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Comprehensive Metabolic Profiling of Age-Related Mitochondrial Dysfunction in the High-Fat-Fed ob/ob Mouse Heart Xinzhu Wang,†,‡ James A. West,†,‡ Andrew J. Murray,§ and Julian L. Griffin*,†,‡ †

Department of Biochemistry & Cambridge Systems Biology Centre, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA, U.K. ‡ MRC, Human Nutrition Research, Elsie Widdowson Laboratory, 120 Fulbourn Road, Cambridge, CB1 9NL, U.K. § Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, U.K. S Supporting Information *

ABSTRACT: The ectopic deposition of fat is thought to lead to lipotoxicity and has been associated with mitochondrial dysfunction and diabetic cardiomyopathy. We have measured mitochondrial respiratory capacities in the hearts of ob/ob and wild-type mice on either a regular chow (RCD) or high-fat (HFD) diet across four age groups to investigate the impact of diet and age on mitochondrial function alongside a comprehensive strategy for metabolic profiling of the tissue. Myocardial mitochondrial dysfunction was only evident in ob/ ob mice on RCD at 14 months, but it was detectable at 3 months on the HFD. Liquid chromatography−mass spectrometry (LC−MS) was used to study the profiles of acylcarnitines and the accumulation of triglycerides, but neither class of lipid was associated with mitochondrial dysfunction. However, a targeted LC−MS/MS analysis of markers of oxidative stress demonstrated increases in GSSG/ GSH and 8-oxoguanine, in addition to the accumulation of diacylglycerols, which are lipid species linked to lipotoxicity. Our results demonstrate that myocardial mitochondria in ob/ ob mice on RCD maintained a similar respiratory capacity to that of wild type until a late stage in aging. However, on a HFD, unlike wild-type mice, ob/ob mice failed to increase mitochondrial respiration, which may be associated with a complex I defect following increased oxidative damage. KEYWORDS: Respiratory capacity, high-fat diet, aging, metabolomics, obesity

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INTRODUCTION

the ability to switch between lipids and carbohydrates in response to hormonal stimulation, substrate availability, oxygenation, and workload.8,9 The balance between fatty acids and glucose metabolism is strongly influenced by diet, and increased fatty acid uptake and concomitantly decreased glucose metabolism results in a greater production of ATP overall per mole of substrate but a reduction in the amount of ATP produced per mole of oxygen consumed.10−12 This ability to switch substrates is impaired in T2DM, with the diabetic heart exhibiting metabolic inflexibility, defined as the impaired adjustment of mitochondrial fuel selection in response to nutritional cues, leading to a lack of substrate switching in response to stress.9 As a result, the maladaptive diabetic heart relies more on lipids as a primary fuel.

The incidence of both obesity and type 2 diabetes (T2DM) is increasing dramatically in both the developed and developing world. In 2008, 10% of men and 14% of women globally were obese (BMI ≥ 30 kg/m2), compared with 5% for men and 8% for women in 1980, whereas 35% of the global population was classified as being overweight (BMI ≥ 25 kg/m2).1 There is a close association between obesity and T2DM, with BMI being the strongest predictor of future risk for development of T2DM as well as contributing to a variety of metabolic diseases including fatty liver disease, atherosclerosis, and dyslipidemia.2−5 This has led to the collective terms metabolic syndrome or syndrome X being associated with the interactions among obesity, high blood pressure, T2DM risk, and dyslipidemia.6 In the normal heart, fatty acid oxidation accounts for 60− 80% of energy demand, with the remainder provided by glucose and pyruvate metabolism.7 Metabolically, the healthy heart has © 2015 American Chemical Society

Received: February 14, 2015 Published: May 19, 2015 2849

DOI: 10.1021/acs.jproteome.5b00128 J. Proteome Res. 2015, 14, 2849−2862

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Journal of Proteome Research

period), 4 months (12 week high-fat feeding), and 10 months (12 week high-fat feeding) (Supporting Information Figure S1). The 14 month old group was omitted from high-fat feeding because of concerns that high-fat feeding may cause significant mortality at this age. The fatty acid composition of the RCD and HFD was analyzed by GC−MS as previously described27 (Supporting Information Table S1). All groups of mice were killed by carbon dioxide asphyxiation at 10 am after a 2 h fasting period. Hearts were rapidly dissected, and a portion of the cardiac tissues was snap frozen with liquid nitrogen and stored at −80 °C until further analysis. All animal protocols were approved by the UK Home Office and the University of Cambridge and carried out by a license holder. Body fat composition and respirometry were performed on the day of dissection. All additional analyses, including gene expression, western blot, and metabolomics, were performed once all of the tissues from all of the animal groups were collected, and analyses were performed in single batches to minimize experimental variation.

Lipid accumulation has been proposed to be responsible for cardiac dysfunction and arises when lipid uptake exceeds lipid oxidation. It has been associated with the accumulation of reactive lipid intermediates such as saturated acyl-CoA species, long-chain acylcarnitine species, diacylglycerols, and ceramides, resulting in lipotoxity.13,14 In recent years, a hypothesis suggesting a direct link between intramyocardial lipid accumulation and mitochondrial dysfunction has emerged. In addition, mitochondria are the major site of reactive oxygen species (ROS) production and are susceptible to ROS-induced lipid peroxidation, which, in turn, damages mitochondrial machinery, particularly during fatty acid oxidation.15 The concept of investigating the complete cardiac metabolic phenotype using metabolic profiling has emerged as a promising approach for unravelling the complexities of cardiac metabolism.16 For example, Kato and colleagues performed a comprehensive metabolomics study in a rat model of congestive heart failure and showed that the pentose phosphate pathway was activated.17 Similarly, in the search for causal factors that explain lipid-induced metabolic dysfunction associated with insulin resistance, Koves and co-workers demonstrated a striking difference in acylcarnitine profiles of skeletal muscle upon high-fat feeding across all of the metabolites evaluated,18 suggesting an important role of incomplete fatty acid oxidation in the development of insulin resistance. Increased myocardial long-chain acylcarnitines have been reported in the hearts of streptozotocin-injected type 1 diabetic animals,19 but it is not clear whether these changes are causes or consequences of the diabetic heart. In the present study, we have investigated whether obesity contributes to mitochondrial respiratory dysfunction in the heart, using metabolomics to investigate cardiac tissue from ob/ ob mice across an aging study of the mouse. We chose to study the cardiac metabolic phenotype of the ob/ob mouse, as its metabolic status as a result of obesity is well-defined in the literature.10,20−25 In addition, we have investigated the effect of an excess supply of lipids on cardiac mitochondrial metabolism in the heart and how this interacts with age. We hypothesized that an excess supply of lipids in the aging heart accelerates mitochondrial dysfunction. We demonstrate that high-fat feeding in the ob/ob mouse results in an impairment in mitochondrial function and fatty acid oxidation, and this arises, in part, through the generation of ROS.

