L type tg m assay

Insulin and glucose tolerance tests were performed after a 4-h fast. Mice were injected intraperitoneally with human insulin (1.0 U/kg body weight; Eli Lilly, Indianapolis, IN) or glucose (1 mg/g body weight) (20) and blood glucose levels were measured at baseline, 15-, 30-, 60-, and 120-minute time points. At the time of sacrifice, blood was collected (post 4-h fast) for insulin, triglyceride, phospholipid, and cholesterol measurements using an ultra-sensitive insulin ELISA (EMD Millipore, Billerica, MA), an L-Type TG M assay (Wako Diagnostics, Richmond, VA), a Phospholipids C assay (Wako Diagnostics, VA), and an Amplex Red Cholesterol Assay Kit (Invitrogen, CA), respectively.

TAG concentration of each fraction was determined using an L Type TG M assay (Wako Diagnostics, Richmond VA, USA). Protein concentration of each fraction was determined by a BCA assay (Thermo Scientific, Rockford IL, USA). Additionally, a Western Blot analysis was performed on each fraction. Briefly, samples from the isolated fractions were delipidated using 10% sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. The membranes were probed overnight at 4°C with antibodies for Plin3 (a gift from Dr. Perry Bickel at the University of Texas Southwestern, Dallas TX, USA), glyceraldehyde 3-phosphate dehydrogenase (Gapdh) (Abcam, Cambridge MA, USA), or calnexin (Cnx) (Santa Cruz Biotechnology, Dallas TX, USA). These proteins were used as markers for the CLD, cytosolic, or membrane fractions, respectively. The blots were then probed with a LI-COR IR DYE 800CW secondary antibody and imaged using Odyssey CLx Infrared imaging system (LI-COR Biosciences, Lincoln NE, USA).

Briefly, immediately following euthanasia whole blood was removed via 27‐gauge needle from the abdominal aorta, centrifuged at 2,000 g for 2 min in EDTA‐coated tubes. Assessment of plasma lactate was determined using the Eton Bioscience (San Diego, CA, USA) l‐Lactate Assay Kit I Protocol Version 7. Plasma iron level was determined using the Iron‐SL Assay (Sekisui Diagnostics, Lexington, MA, USA) following the manufacturer's protocol. Assessment of plasma triglyceride was determined using the L‐Type TG M Assay (Wako Diagnostics, Mountain View, CA, USA). Assessment of plasma glucose was determined using HemoCue Glucose 201 Systems glucometer.