Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics

Abstract

Ketone body metabolism is a central node in physiological homeostasis. In this review, we discuss how ketones serve discrete fine-tuning metabolic roles that optimize organ and organism performance in varying nutrient states and protect from inflammation and injury in multiple organ systems. Traditionally viewed as metabolic substrates enlisted only in carbohydrate restriction, observations underscore the importance of ketone bodies as vital metabolic and signaling mediators when carbohydrates are abundant. Complementing a repertoire of known therapeutic options for diseases of the nervous system, prospective roles for ketone bodies in cancer have arisen, as have intriguing protective roles in heart and liver, opening therapeutic options in obesity-related and cardiovascular disease. Controversies in ketone metabolism and signaling are discussed to reconcile classical dogma with contemporary observations.

Keywords: HMGCS2; NAFLD; SCOT; alternative fuel; cancer metabolism; carbohydrate restriction; cardiac metabolism; extrahepatic ketogenesis; fuel metabolism; ketogenic diet; mitochondrial function.

Fig. 1. Metabolism of ketone bodies

(A) Ketogenesis within hepatic mitochondria is the primary source of circulating ketone bodies, requiring the fate-committing enzyme HMGCS2. Ketone bodies are secreted, and their primary metabolic fate is terminal oxidation within mitochondria of extrahepatic tissues through reactions that require the enzyme SCOT. mThiolase (mitochondrial thiolase); e⁻, electrons emanating from TCA cycle as NADH and FADH₂; ETC, electron transport chain. Question marks reflect uncertainty of the mechanism responsible for transporting ketones across the inner mitochondrial membrane. (B) Ketone body metabolism is integrated through mitochondrial and cytoplasmic metabolic pathways. Cytoplasmic lipogenesis and cholesterol synthesis are nonoxidative metabolic fates of ketone bodies. mThiolase or cThiolase activity is encoded by at least 6 genes: ACAA1, ACAA2 (encoding an enzyme known as T1 or CT), ACAT1 (encoding T2), ACAT2, HADHA, and HADHB. ACSS2, acetyl-CoA synthetase 2 (cytoplasmic).

Fig. 2. Evaluation of extrahepatic ketone body concentrations

(A) Increased steady state abundance of ketone bodies in one biological condition versus another may indicate local ketogenesis, but other interpretations are possible, including selective impairment of ketone oxidation, or global impairment of mitochondrial oxidative function. Experiments that employ isotopically labeled ketone bodies and fatty acids, specifically tracking the fate of the labeled intermediates, are often reliable approaches to demonstrate ketogenesis. Pseudoketogenesis is isotopic dilution without true ketone production, dashed elliptical line. SCOT function can be selectively inhibited by diminished expression or PTM. The SCOT and thiolase reactions are reversible, and can thus support either true ketogenesis or pseudoketogenesis. Only HMGCS2 dependent ketogenesis can support millimolar ketone accumulation (thick elliptical line). Results not clearly circumscribed by this analysis likely indicate that a difference in tissue ketone concentration is attributable to variations of hepatic ketogenesis. (B–E) Extrahepatic tissue ketone concentrations do not exceed that in the circulation. Ten week-old female C57BL/6 mice were bled, and kidneys were harvested in the random fed and 24h fasted states. All measurements (n=3/group) were performed in blinded manner. (B) βOHB concentrations were quantified in serum using standard biochemical enzymatic reagents coupled to a spectrophotometrically-coupled substrate (Wako). βOHB concentrations were also quantified in kidney from fed or 24h fasted mice by (C) LC/MS² or (D) ¹H NMR. For LC/MS², two milligrams of lyophilized and homogenized kidney powder were extracted using optimized protocol in cold (−20°C) 2:2:1 methanol: acetonitrile: water containing sodium β-[U-¹³C]hydroxybutyrate as an internal standard. Quantitation was performed on Dionex 3000 RS liquid chromatography stack coupled to a Thermo Q Exactive Plus mass spectrometer. Separation was optimized on a Phenomenex Luna NH2 column in hydrophilic interaction liquid chromatography mode. Spectrometer was operated in negative Parallel Reaction Mode and MS resolution was set to 17,500. βOHB and its internal standard were quantified using expected m/z transitions of (E) 103.0401 → 59.0133 and 107.0535 → 61.0200 (internal standard’s transition not shown), respectively, with less than 10 ppm mass accuracy. NMR spectra were collected at 25°C in D₂O from perchloric acid extracts of a single snap frozen kidney harvested from fed (bottom) and 24h fasted (top) mice. Data were collected under quantitative steady state conditions using a cryoprobe at 14.1T (Bruker) using trimethylsilylpropionate as an internal chemical shift and concentration reference. Chemical shifts corresponding to renal alanine, lactate, and βOHB are shown. Calculated mean renal βOHB concentrations were 0.08 nmol/mg wet tissue in the fed state, and 0.93 nmol/mg wet tissue in the 24h fasted state. Note that higher apparent βOHB concentrations were quantified via LC/MS², compared to those derived from NMR-based measurements, due to the use of dry versus wet kidney tissue, respectively.

Fig. 3. Non-canonical signaling roles for βOHB

Pleiotropic effects have been observed. Mechanisms of action still require elucidation for many of the observed effects, and ideal experiments discriminate among d-βOHB, l-βOHB, AcAc, and related compounds including butyrate and acetate, and the potential role of altered redox potential and oxidative fate. NLRP3, NACHT, LRR and PYD domains-containing protein 3; PGE₂/PGD₂, prostaglandins E2/D2.

Fig. 4. Hepatic maladaptation to ketogenic insufficiency

(A) Under homeostatic conditions, mitochondrial acetyl-CoA can be directed to ketogenesis, terminal oxidation in the TCA cycle, or exported to the cytoplasm for lipogenesis. (B) In the setting of ketogenic insufficiency, lipogenesis and glucose production are increased. Loss of the ketogenic conduit stimulates increased acetyl-CoA disposal through the TCA cycle prospectively increasing unsafe disposal of elections into reactive oxygen species. Ketogenic impairment also increases acetyl-CoA export to the cytoplasm for lipid-synthesizing pathways. These changes partly reflect the alterations encountered in NAFLD, in which the liver exhibits increased esterification to and lipolysis from lipid droplets, increased β-oxidation of fatty acids, increased terminal oxidation, and increased gluconeogenesis, but diminished ketogenesis relative to the availability of fat.

Fig. 5. Prospective cardioprotection from ketone bodies

The normal heart is omnvirous and flexible among substrate fuels, preferring fatty acids, but oxidizes ketones in proportion to their delivery at the expense of fatty acids. The failing heart becomes reprogrammed and inflexible, diminishing its use of fatty acids. Ketone bodies are mildly elevated in the circulation of human subjects and animal models of heart failure, and myocardial ketone body oxidation is increased. Renal SGLT2 inhibition, a therapy used to lower blood glucose concentrations in diabetics, also increases hepatic ketogenesis and ketonemia, and through unknown mechanisms, improves heart failure mortality rates. Prospective mechanisms that link further enhancement of myocardial ketone oxidation to protection from pathological ventricular remodeling are under investigation.