Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jun 6;35(6):1038-1056.e8.
doi: 10.1016/j.cmet.2023.03.016. Epub 2023 Apr 14.

Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles

Timothy R Koves  1 , Guo-Fang Zhang  2 , Michael T Davidson  3 , Alec B Chaves  3 , Scott B Crown  3 , Jordan M Johnson  3 , Dorothy H Slentz  3 , Paul A Grimsrud  2 , Deborah M Muoio  4
Affiliations

Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles

Timothy R Koves et al. Cell Metab. .

Abstract

Even-chain acylcarnitine (AC) metabolites, most of which are generated as byproducts of incomplete fatty acid oxidation (FAO), are viewed as biomarkers of mitochondrial lipid stress attributable to one or more metabolic bottlenecks in the β-oxidation pathway. The origins and functional implications of FAO bottlenecks remain poorly understood. Here, we combined a sophisticated mitochondrial phenotyping platform with state-of-the-art molecular profiling tools and multiple two-state mouse models of respiratory function to uncover a mechanism that connects AC accumulation to lipid intolerance, metabolic inflexibility, and respiratory inefficiency in skeletal muscle mitochondria. These studies also identified a short-chain carbon circuit at the C4 node of FAO wherein reverse flux of glucose-derived acetyl CoA through medium-chain ketothiolase enhances lipid tolerance and redox stability in heart mitochondria by regenerating free CoA and NAD+. The findings help to explain why diminished FAO capacity, AC accumulation, and metabolic inflexibility are tightly linked to poor health outcomes.

