Glycerol assay kit

These tissues were homogenized in CellLytic (Sigma‐Aldrich Corp., Tokyo, Japan). Supernatant was collected after centrifugation. Protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc., MA). Concentration of FGF21 in the serum and the tissues was measured using a mouse FGF21 immunoassay kit (R&D Systems, MN).
Plasma glucose concentration was measured using the glucose dehydrogenase method (glutest PRO R; Sanwa Kagaku Kenkyusho Co. Ltd., Aichi, Japan). Plasma insulin, glycerol, NEFA, and 3‐HB concentrations were measured using insulin ELISA kit (Mercodia AB, Uppsala, Sweden), glycerol assay kit (Cayman Chemical, Ann Arbor, MI), NEFA‐C kit (Wako Pure Chemical Industries Ltd., Osaka, Japan), and 3‐HB kit (Sanwa Kagaku Kenkyusho Co., Ltd., Aichi, Japan).

Serum levels of cholesterol, TAG, non-esterified fatty acid (NEFA, free fatty acid), glucose, and free glycerol were measured with enzyme assay kits (Cholesterol C-Test, Triglyceride E-Test, NEFA C-Test, Glucose CII-Test: Wako Pure Chemical Industries, Osaka, Japan; Glycerol Assay Kit: Cayman Chemical Company, Ann Arbor, USA). Because amounts of glycerol generated via hydrolysis of serum TAG were measured with an assay kit (GPO-DAOS method [18 ]) to obtain serum TAG levels, collected serum TAG levels were calculated from measured TAG levels and serum free glycerol levels with the following formula:
Collected TAG level (mg/dL) = measured TAG level (mg/dL) – free glycerol level (mg/dL) × 885.4*/92.1** (*molecular weight of glycerol trioleate (triolein), **molecular weight of glycerol). Lipoprotein profiles of pooled blood serum obtained with the fast protein liquid chromatography method were analyzed using the Liposearch analytical lipoprotein profiling service (Skylight Biotech, Tokyo, Japan). Liver lipids were extracted using the method described by Folch et al. [19 (link)] and measured as total as well as free cholesterol, TAGs, and phospholipids (Phospholipid B-Test was also purchased from Wako Pure Chemical Industries).

The dopamine production strain (AN1126, Table 1) was cultured overnight in LB medium at 37°C, and 10 mL cell culture was inoculated into 1 L Terrific Broth with 5 g/L glycerol, 50 mg/L ampicillin, and 25 mg/L kanamycin. The pH, temperature (25°C constant), dissolved oxygen level, and feeding rate of glycerol were controlled as described previously3 (link). Induction was carried out by adding 0.1 mM (final concentration) IPTG and 0.1 mM (final concentration) CuSO₄ at 14 hours after inoculation (OD₆₀₀ was approximately 20, and an OD₆₀₀ of 1.0 was equivalent to a dry cell weight of 0.40 ± 0.01 g/L). The glycerol concentration was measured with a glycerol assay kit (Cayman Chemical) as previously described3 (link). Because glycerol inhibited THP production in the second step of the culture (Fig. S4), the culture was harvested at 105 hours after inoculation, when glycerol had been completely consumed. The supernatant collected after centrifugation contained 12.6 mM dopamine. The supernatant was stored at −80°C until further use.