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Body Fat Composition

After rapidly dissecting hearts and taking blood samples, sacrificed animals (n = 3/group) were analyzed by dual-energy X-ray absorptiometry (DEXA, PIXImus, Lunar, Madison, WI) to determine body fat and lean mass. Plasma Insulin, Glucose, and HbA1C Measurement

Blood was collected rapidly from the chest after dissecting the heart, and blood plasma was separated by centrifugation, snap frozen, and stored at −80 °C. Plasma glucose concentrations were measured by 1H NMR spectroscopy as previously described.28 Plasma insulin was assayed by standard methods with a two-site microtiter plate-based immunoassay with electrochemical luminescence detection. Ten microliters of whole blood was kept in an ETDA-containing tube for measurement of HbA1C by an automated high-performance liquid chromatography system (TOSOH G7) targeted from HbA1C (INSTRU-MED, INC., Atlanta, USA). Histology

Histological staining was conducted in the Cambridge Advanced Imaging Centre at the Department of Physiology, Development and Neuroscience, University of Cambridge. A cross-section of heart was cut, fixed with 10% buffered formalin, and embedded in paraffin. Paraffin-embedded sections were stained with picrosirius red (Sigma-Aldrich) to determine collagen content. Images of the slides were captured with the Nanozoomer 2.0 digital pathology system (Hamamatsu, Hertfordshire, UK). The extent of collagen deposition (picrosirius red staining) was shown as a percentage of the total histological field quantified using ImageJ software (NIH, Bethesda, MD, USA) by randomly selecting 10 fields from the images.

EXPERIMENTAL SECTION

Animals and Diets

Five week old male ob/ob mice and their wild-type (C57BL/6J, WT) controls were purchased from a commercial breeder (Harlan, UK). Mice were housed in a temperature- and humidity-controlled facility, with a 12 h/12 h light−dark cycle and access to water ad libitum. Mice (n = 10/age group/ genotype) fed a regular chow diet (RCD) (caloric content: 11.5% fat, 26.9% protein, 61.6% carbohydrate) (RM1; Special Diet Services, UK) were killed at the ages of 2, 4, 10, and 14 months. A separate group of mice was switched to a customproduced high-fat diet previously described26 (caloric content: 55% fat, 29% protein, 16% carbohydrate; fatty acid composition: 27% saturated fatty acid, 48% monounsaturated fatty acid, and 25% polyunsaturated fatty acid) (diet code: 829197; Special Diet Services, UK) at different stages and for various lengths of time, and they were killed at the age of 2 months (3 week high-fat feeding after a 5 week weaning

Respirometry

Mitochondrial respiratory parameters were studied in saponinpermeablized fibers as described previously.29 Oxygen consumption was measured with a Clark-type oxygen electrode chamber (Strathkelvin Instruments Ltd., Glasgow, UK) at 37 °C using different substrates as follows: 10 mM glutamate and 5 mM malate to measure complex I respiration; subsequently, complex I was inhibited with rotenone, followed by measurement of complex II respiration by addition of 10 mM succinate. In addition, 0.04 mM palmitoyl-carnitine or 10 mM pyruvate, both combined with 5 mM malate, was used as β-oxidation and 2850


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nitine (N-methyl-d3), L-butyrylcarnitine (N-methyl-d9), Lisovalerylcarnitine (N-trimethyl-d9), L-octanoylcarnitine (Nmethyl-d3), L-myristoylcarnitine (N-trimethyl-d9), and L-palmitoylcarnitine (N-methyl-d3) (Cambridge Isotope Laboratories, Inc., MA, USA). The samples were homogenized using a Tissuelyser (Qiagen, UK) before being centrifuged at 13 300 rpm for 5 min. The supernatants were dried and butylated prior to analysis with 100 μL of 3 M HCl in 1-butanol and kept at 60 °C for 15 min. The samples were evaporated to dryness before being reconstituted in 100 μL of acetonitrile with 0.1% formic acid, of which 10 μL was injected for analysis. Chromatographic separations were performed using a Synergi Polar-RP column (Phenomenex, Cheshire, UK) on an ACQUITY UPLC system (Waters Corporation, Elstree, Hertfordshire). The mobile phase consisted of solvent A, 0.1% formic acid in H2O, and solvent B, 0.1% formic acid in acetonitrile. When eluting the column, the mobile phase was increased from 30% solvent B to 100% solvent B in 3 min, held at 100% for 5 min, and decreased back to 30% solvent B in 10 s and held for 1 min and 50 s for re-equilibrium, with a total run time of 10 min. Liquid chromatography−mass spectrometry (LC−MS) data was collected using a Waters Quattro Premier XE Triple Quadrupole MS coupled with an electrospray source. The source temperature was set to 110 °C with a cone gas flow of 46 L/h, a desolvation temperature of 345 °C, and a desolvation gas flow of 700 L/h. A capillary voltage of 3500 V was applied. Mass spectra were generated by allowing only the parent ions of m/z +85 to pass through and be detected as part of a multiple reaction monitoring (MRM) approach. Acquired data was processed with Quanlynx Research Application Manager (Waters Corporation).

pyruvate oxidation substrates, respectively. Cardiac muscle fibers were dried for 48 h at 80 °C before being weighed to estimate dry weight. Real-Time PCR

Total RNA was isolated from frozen ventricular tissues using the RNeasy fibrous tissue mini kit (Qiagen, Hilden, Germany). DNase-treated RNA was reverse-transcribed to cDNA with QuantiTect RT kit (Qiagen, Hilden, Germany). mRNA levels of PPARα, MCAD, LCAD, SCD1, PGC1α, UPC2, UPC3, ANT1, NRF1, and PDK4 were measured by Taqman gene expression assay (Applied Biosystems, UK) with predesigned/ prevalidated FAM-labeled probes and 18s rRNA (VIC-labeled probe, primer-limited) as an endogenous control (housekeeping) gene. Western Blot Analysis of Citrate Synthase and ETC Complexes

