. glutamine) relative to unlabeled carbon sources (e.g. glucose). The fragment ions analyzed for APE were Glu 432, Mal 419, and Asp 418 (A) and PEP 453 and Lac 261 (B). These ions contain the full carbon backbone of their associated parent metabolites (i.e. N = 5 for glutamate, N = 4 for malate and aspartate, and N = 3 for PEP and lactate). Data represent mean ± S.E., N = 3; *, p < 0.05, different from vehicle; †, p < 0.05, different from each other. N refers to the number of carbons. n refers to the number of replicates.

Article Snippet: H4IIEC3 cell culture The H4IIEC3 rat hepatoma cell line was purchased from the ATCC.

Techniques: Extraction, Gas Chromatography-Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Glutamate–oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux

doi: 10.1074/jbc.RA118.004869

Figure Lengend Snippet: 13C MFA reveals that AOA treatment reduces mitochondrial fluxes and reroutes malic enzyme flux in the presence of palmitate. A, reaction network used for 13C flux analysis. B, absolute intracellular CAC fluxes were determined for H4IIEC3 cells treated with BSA (vehicle), 400 μm palmitate (PA), or a combination of 400 μm palmitate and 500 μm AOA (PA+AOA). C, estimated PK flux under each treatment condition. D, relative fluxes (normalized to PK = 100) demonstrate that AOA co-treatment is associated with enhanced glutamate anaplerosis despite a reduction in absolute mitochondrial fluxes. GDH, glutamate dehydrogenase (includes both GOT and Glud1 activity), IDH, isocitrate dehydrogenase; SDH, succinate dehydrogenase; FUS, fumarase, MDH, malate dehydrogenase, PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; LDH, lactate dehydrogenase. Error bars indicate 95% confidence intervals. *, p < 0.05, different from vehicle; †, p < 0.05, different from each other (comparison with the same flux across different treatments).

Article Snippet: H4IIEC3 cell culture The H4IIEC3 rat hepatoma cell line was purchased from the ATCC.

Techniques: Activity Assay, Comparison