Ab178780

Blood samples were collected by puncturing of the V. maxillaris of anesthetized mice. After clotting, blood samples were centrifuged at 2000× g for 10 min. Resulting supernatant (serum) was transferred into clean tubes and stored at −80 °C until use. Measurement of serum albumin was performed using a mouse-specific albumin ELISA kit (ab108792, Abcam). Creatinine and urea measurements were performed using enzymatic kits (Creatinine PAP LT-SYS LT-CR 0106, Urea LT-UR 0010, Labor & Technik, Eberhard Lehmann GmbH). Serum triglyceride (TG) levels were assayed using a fluorometric kit (ab178780, Abcam). Total cholesterol (TC), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) fractions were measured using a calorimetric kit (ab65390, Abcam). Endotoxin (LPS) levels were determined using a chromogenic endotoxin quantification kit (A39552S, Thermo Fisher Scientific, Inc.). All assays were used according to the manufacturer´s instructions.

Cholesterol, triglyceride and free fatty acid contents were analyzed in the plasma and quadriceps muscle isolated from mice after three weeks of treatment. Muscle samples were homogenized with a microdismembrator for 30 s at 3000 rpm (Sartorius Stedim Biotech, Aubagne, France). For the cholesterol and triglyceride contents, muscles were lysed in lysis buffer from the assay kit (ab65390 and ab178780, Abcam, Cambridge, UK) and measured according to the manufacturer’s instructions. For the free fatty acid content, muscles were lyzed in chloroform containing 1% Triton X, centrifuged at 4 °C for 5 min at 16,000× g and the supernatant was vacuum-dried at 50 °C for 30 min. Dried samples were reconstituted and measured according to the manufacturer’s instructions (ab65341, Abcam, Cambridge, UK). Cholesterol, triglyceride and free fatty acid contents were measured directly from the plasma according to the manufacturer’s instructions.

As described in our previous study (Liu et al., 2019 (link); Cen et al., 2020 (link)), cells were firstly washed with phosphate-buffered saline and then fixed with 4% formaldehyde for 20 min. Thereafter, formaldehyde was removed, and the cells were washed with 60% isopropanol and then stained with 0.2% ORO (Sigma-Aldrich, Germany) for 30 min. Afterwards, cells were washed with phosphate-buffered saline and observed under a microscope (Olympus, Japan). Stained oil droplets were dissolved in 100% isopropanol and quantified by detecting the optical absorbance (500 nm) with a spectrophotometer (Thermo Fisher, Germany). Triglyceride content was quantified using a triglyceride quantification kit (ab178780, Abcam, United Kingdom), according to manufacturer’s instructions.