The protein expression of enzymes involved in oxidative phosphorylation and citrate synthase content was measured. Frozen ventricular tissues were homogenized with a Tissuelyser (Qiagen, UK) for 5 min at a frequency of 20/s in buffer containing 20 mM Tris (pH 6.8), 150 mM NaCl, 1 mM Na 2 EDTA, 1% Triton, 2.5 mM Na 4 P 2 O 7 , 1 mM βglycerophosphate, 1 μM Na3VO4, and complete protease inhibitor (Roche Applied Science, UK). Protein concentrations were determined by the BCA protein assay kit (Sigma-Aldrich, MO, USA). Equal protein amounts (20 μg) of each sample were loaded and electrophoresed on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and probed using monoclonal antibodies against oxidative phosphorylation complexes (1:208 dilution; Invitrogen anti-Rt/Ms oxphos complex kit (Life Technologies Ltd., UK)) and polyclonal antibodies against citrate synthase (1:1000 dilution; Abcam UK). The blots were subsequently incubated with a donkey anti-mouse IgG−HRP antibody (1:2000 dilution; Santa Cruz Biotechnology Inc., CA, USA) for oxidative phosphorylation complexes and a goat anti-rabbit IgG−HRP antibody (1:1750 dilution; Santa Cruz Biotechnology Inc., CA, USA) for citrate synthase. Enhanced chemiluminescence (Pierce ECL western blotting substrate, Thermo Scientific, UK) was applied to the blots before being detected using X-ray films. The protein bands were quantified using ImageJ software (NIH, Bethesda, MD, USA). Ponceau S stain (Sigma-Aldrich, MO, USA) was used as a control for loading.

Quantitative Measurement of Oxidative Stress Biomarkers by LC−MS

The dried aqueous fraction was reconstituted in 200 μL of acetonitrile/water (7:3; v/v) containing 20 μM U-13C,15N labeled glutamate. The samples were then vortexed and sonicated for 15 min, before being centrifuged at 15 000 rpm to pellet any remaining undissolved material. Chromatographic separations were performed using a ZIC-HILIC column (Sequant, 100 mm × 2.1 mm, 3.5 μm) kept at room temperature on an ACQUITY UPLC system (Waters Corporation, Elstree, Hertfordshire) coupled with a 5500 QTRAP mass spectrometer (AB Sciex, Warrington, UK). The mobile phase consisted of solvent A, 100 mM ammonium acetate in water, and solvent B, acetonitrile. When eluting the column, the mobile phase was 20% solvent A for 2 min, followed by an increase to 50% solvent A over 10 min, and held for a further 3 min for re-equilibrium, with a total run time of 15 min. The source temperature was 700 °C, cone gas pressure was 25 psi, and the capillary voltage was 4.5 kV. The declustering potential was compound-dependent. Mass spectrometric data was collected in both positive and negative modes with unscheduled MRM approach with a dwell time of 50 ms. For quantitative analysis, acquired data was processed with Analyst 1.6 software (AB Sciex, Warrington, UK). Peaks of both analytes and internal standards were detected and integrated. Peak integrations were then confirmed manually within the software.

Tissue Metabolite Extraction

Metabolites were extracted using a methanol/chloroform/water extraction method described previously.30,31 Briefly, frozen tissues were added to 600 μL methanol/chloroform (2:1; v/v), and the samples were homogenized with a Tissuelyser (Qiagen, UK) for 5 min at a frequency of 20/s and sonicated for 15 min. Water and chloroform (each of 200 μL) were added to the samples before being centrifuged at 13 300 rpm for 7 min. The resulting aqueous and organic phases were separated from the protein pellets. The extraction procedure was repeated on the remaining protein pellets. Both organic and aqueous phases were collected and evaporated to dryness. The dried samples were stored at −20 °C until further analysis. Quantitative Analysis of Acyl-carnitines by Mass Spectrometry

Intact Lipid Analysis by LC−MS

Frozen tissues were added to 200 μL of acetonitrile containing a mixture of isotopically labeled carnitines: L-carnitine (Ntrimethyl-d9), L-acetylcarnitine (N-methyl-d3), L-propionylcar-

The dried organic phase fraction was reconstituted and diluted to the desired concentration with 2-proponol/acetonitrile/ water (2:1:1; v/v/v). Chromatographic separations were 2851


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Figure 1. Physiological characteristics of ob/ob mice and wild-type (WT) controls. Body weight (A) and heart weight (B) are shown (n = 6−10/ group). (C) Body fat composition was determined from 10 month old animals fed on regular chow diet (RCD) or high-fat diet (HFD) by dualenergy X-ray absorptiometry (DEXA) (n = 3−4/group). (D) Representative images showing picrosirius red staining of collagen in left ventricles of 4 month old ob/ob and WT mice on RCD (n = 5/group). (E) Quantification of collagen area/total ventricular area of 4 month old ob/ob and WT mice on RCD using ImageJ software (n = 10−12/group). (F) HbA1C levels of ob/ob and WT controls on high-fat diet (HFD) (n = 5/group). All data are mean ± SEM. Statistical significance with two-way ANOVA is indicated by a, genotype-dependent; b, diet-dependent; and c, interaction between genotype and diet. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with WT animals on RCD. †, p < 0.05; ††, p < 0.01; †††, p < 0.001 compared with ob/ob mice on HFD using Bonferroni’s posthoc analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT animals on the same diet using Student’s t test.

performed on an Acquity UPLC 1.7 μm bridged ethyl hybrid (BEH) C8 column (2.1 × 100 mm) (Waters Corporation, Hertfordshire). The mobile phase consisted of solvent A, HPLC grade acetonitrile (Sigma-Aldrich)/water (SigmaAldrich) (5:2; v/v) and 10 mM ammonium formate, and solvent B, HPLC grade 2-propanol (Merck Millipore)/water (Sigma-Aldrich) (9:1; v/v) and 10 mM ammonium formate. When eluting the column, the mobile phase was increased from 40% solvent B to 99% solvent B in 18 min, decreased back to 40% solvent B in 10 s, and held for 2 min for re-equilibrium, with a total run time of 20 min. Mass spectrometry data was

collected using a Xevo G2 QToF (Waters Corporation, Hertfordshire, UK) with an electrospray source operating in positive ion mode. The full scan mode was 200−2000 m/z with a scan duration of 0.2 s. Acquired data was processed with Micromass Markerlynx Application Manager (Waters Corporation, UK). Using a combination of loading and VIP scores following OPLS-DA, the most perturbed metabolite ions between ob/ob and WT mice were selected. Analysis of variance testing of cross-validated predictive residuals (CV_ANOVA) was used to assess the reliability of OPLS-DA models. The p-value for this analysis indicates the probability level that a 2852


