Dl isoproterenol hydrochloride iso

Lipolysis was measured as described by Dole et al.⁴⁹ (link) Aliquots (300 μl) of a 5% suspension of adipocytes were prepared in Krebs Ringers HEPES (KRH) containing 25 mM HEPES (pH 7.4), 120 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 1% BSA, 2 mM glucose, and 200 nM adenosine and incubated with or without 30 nM DL-Isoproterenol hydrochloride (ISO) (Sigma-Aldrich, St. Louis, MO, USA), 10–30 U/ml adenosine deaminase (ADA) (Roche Diagnostics, Mannheim, Germany), 10 μM OPC3911 (PDE3 inhibitor) (Osaka Inc., Japan) in the presence or absence of 3 and 10 mM propionic acid (Sigma-Aldrich) or butyric acid (Sigma-Aldrich) at 37°C for 30 min in a shaking device (150 rpm). The cell-free medium was transferred to wells and buffer containing 50 mM glycine, 0.05% hydrazinehydrate, 1 mM MgCl₂, 0.75 mg/ml ATP (Sigma-Aldrich), 0.375 mg/ml NAD (Roche, Pleasanton, CA, USA), 25 μg/ml glycerol-3-phosphate dehydrogenase (Roche) and 0.5 μg/ml glycerol kinase (Roche) was added. Lipolysis was measured at optical density of 340 nm as the release of glycerol into the cell-free medium.

The protease inhibitor cocktail used was obtained from Roche Diagnostics GmbH. The iTRAQ Reagent-8plex kit was purchased from AB Sciex. The BCA Protein assay kit was from Thermo Fisher Scientific, Inc., and the anti-apolipoprotein C-III (Apoc3) antibody was purchased from Boster Biological Technology. DL-isoproterenol hydrochloride (ISO; purity ≥98.5%) was purchased from Sigma-Aldrich; Merck KGaA. The other chemicals used were of analytical grade.