Existing techniques for the non-invasive in vivo study of dynamic changes in skeletal muscle metabolism are subject to several limitations, for example, poor signal-to-noise ratios which result in long scan times and low temporal resolution. Hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy (HP-MRS) allows the real-time visualization of in vivo metabolic processes and has been used extensively to study cardiac metabolism, but has not resolved oxidative phosphorylation in contracting skeletal muscle. Combining HP-MRS with an in vivo muscle hindlimb electrical stimulation protocol that modelled voluntary exercise to exhaustion allows the simultaneous real-time assessment of both metabolism and function. The aim of this work was to validate the sensitivity of the method by assessing pyruvate dehydrogenase (PDH) flux in resting vs. working muscle: measuring the production of bicarbonate (H13CO3 -), a byproduct of the PDH-catalysed conversion of [1-13C]pyruvate to acetyl-CoA. Mice (n = 6) underwent two hyperpolarized [1-13C]pyruvate injections with 13C MR spectra obtained from the gastrocnemius muscle to measure conversion of pyruvate to lactate and bicarbonate, one before the stimulation protocol with the muscle in a resting state and one during the stimulation protocol. The muscle force generated during stimulation was also measured, and 13C MRS undertaken at a point of ~50% fatigue. We observed an increase in the bicarbonate/pyruvate ratio by a factor of ~1.5×, in the lactate/pyruvate ratio of ~2.7×, together with an increase in total carbon (~1.5×) that we attribute to perfusion. This demonstrates profound differences in metabolism between the resting and exercising states. These data therefore serve as preliminary evidence that hyperpolarized 13C MRS is an effective in vivo probe of PDH flux in exercising skeletal muscle and could be used in future studies to examine changes in muscle metabolism in states of disease and altered nutrition.

PurposeIn vivo MRS is often characterized by a spectral signal-to-noise ratio (SNR) that varies highly between experiments. A common design for spectroscopic studies is to compare the ratio of two spectral peak amplitudes between groups, e.g. individual PCr/-ATP ratios in P-31-MRS. The uncertainty on this ratio is often neglected. We wished to explore this assumption. TheoryThe canonical theory for the propagation of uncertainty on the ratio of two spectral peaks and its incorporation in the Frequentist hypothesis testing framework by weighted averaging is presented. MethodsTwo retrospective re-analyses of studies comparing spectral peak ratios and one prospective simulation were performed using both the weighted and unweighted methods. ResultsIt was found that propagating uncertainty correctly improved statistical power in all cases considered, which could be used to reduce the number of subjects required to perform an MR study. ConclusionThe variability of in vivo spectroscopy data is often accounted for by requiring it to meet an SNR threshold. A theoretically sound propagation of the variable uncertainty caused by quantifying spectra of differing SNR is therefore likely to improve the power of in vivo spectroscopy studies. Magn Reson Med 78:2082-2094, 2017. (c) 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Ketone bodies are the most energy-efficient fuel and yield more ATP per mole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high-fat, low carbohydrate diets has been used for the treatment of drug-refractory epilepsy and for neurodegenerative diseases, such as Parkinson's disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)-3-hydroxybutyl (R)-3-hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a treadmill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palm oil (P < 0.05). Ketone-fed rats completed an 8-arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts from rats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [P-31]-NMR spectroscopy. The novel ketone diet, therefore, improved physical performance and cognitive function in rats, and its energy-sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.-Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M. K., Atherton, H. J., Schroeder, M. A., Deacon, R. M. J., Kashiwaya, Y., King, M. T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance.