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Figure 2. Mitochondrial respiratory function in the heart. Substrate-specific mitochondrial respirations in saponin-permeabilized cardiac fibers from ob/ob mice and wild-type (WT) controls on a regular chow diet (RCD) or high-fat diet (HFD) (n = 6−10/group). State 2 (no ADP) (A) and state 3 (maximally ADP stimulated) (B) respiration rates with palmitoyl-carnitine/malate as substrates. State 2 (no ADP) (C) and state 3 (maximally ADP stimulated) (D) respiration rates with pyruvate and malate as substrates. State 2 (no ADP) (E) and state 3 (maximally ADP stimulated) (F) respiration rates with complex I-supported substrates: glutamate and malate. (G) State 3 (maximally ADP stimulated) respiration rates with complex II-supported substrates: succinate and malate. (H) Coupling of complex I, as measured by the respiratory control ratio (defined as state 3/state 2). n = 6−10/group. All data are mean ± SEM. Statistical significance with two-way ANOVA is indicated by a, genotype-dependent; b, diet-dependent; and c, interaction between genotype and diet. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with WT animals on a RCD. †, p < 0.05; ††, p < 0.01; †††, p < 0.001 compared with ob/ob mice on a HFD using Bonferroni’s posthoc analysis. *, p < 0.05 compared with WT animals on a RCD using a Student’s t test. 2853


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The ob/ob Mouse Heart Fails To Stimulate Mitochondrial Oxygen Consumption on a High-Fat Diet

given model has been built by chance, and the convention is that a p-value lower than 0.05 is associated with a significant model. TAGs were identified using the LIPID MAPS database (LIPID Metabolites And Pathway Strategy; http://www. lipidmaps.org) according to their m/z ratios and retention time and confirmed by subsequent MS/MS. Total TAG content was determined by summing all of the regions of TAGs from the chromatogram (between 12 and 18 min) and normalizing against an internal standard (N-heptadecanoyl-Derythro-sphingosylphosporylcholine).

We initially investigated whether mitochondrial dysfunction exists in the heart of ob/ob mice on RCD, applying a relatively novel protocol for respirometry involving permeabilized fibers at a physiological 37 °C. State 2 basal respiration rates in mitochondria from ob/ob mice with pyruvate, or with palmitoyl-carnitine, as substrate plus malate were not significantly different when compared with those of their agematch WT controls across all groups (Supporting Information Figure S2A,C). However, a significant decrease in state 3 respiration rate was observed using pyruvate/malate as substrates in ob/ob mice in the 14 month old group on a RCD (Supporting Information Figure S2D). Across all of the mitochondrial respiratory studies, we did not see any changes after addition of cytochrome c (data not shown), indicating that the integrity of the outer mitochondrial membrane was maintained during sample preparation. To measure the effect of HFD on mitochondrial function in the mouse heart, we investigated age-matched ob/ob and WT mice on a HFD. State 2 (no ADP) basal respiration rates and state 3 (maximally ADP-stimulated) respiration rates with palmitoyl-carnitine plus malate for mitochondria from ob/ob mice were not significantly different when compared with those of their age-matched WT controls across all groups on the HFD (Figure 2A,B). Two-way ANOVA revealed a significant HFD effect compared with the RCD (P < 0.05) in the 2 and 4 month old groups, indicating that HFD enhances the respiratory capacity of mitochondrial metabolism of fatty acids in younger animals when switched to a HFD at a relatively early stage (Figure 2A,B). To further investigate the changes associated with mitochondrial function upon HFD, we also looked into substrate-specific mitochondrial respiration of ob/ob and WT mice, with pyruvate/malate as substrates to model glucose oxidation, and used the substrate/inhibitor titration approach to assess the function of complexes I and II of the electron transport chain (ETC). In the 2 month old group, HFD stimulated the state 2 respiration rate with pyruvate/malate in WT animals compared with that in WT animals on a RCD, but it did not in ob/ob mice (Figure 2C), and this produced a significant genotype versus diet interaction (p < 0.01), as measured by two-way ANOVA. The same pattern was also detected in state 3 respiration rates with pyruvate/malate. HFD increased the respiratory capacity for oxidizing pyruvate in WT animals across the three age groups compared with that of animals on a RCD, but it failed to exert the same effect on ob/ob mice (Figure 2D). This resulted in a reduction of myocardial respiration rates in ob/ob mice when compared with those in WT controls for the HFD. This elevation in the respiratory capacity in WT animals due to HFD is less profound when examining respiration rates with glutamate/malate (both state 2 and state 3) and was significant only in 10 month old animals (Figure 2E,F). Nevertheless, respiration rates of ob/ob mice were either decreased or unchanged across the time points, suggesting a defect in complex I of the ETC. We also observed a defect in complex II of the ETC, which is again associated with increased state 3 respiration rates with succinate/malate in 4 and 10 month old WT animals (Figure 2G). Although the HFD did not affect RCR with palmitoyl-carnitine and pyruvate (data not shown), diet had a significant effect (p < 0.01) on RCR with glutamate

Univariate and Multivariate Statistics

Data in figures and tables are expressed as the mean ± SEM unless otherwise specified. Group comparisons were performed using Student’s t test or two-way analysis of variance (ANOVA). A two-way ANOVA with a Bonferroni posthoc test was used to determine effects of genotypes (ob/ob and wild type), diet (RCD and HFD), or their interaction (GraphPad Prism, La Jolla, CA). The posthoc test compared ob/ob and WT animals on the same diet or RCD and HFD on the same strain of animals. Significance was accepted at p < 0.05. The intact lipid data set obtained from MS-based measurements was preprocessed where the peaks are picked to generate a data set with optimized resolution. The processed data set was meancentered, normalized, and Pareto-scaled prior to multivariate statistical analysis within SIMCA-P+ (version 13, Umetrics, Umea, Sweden). Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify the discriminant lipid species between the ob/ob and WT groups.

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RESULTS

Physiological Characteristics

Both genotype and diet had significant effects on the body weight of the animals, with genotype being dominant across the three age groups where direct comparisons were possible (>70% of the total variation, revealed by two-way ANOVA). As expected, high-fat feeding significantly increased body weight in both WT and ob/ob mice across all three age groups (2, 4, and 10 month) compared to that of mice fed on a RCD (Figure 1A). However, the heart weights of ob/ob mice fed on a RCD were either decreased or were not different than those of agematched controls (Figure 1B), whereas ob/ob and WT mice on HFD had similar heart weights until they reached 10 months old, at which point an increase in heart weight was observed in ob/ob mice compared with that of age-matched controls on a HFD (genotype and diet interaction: p < 0.0001). The lean mass of 10 month old animals on RCD and HFD were not altered (Figure 1C). High-fat feeding did not induce additional fat weight gain in ob/ob mice compared with that of ob/ob mice on the RCD (ob/ob RCD = 43.5 ± 2.6 g; ob/ob HFD = 48 ± 3.1 g, not significant), in contrast with WT mice, where a 2-fold increase in fat weight was observed across the two diets (WT RCD = 9.6 ± 1.2 g; WT HFD = 21 ± 1.1 g; p = 0.002). The diabetic status of the animals was assessed in terms of hyperinsulinemia and hyperglycemia in ob/ob mice and compared with that of age-matched controls on either RCD or HFD (Supporting Information Table S2). Glycosylated hemoglobin levels were also measured and showed that ob/ob mice on a HFD have significantly higher values compared to those of WT controls, demonstrating the long-term effect of having a raised blood glucose level (Figure 1F). In addition, cardiac fibrosis was established in the diabetic heart of ob/ob mice (Figure 1D,E). 2854


