Stock solutions of CPT1 inhibitor Etomoxir (2[6(4-chlorophenoxy)hexyl]oxirane-2-carboxylate; Tocris Bioscience) and FAS inhibitor C75 (4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid; ENZO Life Sciences) were prepared by dissolving in ddH2O and DMSO respectively. Before use, both stocks were diluted with PBS to the required concentration.
Etomoxir
Manufactured by Bio-Techne
Sourced in United Kingdom
Etomoxir is a chemical compound used in research laboratories. It functions as an inhibitor of carnitine palmitoyltransferase 1 (CPT1), an enzyme involved in the transport of long-chain fatty acids into mitochondria for β-oxidation. Etomoxir is utilized in research studies to investigate cellular energy metabolism and the role of fatty acid oxidation in various biological processes.
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14 protocols using etomoxir
1
CPT1 and FAS Inhibitor Preparation
2
Antibody Panel for Protein Analysis
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The following antibodies were used: anti-ATF4/CREB-2 (c-20) (Santa Cruz Biotechnology, Cat. No. sc-200); anti-eIF4E1 (P-2) (Santa Cruz Biotechnology, Cat. No. sc-9976); anti-eIF4E3 (Proteintech, Cat. No. 17282-1-AP); anti-FAM129A/Niban (Signalway Antibody, Cat. No. 21401-2); anti-Keap1 (H-190) (Santa Cruz Biotechnology, Cat. No. sc-33569); anti-Nrf2 (H-300) (Santa Cruz Biotechnology, Cat. No. sc-13032); anti-RhoE/Rnd3 clone 4 (EMD Millipore Corporation, Cat. No 05-723); anti-SQSTM1/p62 (Clone 3) mouse mAb (BD Transduction Laboratories, Cat. No. 610832); and anti-α-tubulin mouse mAb (DM1A) (EMD Millipore Corporation, Cat. No. CP06). Carfilzomib (Cat. No. A-1098) was obtained from Active Biochem; chloroquine (Cat. No. S4157) and GSK2656157 (Cat. No. S7033) were purchased from Selleck Chemicals; (S)-4-carboxyphenylglycine (Cat. No. 0323), CGP57380 (Cat. No. 2731), 4EGI-1 (Cat. No. 4800) and MG-132 (Cat. No. 1748) were ordered from Tocris Bioscience; etomoxir (Cat. No. 11969) was obtained from Cayman Chemical; and trigonelline hydrochloride (Cat. No. T5509-1G) was from Sigma-Aldrich.
3
Pharmacological Modulation of α1-AR Signaling
4
Mitochondrial Function Optimization
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Etomoxir (ETO) and STO-609 was purchased from TOCRIS (Tocris Bioscience, UK). Bezafibrate (BEZA), CCCP, and H2O2 were obtained from Sigma (St. Louis, MO, USA). Oligomycin was purchased from Cell Signaling Technology (USA). cyclosporin A (CsA) was obtained from Selleck Chemicals (USA) and BAPTA-AM was purchased from MedChemexpress Co. (USA). The calcium-sensitive indicator, Fluo-3 AM was purchased from Beyotime Co. (China). XF Palmitate-BSA FAO Substrate was obtained from seahorse Bioscience.
5
Measuring Cellular Bioenergetics in XF Media
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OCR and ECAR were measured in XF media (non-buffered RPMI 1640 containing 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) under basal conditions and in response to 3 μM etomoxir (Tocris), 1 μM oligomycin, 1.5 μM FCCP, and 100 nM rotenone + 1 μM antimycin A, using a 96 well XF or XFe extracellular flux analyzer (Seahorse Bioscience).
6
Adipocyte Lipolysis Regulation Protocols
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Lipase inhibitor, Triascin C, Etomoxir treatment: On day 7 of differentiation, media was aspirated and replaced with maintenance media supplemented with lipase inhibitors (10 μM Atglistatin (Cayman Chemicals, 15284) and 20 μM CAY10499 compound (Cayman Chemicals, 10007875) or 5 μM Triascin C (Sigma-Aldrich, T4540) or 50 μM Etomoxir (Tocris, 4539). After 1 h preincubation, cells were stimulated with 100 nM isoproterenol (ISO) (Sigma-Aldrich, I6504) or 20 μM SR-3420 (Rondini et al., 2017 (link)). Cells were harvested for RNA extraction after 3 h stimulation.
NE stimulation: On day 7 of differentiation, cells were stimulated with 1 μM NE (Sigma Aldrich, A9512) or sterile H2O. Cells were harvested for RNA extraction after 1 h stimulation.
NE stimulation: On day 7 of differentiation, cells were stimulated with 1 μM NE (Sigma Aldrich, A9512) or sterile H2O. Cells were harvested for RNA extraction after 1 h stimulation.
7
Spheroid Culture Assay for Metabolic Modulators
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Cells were seeded at 1000 cells per mL in their respective growth medium with 2% matrigel on low adherence plates (Corning, Corning, NY, USA). Control BSA treated cells were grown with 34 µM BSA (Sigma-Aldrich, St. Louis, MO, USA) final concentration, and treatments contained 0.5 µM of L-carnitine with BSA or 30 µM final concentration BSA-conjugated Palmitic Acid (Tocris, Bristol, UK). Etomoxir (Tocris, Bristol, UK) was used at 10 and 100 µM in the spheroid growth medium. Spheroid cultures were maintained for 2 weeks; the treatments were added on day 0 and renewed on day 7. Spheroid diameter was determined before harvest by counting 10 separate 100× fields with ImageJ. After imaging and counting, 1st generation spheroids were dissociated using Trypsin and 1000 cells seeded with 2% matrigel on low adherence plates for 2nd generation spheroids. These were maintained for another 3 weeks in the absence of new Etomoxir treatment but with renewing of growth media. At the end of the incubation period, spheroid diameter was determined after imaging.
8
Investigating Immune Modulation in Preclinical Models
9
Reagents and Suppliers for Biochemical Assays
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Reagents were purchased from the following suppliers: GAPDH and catalase antibodies from Cell Signaling Technologies; horseradish peroxidase–conjugated goat anti-rabbit secondary antibody from Thermo Fisher Scientific; bile acid standards from CDN Isotopes, Cambridge Isotope Labs, and Avanti Polar Lipids; MS-grade acetonitrile from Honeywell; TRI Reagent from Molecular Research Center, Inc.; 14C-palmitic acid from American Radiolabeled Chemicals, and etomoxir from Tocris Bioscience. The NUDT7 antibody was generated as described previously (18 (link)). All other chemicals were of analytical grade or better and were purchased from Sigma-Aldrich or Thermo Fisher Scientific, unless stated otherwise.
10
Modulating Metabolic Pathways in Mice
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To block RA signaling, a total of 220 mg of the pan RA receptor inverse agonist BMS 493 (R&D Systems; RAi) was resuspended in 30 µl of biotechnology performance–certified DMSO (Sigma-Aldrich) and administered intraperitoneally to C57BL/6 WT mice every day for 8 d as previously described (Kastner et al., 2001 (link)). To block FAO, mice were administered 15 mg/kg etomoxir (Tocris Bioscience) every other day throughout the course of infection or for 7 d under steady-state conditions. To block glycolysis, mice were administered 1 g/kg 2-DG (Cayman Chemicals) every other day starting upon infection with T. muris for 7 d and then every day thereafter. For simultaneous treatment, mice were treated with the indicated combination in a total volume of 30 µl DMSO or DMSO only as a vehicle control. To block FA uptake, mice were treated with 200 mg/kg orlistat every other day starting 7 d after infection. Control mice received DMSO only.
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