AIMS Hypertrophic cardiomyopathy (HCM) is the commonest cause of sudden cardiac death in the young, with an excess of exercise-related deaths. The HCM sarcomere mutations increase the energy cost of contraction and impaired resting cardiac energetics has been documented by measurement of phosphocreatine/ATP (PCr/ATP) using (31)Phosphorus MR Spectroscopy ((31)P MRS). We hypothesized that cardiac energetics are further impaired acutely during exercise in HCM and that this would have important functional consequences. METHODS AND RESULTS (31)P MRS was performed in 35 HCM patients and 20 age- and gender-matched normal volunteers at rest and during leg exercise with 2.5 kg ankle weights. Peak left-ventricular filling rates (PFRs) and myocardial perfusion reserve (MPRI) were calculated during adenosine stress. Resting PCr/ATP was significantly reduced in HCM (HCM: 1.71 ± 0.35, normal 2.14 ± 0.35 P < 0.0001). During exercise, there was a further reduction in PCr/ATP in HCM (1.56 ± 0.29, P = 0.02 compared with rest) but not in normals (2.16 ± 0.26, P = 0.98 compared with rest). There was no correlation between PCr/ATP reduction and cardiac mass, wall thickness, MPRI, or late-gadolinium enhancement. PFR and PCr/ATP were significantly correlated at rest (r = 0.48, P = 0.02) and stress (r = 0.53, P = 0.01). CONCLUSION During exercise, the pre-existing energetic deficit in HCM is further exacerbated independent of hypertrophy, perfusion reserve, or degree of fibrosis. This is in keeping with the change at the myofilament level. We offer a potential explanation for exercise-related diastolic dysfunction in HCM.

Background— Whether the myocardium in nonischemic heart failure experiences oxygen limitation remains a long-standing controversy. We addressed this question in patients with dilated cardiomyopathy (DCM) using a dual approach. First, we tested the changes in myocardial oxygenation between rest and stress states, using oxygenation-sensitive cardiovascular magnetic resonance. Second, we sought to assess the functional consequences of oxygen limitation at rest by measuring myocardial energetics before and after short-term oxygen supplementation. Methods and Results— Twenty-six subjects (14 DCM and 12 normal) underwent cardiac magnetic resonance imaging at 3 Tesla to assess cardiac volumes, function, oxygenation, and first-pass perfusion (0.03 mmol/kg Gd-DTPA bolus) at stress and rest (4–6 minutes IV adenosine, 140 μg/kg per minute). Signal intensity change (SIΔ) and myocardial perfusion reserve index (MPRI) were measured from oxygenation and perfusion images, respectively. Furthermore, the effect of oxygen supplementation on resting myocardial energy metabolism was tested using 31 P MR spectroscopy, measuring PCr/ATP ratios in both groups at baseline and after 4 hours of oxygen via facemask in the DCM group. During stress, there were equivalent rises in rate pressure product in both groups (DCM, 76±15% and normal, 79±9%; P =0.84). MPRI was significantly reduced in DCM (1.51±0.11 versus normal 1.86±0.10; P =0.03). However, there was no difference in oxygenation between groups: SIΔ in DCM 17±3% versus normal 20±2% ( P =0.38). Furthermore, at a left ventricular segmental level, there was no correlation between oxygenation-sensitive SIΔ and MPRI ( R =0.06; P =0.43). Resting PCr/ATP was reduced in DCM (1.66±0.07 versus normal 2.12±0.06; P =0.002). With oxygen supplementation, there was no change in PCr/ATP (1.61±0.08; P =0.58; Δ=0.04±0.05). There was also no effect of oxygen on systolic function (ejection fraction pre oxygen, 34±1%; post oxygen, 36±2%; P =0.46; Δ 2±1%). Conclusions— Our results demonstrate dissociation between microvascular dysfunction and oxygenation in DCM, suggesting that the impairment of perfusion is not sufficient to cause deoxygenation during stress. Cardiac energetics are unaffected by oxygen supplementation, indicating the absence of relevant myocardial hypoxia at rest. Our study suggests that novel treatments for nonischemic heart failure should focus on efforts to directly target cardiomyocyte function and metabolism rather than oxygen delivery and microvascular function.

