Detecting de novo Hepatic Ketogenesis Using Hyperpolarized [2-13C] Pyruvate

Abstract

The role of ketones in metabolic health has progressed over the past two decades, moving from what was perceived as a simple byproduct of fatty acid oxidation to a central player in a multiplicity of disease states. Previous work with hyperpolarized (HP) ¹³C has shown that ketone production can be detected when using precursors that labeled acetyl-CoA at the C1 position, often in tissues that are not normally recognized as ketogenic. Here, we assay metabolism of HP [2-¹³C]pyruvate in the perfused mouse liver, a classic metabolic testbed where nutritional conditions can be precisely controlled. Livers perfused with long-chain fatty acids or the medium-chain fatty acid octanoate showed no evidence of ketogenesis in the ¹³C spectrum. In contrast, addition of dichloroacetate, a potent inhibitor of pyruvate dehydrogenase kinase, resulted in significant production of both acetoacetate and 3-hydroxybutyrate from the pyruvate precursor. This result indicates that ketones are readily produced from carbohydrates, but only in the case where pyruvate dehydrogenase activity is upregulated.

Keywords: dichloroacetate; hepatic metabolism; hyperpolarization; ketogenesis; octanoate metabolism.

Figure 1

Overlay of representative ¹³C sum spectra following injection of HP [2-¹³C] pyruvate. AcAc, acetoacetate; AcCar, acetyl carnitine; ALA, alanine; BHB, 3-hydroxy butyrate; CIT, citrate; GLU, glutamate; LAC, lactate; and PYR, pyruvate. ^# and * represent unidentified and natural abundance signals, respectively. “DCA” is Dichloroacetate. Perfusion conditions and NMR parameters are described under Materials and Methods section. Resonance assignments were confirmed using [¹H, ¹³C] HSQC and [¹H, ¹³C] HMBC experiments.

Figure 2

Ratios of signal intensities of individual resonances to sum of all ¹³C resonances in the spectrum. Pyruvate-hydrate resonance was excluded from total ¹³C intensities sum. *Indicates statistical significance (p < 0.05; see section Materials and Methods). AcAc sum includes signal intensities at both C-1 and C-3 chemical shifts. AcAc, acetoacetate; AcCar, acetyl carnitine; ALA, alanine; BHB, 3-hydroxy butyrate; CIT, citrate; GLU, glutamate; MAL, malate; LAC, lactate; PEP, phosphoenolpyruvate; and PYR, pyruvate. n = 5 (DCA), 4 (Octanoate), and 5 (FFA).

Figure 3

Fractional enrichments for the ketones Acetoacetate and 3-hydroxybutyrate based on GC-MS analysis of efferent perfusate. The data shown is presented after natural abundance correction using the INCA software, which results in no excess enrichment prior to the injection. (A,B) Enrichments for ketones produced prior to [2-¹³C] pyruvate were confirmed to contain no label while ketone enrichment was observed to increase in the octanoate and DCA groups considerably indicating labeled ketone production from [2-¹³C] pyruvate. (C,D) The fractional enrichment profile for Acetoacetate (M + 0–1) and BHB (M + 0–3) showed significantly higher label in the DCA group than the FFA and octanoate groups for all but BHB M + 3. BHB M + 3 cannot be produced from [2-¹³C] pyruvate and hence has an enrichment within the error of measurement ~0.1% or 0.001. *Indicates statistical significance (p < 0.05; see section Materials and Methods).

Figure 4

(A) Total amount of BHB and Lactate from efferent perfusate post [2-¹³C] pyruvate injection in mg using GC-MS standard curve quantification. (B) Amount of ¹³C labeled BHB and Lactate that are (total mg metabolites * total enrichment) in efferent perfusate post [2-¹³C] pyruvate injection. *Indicates statistical significance (p < 0.05; see section Materials and Methods).

Figure 5

(A) Pool sizes of metabolites extracted from the liver estimated using GC-MS. (B–I) ¹³C enrichment of all metabolites shown in (A). M + 0, M + 1, M + 2, and M + 3 represent individual mass isotopologues. *Indicates statistical significance (p < 0.05; see section Materials and Methods).