One touch ultra

Mice were fasted for 6 h prior to intraperitoneal injection with 2 g glucose/kg body weight of 40% w/v solution D-glucose (Sigma). Blood glucose concentrations were quantified using a glucometer (One Touch Ultra, Roche). Insulin concentrations were measured using the Mercodia Ultrasensitive Mouse Insulin ELISA kit (Mercodia). HOMA-IR was calculated using the formula HOMA-IR =  (fasting plasma insulin x fasting plasma glucose)/22.5 [39].
For histology, liver sections were fixed in formalin 10% solution for 24 h, transferred to 100% ethanol and mounted in paraffin blocks prior to staining with haematoxylin and eosin or picosirius red. Stained sections were independently assessed by a consultant transplant liver histopathologist from the national liver transplant centre, experienced in evaluation of histology in clinical trials and translational models of NAFLD. The NAFLD activity score (NAS) [40] can be used to quantify features of NAFLD in human disease. The same features can be evaluated in rodent models of NAFLD, as previously described [41]. Features of steatotic injury were scored on a scale from 0 to 8 (steatosis 0–3, lobular inflammation 0–3 and hepatocyte ballooning 0–2) on H&E stained sections. Fibrosis was assessed from the PSR-stained sections by applying the fibrosis score used with NAS. Analysis was undertaken blinded to all other data.

At P80 (MID) and P150 (END), F1 mice were fasted for 6 h prior to the glucose test and 4 h prior to the insulin test (n = 7–10 animals per sex) and performed as detailed^{49 (link)}. Matsuda index (whole body IS index) and direct measurement of hepatic IR (HOMA index) were calculated as described^{50 (link),51 (link)}. Briefly, Matsuda index was calculated as follows = 100/(√[G₀ × I₀ × G_mean × I_mean]), the suffix mean indicates the average value of glucose and insulin concentration measured during the whole length of the glucose test. HOMA index was calculated as HOMA = (I₀ × G₀)/22.5. Evaluation of β-cell function was calculated by dividing the area under the curve (AUC) of insulin and glucose levels during the glucose test (AUCins:AUCglc).
Prior to sacrifice, mice were fasted for 2 h and anesthetized with 4% isoflurane. Blood glucose level was measured with a OneTouch Ultra glucometer (AccuChek Sensor, Roche Diagnostics). Subsequently, mice were exsanguinated via cardiac puncture and blood saved for plasma analysis. The whole liver was quickly removed and washed into PBS. A piece of left lobe of the liver was collected, fresh-frozen into liquid nitrogen and stored at −80 °C until further analysis. The rest of the liver was fixed into paraformaldehyde for histology.