Background Hypertension (HPT) is an important risk factor for heart disease worldwide. It is already established that in hypertension there is an abnormal oxygenation response to stress (by exploiting the paramagnetic properties of deoxyhemoglobin, blood oxygen level-dependent, BOLD). Additionally, abnormal resting cardiac energetics (phosphocreatine/adenosine triphosphate, PCr/ATP, as measured by 31Phosphorus MR Spectroscopy, 31P MRS) has been reported. However, it is not known if this energetic profile worsens with the increasing energy demand of exercise, and if so, if the blunted Oxygen supply response may contribute to this. Understanding this relation can lend new insights into the pathophysiology and management of hypertensive heart disease. We hypothesized that cardiac energetic are abnormal at rest and are further impaired during acute exercise in HPT, and that this impairment is related to abnormal oxygenation during stress.

In the current work, we labelled human hematopoietic stem cells with polymeric nanoparticles (NPs) that can be tracked by Magnetic Resonance Imaging (MRI) and studied their effect on cell metabolism, proliferation, secretomics, genomics and differentiation. We showed that NPs had no effect on the stem cell differentiation program but affected their paracrine activity.

Herein, we report the use of biodegradable nanoparticles (NPs) containing perfluoro-1,5-crown ether (PFCE), a fluorine-based compound (NP170-PFCE) with the capacity to track cells in vivo by magnetic ressonance imaging (MRI) and efficiently release miRNA. NP170-PFCE complexed with miRNAs accumulate whitin the cell's endolysosomal compartment and interact with higher frequency with argonaute2 (Ago2) and GW182 proteins, which are involved in the biological action of miRNAs, than commercial complexes formed by commercial reagents and miRNA, which in turn accumulate in the cell cytoplasm. The release of miRNA132 (miR132) from the NPs increased 3-fold the survival of endothelial cells (ECs) transplanted in vivo and 3.5-fold the blood perfusion in ischemic limbs relatively to control.

Background 31P MRS is an important tool for the measurement of in vivo energetics via the ratio PCr/ATP. Low signal-to-noise (31P molar receptivity is 1/14 that of 1H) coupled with low concentration of high-energy phosphates can result in high measurement variability. 31P MRS of human heart often contains signals originating from blood i.e. 2,3DPG and ATP. Measurement of myocardial PCr/ATP thus requires subtraction of blood derived ATP signal. The aim of this work was to improve blood correction by applying prior knowledge (PK) of molecular spectral parameters to the 2,3DPG signal. We demonstrate that accurate spectral fitting of 2,3DPG prevents proliferation of fitting errors through blood correction, resulting in improved accuracy of PCr/ATP measurement. Methods

Using P-MRS to Explore the Effects of Iron Deficiency on Murine Skeletal Muscle Function and Metabolism During Exercise M. Kate Curtis, Lowri E. Cochlin, Mark A. Cole, David P. O'Neill, Michael S. Dodd, Damian J. Tyler, Kieran Clarke, and Peter A. Robbins Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom, Department of Physiology, Anatomy and Genetics, PulseTeq Ltd, Oxford, United Kingdom, Department of Life Sciences, University of Nottingham Medical School, Nottingham, United Kingdom, Oxford Centre for Clinical Magnetic Resonance Research, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom

Neuronal glucose uptake was thought to be independent of insulin, being facilitated by glucose transporters GLUT1 and GLUT3, which do not require insulin signaling. However, it is now known that components of the insulin-mediated glucose uptake pathway, including neuronal insulin synthesis and the insulin-dependent glucose transporter GLUT4, are present in brain tissue, particularly in the hippocampus. There is considerable recent evidence that insulin signaling is crucial to optimal hippocampal function. The physiological basis, however, is not clear. We propose that while noninsulin-dependent GLUT1 and GLUT3 transport is adequate for resting needs, the surge in energy use during sustained cognitive activity requires the additional induction of insulin-signaled GLUT4 transport. We studied hippocampal high-energy phosphate metabolism in eight healthy volunteers, using a lipid infusion protocol to inhibit insulin signaling. Contrary to conventional wisdom, it is now known that free fatty acids do cross the blood-brain barrier in significant amounts. Energy metabolism within the hippocampus was assessed during standardized cognitive activity. (31)Phosphorus magnetic resonance spectroscopy was used to determine the phosphocreatine (PCr)-to-adenosine triphosphate (ATP) ratio. This ratio reflects cellular energy production in relation to concurrent cellular energy expenditure. With lipid infusion, the ratio was significantly reduced during cognitive activity (PCr/ATP 1.0 ± 0.4 compared with 1.4 ± 0.4 before infusion, P = 0.01). Without lipid infusion, there was no reduction in the ratio during cognitive activity (PCr/ATP 1.5 ± 0.3 compared with 1.4 ± 0.4, P = 0.57). This provides supporting evidence for a physiological role for insulin signaling in facilitating increased neuronal glucose uptake during sustained cognitive activity. Loss of this response, as may occur in type 2 diabetes, would lead to insufficient neuronal energy availability during cognitive activity.

