Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy

Abstract

Despite the central role of the liver in the regulation of glucose and lipid metabolism, there are currently no methods to directly assess hepatic oxidative metabolism in humans in vivo. By using a new (13)C-labeling strategy in combination with (13)C magnetic resonance spectroscopy, we show that rates of mitochondrial oxidation and anaplerosis in human liver can be directly determined noninvasively. Using this approach, we found the mean rates of hepatic tricarboxylic acid (TCA) cycle flux (VTCA) and anaplerotic flux (VANA) to be 0.43 ± 0.04 μmol g(-1) min(-1) and 0.60 ± 0.11 μmol g(-1) min(-1), respectively, in twelve healthy, lean individuals. We also found the VANA/VTCA ratio to be 1.39 ± 0.22, which is severalfold lower than recently published estimates using an indirect approach. This method will be useful for understanding the pathogenesis of nonalcoholic fatty liver disease and type 2 diabetes, as well as for assessing the effectiveness of new therapies targeting these pathways in humans.

Figure 1

Plasma acetate concentration (black diamonds) and 1-¹³C enrichment (grey diamonds) during the [1-¹³C] acetate infusion protocol (n = 12).

Figure 2

(a) Axial gradient-echo image of the torso acquired during a single breath-hold. ¹H-decoupled ¹³C MR spectra were acquired from a localized region within the liver. Voxel selection was accomplished using outer volume suppression (shaded regions) applied in 3 dimensions to suppress signals arising from outside the liver and define the volume of interest (white box). (b) Localized ¹³C spectra of the human liver acquired prior to and at the end of a 120min infusion of 1-¹³C-acetate. ¹³C-labeling at the C₅ position of glutamate (C₅-Glu) was observed due to the oxidation of [1-¹³C] acetate via the TCA cycle. Labeling at C₁-glutamate (C₁-Glu) and of the bicarbonate (HCO₃⁻) pool was also observed as the ¹³C label traversed a 2nd turn of the TCA cycle.

Figure 3

Time-courses of hepatic C₅- and C₁-glutamate enrichment determined by localized in vivo ¹³C-MRS during an infusion of [1-¹³C] acetate (n = 12). The kinetics of liver glutamate enrichment for each subject was determined by the metabolic model of hepatic metabolism. The average fit of the model to the glutamate enrichment data is superimposed.

Figure 4

Metabolic model of liver acetate oxidative metabolism used to estimate hepatic TCA cycle flux (V_TCA) and anaplerosis (V_ANA). Carbon positional enrichment denoted in red occurs during the initial incorporation of label from 1-¹³C-acetate to glutamate on the first turn of the TCA cycle. Positional enrichment denoted in blue occurs during the 2^nd turn of the TCA cycle, with label originating from internal scrambling at succinate or from bicarbonate (HCO₃⁻)/¹³CO₂ via anaplerosis. A full description of the metabolic model can be found in the Online Methods section.