Cay10499

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.

Commercially available chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Alfa Aesar-Thermo Fisher Scientific (Karlsruhe, Germany) and used without further purification. JZL-184 and CAY10499 were purchased from Cayman Chemical. NMR spectra were obtained with a Bruker Avance III 400 MHz spectrometer. Chemical shifts (δ) are reported in parts per million downfield from tetramethylsilane and referenced from solvent references. HPLC analysis: all target compounds (i.e. assessed in biological assays) were ≥94% pure by HPLC, confirmed via UV detection (λ = 310 nm). Analytical reversed-phase HPLC was conducted using a Kinetex EVO C18 column (5 µm, 150 × 4.6 mm, Phenomenex, Inc.); eluent A, water; eluent B, CH₃CN; after 3 min at 25% B, a gradient was formed from 25 to 85% of B in 4 min and held at 85% of B for 8 min; flow rate was 1 ml/min. Chromatographic separations were performed on silica gel columns by flash chromatography (Kieselgel 60, 0.040–0.063 mm; Merck). Reactions were followed by thin-layer chromatography (TLC) on Aldrich aluminum silica gel (F254) sheets that were visualised under a UV lamp. Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was always used as the drying agent. Elemental analysis has been used to determine the purity of target compounds. Analytical results are within ±0.40% of the theoretical values.

FK565 (heptanoyl-γ-D-glutamyl-L-meso –diamino-pimelyl-D-alanine) was obtained from Fujisawa Pharmaceuticals (Osaka, Japan). H89, SP600125, U0126, pERK (#4370), ERK (#4695), p-p38 (#9215), p38 (#9212) β-Actin (#5125) and phospho-PKA substrate (#9624) antibodies were from Cell Signaling Technology (Boston, MA, USA). Fatty acid-free bovine serum albumin (BSA), ammonium pyrrolidinedithiocarbamate (PDTC) and isoproterenol were from Sigma Aldrich (St. Louis, MO). CAY10499 and CAY10470 were from Cayman Chemicals (Ann Arbor, MI). Myristoylated PKI (14–22) amide and ML130 were from Tocris Bioscience (Bristol, UK). Dulbecco's modified Eagle medium (DMEM), Dulbecco's Phosphate-Buffered Saline (DPBS) and fetal bovine serum (FBS) were from Life Technologies (Burlington, ON, CA). c12-iEDAP and MDP were from Invivogen (San Diego, CA).

12 well plates of differentiated 3T3-L1 adipocytes were washed with Krebs Ringer buffer (KRB; 12 mM HEPES, 121 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO₄, 0.33 mM CaCl₂) and incubated at 37 °C in 300 μl of Krebs Ringer buffer containing 2% FA-free bovine serum albumin (BSA) and 0.1% glucose in the presence or absence of 10 μM isoproterenol (Sigma) for 1 hr.
Primary adipocytes were isolated from epi-WAT after digestion at 37 °C for 1 h with collagenase (Roche) in KRB supplemented with 3 mM glucose and 1% FA-free BSA. Digestion products were filtered through nylon mesh and centrifuged. Adipocytes were collected from the upper phase. Cells were then incubated at 37 °C in 500 μl of KRB in the presence or absence of 10 μM isoproterenol (Sigma) for 1 h or with 50 μM ATGL inhibitor, Atglistatin (Sigma), and 10 μM HSL inhibitor CAY10499 (Cayman). Lipolysis was assayed by the release of FFAs and glycerol into the media as described previously using the NEFA-HR (Wako, #997-76491) and glycerol reagent (Sigma), respectively.
For in vivo lipolysis, mice were injected intraperitoneally with isoproterenol in PBS at 10 mg/kg. Blood samples from tails were collected at 0, 15, 30, 60 and 90 min after injection. Serum was subjected to glycerol measurement.