Keywords: acylcarnitines; bioenergetics; exercise; fatty acid oxidation; heart; ketothiolase; metabolic flexibility; mitochondria; pyruvate; skeletal muscle.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Mitochondria from mixed SkM are metabolically inflexible and intolerant to long chain lipid fuel.
A. The CK clamp technique permits assessment of energy transduction in the context of near-physiological energy demands. Measures of (1) NAD(P)H/NAD(P)+ redox potential, (2) membrane potential (ΔΨm)–the major component of the proton motive force (PMF), and (3) overall respiratory flux (JO2) provide metrics of energy transfer and ATP turnover as a function of the ATP free energy charge (ΔGATP) established by the clamp. B. Respiration plot of PM (5mM/2.5mM) versus SR (5mM/5μM) as a function of ΔGATP. C. Respiratory efficiency plot in SkM mitochondria fueled by PM versus SR. D. Comparison of heart and SkM mitochondria respiratory responses to PM, OcM or PcM. E. Respiratory responses by heart and SkM mitochondria when Pc is added to PM. F. Addition of Pc to PM augments respiratory conductance (slope) in heart but is inhibitory in SkM. G. Addition of Pc to PM inhibits respiratory efficiency in SkM but not heart mitochondria (red arrows). H. Redox response plot illustrating the relationship between NAD(P)H/NAD(P)+ redox potential and JO2 in heart and SkM mitochondria fueled by PM or PMPc. Curved arrow highlights redox instability with PMPc. See also Figure S1. CK clamp assay conditions: Mitochondria (0.05 mg) from mouse Heart (Hrt), Mixed Skeletal Muscle (SkM), Red Gastrocnemius (RG) or White Gastrocnemius (WG) were added to 2 ml Buffer Z containing 5mM Creatine, 20U/ml Creatine Kinase, and 1.5mM phosphocreatine (PCr) at 37°C. The CK clamp was engaged upon addition of 5mM ATP. PCr was added to final concentrations of 3, 6, 9, 12, and 15 mM. Mitochondrial membrane potential (ΔΨm) and redox status (NAD(P)H) were assessed in parallel in 0.2 ml Buffer Z containing 0.02 mg mitochondria and 0.2μM TMRM. Pyruvate/Malate (5mM/2.5mM; PM), Succinate/Rotenone (5mM/5μM; SR), Octanoyl-L-Carnitine/Malate (0.2mM/2.5mM; OcM), Palmitoyl-L-Carnitine/Malate (20μM/2.5mM; PcM), Pyruvate/Malate/Palmitoyl-L-Carnitine (5mM/2.5mM/20μM;PMPc). All substrates were provided at concentrations that are saturating for JO2. Respiratory efficiency is defined as the relationship between oxygen flux (JO2) and mitochondrial membrane potential (ΔΨm). Arrows denote a shift in efficiency. Redox response refers to changes in (NAD(P)H/NAD(P)+ redox potential in relation to changes in JO2. Statistics: n=5 biological replicates for PM/SR study. Data are presented as means ± SEM. Conductance slopes and maximal JO2 responses were tested with unpaired t-tests with Welch’s correction and Holms-Šídák correction for multiple comparisons. * denotes differences in conductance slope, P<0.05, # = Maximal JO2 differences P<0.05.
Figure 2.
Figure 2.. Mitochondrial flexibility and lipid tolerance are regulated by fiber type and Pgc1a.
A. Mitochondria were isolated from red (RG) and white (WG) gastrocnemius SkMs. B. Respiratory responses to PM and PMPc in mitochondria from red gastrocnemius (RG) as compared with white gastrocnemius (WG). C. Pc-induced inhibition of respiratory conductance is more pronounced in mitochondria from WG as compared with RG. D. PMPc-supported respiratory efficiency is diminished in mitochondria from WG as compared with RG. E-G. Addition of Pc but not Oc to PM inhibits respiratory conductance and efficiency in SkM mitochondria. H. Mitochondria were isolated from SkM of non-transgenic (NT) mice and Mck-Pgc1a transgenic (Tg) littermates. I. Mitochondrial input was normalized based on mitochondrial protein content. Expression of several proteins belonging to OXPHOS complexes were similar between groups. J. Respiratory responses were measured in SkM mitochondria exposed to PM, PMPc and PMOc. K-L. Genotype-specific responses in respiratory conductance (K) and/or respiratory efficiency (L) were observed in SkM mitochondria provided with PMPc or PMOc. See also Figure S2. CK clamp assay conditions: Mitochondria (0.05 mg) mitochondria from non-transgenic mouse mixed skeletal muscle (NT) or the same SkM from mice with muscle-specific Pgc1a overexpression (Tg) were added to 2 ml Buffer Z containing 5mM Creatine, 20U/ml Creatine Kinase, and 1.5mM phosphocreatine at 37°C. The CK clamp was engaged upon addition of 5mM ATP. PCr was added to final concentrations of 3, 6, 9, 12, and 15mM. Mitochondrial membrane potential (ΔΨm) and redox status (NAD(P)H) were assessed in parallel in 0.2ml Buffer Z containing 0.02mg mitochondria and 0.2μM TMRM. Pyruvate/Malate (5mM/2.5mM; PM), Pyruvate/Malate/Palmitoyl-L-Carnitine (5mM/2.5mM/20μM;PMPc), Pyruvate/Malate/Octanoyl-L-Carnitine (5mM/2.5mM/0.2mM;PMPc). All substrates were provided at concentrations that are saturating for JO2. Respiratory efficiency is defined as the relationship between oxygen flux (JO2) and mitochondrial membrane potential (ΔΨm). Arrows denote a shift in efficiency. Statistics: n=6–7 biological replicates with data presented as means ± SEM. Conductance slopes and maximal JO2 responses were tested with unpaired t-tests with Welch’s correction and Holms-Šídák correction for multiple comparisons. * = conductance slopes differences P<0.05, # = Maximal JO2 differences P<0.05.
