Fig 1.
Measurement of hepatic glucose and oxidative metabolism in vivo.
The workflow depicted in Fig 1 transitions from in vivo isotope delivery and plasma sampling to in silico flux analysis. 2H and 13C isotopes delivered intravenously in consciously catheterized mice enrich glucose produced from the liver. Plasma glucose samples obtained during the isotopic steady-state are derivatized and analyzed through GC-MS analysis [30]. Glucose-fragment MIDs—m/z 173–176, 259–263, 284–287, 370–374, 145–147, and 301–311—are integrated from MS peaks. A previously generated model of hepatic metabolism is used to simulate glucose fragments MIDs using INCA (custom MFA software) [29]. Flux estimates are regressed by minimizing the difference between simulated and empirically measured MIDs.
Fig 2.
Scheme of glucose producing and CAC-related fluxes for MFA.
Metabolites modeled for intermediary metabolic exchange include lactate, glycerol, glycogen, amino and fatty acids. SucCoA serves as the entry point for [13C3]propionate into the CAC and 2H from 2H2O enters at multiple loci in the flux model. A complete description of the molecular reaction network, assumptions, limitations, and flux analysis used in these studies has been elaborated on elsewhere (31). Multiple substrates shuttle through Pyr to the CAC and, thus, VLDH encompasses all non-PEP derived, unlabeled sources of anaplerotic flux. Total anaplerotic flux is the sum of anaplerotic inputs (VPC + VPCC) and is equal to cataplerosis (VPCK). Abbreviations and a description of metabolites and central reactions in the flux model are listed in the Abbreviations subsection.
Fig 3.
Abnormal glucose and oxidative fluxes in mice lacking hepatic AMPK.
Absolute fluxes (μmol•kg-1•min-1) (A) were determined for short (S) and long (L) term fasted WT and L-KO mice. Relative contributors to VEndoRa (B) were determined by dividing VPYGL, VGK, and VEnol by VEndoRa; VGK and VEnol are presented in hexose units such that VPYGL+VGK+VEnol = VEndoRa. Data are presented as means ± SEM, n = 5–7 in each group. *p≤0.05 vs. short term fasting; †p≤0.05 vs. WT mice.
Table 1.
Metabolites of the CAC and glucose producing pathways.
Fig 4.
AMPK protects against fasting-mediated deficits in liver ATP.
Hepatic adenine nucleotides (A), the total adenine nucleotide pool (TAN = ATP + ADP + AMP) (B), energy charge (EC = [ATP + 0.5ADP]/[TAN]) (C.), and the AMP/ATP ratio (D) were determined for WT and L-KO mice in short (S) and long (L) term fasting. Liver AMPK (E and F) and Akt (G) signaling in short and long term fasted mice. Phosphorylated to total AMPK, ACC, Akt are provided as ratios (A.U.). Black lines separating lanes denote images obtained either from portions of the same or separate blots. Data are expressed as means ± SEM, n = 6–7 in each group. *p≤0.05 vs. short term fasting; †p≤0.05 vs. WT mice.
Fig 5.
Liver AMPK-dependent and independent effects of fast duration on liver lipids.
Liver triglycerides (TGs) (A), diglycerides (DGs) (B), cholesterol esters (CEs) (C), and phospholipids (PLs) (D) in WT and L-KO mice following a short (S) and long (L) term fast. Data are expressed as means ± SEM, n = 6–7 in each group. *p≤0.05 vs. short term fasting; †p≤0.05 vs. WT mice.
Table 2.
Hepatic long-chain fatty acids, linoleic and arachidonic acid derivatives are elevated in AMPK-deficient livers of short term fasted mice.
Table 3.
AMPK deletion results in aberrant BCAA/BCKA-related metabolism.