Mak046

Glucose and insulin tolerance tests were performed on overnight fasted, or 4-h fasted mice, respectively. Blood glucose levels were measured over a period of 3 h after injection of glucose (Roth X997.2) 2 mg/g body weight or insulin 0.5 µ i.U./g body weight (Huminsulin Normal 100 Lilly, HI0210) using an Aviva Accu-Check, and Accu-Check test strips (Roche). Fasted serum insulin levels were determined using Ultrasensitive mouse insulin ELISA (Mercodia 10-1249-01). Serum non-esterified fatty acids (NEFA) were measured on serum from fed mice (NEFA-HR(2) Assay, Wako Chemicals Europe). ALT (Sigma, MAK052) and AST (Sigma MAK055) measurements were made on serum isolated from fed mice. Lipase activity assay was performed on frozen eWAT isolated from obese mice according to the manufacturer’s instructions (Sigma, MAK046). Corticosterone was measured using mass spectrometry as described before^{81 (link)}.

Blood glucose levels (mg/dl) were quantified using the Bayer Contour Next One Glucose Meter. Mice were either fed ad libitum or fasted overnight. Levels of amylase and lipase in the blood were measured using as reference the ethylidenep-NP-G7 (catalog # MAK009, Sigma-Aldrich) and glycerol (catalog # MAK046, Sigma-Aldrich) substrates in kinetic activity assays, respectively. Serum (2 μL) was diluted in to 1 : 1 ratio with normal saline and mixed with either the amylase or lipase assay buffer to initiate the reaction. The increase in absorbance due to the release of p-nitrophenol and glycerol was monitored at 405 nm and 570 nm for amylase and lipase, respectively. Activity was recorded as nmole/min/ml and reported as Units/Liter (U/L). One unit of amylase is the amount of the enzyme that cleaves ethylidene-pNP-G7 to generate 1.0 μmol of p-nitrophenol per minute at 25 °C. One unit of lipase is the amount of enzyme that will generate 1.0 μmol of glycerol from triglycerides per minute at 37 °C.

Levels of the serum cytokines TNF-α, IL-1β, and IL-6 were measured by the Cytokine Reference Lab at the University of Minnesota (MAGPIX, Luminex, Austin, TX, USA). Amylase (Abcam, ab102523), lipase (Sigma-Aldrich, MAK046), and LDH (Sigma-Aldrich, MAK066) activity assays were performed according to kit instructions. Western blotting was performed in pancreatic lysates as previously described [28 (link)]. Antibodies against OGT and Vinculin were purchased from Cell Signaling Technology.