Total cholesterol

Mice were anaesthetised with isoflurane (2–3% carried in 2–3% O₂) and blood samples were obtained via a cardiac puncture, using a heparinised syringe and needle (100 U/ml). Plasma was separated with centrifugation (400 × g, 20 min), snap frozen in liquid nitrogen and stored at −80 °C until processing. Whole liver was weighed and snap frozen in liquid nitrogen for biochemical assays. Plasma and hepatic triglyceride (TG) (Cayman, UK) and total cholesterol (Abcam, UK) were quantified as per manufacturer’s instructions. Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma glutamyltransferase (GGT) levels were determined following manufactuer’s instructions (Sigma-Aldrich, UK). Plasma albumin was measured according to manufacturer’s instructions (Abcam, UK). Liver samples were homogenised in 0.125 M potassium phosphate buffer with protease inhibitors, as previously described^{19 (link)}; results were normalised to protein content.

Plasma total cholesterol (Abcam® Inc., Cambridge, MA, USA), triglyceride (Abcam® Inc., Cambridge, MA, USA), HDL-C (Abcam® Inc., Cambridge, MA, USA) and LDL-C + VLDL-C (Abcam® Inc., Cambridge, MA, USA) concentrations were determined using assay kits, according to manufacturer’s instructions. Sample and standard(s) optical density values were measured at 450 nm using a BioTek® Microplate Reader (Winooski, VT, USA).

Seven-week-old mice (nine/group) fed a standard chow were administered glucose in the drinking water without (controls) or with synthetic (±)-2-cis, 4-trans abscisic acid (ABA) (Sigma Aldrich, Milano, Italy). To achieve the required daily dose of glucose (1 g/Kg BW) and of ABA (1 µg/Kg BW), the daily volume of water drank by the animals was preliminarily established. Based on this volume (5 mL/day) and taking into account an average weight of the mice of 25 g, the water administered to the animals contained 0.005 g/mL of glucose and 0.005 µg/mL of ABA. The animals were weighed weekly and the concentrations of glucose and of ABA in the water were adjusted to the mean BW in each cage. After 4 months of treatment, blood was drawn after overnight fasting to measure HbA1c (Crystal Chem Inc., Elk Grove Village, IL, USA), total cholesterol, and triglycerides (Abcam, Cambridge, UK) and body weight was measured.