Etomoxir

Tg(fabp10a:pt-β-catenin) larvae were treated with 0.1% dimethyl sulfoxide (DMSO), 1 µM GW7647 (Cayman Chemical, Ann Arbor, MI), 2 µM K-975 (MedChemExpress, Princeton, NJ), 15 µM Etomoxir (Cayman Chemical, Ann Arbor, MI), 2 µM AG1478 (ApexBio, Houston, TX), or 7 μM CAY10599 (Cayman Chemical, Ann Arbor, MI) for 48 h from 12 or 13 days post-fertilization (dpf). Compounds were refreshed every 24 h. The larvae were fed prior to the drug treatments but fasted during the treatments. They were harvested at 14 or 15 dpf for subsequent analyses.

All the cell culture reagents were provided by HyClone Laboratories (South Logan, UT, USA). Recombinant mouse leptin, Bafilomycin A1, and fatostatin were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Oligomycin and Etomoxir were obtained from Cayman Chemical (Ann Arbor, MI, USA). Bodipy 493/503 was purchased from Invitrogen (Carlsbad, CA, USA). CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS) was procured from Promega Corporation (Madison, WI, USA). Cycle test plus DNA reagent kit was provided by BD Biosciences (San Jose, CA, USA). Primary antibodies against phospho‐Akt, total Akt, LC3B, p27 Kip1, and FASN were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti‐cyclin D1 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against β‐actin and SREBP‐1 were procured from Thermo Scientific (Waltham, MA, USA) and Abcam (Cambridge, MA, USA), respectively. The anti‐rabbit and anti‐mouse secondary antibodies conjugated with horseradish peroxidase (HRP) were provided by Thermo Scientific. The biotinylated anti‐rabbit secondary antibody was acquired from Vector Laboratories Inc (Burlingame, CA, USA). All other chemicals and reagents were purchased from Sigma‐Aldrich unless mentioned elsewhere.

Inhibition assays were performed in a range of 0.125 to 12.5 µM FATP1 inhibitor, compound 5k and 12a^{43 (link),44 (link)} (kindly provided by Tsuyoshi Shinozuka, Japan), 0.02 to 40 µM CPT1a inhibitor, ST1326 (Avanti Lipids, Croda International, Snaith, UK), 5 µM CPT1a inhibitor, Etomoxir (Cayman Chemical, Michigan, USA) and 0.4 to 10 µM ACSL1 inhibitor, Triacsin C (Abcam, Cambridge, UK) in RPMI1640 with 10% FBS, 1% UG, and 1% AA. For the compound 5k studies, WT and CD37KO cells of the same genotypic background (BJAB and OciLy1) were pooled prior to treatment, and later identified by CD37 FACS-signal to exclude differences in culture conditions. About 5 µM CellTrace CFSE (Thermo Fisher Scientific, Waltham, MA, USA) was used to assess proliferation. Cell division numbers were established by assessing peak intensities. AnnexinV/7-AAD (1:100, BioLegend, San Diego, CA, USA, 640930) stainings were used to determine viability after inhibition using flow cytometry (MACS Quant, Miltenyi Biotec, Bergisch Gladbach, Germany).

On day 8, the culture medium of mature 3T3-L1 adipocytes was replaced with Seahorse XF DMEM medium (103575-100) supplemented with 1 mM sodium pyruvate (Wisent), 2 mM glutamine (Wisent), 4.5 g/L glucose (Wisent), 20 nM insulin, and (D-Ala²)GIP[Lys³⁷PAL], then incubated at 37 °C in a non-CO₂ incubator for 60 min. After hydration, the Seahorse cartridge was loaded with 1 µM oligomycin (port A), 1 µM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP; port B), 4 µM etomoxir (11969, Cayman) in DMSO 0.1% (port C), 1 µM rotenone (ab143145), and 1 µM antimycin (A8674, Sigma-Aldrich) in DMSO 0.001% and ethanol 0.002% (R&A; port D). OCR and ECARs were measured using an XF96 extracellular flux analyzer (Seahorse Bioscience). Cellular bioenergetics and parameter calculations were performed according to the manufacturer’s instructions, with the etomoxir effect (OCR dedicated to FAO) calculated as maxOCRFCCP-meanOCRetomoxir.

Cultured adipocytes were washed 1× with PBS and then incubated with DMEM (following manufacturer’s guidelines; catalog #D5030; Sigma-Aldrich) containing BSA-conjugated approximately 3 μCi/ml of either [9,10-3H(N)]-palmitic acid, [9,10-3H(N)]-oleic Acid (PerkinElmer) or n-[2,2′,3,3′-3H] octanoic acid (American Radiolabeled Chemicals, St. Louis, MO, USA) and 22 μM of unlabeled NEFAs, respectively (Sigma-Aldrich). For long-chain fatty acid oxidation, assay media was contained with 200 μM L-carnitine (Sigma-Aldrich), and a subset of wells were treated with 40 μM etomoxir (Cayman Chemical) 2 hours before and duration of the assay. After 3 hours, conditioned media from cells was passed through columns containing AG1-X8 Anion Exchange Resin (Bio-Rad, Hercules, CA, USA) and collected in scintillation vials, then mixed with scintillation cocktail Bio-Safe II (RPI Research Product International, Mount Prospect, IL, USA). Radioactivity in the supernatant was measured using a scintillation counter.

Reagents used were as follows: adenosine 5′-diphosphate (Sigma-Aldrich, St. Louis, MO, USA), A-939572 (Cayman Chemical), CAY10566 (Cayman Chemical), Cell-Tak (Corning, Corning, NY, USA), CL-316,243 (Tocris, Minneapolis, MN, USA), C75 (Cayman Chemical), etomoxir (Cayman Chemical), forskolin (Tocris), MF-438 (Millipore Sigma, St. Louis, MO, USA), octanoic acid (Sigma-Aldrich), oleic acid (Sigma-Aldrich), rotenone (Tocris), palmitoleic acid (Sigma-Aldrich), palmitoyl carnitine (Sigma-Aldrich), pyruvate (Sigma-Aldrich), sodium palmitate (Sigma-Aldrich), and TOFA (Cayman Chemical). Additional information is available in S1 Table.