Atglistatin

All chemicals were obtained from Sigma (St Louis, MO) or Fisher (Waltham, MA) unless otherwise stated. [9,10-³H]oleic acid (sp act: 50 Ci/mmol) was purchased from Moravek (Brea, CA), [³H]uracil from PerkinElmer (Shelton, CT), 5-Butyl-4,4-Difluoro-4-Bora-3a,4a-Diaza-s-Indacene-3-Nonanoic Acid (C4-BODIPY-C9) from ThermoFisher Scientific (Waltham, MA) and 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene (BODIPY 493/503) from Life Technologies (Carlsbad, CA). Non-polar lipid mixture for thin-layer chromatography was purchased from Matreya LLC (State College, PA). Atglistatin was purchased from Cayman Chemical (Ann Arbor, MI). The primary antibodies include: rabbit polyclonal anti-GRA7 [34 (link)], the rabbit polyclonal anti-aldolase and mouse monoclonal anti-Hsp70 (from Fidel Zavala, Johns Hopkins university, Baltimore), and the mouse monoclonal anti-SAG1 (from Jean-Francois Dubremetz, University of Montpellier, France). Secondary antibodies used for immunofluorescence were conjugated to Alexa⁴⁸⁸, Alexa⁵⁹⁴ or Alexa³⁵⁰ (Invitrogen, Carlsbad, CA). To prepare oleic acid (OA)-albumin complexes, sodium oleate was dissolved in H₂O at a concentration of 100 mM, then thoroughly mixed by vortexing for 3 min with 5% Fatty Acid Free BSA to reach a final concentration of 10 mM OA-BSA complexes.

To study the role of lipolysis in vivo, mice were given two different lipolysis inhibitors: Acipimox or Atglistatin. Acipimox is a niacin derivate that suppresses cyclic adenosine monophosphate (cAMP), leading to a general inhibition of lipolysis [48 (link)]. Atglistatin is a selective and competitive inhibitor of ATGL, leaving other lipases unaffected [49 (link)]. Mice received either vehicle, Acipimox (0.5 g/L, Sigma-Aldrich, St. Louis, MO, USA) in drinking water or Atglistatin (0.1 μmol/g, Cayman Chemical Company, Ann Arbor, MI, USA) by oral gavage for 14 days after E0771 transplantation. In the Acipimox study, the drinking water was replaced two times per week and the water consumption of each cage was monitored every 24 h.

GW6471 and Atglistatin were purchased from Cayman Chemical (Ann Arbor, MI, USA). GSK3787 was purchased from Abcam (Cambridge, UK). GW9662 was purchased from FUJIFILM Wako Chemicals (Osaka, Japan). A922500 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against carnitine palmitoyltransferase 2 (CPT2), electron transfer flavoprotein subunit α (ETFα), electron transfer flavoprotein dehydrogenase (ETFDH), Acox1, and VLCAD were kindly gifted by T. Osumi (University of Hyogo) [25 (link),26 (link),27 (link)]. ADRP (PRIN2; sc-377429) and adipose triglyceride lipase (ATGL) (sc-365278) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against SOX2 (#3579), Nanog (#4903), Oct4 (#2890), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

Experiments were carried out using the xCELLigence RTCA DP instrument (Roche Diagnostics GmbH, Mannheim, Germany) which was placed in a humidified incubator at 37°C and 5% CO2. Invasion assays were performed using CIM-16 plates (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacture protocols. 5×10⁴ cells were seeded in the upper chamber and each experimental condition was performed in quadruplicate with a programmed signal detection every five minutes over 72 hours of incubation. Experiments measuring cytotoxicity of Atglistatin were performed using E-plates (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacture's protocols. 5000 HOP62 or 4000 A549 cells undergoing exponential growth were seeded in each well. Cell Index value (CI) of each well was monitored every 15 min. For drug treatment, 22 hours after seeding, cells were treated over a period of 72 hours with the indicated concentrations of Atglistatin (Cayman Chemical Company, MI, USA). All data was recorded using the supplied RTCA software (vs. 1.2.1). CI-data from cell growth experiments have been normalized using the RTCA software prior to the start of treatment.

Inguinal WAT from wild-type mice and Adipo-ATGL KO mice were cut into pieces weighing approximately 10 mg. For human subcutaneous WAT culture, a total of 120 mg of human adipose tissue biopsies were divided into 6 pieces, and each piece was cut into pieces weighing approximately 10 mg. Adipose tissue samples were quickly washed three times with Phosphate-buffered saline (PBS) and centrifuged for 1 min at 300 ×g at room temperature. Subsequently, adipose tissues were cultured overnight in DMEM/F12 medium containing 1%GlutaMAX-I and 10% FBS. After the preincubation, adipose tissue biopsies were washed three times with PBS and centrifuged for 1 min at 300 × g at room temperature (20–25°C). Then, adipose tissue samples were cultured with vehicle or CL316,243 at doses ranging from 0.1 μM to 1 μM in DMEM/F12 without FBS for 12 h (mice) or 24 h (human) for FACS analyses. To pharmacologically inhibit ATGL, Atglistatin (50 μM, Cayman) or vehicle (DMSO) was dissolved in DMEM/F12 containing 10% FBS.

Each LD homeostasis inhibitor used was diluted in DMSO based on manufacturer’s instructions and optimum inhibitor concentration was determined based on 100% host cell viability determined by trypan blue staining, and changes in LD numbers per cell. The optimum concentrations determined for each inhibitor was: Triacsin C (Enzo Life Sciences, Farmingdale, NY, USA)– 10 µM, T863 (Sigma-Aldrich)– 10 µM, CAY10499 (Cayman Chemicals, Ann Arbor, MI, USA) - 10 µM, Atglistatin (Cayman Chemicals)– 20 µM.

MEFs without or with the expression of GFP-ULK1, GFP-WIPI1, GFP-DFCP1, or GFP-LC3^{20 (link)} were kindly provided by Dr. Noboru Mizushima (University of Tokyo). U2OS and Huh7 cells were kindly donated by Dr. Hidemasa Goto (Mie University) and Dr. Eija Jokitalo (University of Helsinki), respectively. MEFs and U2OS were cultured in Dulbecco’s modified Eagle medium supplemented with 10% FBS and antibiotics. For Huh7, Eagle’s minimum essential medium supplemented with 10% FBS and antibiotics were used. The cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The total choline concentration in the medium was measured using LabAssay^TM Phopholipid (296-63801, Fujifilm Wako Pure Chemical). Autophagy was induced by culturing cells in a serum-free EBSS (6.8 mg ml^–1 NaCl, 0.4 mg ml^–1 KCl, 0.16 mg ml^–1 NaH₂PO₄•2H₂O, 0.26 mg ml^–1 CaCl₂•2H₂O, 0.21 mg ml^–1 MgSO₄•7H₂O, 2.2 mg ml^–1 NaHCO₃, 1 mg ml^–1 glucose). OA was conjugated with fatty acid-free BSA (Fujifilm Wako Pure Chemical) at a 6:1 molar ratio before being added to the medium at a final concentration of 0.4 mM^{60 (link)}. Hypoxic conditions were produced using the BIONIX-3 hypoxic culture kit (Sugiyama-gen). T863 (SML0539-5MG, Sigma), avasimibe (A794680, Toronto Research Chemicals), and Atglistatin (15284, Cayman Chemical) were used for the inhibition of DGAT1, ACAT, and ATGL, respectively.