2.5. Tissue metabolite extraction for NMR-based metabolomic analysis

Frozen liver and muscle tissue samples were homogenized using a cryogenic mill (Retsch, Inc., Newtown, PA, USA) and extracted using the chloroform-methanol–water extraction protocol by Bligh and Dyer (Bligh and Dyer, 1959; Lin et al., 2007; Viant, 2007) as described in detail elsewhere (Casu et al., 2017; 2019; Schock et al., 2012; 2013) (see Supplementary methods). Every step in the homogenization protocol was performed inside a Cryocart (Chart Industries, Inc., Garfield Heights, OH, USA) to prevent the tissue samples from thawing. The tissue sample homogenates were aliquoted by weighing 100 mg (±3 mg) per sample, transferred into ceramic bead tubes (2.8 mm) (Mo Bio Laboratories, Carlsbad, CA, USA), and subsequently stored at −80 °C until further processing. Details on the NMR spectra acquisition and processing are provided (see Supplementary methods).