Journal: American Journal of Physiology - Cell Physiology

Article Title: Synergistic activation of mitochondrial metabolism and the glutathione redox couple protects HepG2 hepatocarcinoma cells from palmitoylcarnitine-induced stress

doi: 10.1152/ajpcell.00366.2019

Figure Lengend Snippet: Palmitoylcarnitine promotes selective growth in HepG2 cells compared with HT29 and HCT 116 cells and increases mitochondrial respiratory capacity in HepG2 cells. A: schematic showing palmitoylcarnitine bypassing CPT-1, whereby it enters the mitochondria and stimulates β-oxidation resulting in ATP and reactive oxygen species (ROS) production. B: relative cell survival was measured in HepG2 (n = 14), HT29 (n = 6), and HCT 116 (n = 6) cells following 24 and 48 h of 100 µM palmitoylcarnitine relative to 0 µM palmitoylcarnitine at the same time points. Data are reported as means ± SE. *P < 0.05, significant decrease relative to 0 µM palmitoylcarnitine of the same cell type within the same time point. #P < 0.05, significant increase relative to 0 µM palmitoylcarnitine of the same cell type within the same time point. C–F: HepG2 and HT29 cells were incubated with 0 µM or 100 µM palmitoylcarnitine for 24 h. Mitochondrial respiration (C and D) was measured following ADP titration supported by 5 mM pyruvate and 2 mM malate (NADH, complex I; not shown), 5 mM glutamate (G; NADH), and 20 mM succinate (S; FADH2, complex II) (n = 4), and maximal uncoupled rate of respiration (E and F) was measured following FCCP as an index of electron transport chain content (n = 4). Data are reported as means ± SE. #P < 0.05, main effect for palmitoylcarnitine.

Article Snippet: Cells were cultured with 2 mM l -carnitine (Sigma-Aldrich, St. Louis, MO) and 0 μM or 100 μM palmitoylcarnitine (Toronto Research Chemicals, Toronto, QC, Canada).

Techniques: Incubation, Titration

Journal: American Journal of Physiology - Cell Physiology

Article Title: Synergistic activation of mitochondrial metabolism and the glutathione redox couple protects HepG2 hepatocarcinoma cells from palmitoylcarnitine-induced stress

doi: 10.1152/ajpcell.00366.2019

Figure Lengend Snippet: Redox stress following palmitoylcarnitine exposure: maintenance of overall redox conditions (GSH/GSSG) in HepG2 cells but not in HT29 cells. HepG2 (n = 5) and HT29 (n = 3) cells were incubated with 0 µM and 100 µM palmitoylcarnitine for 24 h. Reduced glutathione (GSH; A), oxidized glutathione (GSSH; B), and the ratio of reduced-to-oxidized glutathione (GSH/GSSG; C) were assessed. Data are reported as means ± SE. *P < 0.05, significant decrease with 100 µM palmitoylcarnitine compared with 0 µM. #P < 0.05, significant increase with 100 µM palmitoylcarnitine compared with 0 µM.

Article Snippet: Cells were cultured with 2 mM l -carnitine (Sigma-Aldrich, St. Louis, MO) and 0 μM or 100 μM palmitoylcarnitine (Toronto Research Chemicals, Toronto, QC, Canada).

Techniques: Incubation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Synergistic activation of mitochondrial metabolism and the glutathione redox couple protects HepG2 hepatocarcinoma cells from palmitoylcarnitine-induced stress

doi: 10.1152/ajpcell.00366.2019

Figure Lengend Snippet: Palmitoylcarnitine alters H2O2, glutathione and cell growth. A and B: intracellular lactate (n = 5; A) and NAD(p)H (n = 9; B) were measured in HepG2 cells following 24 h of 0 µM or 100 µM palmitoylcarnitine. C: H2O2 was assessed following 10 min and 24 h of 0 µM and 100 µM palmitoylcarnitine (n = 15). D: total glutathione was measured in HepG2 and HT29 cells following 24 h of 0 µM or 100 µM palmitoylcarnitine (n = 3–5). E and F: relative cell survival was assessed in HepG2 (n = 6; E) and HT29 (n = 3; F) cells following 48 h of 0 µM or 100 µM palmitoylcarnitine, as well as concurrent incubations with 50 µM buthionine sufloximine or 100 µM genipin. G and H: HepG2 cells were incubated with 0 µM and 100 µM palmitoylcarnitine for 24 h and uncoupling protein-2 (UCP2) protein content was visualized (G) and determined (H) (n = 5). I: schematic depicting the selective inhibition of UCP2 by genipin and the depletion of glutathione with buthionine sulfoximine (BSO). J: schematic of palmitoylcarnitine acutely triggering an increase in H2O2 emission, resulting in UCP2 activation and an increase in GSH, which, in turn, lowers H2O2, as well as stimulates an increase in growth. Data are reported as means ± SE. *P < 0.05, significant decrease with 100 µM palmitoylcarnitine relative to 0 µM palmitoylcarnitine. #P < 0.05, significant increase with 100 µM palmitoylcarnitine relative to 0 µM palmitoylcarnitine.

Article Snippet: Cells were cultured with 2 mM l -carnitine (Sigma-Aldrich, St. Louis, MO) and 0 μM or 100 μM palmitoylcarnitine (Toronto Research Chemicals, Toronto, QC, Canada).

Techniques: Incubation, Inhibition, Activation Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Synergistic activation of mitochondrial metabolism and the glutathione redox couple protects HepG2 hepatocarcinoma cells from palmitoylcarnitine-induced stress

doi: 10.1152/ajpcell.00366.2019

Figure Lengend Snippet: Proposed model of HepG2 cell adaptation to palmitoylcarnitine. The mitochondrial substrate, palmitoylcarnitine, stimulates an acute increase in H2O2. A compensatory increase in glutathione and activation of UCP2 eventually lead to lower H2O2 emission. These hormetic responses to palmitoylcarnitine result in an increase in glutathione redox-buffering capacity. An increase in oxidative capacity also improves ATP synthesis. Collectively, the metabolic and redox flexibility of HepG2 cells results in improved proliferation in response to palmitoylcarnitine in contrast to the abrogations observed in HT29 and HCT 116 cells.

Article Snippet: Cells were cultured with 2 mM l -carnitine (Sigma-Aldrich, St. Louis, MO) and 0 μM or 100 μM palmitoylcarnitine (Toronto Research Chemicals, Toronto, QC, Canada).

Techniques: Activation Assay