Figure 3.
Figure 3.. Surplus L-carnitine rescues lipid-induced respiratory inefficiency in SkM mitochondria.
A. CrAT is a carnitine-dependent acyltransferase enzyme with specificity for short chain carbon substrates. B. Mixed SkM mitochondria isolated from CrATfl/fl control mice (Cont) or muscle-specific CrAT knockout (KO) littermates were provided with PM or PMPc ± surplus L-carnitine and respiratory responses were assayed using the CK clamp technique. C-F. Respiratory response (C), respiratory conductance (D), respiratory efficiency (E), and redox stability (F) were enhanced in control mitochondria when surplus L-carnitine was added to PMPc but not PM. G-J. Respiratory response (G), respiratory conductance (H), respiratory efficiency (I), and redox stability (J) were enhanced in control mitochondria when surplus L-carnitine was added to PMPc but not PM. CK clamp assay conditions: Mitochondria (0.05 mg) from Cont or KO mouse mixed SkM were added to 2 ml Buffer Z containing 5mM Creatine, 20U/ml Creatine Kinase, and 1.5mM phosphocreatine at 37°C. CK clamp was engaged by the addition of 5mM ATP. PCr was added to final concentrations of 3, 6, 9, 12, and 15mM. Mitochondrial membrane potential (ΔΨm) and redox status (NAD(P)H) were assessed in parallel in 0.2ml Buffer Z containing 0.02mg mitochondria and 0.2μM TMRM. Pyruvate/Malate (5mM/2.5mM; PM), Pyruvate/Malate/Palmitoyl-L-Carnitine (5mM/2.5mM/20μM; PMPc) ± 2mM L-Carnitine (Carn). All substrates were provided at concentrations that are saturating for JO2. Respiratory efficiency is defined as the relationship between oxygen flux (JO2) and mitochondrial membrane potential (ΔΨm). Arrows denote a shift in efficiency. Redox response refers to changes in (NAD(P)H/NAD(P)+ redox potential in relation to changes in JO2. Statistics: n=6 Cont and 5 KO biological replicates per group with data presented as means ± SEM. Conductance slopes and maximal JO2 responses were tested with unpaired t-tests with Welch’s correction and Holms-Šídák correction for multiple comparisons. * = conductance slopes differences P<0.05, # = Maximal JO2 differences P<0.05. For conductance comparisons in (D) and (H), all substrate conditions were tested for significance versus the PM condition and using Dunnett post-hoc testing with correction for multiple comparisons.
Figure 4.
Figure 4.. Mitochondrial metabolomics and MFA link lipid tolerance to free CoA and reverse SC carbon flux.
A. Schematic of 13C pyruvate tracer experiment performed with isolated heart and SkM mitochondria from non-transgenic (NT) and Mck-Pgc1a transgenic (Tg) mice. Mitochondria were incubated with 5 mM [UL-13C]pyruvate and 2.5mM malate (PM) ± 20μM Pc ± 2mM L-carnitine in the context of 6 mM PCr to mimic moderate intensity exercise. The CK clamp was engaged upon addition of 5mM ATP. Five minutes after ATP addition, mitochondrial pellets were collected by centrifugation. Metabolites were extracted and measured by mass spectrometry. B. Heat map representing log2 transformed row normalized Z-scores of log2 normalized metabolite abundances. C-D. Free CoA (C) and Succinyl CoA (D) measured in pelleted heart and SkM mitochondria. E-H. Acyl CoA metabolites measured in mitochondrial pellets. I-L. Acylcarnitines measured in mitochondrial pellets. M-O. Label incorporation from [UL-13C]pyruvate (M+2) into short chain metabolites. P. Reverse short chain carbon flux estimated by measuring label incorporation into C4OH AC relative to C2. See also Figure S3. Statistics: Data are presented as means ± SEM and were analyzed by two-way ANOVA (Tissue x Substrate) with no assumptions for equal variance and Dunnet’s correction applied for multiple comparisons. a = P<0.05 for Heart versus NT SkM. b = P<0.05 for Tg SkM versus NT Skm. $ = P<0.05 when PM+Pc condition was different from all other substrate combinations.
Figure 5.
Figure 5.. MS-based proteomics identifies MKT as a common feature of lipid-tolerant mitochondria.