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on either RCD or HFD. There were no significant differences in protein levels of five complexes of ETC (Supporting Information Figure S3A−E). Therefore, the changes we observed in oxidative capacities were not due to the changes in expression level of the complexes. The expression of citrate synthase remained similar across all of the age groups on both diets, indicating that the changes we detected with mitochondrial respiratory capacity are not associated with overall changes in myocardial mitochondrial content (Supporting Information Figure S3F).

in the 2 and 4 month old groups (Figure 2H). HFD reduced RCR, suggesting an increase in mitochondrial uncoupling. Myocardial Gene and Protein Expression

To elucidate the molecular basis for the changes in myocardial mitochondrial function and cardiac metabolism associated with a HFD in ob/ob mice, we measured myocardial gene expression of 10 month old animals on either RCD or HFD. Two-way ANOVA demonstrated a significant effect of diet on the myocardial mRNA expression of all genes measured except NRF1. The HFD had the most profound effect on the expression level of PDK4, which led to a 10-fold increase in both ob/ob and WT mice. Under RCD, mRNA expression levels of fatty acid oxidation regulatory genes including PGC1α (p < 0.05), MCAD (p < 0.01), and LCAD (p < 0.01) were increased in the hearts of ob/ob mice (Figure 3). These fatty

Long-Chain Acylcarnitines Are Not Associated with Mitochondrial Dysfunction

Long-chain acylcarnitines have been suggested to play a role in the uncoupling of mitochondria in animal models of T2DM and obesity, so we investigated whether alterations in these species could explain the mitochondrial dysfunction detected in hearts from ob/ob mice on a HFD. LC−MS was used to quantify myocardial acylcarnitines from ob/ob and WT control mice fed either RCD or HFD to monitor the uptake of fatty acids as carnitine derivatives into mitochondria. The averaged concentrations of individual acylcarnitine species for each group are listed in Supporting Information Table S3. Two and 4 month old ob/ob and WT control mice on a RCD shared similar acylcarnitine profiles (Supporting Information Table S4). Medium- and long-chain acylcarnitine species were decreased in 14 month old ob/ob mice when compared to those in the WT animals on a RCD (Figure 4D,E). Similarly, acylcarnitine species including C6, C8, C20:2 were decreased in the 10 month old group for ob/ob mice (Supporting Information Tables S4 and S5). The decrease in acylcarnitine concentrations was most evident in the 14 month group, where the majority of acylcarnitine species were reduced in the ob/ob heart. Interestingly, unlike RCD, HFD induced an increase in medium- and long-chain acylcarnitines in the 2 month ob/ob group (Figure 4D,E), except for selected 3-hydroxylated acylcarnitines including C16:1-OH, C16-OH, and C18-OH, which were reduced in ob/ob mice compared to those in WT controls. However, the decrease in total acylcarnitine species detected in ob/ob mice on a RCD was detected at earlier time points (younger mice) for animals on a HFD, starting from the 4 month group when placed on a HFD. When comparing within a genotype (either ob/ob or WT), the effect of HFD on the same strain of animals was associated with increased medium- and long-chain acylcarnitines for a given age group (Figure 4D,E). Among the changes, the most dramatic increase was C8:1 acylcarnitine, which could be related to the specific composition of high-fat diet, which contains a higher percentage of monounsaturated fatty acid compared with that in RCD (Supporting Information Table S1). In contrast, the concentration of free carnitine and C2 and C3 carnitines was reduced in both WT and ob/ob mice in the 10 month group on a HFD. Overall, this suggests that, regardless of diet as the animals age, the ob/ob mouse heart reduces the transport of fatty acids into the mitochondria, as evidenced by the global decreases in carnitine species.

Figure 3. Relative mRNA expression of key transcripts involved in mitochondrial function. Myocardial mRNA from ob/ob and wild-type (WT) controls (n = 5/group) from the 10 month old group on a regular chow diet (RCD) and high-fat diet (HFD) was amplified by quantitative real-time PCR and normalized to 18s rRNA expression level. WT RCD values are normalized to 1 and shown as the dashed line. MCAD, medium-chain acyl CoA dehydrogenase; LCAD, longchain acyl CoA dehydrogenase; PGC1α, peroxisome proliferatoractivated receptor gamma coactivator 1 alpha; PPARα, peroxisome proliferator-activated receptor alpha; UCP2, uncoupling protein 2; UCP3, uncoupling protein 3; ANT1, adenine nucleotide translocator 1; NRF1, nuclear respiratory factor 1; SCD1, stearoyl-coenzyme A desaturase 1; All data are mean ± SEM. Statistical significance with two-way ANOVA is indicate by a, genotype-dependent; b, dietdependent; and c, interaction between genotype and diet. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with WT animals on a RCD. †, p < 0.05; ††, p < 0.01; †††, p < 0.001 compared with ob/ob mice on a HFD using Bonferroni’s posthoc analysis.

acid oxidation regulatory genes, together with PPARα, were all upregulated when the mice were switched to a HFD. Genes associated with mitochondrial uncoupling such as UCP2 and UCP3 were differentially expressed by HFD. UCP2 gene expression was decreased by HFD, whereas UCP3 expression was significantly induced. The expression of SCD1, involved in the biosynthesis of monounsaturated fatty acids, was reduced by HFD. Thus, in both ob/ob and WT mice, HFD had a similar effect on the transcription of key genes involved in fatty acid metabolism and thus did not explain the differences detected in mitochondrial function between the two genotypes. To determine whether defects in expression of specific complexes in ETC accounted for changes in mitochondrial respiratory capacity, we measured protein levels of five complexes using western blotting in 10 month old animals

Myocardial Triacylglycerol (TAG) Profile

To further investigate how age and HFD affect lipid metabolism, TAGs, which serve as an important intramyocardial lipid reserve, were measured using LC−MS, and the resultant data set was analyzed by OPLS-DA. All group 2855


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Figure 4. Effect of genotype and diet on myocardial acylcarnitine content under different ages. (A) Total acylcarnitine content. (B) Free carnitine content. Summed acylcarnitine content for short-chain species (C2−C14) (C), medium-chain species (C16−C18) (D), and long-chain species (C20) (E). All data are mean ± SEM. Statistical significance with two-way ANOVA is indicated by a, genotype-dependent; b, diet-dependent; and c, interaction between genotype and diet. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with WT animals on a RCD. †, p < 0.05; ††, p < 0.01; †††, p < 0.001 compared with ob/ob mice on a HFD using Bonferroni’s posthoc analysis. *, p < 0.05; **, p < 0.001 compared with WT animals on a RCD using Student’s t test.

