Hepatic ketogenic insufficiency reprograms hepatic glycogen metabolism and the lipidome
D. André d’Avignon, … , Xianlin Han, Peter A. Crawford
D. André d’Avignon, … , Xianlin Han, Peter A. Crawford
Published June 21, 2018
Citation Information: JCI Insight. 2018;3(12):e99762. https://doi.org/10.1172/jci.insight.99762.
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Research Article Hepatology Metabolism

Hepatic ketogenic insufficiency reprograms hepatic glycogen metabolism and the lipidome

Abstract

While several molecular targets are under consideration, mechanistic underpinnings of the transition from uncomplicated nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) remain unresolved. Here we apply multiscale chemical profiling technologies to mouse models of deranged hepatic ketogenesis to uncover potential NAFLD driver signatures. Use of stable-isotope tracers, quantitatively tracked by nuclear magnetic resonance (NMR) spectroscopy, supported previous observations that livers of wild-type mice maintained long term on a high-fat diet (HFD) exhibit a marked increase in hepatic energy charge. Fed-state ketogenesis rates increased nearly 3-fold in livers of HFD-fed mice, a greater proportionate increase than that observed for tricarboxylic acid (TCA) cycle flux, but both of these contributors to overall hepatic energy homeostasis fueled markedly increased hepatic glucose production (HGP). Thus, to selectively determine the role of the ketogenic conduit on HGP and oxidative hepatic fluxes, we studied a ketogenesis-insufficient mouse model generated by knockdown of the mitochondrial isoform of 3-hydroxymethylglutaryl-CoA synthase (HMGCS2). In response to ketogenic insufficiency, TCA cycle flux in the fed state doubled and HGP increased more than 60%, sourced by a 3-fold increase in glycogenolysis. Finally, high-resolution untargeted metabolomics and shotgun lipidomics performed using ketogenesis-insufficient livers in the fed state revealed accumulation of bis(monoacylglycero)phosphates, which also accumulated in livers of other models commonly used to study NAFLD. In summary, natural and interventional variations in ketogenesis in the fed state strongly influence hepatic energy homeostasis, glucose metabolism, and the lipidome. Importantly, HGP remains tightly linked to overall hepatic energy charge, which includes both terminal fat oxidation through the TCA cycle and partial oxidation via ketogenesis.

Authors

D. André d’Avignon, Patrycja Puchalska, Baris Ercal, YingJu Chang, Shannon E. Martin, Mark J. Graham, Gary J. Patti, Xianlin Han, Peter A. Crawford

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Figure 5

Acetyl-CoA fates in ketogenic insufficiency.

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Acetyl-CoA fates in ketogenic insufficiency. (A) Octanoate consumption r...
(A) Octanoate consumption rates and (B) derived acetyl-CoA fates of livers of animals perfused with 0.2 mM unlabeled octanoate–containing oxygenated perfusion buffer from the experiments whose data are presented in Figures 2–4. Note that little octanoate is esterified to glycerol, and much of the unaccounted fraction in controls is attributable to AcAc, while very little of the unaccounted fraction can be sourced to AcAc in ketogenic insufficiency. (C) LC-MS/MS measurements of acetyl-CoA from extracts of isolated mitochondria, normalized to mitochondrial protein content, or (D) total liver tissue, normalized to tissue mass, from livers harvested from unperfused mice. (E–H) Untargeted LC/MS metabolomics from liver extracts derived from portal vein perfusions using oxygenated buffer containing 0.2 mM sodium [1,2,3,4-13C4]octanoate. Collections were acquired in the negative-ion mode after chromatographic separation. Isotopologues reflect 13C incorporation in 2-atom units consistent with products from labeled acetyl-CoA export from mitochondria. (E) 3-Oxo-octanoic acid, whose monoisotopic mass is 158.0943, putatively assigned to m/z 217.1081 [M+CH3COO]– (acetylated molecular ions are common under the HILIC mobile phase conditions employed); M+0, M+2, M+4, and M+6 isotopologues are equally abundant in both genotypes, suggestive that ketogenic insufficiency does not alter this metabolite’s labeling. (F) 3-Oxo-decanoic acid, whose monoisotopic mass is 186.1256, putatively assigned to m/z 245.1393 [M+CH3COO]–; M+0, M+2, M+4, M+6, and M+8 isotopologues are equally abundant in both genotypes. (G) Butyryl-CoA, whose monoisotopic mass is 837.1571, putatively assigned to m/z 417.5715 [M-2H]2–; M+0 and M+2 isotopologues are equally abundant in both genotypes (parent monoisotopic masses that acquire 2e– in ionization result in a m/z that is half of the expected monoisotopic mass for the molecular ion). (H) Hydroxybutyryl-CoA, whose monoisotopic mass is 853.1520, putatively assigned to m/z 425.5691 [M-2H]2–; M+0, M+2, and M+4, isotopologues are equally abundant in both genotypes. n = 4–6/group. ***P < 0.001; ****P < 0.0001 by Student’s t test.