Objective To determine the effects of short-term exercise training on cardiac function and metabolism during rest and physical exercise in patients with heart failure from dilated cardiomyopathy (DCM).Design Patients with DCM (n=15, age 58 +/- 2 years, NYHA class I-III) were studied before and after 8 weeks of cycle exercise for 20 min, five times per week.Main outcome measures Cardiac volumes, function and high energy phosphate metabolism were measured using cardiac magnetic resonance during rest and 7 min of acute physical exercise (leg-raising).Results At baseline, average left ventricular ejection fraction (LVEF) was 38 +/- 3%, which did not alter during 7 min of exercise. After 8 weeks of home exercise training, there was a 16% improvement in resting LVEF to 44 +/- 3% (p<0.01). Training caused a further 20% improvement in LVEF (p<0.05) during acute physical exercise. There was a negative correlation between subjects' baseline level of exercise and change in LVEF (r=-0.67, p<0.05), with sedentary patients having the greatest improvement. Cardiac phosphocreatine (PCr) to ATP ratio did not change during acute physical exercise or after exercise training.Conclusions Short-term exercise training improves resting LVEF and LVEF with acute physical exercise with sedentary patients having the greatest improvement. There were no changes in cardiac PCr to ATP, before or after exercise training, suggesting that the improved cardiac function was not caused by improved energetics. Therefore, peripheral factors likely underlie the improved cardiac function in patients with heart failure after short-term exercise.

Stem-cell therapy holds great promise for treatment of disease. However, optimal stem-cell type, number, route, timing of administration have yet to be quantitatively determined. Non-invasive methods of monitoring cell retention are therefore in demand. Fluorine-MRI offers the facility for unambiguous localisation of fluorine-labelled cells as they migrate in-vivo. We achieve high fluorine sensitivity by simple modification to a standard MRI-coil. We monitored stem-cell retention in a rat model of myocardial infarction (MI), for 13 days after administration of fluorine-labelled stem-cells Methods MRI-coil: 65mm diameter, low-pass birdcage fitted with two RF-shields; 85mm removable, 79mm fixed. Inner shield removal causes the resonant frequency window to shift from proton to fluorine. Labelled cell preparation and administration: cardiac-derived stem-cells were incubated with polylactic-co-glycolic acid (PLGA) nanoparticles containing perfluoro(crown)ether (PFCE) and fluoresceinamine. Five million labelled cells were administered immediately post MI, induced by occlusion of the left descending coronary artery. MRI at 1, 5, 7 and 13 days post MI, using a 7 Tesla magnet. Cardiac-gated gradient-echo cardiac long-axis and short-axis stacks: 1 proton average (Texp~25s), 15 fluorine averages, (Texp~380s) TE=2.9ms, FA=Ernst angle (TR=RRinterval~200ms, T1(PFCE)=1090±15ms in-vivo), FOV=50x50x1.5mm, matrix=128x128. Results In-vivo, fluorine signal was easily located for setup, and high-resolution fluorine images showed signal co-registered with infarction and operation wound. At day 13, hearts were excised for histological verification of MRI findings. Conclusion We demonstrate high proton/fluorine sensitivity using a standard MRI-coil, modified for inductance tuning to fluorine. Labelled cardiac stem-cells were imaged for 13 days post MI. Whole-heart proton and fluorine image stacks of matched resolution were acquired within one-hour scan-time.

