Free fatty acid kit

Lipolysis assays were conducted on adipocytes grown in 12-well plates. Adipocytes were placed in an incubation medium with or without triacsin C (containing phenol red-free DMEM, low glucose, and fatty acid-free BSA, and allowed to rest for one hour. Cells were then exposed to conditions as described here for each experiment. For the experiment in Figure 1, vehicle, OSM (0.5 nM), isoproterenol (ISO, positive control; 2nM; Sigma-Aldrich, St. Louis, MO, USA; #I-5752), or bovine insulin (17.5 nM; Sigma-Aldrich; I-5500) were used. For the experiment in Figure 4, vehicle, OSM, the SHC1 inhibitor PP2 (10μM; Selleck Chemicals, Houston, TX, USA; #S7008), or 10 nM ISO were used. For the experiment in Figure 5, vehicle, OSM, triacsin C (5 μM; Cayman Chemical, Ann Arbor, MI, USA; # 10007448), the MEK inhibitor, U0126 (50mM; Cayman Chemical, #70970), or an anti-OSMR antibody (10 μg/well; Santa Cruz Biotechnology, Dallas, TX, USA; #sc-8493), were used. Adipocytes were exposed to conditions as noted in figure legends, for 4–6 h. Media was collected from each well and assayed for glycerol content using free glycerol reagent and glycerol standard (Sigma-Aldrich, #F6428 and #G7793, respectively). NEFA release into triacsin C-free culture media was quantified using a Free Fatty Acid Kit (Sigma-Aldrich, #MAK044) according to the manufacturer’s instructions.

The control or SENP1-aP2KO mice were fed with normal chow, water consumption and food intake was recorded daily. For the GTT and ITT, the mice were fasted overnight, followed by an intraperitoneal injection of glucose (GTT; 1 g kg⁻¹ body weight) or insulin (ITT; 0.75 U kg⁻¹ body weight). Blood glucose levels at various times after the injections were determined by an electronic glucometer (Abbott Diabetes Care, Alameda, CA, USA). Lipids analysis and lipoprotein profile measurement were performed60 (link). In brief, mice were fasted for 12–14 h before blood samples were collected by retro-orbital venous plexus puncture. Plasma was separated by centrifugation and stored at −80 °C. Total plasma cholesterol, TGs and FFAs were enzymatically measured with the Amplex red cholesterol assay kit (Molecular Probes), serum triglyceride determination kit and free fatty acid kit (Sigma), respectively, according to the manufacturer's instructions. The lipid distribution in plasma lipoprotein fractions were assessed by fast-performance liquid chromatography gel filtration with two Superose 6 HR 10/30 columns (Pharmacia).