Nonesterified fatty acids

After an overnight fast, rats were briefly gassed with CO₂ and euthanized by cervical decapitation. Mixed blood was collected in chilled tubes containing EDTA and centrifuged at 12,000 rpm for 4 min. The plasma was removed and stored at −80 °C for measurement of glucose (Abcam, Cambridge, MA, USA), insulin (Alpco Diagnostics, Salem, NH, USA), non-esterified fatty acids (Abcam, Cambridge, MA, USA), and FC (Abcam, Cambridge, MA, USA) following the manufacturer’s guidelines and using a multi-mode microplate reader (Biotek Instruments Inc., model Synergy 2, Winooski, VT, USA). Livers and kidneys were excised, weighed, and snap frozen in liquid nitrogen. Inguinal, peritoneal, and visceral fat pads were carefully excised and weighed to determine total fat pad weight.

For glucose tolerance testing (GTT), the amount of glucose administered was normalized to body weight at 2.5 mg gm glucose/kg body weight. For insulin tolerance testing, 0.02 units of insulin/mouse (HumulinR) was administered. The female mice were fasted 4 h prior to administering the glucose or insulin solution by intraperitoneal injection. Fasting glucose levels were measured in whole blood using a Breeze2 glucometer (Bayer, Leverkusen, Germany). Serum non-esterified fatty acids (Abcam, Cambridge, MA, USA), triglyceride (Eagle Diagnostics, Cedar Hill, TX, USA), and cholesterol (Cell Biolabs, San Diego, CA, USA) levels were assayed according to the manufacturers’ instructions. Serum ALT, AST, total bilirubin and albumin were assayed in the Pennington Biomedical Clinical Core. Hepatic triglycerides were assayed as previously described [16 (link)].