comparisons were then validated using CV-ANOVA (Supporting Information Table S6), and all group comparisons passed cross-validation except for the 10 month old WT controls and ob/ob on a HFD. For both 2 and 4 month old animals, more TAGs were accumulated in the hearts of ob/ob mice on either RCD or HFD when compared with those in WT controls on the same diet. Although neither genotype nor diet had an effect on the TAG concentrations in 10 month old animals between genotypes, more TAGs were found in ob/ob hearts in the 14 month old group on the RCD (Figure 5A). When considering just the WT animals, myocardial TAG levels were unaffected by

either age or diet. Across the study, the TAG concentration was highest in the 2 month old ob/ob mice on either a RCD or HFD, and these TAGs decreased as the animals aged. For both 2 and 4 month old ob/ob mice, more TAGs were found in the RCD fed animals compared with those in the HFD fed animals. When examining the individual TAG species themselves, TAG species from 2 month old ob/ob mice had longer acyl chains but a similar number of double bonds compared with those in WT mice on a RCD (Figure 5C). However, the TAG composition changed dramatically in 14 month old ob/ob mice when compared with that in WT controls on a RCD, with an 2856


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Figure 5. Myocardial triacylglycerol (TAG) profile. (A) Total TAG content was measured in ob/ob mice and wild-type (WT) controls on a regular chow diet (RCD) or high-fat diet (HFD) across age groups (n = 6−10/group). The most perturbed metabolite ions between ob/ob and WT mice were selected by a combination of loading and VIP scores following OPLS-DA (except for the 10 month old group on a HFD, where all TAGs were used to calculate the short-chain/long-chain TAG ratio) and subsequently identified according to their m/z ratios, retention time, and fragmentation pattern, as measured by MS/MS. (B) Ratio of short-chain TAGs (sum of 52 carbon and less)/long-chain TAGs (sum of 60 carbon and more). The composition of identified TAG species with number of carbons is plotted against the number of double bonds in the 2 month old group (C) and 14 month old group (D) on a RCD. Data are mean ± SEM. Statistical significance with two-way ANOVA is indicated by a, genotype-dependent; b, diet-dependent; and c, interaction between genotype and diet. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with WT animals on a RCD. †, p < 0.05; ††, p < 0.01; †††, p < 0.001 compared with ob/ob mice on a HFD using Bonferroni’s posthoc analysis. *, p < 0.05 compared with WT animals on a RCD using a Students t test.

increases in acyl chain lengths with more double bonds in the TAGs from heart tissue from ob/ob mice. (Figure 5D). A similar observation was made when comparing the ratio of short-chain TAGs and long-chain TAGs for the two mouse strains (Figure 5B). In addition, we assessed the concentration of DAGs as potential lipotoxic intermediates that may mediate insulin resistance. The concentration of DAG (40:4) was significantly increased in 4 and 10 month old ob/ob mouse hearts when compared with that in WT mice on a HFD. Similarly, another DAG species, DAG (38:2), was significantly increased in 10 month old ob/ob mouse hearts when compared with that in WT mice on a HFD (data not shown). This data demonstrates

that both genotype and diet influence the intact lipid profile of the heart in these two mouse strains, and there is evidence of an increase in concentration of potentially lipotoxic DAG species. Oxidative Stress Increases on a HFD and in ob/ob Mice

To determine the oxidative stress in the heart associated with genotype or high-fat diet, we measured the myocardial content of 8-oxoguanine, GSH, and GSSG as markers of oxidative damage. ob/ob mice showed comparable concentrations of 8oxoguanine with those in WT when fed a RCD (Figure 6A). Mice on a HFD exhibited increased myocardial content of 8oxoguanine, indicating increased oxidative stress. Diet was the dominant effect across the three common age groups, as revealed by two-way ANOVA. Two month old ob/ob mice on a 2857


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Figure 6. Myocardial content of oxidative stress biomarkers. 8-Oxoguanine (A), reduced glutathione (GSH) (B), GSH/GSSG ratio (C), and total glutathione (D) in ob/ob and wild-type (WT) controls on a regular chow diet (RCD) or high-fat diet (HFD) (n = 6−10/group). All data are mean ± SEM. Statistical significance with two-way ANOVA is indicated by a, genotype-dependent; b, diet-dependent; and c, interaction between genotype and diet. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with WT animals on a RCD. †, p < 0.05; ††, p < 0.01; †††, p < 0.001 compared with ob/ ob mice on a HFD using Bonferroni’s posthoc analysis.

sufficient to match the increased influx of acyl-CoA provided from HFD, resulting in incomplete β-oxidation, which is evident from increased levels of medium- and long-chain acylcarnitines in the heart compared with those in mice on the RCD. The gene expression of PPARα and its downstream targets were increased upon HFD, further supporting the increased FAO observed upon HFD. To confirm that these changes in carnitines derivatives and gene expression were associated with changes in fatty acid oxidation, we examined the mitochondrial respiratory capacities for metabolizing pyruvate, glutamate/malate (complex I supported), and succinate/malate (complex II supported) in animals on RCD and HFD. Interestingly, HFD stimulated mitochondrial respiratory capacities for metabolizing pyruvate and glutamate in WT mice, but it failed to exert the same effect on ob/ob mice. This metabolic inflexibility in response to HFD results in mitochondrial dysfunction, in terms of decreased respiratory capacity, in 2 month old ob/ob mice when compared with that of age-matched controls on HFD. The primary site of metabolic defect appeared to be complex I, since both pyruvate and glutamate are comple I supported substrates. In addition, HFD led to significant oxidative stress, as indicated by oxidative stress biomarkers 8-oxoguanine and GSH. ob/ob mice on a HFD appeared to be under even more oxidative stress when compared with that of age-matched WT mice on the same diet, suggesting its potential role in the complex I defect observed in ob/ob mice under HFD. Our current finding suggests that mitochondrial function remained normal in the ob/ob heart on RCD until a relatively late stage, whereas others have shown decreased oxidative capacity in rodent models of diabetes.22,32,33 The reason behind

HFD were under more oxidative stress when compared with that of WT mice. There was an age-dependent decrease in GSH content and the GSH/GSSG ratio in animals on a RCD (Figure 6B,C). Similar to 8-oxoguanine, diet had a significant effect on GSH content and the GSH/GSSG ratio across the three age groups. Animals on a HFD showed decreased concentrations of GSH, associated with increased oxidative stress. However, the total glutathione content remained similar between ob/ob and WT mice on the two different diets across four age groups, except in 2 month old WT animals on a RCD, which exhibited a higher content of total glutathione compared to that in the rest of the groups (Figure 6D). These results indicate that both a high-fat diet and ob/ob status are associated with increased oxidative stress, providing a putative mechanism for the defects detected in complex I and the resultant alterations in myocardial metabolism.