Background— Carnitine acetyltransferase catalyzes the reversible conversion of acetyl-coenzyme A (CoA) into acetylcarnitine. The aim of this study was to use the metabolic tracer hyperpolarized [2- 13 C]pyruvate with magnetic resonance spectroscopy to determine whether carnitine acetyltransferase facilitates carbohydrate oxidation in the heart. Methods and Results— Ex vivo, following hyperpolarized [2- 13 C]pyruvate infusion, the [1- 13 C]acetylcarnitine resonance was saturated with a radiofrequency pulse, and the effect of this saturation on [1- 13 C]citrate and [5- 13 C]glutamate was observed. In vivo, [2- 13 C]pyruvate was infused into 3 groups of fed male Wistar rats: (1) controls, (2) rats in which dichloroacetate enhanced pyruvate dehydrogenase flux, and (3) rats in which dobutamine elevated cardiac workload. In the perfused heart, [1- 13 C]acetylcarnitine saturation reduced the [1- 13 C]citrate and [5- 13 C]glutamate resonances by 63% and 51%, respectively, indicating a rapid exchange between pyruvate-derived acetyl-CoA and the acetylcarnitine pool. In vivo, dichloroacetate increased the rate of [1- 13 C]acetylcarnitine production by 35% and increased the overall acetylcarnitine pool size by 33%. Dobutamine decreased the rate of [1- 13 C]acetylcarnitine production by 37% and decreased the acetylcarnitine pool size by 40%. Conclusions— Hyperpolarized 13 C magnetic resonance spectroscopy has revealed that acetylcarnitine provides a route of disposal for excess acetyl-CoA and a means to replenish acetyl-CoA when cardiac workload is increased. Cycling of acetyl-CoA through acetylcarnitine appears key to matching instantaneous acetyl-CoA supply with metabolic demand, thereby helping to balance myocardial substrate supply and contractile function.

This work demonstrates that cardiac energetcics is further impaired during exercise in hypertrophic cardiomyopathy.This may be a possible reason for exercise related death in HCM.

(R)-3-Hydroxybutyl (R)-3-hydroxybutyrate (ketone monoester) has been developed as an oral source of ketones, which may be utilized for energy. In a 28-day toxicity study, Crl:WI (Wistar) rats received diets containing, as 30% of the calories, ketone monoester (12 and 15g/kg body weight/day for male and female rats, respectively). Control groups received either carbohydrate- or fat-based diets. Rats in the test group consumed less feed and gained less weight than control animals; similar findings have been documented in studies of ketogenic diets. Between-group differences were noted in selected hematology, coagulation, and serum chemistry parameters; however, values were within normal physiological ranges and/or were not accompanied by other changes indicative of toxicity. Upon gross and microscopic evaluation, there were no findings associated with the ketone monoester. In a developmental toxicity study, pregnant Crl:WI (Han) rats were administered 2g/kg body weight/day ketone monoester or water (control) via gavage on days 6 through 20 of gestation. No Caesarean-sectioning or litter parameters were affected by the test article. The overall incidence of fetal alterations was higher in the test group; however, there were no specific alterations attributable to the test substance. The results of these studies support the safety of ketone monoester.

Magnetic resonance imaging (MRI) is a powerful non-invasive imaging platform which, in combination with contrast agents, can be used to track stem cell fate after transplantation. Here, we developed a novel formulation of nanoparticles (NPs), based on poly(lactic-co-glycolic acid) (PLGA) which is a biocompatible polymer approved for human use. PLGA NPs are bioluminescent and contain fluorine, enabling cell tracking using optical imaging or 19F MRI, and can be used as vectors for microRNA (miR) delivery. The PLGA NPs (average diameter 170nm, positive (7.0±1.7 mV) zeta potential) successfully labelled HUVECs (63.18±0.96%) and mononuclear cells (MNCs; 80.75±1.21%) with only 10% of cells dying after 24 h of NP exposure. Labelled MNCs, administered to the infarcted rat heart, could be detected in vivo using 19F MRI. HUVECs were incubated with miRNA-complexed NPs to deliver pro-survival and pro-angiogenic miRs (miR-132, miR-424 and antagomir-92a (amiR-92a)) and cultured on matrigel to assess their capacity to form vascular networks. Cells transfected with miR-132 NPs and miR-424 NPs yielded vascular networks with greater tube length and more branching points than those transfected with amiR-92a NPs. Furthermore, NPs were more efficient at transfection than the commercially available reagent, SIPORT. Endothelial cells labelled with miR-132 NPs were administered to rodent models of hind limb ischemia. Labelled cells could be tracked in vivo using 19F MRI, improved cell survival and induced neo-angiogenesis (measured using ultrasound and histology). This work shows for the first time a NP formulation that can be used both to track cells and successfully deliver miRs.

