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. 2018 Mar 31;12:12–24. doi: 10.1016/j.molmet.2018.03.014

Figure 4.

Figure 4

13C NMR Isotopomer analysis from tissue extracts perfused isolated mice livers and flux analysis. The following three groups (n = 3 per group) of DIO mice (6–8 week old) were studied: 1. DIO control, 2. DIO DKO, 3. PS10-treated DIO. Mice in DIO PS10-treated group was injected into the subcutaneous cavity with PS10 dose at 40–45 mg/kg. Mice were anesthetized by intramuscular injection of a ketamine/xylazine solution (0.1 mL, 85:15 w/w). Livers were cannulated via hepatic portal vein, isolated, connected to a perfusion column apparatus, and perfused at 25 cm H2O pressure with a modified Krebs-Henseleit (KH) buffer containing 5.5 mM [1,6–13C]glucose, 1.2 mM [3–13C]lactate, 0.12 mM [3–13C]pyruvate, 0.4 mM [U-13C] free fatty acids (FFA), and 0.75% BSA. The perfusion buffer for the PS10-treated group also contained 25 μL/L PS10. Oxygen consumption was measured at 25 min using blood gas analyzer (Instrumentation Laboratory, Lexington, MA) from the perfusate collected into a gas-tight syringe. Livers were perfused for 30 min before being freeze-clamped. The frozen tissues were pulverized in liquid nitrogen and extracted with perchloric acid (4–6%), neutralized, and reconstituted in D2O containing 1 mM EDTA and 0.5 mM 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) standard. Proton-decoupled 13C-NMR spectra of heart extracts were acquired at Bruker 600 MHz spectrometer equipped with 10-mm CPDUL cryoprobe. Glutamate resonances were measured to determine the relative oxidation of [1,6–13C2]glucose, [U-13C]FFA, and unlabeled endogenous substrates (e.g., triglycerides and glycogen) and submitted as input for an isotopomer analysis using the program TCACALC v.2.07. A. Glutamate C-2 (55.35 ppm) B. Glutamate C-4 (34.20 ppm) and C. C-3 (27.60 ppm) spectra, D. Fractional Oxidation, E. TCA flux, and F. O2 consumption. HVO2, Hepatic venous oxygen. The letters S, D, T, and Q refer to a singlet, doublet (with the relevant J-coupled spins), triplet (a degenerate doublet of doublets) and quartet respectively. Data are presented as the mean ± SD (n = 3 per group) with significance indicated by “*” (P < 0.05).