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DISCUSSION The present study was conducted to investigate mitochondrial function and cardiac metabolism in the heart of ob/ob mice by following the changes across four different time points as the animals become more obese. First, our aim was to establish the degree of mitochondrial dysfunction that exists in the hearts of ob/ob mice by measuring the respiratory capacity in these mice and in WT mice as the animals age on a RCD. To our surprise, myocardial mitochondrial dysfunction was only evident in 14 month old ob/ob animals on a RCD. To extend this investigation, we further explored the effect of high-fat diet on mitochondrial function. An early switch to a HFD increased mitochondrial respiratory capacity for metabolizing fatty acids. It is worth noting that the enhanced respiratory capacity is not 2858


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due to increased intake of HFD or maladaptive processes associated with diabetes warrants further investigation. The gene expression of PGC1α, an important regulatory component of mitochondrial biogenesis and regulation of FAO, was upregulated in response to HFD feeding, in agreement with previous studies.39,44,45 Together with upregulation of PPARα and its downstream targets, we speculated that mitochondrial respiratory capacities would increase when mice were switched to a HFD. This was confirmed in WT animals, which showed enhanced mitochondrial respiratory capacities to metabolize pyruvate/malate and glutamate/malate in response to HFD across three age groups. However, this stimulatory effect was absent in ob/ob mice. This suggests that mitochondrial dysfunction becomes evident at a relatively early age in ob/ob mice when switching to a high-fat diet, even for a relatively short duration of the feeding period (3 weeks on a HFD compared with 14 months on a RCD). This indicates that ob/ob mice have a lower cardiac plasticity to adapt to fat metabolism, i.e., metabolic inflexibility, following high-fat exposure compared to that of WT mice. The level of citrate synthase remained similar in all animals on both diets, indicating that the number of mitochondria is not attributable to the changes in mitochondrial function. However, the HFD increased mitochondrial uncoupling in 2 month old WT and 4 month old ob/ob mice. The gene expression of UCP3 was also upregulated in 10 month old animals on HFD, as has been previously reported in other high-fat feeding studies in the ob/ ob mouse.36 This increase in FAO has been shown to increase the production of ROS by oversupplying reducing equivalents, especially at complex I,46 which, in turn, activates mitochondrial uncoupling proteins, possibly as an adaptive mechanism to reduce oxidative damage.47 The increased fatty acid oxidation was also evident in the hearts of ob/ob mice on a RCD, as the gene expression of MCAD and LCAD was increased. The accumulated ROS production due to this persistent increase in FAO, at some point in the etiology of obesity and diabetes, eventually causes damage at complex I of ETC, thus leading to a defect in this specific complex. In this particular model, this defect at complex I occurred at a relatively late stage in the animal’s life on a RCD. We demonstrated that animals on a HFD showed increased oxidative stress, as indicated by oxidative stress biomarkers 8-oxoguanine48 and GSH/ GSSG.49 Although the GSH/GSSG ratio that we measured is not physiologically accurate in terms of the cellular concentrations, as post mortem oxidation of GSH to GSSG was introduced during tissue extraction, we can assume that GSH is oxidized at the same rate for all groups, as the samples were prepared as a single batch. Therefore, the reduced GSH/ GSSH ratio may still be used as an indicator for increased oxidative stress. Furthermore, lipotoxic DAG species were increased in 4 and 10 month old ob/ob mice. Therefore, ob/ob mice on a HFD were under even more oxidative stress when compared with that of age-matched controls, suggesting that the HFD accelerates the mitochondrial dysfunction in ob/ob mice by generating increased ROS to damage complex I at a much higher rate, hence resulting in the lack of a stimulatory effect on mitochondrial respiratory capacities in ob/ob mice in response to HFD. Furthermore, glutamate and pyruvate are both complex I supported substrates, and the respiratory capacities of both substrates were decreased even in 2 month old ob/ob mice when fed a HFD for only 3 weeks.

such divergent observations may stem from variations in the protocols used to measure mitochondrial respiratory parameters. We measured mitochondrial respiratory capacity in saponin-permeabilized myofibers rather than in isolated mitochondria. The process of isolating mitochondria has been shown to induce artificial stress on mitochondria, which suggests that cardiac mitochondria in the diabetic state have an impaired ability to tolerate the stress of isolation or stress in general.34,35 Therefore, the defects in mitochondrial machinery reported by others using isolated mitochondria might not be evident in situ in our study. Saponin-permeabilized fibers may be more adaptive to stress, as the morphology and intracellular interactions were preserved. Furthermore, while some studies have measured mitochondrial function in saponin-permeabilized fibers at 30 °C, we conducted the experiments at 37 °C by following the protocol reported by Kuznetsov et al.29 Taken together, our approach is more physiologically relevant, providing better insight into mitochondrial functionality in the heart. We found that HFD increased state 2 and state 3 mitochondrial respiration rates in the presence of palmitoylcarnitine and malate in younger ob/ob and WT mice when they were switched to HFD at a relatively early stage. Consistent with other studies,36−39 it is evident that HFD leads to enhanced fatty acid flux through β-oxidation and the mitochondrial ETC. This metabolic flexibility is particularly evident in soleus and heart from 2 month old animals. The respiratory capacity of these animals for metabolizing fat was increased dramatically by high-fat feeding. Accumulation of TAGs within the diabetic heart of ob/ob mice was observed, which is in agreement with previous findings.24 Consistent with a previous study examining age-related changes in myocardial TAG content in ob/ob mice,40 the highest amount of TAG content was found in the youngest ob/ob mice on RCD, which at first seems to be counterintuitive. However, when correlating this with the respiratory capacities of these mice to metabolize fatty acids, 2 month old ob/ob mice on a RCD had the lowest state 3 respiration rates with palmitoyl-carnitine/malate as substrates; therefore, more lipids may accumulate in the heart due to limited fatty acid oxidation. As the animals age, the respiratory capacity increases, increasing the turnover of fatty acids, which led to a progressive decrease in myocardial TAG content. This mechanism may also explain the unchanged or reduced myocardial TAGs in ob/ob mice when they were switched to a HFD.41 Myocardial medium- and long-chain acylcarnitines, representing the byproducts of β-oxidation, were clearly increased in both ob/ ob and WT animals on HFD when compared with those in their age-matched groups on a RCD. This is consistent with previous reports,18,42,43 suggesting that, although high-fat feeding increased myocardial respiratory capacity for fat metabolism, the increased influx of acyl-CoA species into mitochondria associated with high-fat feeding still exceeds the enhanced capacity for complete β-oxidation in ob/ob mice. Interestingly, this increase in acylcarnitines was also evident in 2 month old ob/ob mice when compared with that in WT mice on a HFD, suggesting that 2 month old ob/ob hearts, which have been exposed to HFD for 3 weeks, were subject to increased substrate delivery as there were more byproducts of β-oxidation. This increased substrate availability in the heart of obese mice may underline the mitochondrial dysfunction that we observed upon high-fat feeding. Whether this dysfunction is 2859


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findings suggest that long-chain acylcarnitines are not responsible for myocardial mitochondrial dysfunction associated with either obesity or HFD per se and that increases in long-chain acylcarnitines are an early stage change, most likely reflecting increased substrate delivery.