Background— Noncontrast magnetic resonance T1 mapping reflects a composite of both intra- and extracellular signal. We hypothesized that noncontrast T1 mapping can characterize the myocardium beyond that achieved by the well-established late gadolinium enhancement (LGE) technique (which detects focal fibrosis) in both hypertrophic (HCM) and dilated (DCM) cardiomyopathy, by detecting both diffuse and focal fibrosis. Methods and Results— Subjects underwent Cardiovascular Magnetic Resonance imaging at 3T (28 HCM, 18 DCM, and 12 normals). Matching short-axis slices were acquired for cine, T1 mapping, and LGE imaging (0.1 mmol/kg). Circumferential strain was measured in the midventricular slice, and 31 P magnetic resonance spectroscopy was acquired for the septum of the midventricular slice. Mean T1 relaxation time was increased in HCM and DCM (HCM 1209±28 ms, DCM 1225±42 ms, normal 1178±13 ms, P <0.05). There was a weak correlation between mean T1 and LGE ( r =0.32, P <0.001). T1 values were higher in segments with LGE than in those without (HCM with LGE 1228±41 ms versus no LGE 1192±79 ms, P <0.01; DCM with LGE 1254±73 ms versus no LGE 1217±52 ms, P <0.01). However, in both HCM and DCM, even in segments unaffected by LGE, T1 values were significantly higher than normal ( P <0.01). T1 values correlated with disease severity, being increased as wall thickness increased in HCM; conversely, in DCM, T1 values were highest in the thinnest myocardial segments. T1 values also correlated significantly with circumferential strain ( r =0.42, P <0.01). Interestingly, this correlation remained statistically significant even for the slices without LGE ( r =0.56, P =0.04). Finally, there was also a statistically significant negative correlation between T1 values and phosphocreatine/adenosine triphosphate ratios ( r =−0.59, P <0.0001). Conclusions— In HCM and DCM, noncontrast T1 mapping detects underlying disease processes beyond those assessed by LGE in relatively low-risk individuals.

Short-term consumption of a high-fat diet impairs exercise capacity in both rats and humans, and increases expression of the mitochondrial uncoupling protein, UCP3, in rodent cardiac and skeletal muscle via activation of the transcription factor, peroxisome proliferator-activated receptor α (PPARα). Unlike long-chain fatty acids however, medium-chain fatty acids do not activate PPARα and do not increase muscle UCP3 expression. We therefore investigated exercise performance and cardiac mitochondrial function in rats fed a chow diet (7.5% kcal from fat), a long-chain triglyceride (LCT) rich diet (46% kcal from LCTs) or a medium-chain triglyceride (MCT) rich diet (46% kcal from MCTs). Rats fed the LCT-rich diet for 15 days ran 55% less far than they did at baseline, whereas rats fed the chow or MCT-rich diets neither improved nor worsened in their exercise capacities. Moreover, consumption of an LCT-rich diet increased cardiac UCP3 expression by 35% and decreased oxidative phosphorylation efficiency, whereas consumption of the MCT-rich diet altered neither UCP3 expression nor oxidative phosphorylation efficiency. Our results suggest that the negative effects of short-term high-fat feeding on exercise performance are predominantly mediated by long-chain rather than medium-chain fatty acids, possibly via PPARα-dependent upregulation of UCP3.