1 13c acetate

Hepatic de-novo lipogenesis (DNL) was measured as we previously reported^{45 (link)}. Briefly, the indicated mice received 2% [1-¹³C]acetate (Sigma) in their drinking water starting 3 days before killing. Hepatic lipids were extracted from homogenates (10% w/w in PBS) according to Bligh and Dyer^{46 (link)}. Fatty acids were then methylated by incubating the lipid extracts in a 5:1 (v/v) mixture of methanol/HCL (6N) for 4 h at 90 °C and extracted twice using hexane. Isotopomer distributions of the C16:0, C16:1, C18:0, and C18:1 fatty acid methyl esters were then analyzed by GC/MS (Agilent 5975 C GC/MSD, Agilent Technologies, Santa Clara, CA) using ammonia-induced chemical-ionization (CI). The measured isotopomer distribution patterns were then corrected for the natural abundance of ¹³C to obtain the excess fractional distribution of the isotopomers (m₊₀ - m_+x) due to incorporation of the labeled ¹³C-acetate into the fatty acid molecules^{47 (link)}. Mass isotopomer distribution analysis was then used to calculate fatty acid synthesis, as previously described^{45 (link)}.

Optical rotations were measured on a Jasco P2000 digital polarimeter (Jasco, Cremella, Italy). UV spectra were acquired on a Jasco V-650 spectrophotometer, NMR spectra were recorded on a Bruker Avance DRX 600 (Bruker, Milan, Italy) equipped with a cryoprobe operating at 600 MHz for protons. Chemical shift values are reported in ppm (δ) and referenced to internal signals of residual protons (CD₃OD ¹H δ3.34, ¹³C 49.0 ppm). High-resolution mass spectra were acquired on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Milan, Italy); HPLC analyses have been performed on a Jasco system (PU-2089 Plus-quaternary gradient pump equipped with a Jasco MD-2018 Plus photodiode array detector). [1-¹³C]-acetate, [2-¹³C]-acetate and [1,2-¹³C₂]-acetate, [1-¹³C]-glycolate (all sodium salts), and salicylhydroxamic acid (SHAM) were obtained from Sigma Aldrich (Milan, Italy). Solvents were purchased from VWR (Milan, Italy) and were HPLC-grade.