Mak117

Glycerol accumulation in culture medium of fully differentiated ASCs (Day 11 of differentiation) was measured as an indicator of lipolysis. Basal lipolytic rates were defined as glycerol accumulation after 24 h and stimulated lipolysis was measured with isoproterenol (10⁻⁶ M) during 24 h. Glycerol concentration in culture media was measured with a standard enzymatic fluorometric assay (Sigma-Aldrich, St. Louis, MI, USA, cat#MAK117).

Samples were obtained from the diffusion of R2YE agar medium in water as described above. Different molecules were quantified from the eluates. Enzymatic assays were used to determine glucose and glycerol concentrations (Kits GAHK-20 and MAK117, Sigma-Aldrich) and a colorimetric assay kit (MAK030, Sigma-Aldrich) was used to assay total free phosphate. A colorimetric method was used for proline quantification with isatin as derivatizing agent^{119 (link)}. These assays were carried out in four independent biological replicates.

We performed all metabolite assays at the Laboratorio Clínico Veterinario (Universidad Austral de Chile) using an auto-analyzer (Metrolab 2300, Wiener Lab). Briefly, total triglycerides (triglycerides plus free glycerol) was measured by enzymatic colorimetric assay (Triglycerides liquicolormono GPO-PAP kit, HUMAN GmbH) adapted to small sample volumes in 300 μL flat-bottom microplates. Glycerol concentration was determined by a coupled enzyme assay (Sigma-Aldrich MAK117). Concentration of uric acid was determined using the enzymatic colorimetric test with lipid clearing factor (uric acid liquicolor PAP kit, HUMAN GmbH). β-hydroxybutyrate was measured using colorimetric enzymatic reaction (D-3-hydroxybutyrate kit; Ranbut, Randox Laboratories). Triglyceride levels were calculated by subtracting free glycerol from total triglyceride levels^{23 (link),39 (link)}.

Intracellular glycerol concentration was measured as described previously (Dunayevich et al., 2018 (link)). Yeast cells were grown in SC media, inoculated from an overnight grown culture, till the mid-log phase. An aliquot of 1 ml culture was taken and OD₆₀₀ was measured for all the replicates. The cells were harvested by centrifuging at 3000 rpm for 5 min at RT and washed twice with PBS. Cells were resuspended in 1  ml of boiling water and incubated at 100°C for 10 min. Next, the resuspension was cooled on ice for 10 min and centrifuged at 15,000 × g for 5 min. The supernatants were used to determine glycerol concentration using a commercial kit following the manufacture’s indications (MAK117, Sigma). The standard curve was measured between 0 and 1 mM of glycerol. Samples were diluted as necessary to obtain data in this range. The amount of glycerol was normalized to OD₆₀₀ and the mean value of intracellular glycerol concentration/OD₆₀₀ ± standard deviation SD (n = 4) was plotted.