Mass spectrometry (MS)-based proteomics was performed on semi-purified mitochondria isolated from heart as compared to SkM (n=5 biological replicates per group) and SkM of Mck-Pgc1a transgenic (Tg; n=6) mice compared to Non-transgenic (NT; n=5) littermate controls. A. Volcano plot (FDR Adjusted P-Values as a function of Log2 fold change) showing proteins that are differentially abundant in mitochondria from heart vs. SkM. Fatty acid oxidation (FAO) proteins are highlighted in red and yellow denotes FAO proteins of high interest. B. List of the top 15 FAO proteins that were more abundant in mitochondria from heart vs. SkM, ranked by Padj. C. Volcano plot showing proteins that are differentially abundant in mitochondria from SkM of NT vs. Pgc1a Tg mice. FAO proteins are highlighted in red and yellow denotes FAO proteins of interest. D. List of the top 15 proteins that were more abundant in SkM mitochondria from Tg vs. NT mice, ranked by Padj. E. Relationship between proteins differentially abundant in Heart:SkM mitochondria and those changing in response to chronic transaortic constriction relative to sham controls (TAC:Sham) in mouse hearts. The dataset shows 110 proteins that were commonly identified in both sets and differentially abundant at FDR<1% and Log2 fold-change cutoff of at least ±0.5 in heart/SkM. F. Quantitative results from western blots performed to measure abundance of MKT, SCHAD, CrAT, and CV subunit ATP5A1 in semi-purified mitochondria isolated from heart, RG and WG. semi-purified mitochondria isolated from heart, RG and WG. G. Quantitative results from western blots performed to measure abundance of MKT, SCHAD, and CrAT in semi-purified SkM mitochondria isolated from NT compared Tg mice. H. mRNA expression of Acaa2 (MKT) measured in mouse TA muscles harvested at rest or 5 min, 3 h or 24 h after a 90 min bout of acute treadmill exercise. See also Figure S4.
Figure 6.
Figure 6.. Reverse short chain carbon flux is substantial in cardiac and skeletal muscle tissues and contributes to CrAT-dependent production of 3OH-butyrylcarnitine.
A. Tracer strategy used for metabolic flux analysis (MFA) performed in perfused hearts harvested from MckCrAT KO mice and fl/fl littermate controls (B-G); or incubated soleus and EDL muscles (H-J). B-C. [U-13C]glucose incorporation into 3-OH-butyrylcarnitine (B) and 3OH-butyryl-CoA (C) measured in perfused hearts isolated from Mck-CrAT KO mice and fl/fl controls. D. Heart concentrations of total 3OH-butyrlcarnitine after perfusion with 11 mM [U-13C]glucose. E-F. [U-13C]acetate incorporation into 3-OH butyrylcarnitine (E) and 3-OH butyryl CoA (F) measured in perfused hearts isolated from Mck-CrAT KO mice and fl/fl controls. G. Heart concentrations of total 3-OH-butyrlcarnitine after perfusion with 1 mM [U-13C]acetate. H-I. [U-13C]glucose incorporation into short chain acylcarnitines, including 3-OH-butyrlcarnitine (C4OH), measured in soleus (H) and EDL (I) muscles isolated from wildtype mice and incubated ex vivo in the absence and presence of 100 nM insulin. J-K. Reverse short chain carbon flux estimated by measuring label incorporation into short chain acylcarnitines (AC) relative to acetylcarnitine (C2) in soleus (J) and EDL (K) muscles. Statistics: Heart perfusions were performed with n=4 biological replicates per group for 13C-glucose and 6 biological replicates for [U-13C]acetate tracer experiments. Means were tested using Unpaired t-tests with Welch’s correction and Holms-Šídák correction for multiple comparisons. Small muscle incubations were performed with n=6 biological replicates for soleus and EDL. ‡ = main effect of insulin across the reported species.
Figure 7.
Figure 7.. Insufficient MKT flux results in lipid-induced respiratory inefficiency in heart mitochondria.
A-C. Isolated heart and skeletal muscle (SkM) mitochondria were exposed to aKG±Pc followed by assessments of respiration (A), respiratory efficiency (B), and conductance (C). In the absence of PM, addition of Pc inhibits respiratory conductance and disrupts efficiency and redox stability in both heart and SkM mitochondria. CK clamp assay conditions: Mitochondria (0.05 mg) isolated from mouse Heart (Hrt) or Mixed Skeletal Muscle (SkM) were added to 2 ml Buffer Z containing 5mM Creatine, 20U/ml Creatine Kinase, and 1.5mM phosphocreatine at 37C. CK clamp was engaged upon addition of 5mM ATP. PCr was added to final concentrations of 3, 6, 9, 12, and 15mM. Mitochondrial membrane potential (ΔΨm) and redox status (NAD(P)H) were assessed in parallel in 0.2 ml Buffer Z containing 0.02mg mitochondria and 0.2μM TMRM. Alpha-Ketoglutarate (5mM; aKG), alpha-Ketoglutarate/Palmitoyl-L-Carnitine (5mM/20μM; aKG+Pc). All substrates were provided at concentrations that are saturating for JO2. Respiratory efficiency is defined as the relationship between oxygen flux (JO2) and mitochondrial membrane potential (ΔΨm). Redox response refers to changes in (NAD(P)H/NAD(P)+ redox potential in relation to changes in JO2. Redox transfer refers to the transfer of electron energy potential (NAD(P)H/NAD(P)+ redox) to proton energy potential, which is informed by the relationship between NAD(P)H/NAD(P)+ redox potential and