Ab65336

Glucose levels were determined in whole blood from the tail vein using an automatic glucose monitor (TRUE2go Glucose Test Meter). For GTT, blood glucose levels were determined in 5 μl of blood from the tail vein in mice fasted overnight and monitored for 240 min following a glucose injection (1.5 g kg⁻¹ body weight, IP). Mixed linear modelling was used to analyse GTT profiles. Areas under the GTT curves were calculated using trapezoid method using R (trapz with the default parameters) and compared using Mann–Whitney test. For insulin tolerance test, mice were fasted for 4 h and injected with insulin (0.75 U kg⁻¹ body weight, IP). Blood glucose was measured as above right before the insulin injection (time 0) and over 120 min post-injection. Serum FFA and triglycerides were measured using quantification assay kits (Abcam, ab65341; ab65336) using mouse standards according to the manufacturer guidelines. Serum corticosterone and insulin levels were measured by ELISA (Abcam, ab108821; Crystal Chem Inc, 90080) using mouse standards according to the manufacturer guidelines.

Hepatic and faecal lipid extraction has been performed according to Folch’s method^{66 (link)}. Triglyceride quantification in the liver and in the stool were assessed using commercially available kits (respectively #ab65336, Abcam, Cambridge, UK and #K622-100, Biovision Inc, Milpitas, CA, USA). The absorbance has been quantified by using Sunrise spectrophotometer instrument, (Tecan Austria Gmbh. Untersbergstr, 1 A. A-5082 Grodig, Austria) at λ = 570 nm.

Serum samples were prepared at the end of each experiment and stored at −70 °C until further analysis. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined with an automated blood chemistry analyzer (Hitachi 7150; Tokyo, Japan). Liver triglycerides (TGs) were measured using an enzymatic assay kit (ab65336; Abcam, Cambridge, UK) following the manufacturer’s instructions.

Oil red O (Abcam) staining was done on frozen OCT embedded tissue as previously described.^{56 (link)} Oil red O quantification was performed using ImageJ, averaging the percentage of areas stained across four liver sections per mouse. Hematoxylin and eosin staining were performed by the histology core at the University of California, Los Angeles. NAFLD activity scores based on hematoxylin and eosin staining were calculated by a blinded pathologist for each mouse. Triglyceride content of the liver was quantified using a calorimetric triglyceride assay kit (Abcam, ab65336).