Liver Metabolism Flux Analysis

Whole-body glucose turnover was measured by determining the specific activity of glucose in the steady-state plasma using a scintillation counter. Hepatic glucose production was assumed to represent ∼90% of the measured whole-body glucose turnover based on our previously published data (7 (link)). Liver-specific metabolic flux rates were calculated using a combined NMR-LC-MS/MS method. We corrected for the natural abundance of each metabolite included in the flux calculations, measuring all possible enrichments (for instance, m+0, m+1, m+2, m+3, and m+4 for malate) and correcting the measured peak areas to account for the fact that once a carbon is labeled it can no longer contribute to the natural abundance (10 (link)). Samples were prepared for NMR by homogenizing 2–3 g of liver in 5 volumes of 7% perchloric acid. The pH of the samples was adjusted to 6.8–7.3 using 30% potassium hydroxide and 7% perchloric acid as necessary, and the samples were centrifuged at 4000 × g for 10 min. The supernatant was frozen in liquid N₂ and lyophilized. ¹³C NMR analysis was performed as described by Befroy et al. (6 (link)). Total glucose and alanine enrichment was measured by GC/MS and glutamate by LC-MS/MS, with ¹³C NMR used to algebraically divide the total enrichment to determine the enrichment of each carbon of these metabolites.
We calculated the [2-¹³C]malate enrichment by relating the positional enrichments of malate to those measured in glutamate assuming (and validating) full equilibration across fumarase as shown in Equations 1 and 2.


For calculation of the liver-specific metabolic flux ratio V_Pyr-Cyc/V_Mito = (V_PK + V_{ME, out})/(V_PC + V_{ME, in}+ V_PDH), we used our previously published isotopic labeling model (6 (link), 7 (link)) extended using a mass isotopomer multiordinate spectral analysis approach to take into account V_PK and unlabeled mass entry from propionate at the succinyl-CoA step of the TCA cycle (10 (link)). Here, V_{ME, out} refers to ME flux in the direction of pyruvate synthesis; V_{ME, in} refers to the reverse reaction of pyruvate into malate, and V_LDH refers to pyruvate synthesis via LDH. With these fluxes taken into account, we can describe the steady-state mass balance at pyruvate with Equation 3,
and isotope balance at [2-¹³C]pyruvate with Equation 4,

Because the positional enrichments of pyruvate and PEP cannot be measured reliably using our NMR-LC-MS/MS techniques, we use the following label substitutions shown in Equations 5 and 6,


Substituting Equations 5 and 6 into Equation 4 and rearranging, we derive Equation 7,

By separating out like terms, we get Equation 8,
where V_PDH denotes flux through pyruvate dehydrogenase; V_CS denotes flux through citrate synthase; V_PK denotes flux through pyruvate kinase; V_{ME, out} denotes flux through malic enzyme from malate to pyruvate; V_{ME, in} denotes flux through malic enzyme from pyruvate to malate; and V_PC denotes flux through pyruvate carboxylase. Additional fluxes shown in the complete flux diagram in Fig. 1 are V_{GNG OAA} gluconeogenesis from oxaloacetate, V_GNG total gluconeogenesis, and V_prop the rate of propionate entry into the TCA cycle. V_PDH/V_CS was measured using the ratio [4-¹³C]glutamate/[3-¹³C]alanine, as described by Alves et al. (11 (link)).