mitochondrial membrane potential (ΔΨm). Statistics: n=6 biological replicates per condition. Data are presented as means ± SEM. Conductance slopes and maximal JO2 responses were tested with unpaired t-tests with Welch’s correction and Holms-Šídák correction for multiple comparisons. * = conductance slopes differences P<0.05, # = Maximal JO2 differences P<0.05. D-G. Lipid tolerance was assessed in differentiated primary human skeletal myocytes (HSkMC) using a CK clamp adapted for use with the Seahorse Flux Analyzer. Western blot analysis (15 μg protein per lane) of MKT protein abundance in HSkMC is comparable to heart tissue (D). Addition of 25 uM Pc to PM does not affect respiratory conductance whereas 50 Pc is inhibitory, while 100 uM Oc has no effect (E). MKT-targeted siRNAs diminished protein abundance in HSkMC as compared with cells treated with non-targeting control siRNA (NTC) (F). Knockdown (KD) of MKT protein in HSkMC diminishes respiratory conductance measured with PM+25 uM Pc (G). Statistics: Conductance measures are from n=4 (E) and n=7 (G) independent Seahorse flux experiments, each with at least 4 technical replicates per condition (i.e. 2 siRNA treatments x 2 substrate mixtures). Data are presented as means ± SEM of n=4 and n=7, respectively. Conductance slopes calculated from protein normalized JO2 measures were analyzed by paired t-tests; * = P<0.05. Quantification of MKT western analysis (F) represents n=7 biological replicates per treatment, each representative of 15–30 technical replicates pooled from 96-well Seahorse plates after permeabilization and JO2 assessment shown in (G). H-N. Heart mitochondria were isolated from mice that were fed ad libitum or fasted for 6, 12 or 24 hours (H). Blood metabolites (I) were measured by glucometer and ketometer. Mitochondrial assays were performed in parallel (i.e. 4 mice per day, one from each of the 4 timepoints) upon exposure to 0.55 mM aKG + 2 mM beta-hydroxy butyrate (3OHB) followed by assessments of respiratory efficiency (J) and redox response (K). L-M. Western blot (L) and the corresponding relative quantitation (M) of pAMPK relative to total AMPK in hearts of mice that were fed ad libitum or fasted for 6, 12 or 24 hours. N. Cardiac function assessed by echocardiography performed in mice that were fed or starved overnight (OS). O. Working model of a short chain carbon circuit that confers lipid tolerance and metabolic flexibility. AC, acylcarnitine; CoQ, Coenzyme Q; CrAT, carnitine acetyltransferase; ETC, electron transport chain; ETF; electron-transfer flavoprotein system; LC, long chain, MC, medium chain; MKT, medium chain ketothiolase; SC, short chain; SCHAD, short chain hydroxyacylCoA dehydrogenase. See also Figure S5. Fasting time course: Mitochondria (0.05 mg) isolated from mouse heart mitochondria were added to 2 ml Buffer Z containing 5mM Creatine, 20U/ml Creatine Kinase, and 1.5mM phosphocreatine at 37°C. CK clamp was engaged upon addition of 5mM ATP. PCr was added to final concentrations of 3, 6, 9, 12, and 15mM. Mitochondrial membrane potential (ΔΨm) and redox status (NAD(P)H) were assessed in parallel in 0.2ml Buffer Z containing 0.02mg mitochondria and 0.2μM TMRM. N=6 biological replicates per timepoint. alpha-Ketoglutarate/3OH-butyrate (0.55mM/1mM; aKG/3OHB). Statistics: n=6 biological replicates per condition. Data are presented as means ± SEM. Differences in blood metabolites were tested with a one-way ANOVA for differences versus 0 h using Dunnett’s correction for multiple comparisons. Conductance slopes and maximal JO2 responses were tested with unpaired t-tests with Welch’s correction and Holms-Šídák correction for multiple comparisons. * = conductance slopes differences P<0.05, # = Maximal JO2 differences P<0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fisher-Wellman KH, Davidson MT, Narowski TM, Lin CT, Koves TR, and Muoio DM (2018). Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes. Cell Rep 24, 3593–3606 e3510. 10.1016/j.celrep.2018.08.091. - DOI - PMC - PubMed
    1. Willis WT, Jackman MR, Messer JI, Kuzmiak-Glancy S, and Glancy B. (2016). A Simple Hydraulic Analog Model of Oxidative Phosphorylation. Med Sci Sports Exerc 48, 990–1000. 10.1249/MSS.0000000000000884. - DOI - PMC - PubMed
    1. Muoio DM (2014). Metabolic inflexibility: when mitochondrial indecision leads to metabolic gridlock. Cell 159, 1253–1262. 10.1016/j.cell.2014.11.034. - DOI - PMC - PubMed
    1. Zhou B, and Tian R. (2018). Mitochondrial dysfunction in pathophysiology of heart failure. J Clin Invest 128, 3716–3726. 10.1172/JCI120849. - DOI - PMC - PubMed
    1. Kelley DE, Goodpaster B, Wing RR, and Simoneau JA (1999). Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss. Am J Physiol 277, E1130–1141. 10.1152/ajpendo.1999.277.6.E1130. - DOI - PubMed

Publication types

LinkOut - more resources