Changes in L-carnitine and acylcarnitine species are of importance in cardiac metabolism, as the heart relies on longchain fatty acid oxidation as the primary source of ATP production.7,50 Carnitine transports fatty acids across the inner mitochondrial membrane for subsequent β-oxidation and removes accumulated toxic acyl-coenzyme A metabolites. Many studies have focused on the acylcarnitine profiles in skeletal muscle and plasma in the diabetic state.18,51−53 To date, only a few studies have been conducted to investigate acylcarnitine changes in the obese or diabetic heart, and still fewer studies have examined how these profiles change with age. For example, Su et al.19 and others54 demonstrated a dramatic increase in acylcarnitine species in the diabetic rat heart. The present study illustrated that the concentration of acylcarnitines remained similar at an early stage of obesity, whereas toward the late time points, these species were decreased in ob/ob mice across the chain lengths when compared with those in age-matched WT mice on the same diet, reflective of decreased acyl-CoA transport into the mitochondrial matrix. It is important to note that the inconsistency between the two studies may originate from the different animal models used. We used the insulin-resistant ob/ ob diabetic mouse model across a time course, whereas Su et al. used a streptozotocin-induced diabetic rat model. This difference suggests that the increase in acylcarnitine species is associated with an absence of insulin itself or an acute insult rather than imbalanced fuel utilization in the chronic insulinresistant/type 2 diabetic state. Indeed, the changes in acylcarnitines are reversible upon insulin treatment in streptozotocin-induced diabetic rats.19 The reason for this reduction in many acylcarnitine species remains unknown, but it could be speculated to be due to decreased activity of CPT1 activity, probably due to malonyl-CoA inhibition, which has previously been reported.55,56 Son et al.57 crossed mice with dilated cardiomyopathy due to PPARγ overexpression with PPARα-deficient mice, and the resultant mouse model showed rescued cardiac dysfunction and improved survival. The improved cardiac function was correlated with decreased cardiomyocyte total acylcarnitine levels; therefore, the authors suggested a potential role of acylcarnitine as a toxic intermediate for cardiolipotoxicity. The most important difference in the two study designs is that we looked at the myocardial acylcarnitine levels across a time course, whereas Son et al. measured functional and biochemical data in one single time point in 3 month old mice. In the 10 and 14 month ob/ob mice, we clearly demonstrated a dramatic decrease in many acylcarnitine species, which, to our knowledge, has not been measured before in any obese models of similar ages. In addition, any metabolic changes associated with mitochondrial machinery must be considered in the context of energy demand, as suggested by Muoio and Neufer.58 ob/ob mice become less active as they become older and more obese, and with the lower activity, they have less energy demands in the form of β-oxidation for contractile activity. Indeed, a reduction in acylcarnitines has been reported recently as a biomarker for aging and health span.59 In that study, it was shown that the medium- and long-chain acylcarnitines were decreased in the plasma of 24 month old C57BL/6J mice. In the present study, this age-related decrease in acylcarnitine levels in the diabetic heart is accelerated when the mice were placed on a HFD, with acylcarnitine levels starting to decrease in the 4 month old ob/ob mouse heart compared with that in WT mice on the same HFD. Our

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CONCLUSIONS In summary, our results demonstrated that myocardial mitochondria in ob/ob mice on a RCD maintained similar respiratory capacity when compared with that of their agematched controls until a very late stage in aging. HFD stimulated mitochondrial respiratory capacity in WT animals, but it did not in ob/ob mice, thus leading to accelerated mitochondrial abnormalities in 2 month old ob/ob mice. This metabolic defect is associated with changes in complex I of the ETC, which is highly susceptible to ROS-induced oxidative damages following persistently elevated FAO. Another novel finding of this study is that there was an age-dependent decrease in myocardial acylcarnitine concentrations in ob/ob mice fed either RCD or HFD compared with those in the corresponding WT mice, suggesting that the accumulation of long-chain acylcarnitines is not responsible for mitochondrial dysfunction, as had been previously suggested.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1: Schematic of the study design. Figure S2: Mitochondrial respiratory capacity of metabolizing fatty acids in saponin-permeabilized cardiac fibers and soleus fibers from ob/ob mice and wild-type (WT) controls on a regular chow diet. Figure S3: Summary of protein expressions of complex I, II, III, IV, and V of the electron transport chain and citrate synthase in 10 month old animals fed either regular chow diet (RCD) or high-fat diet (HFD). Table S1: The composition of total fatty acids from regular chow diet (RCD) and high-fat diet (HFD) measured by GC−MS. Table S2: Plasma insulin and glucose levels in ob/ob and wild-type mice. Table S3: Semiquantitative analysis of acyl-carnitines by LC−MS. Table S4: Biochemical names of acylcarnitines. Table S5: Genotypeand diet-induced changes of acylcarnitines profiles under different ages. Table S6: To examine lipidomic changes, LC− MS data was processed using OPLS-DA, CV-ANOVA was applied to ensure that these models were not overfitted, and the statistics for each model are displayed in this table. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00128.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: jules.griffi[email protected]. Tel.: +44 (0)1223 437503. Author Contributions

X.W., A.J.M., and J.L.G. conceived the project. X.W. and J.A.W. performed experiments and data analysis. X.W. and J.L.G. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is funded by grants from the UK Medical Research Council (UD99999906) and the Biotechnology and Biological 2860


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Sciences Research Council (BBSRC). We are grateful to Dr. Tom Ashmore and James Horscroft from University of Cambridge for their expert technical assistance.

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ABBREVIATIONS RCD, regular chow diet; HFD, high-fat diet; LC−MS, liquid chromatography−mass spectrometry; T2DM, type 2 diabetes; ROS, reactive oxygen species; DEXA, dual-energy X-ray absorptiometry; MRM, multiple reaction monitoring; ETC, electron transport chain; TAG, triacylglycerol; DAG, diacylglycerol

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