Scand J Med Sci Sports 2014: ••: ••–•• doi: 10.1111/sms.12252

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

The effect of high-intensity training on mitochondrial fat oxidation in skeletal muscle and subcutaneous adipose tissue S. Larsen, J. H. Danielsen, S. D. Søndergård, D. Søgaard, A. Vigelsoe, R. Dybboe, S. Skaaby, F. Dela, J. W. Helge Xlab, Center for Healthy Aging, Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Corresponding author: Steen Larsen, DMSci, Center for Healthy Aging, Department of Biomedical Sciences, Faculty of Health Sciences, Copenhagen University, Blegdamsvej 3b, 2200 Copenhagen N, Denmark. Tel: +45 21 15 19 24, Fax: +45 35 32 74 20, E-mail: [email protected] Accepted for publication 16 April 2014

High-intensity interval training (HIT) is known to increase mitochondrial content in a similar way as endurance training [60–90% of maximal oxygen uptake (VO2peak)]. Whether HIT increases the mitochondria’s ability to oxidize lipids is currently debated. We investigated the effect of HIT on mitochondrial fat oxidation in skeletal muscle and adipose tissue. Mitochondrial oxidative phosphorylation (OXPHOS) capacity, mitochondrial substrate sensitivity (Kmapp), and mitochondrial content were measured in skeletal muscle and adipose tissue in healthy overweight subjects before and after 6 weeks of HIT (three times per week at 298 ± 21 W). HIT significantly increased VO2peak from 2.9 ± 0.2 to 3.1 ± 0.2 L/min.

No differences were seen in maximal fat oxidation in either skeletal muscle or adipose tissue. Kmapp for octanoyl carnitine or palmitoyl carnitine were similar after training in skeletal muscle and adipose tissue. Maximal OXPHOS capacity with complex I- and II-linked substrates was increased after training in skeletal muscle but not in adipose tissue. In conclusion, 6 weeks of HIT increased VO2peak. Mitochondrial content and mitochondrial OXPHOS capacity were increased in skeletal muscle, but not in adipose tissue. Furthermore, mitochondrial fat oxidation was not improved in either skeletal muscle or adipose tissue.

It is well known that regular endurance training increases mitochondrial fat oxidation in both healthy subjects (Pesta et al., 2011) and patients with type 2 diabetes (Hey-Mogensen et al., 2010) as well as fat oxidation during whole-body exercise (Mogensen et al., 2009). In the last 5–10 years, a renewed interest has been directed toward a different training method, where highintensity training is performed for shorter durations. It has been reported that high-intensity interval training (HIT) leads to similar metabolic adaptations compared with regular endurance training when it comes to improvement in maximal oxygen uptake and increase in mitochondrial content in human skeletal muscle (Gibala et al., 2006; Burgomaster et al., 2008). The majority of studies investigating HIT have reported adaptations in both the oxidative and glycolytic pathway (MacDougall et al., 1998; Burgomaster et al., 2005, 2007, 2008; Gibala et al., 2006; Perry et al., 2007, 2008; Jacobs et al., 2013). When it comes to the adaptations of HIT on lipid metabolism, the literature is sparse and inconsistent (MacDougall et al., 1998; Burgomaster et al., 2008; Perry et al., 2008). The explanation behind the different results may be ascribed to different training conditions (intensity, duration, and energy expenditure). Studies have reported an increased activity of β-hydroxyacyl-CoA-dehydrogenase (HAD;

Burgomaster et al., 2008; Perry et al., 2008), but no consensus have been reached (MacDougall et al., 1998). In a study by Perry and colleagues, an increased protein content of fatty acid transport proteins (FAT/CD36 and FABPpm) with no change in hormone-sensitive lipase content (HSL) was observed after HIT (Perry et al., 2008), which is in contrast to a study by Burgomaster and colleagues, where fatty acid transport proteins (FAT/ CD36 and FABPpm) were unchanged after training (Burgomaster et al., 2007). These findings indicate that the proteins responsible for the transport of lipids and the enzymes responsible for mobilization of stored lipids for β-oxidation in the mitochondria are not always improved by HIT, and that the intensity of the training seems to be important for the outcome. Perilipin 2 and 5 regulate intramuscular triglyceride (IMTG) lipolytic rates, with perilipin 5 (PLIN5) being the most important. PLIN5 expression is increased in athletes compared to controls (Amati et al., 2011), and it has been reported that endurance and sprint interval training increases PLIN5 expression (Shepherd et al., 2013). PLIN5 expression has also been linked to insulin resistance (Shepherd et al., 2013), but not all studies support this (Vigelsø et al., 2013). A study by Jacobs and colleagues investigated mitochondrial respiratory capacity after HIT, and reported an

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Larsen et al. increased mitochondrial fat oxidative capacity (using a medium-chain fatty acid, octanoyl carnitine) after 2 weeks of HIT (Jacobs et al., 2013). They also reported an increased maximal mitochondrial oxidative phosphorylation (OXPHOS) capacity, with no difference in intrinsic mitochondrial OXPHOS capacity [mitochondrial OXPHOS capacity/cytochrome C oxidase (COX) activity (measure of mitochondrial content)]. Mitochondrial substrate sensitivity for medium- and long-chain fatty acids has not been investigated in a longitudinal study before. One study reported no differences in mitochondrial substrate sensitivity for medium-chain fatty acids between lean and obese subjects that differed in physical fitness (Larsen et al., 2011); the same group reported no differences between young and middle-aged subjects with the same physical fitness level (Larsen et al., 2012a). The literature on mitochondrial measurements in adipose tissue is sparse, especially when it comes to training intervention studies. One study investigated 10 weeks of swim training in rats, and found an increased mitochondrial content (Stallknecht et al., 1991), whereas a study conducted in humans found no difference in citrate synthase (CS) activity after 10 days of training (Camera et al., 2010). To our knowledge, only one study has investigated mitochondrial OXPHOS capacity in human adipose tissue (visceral and subcutaneous abdominal adipose tissue) (Kraunsoe et al., 2010). Recently, focus has been placed on the browning phenomena of white adipose tissue, where a transition from a white adipocyte phenotype where storage is the primary function to a more oxidative phenotype (brown adipose tissue) takes place (Smorlesi et al., 2012). It has been reported that cold exposure (Rosen & Spiegelman, 2014) as well as physical exercise (Xu et al., 2011a) are responsible for the conversation of adipose tissue from white to brown. Furthermore, it has been reported that mitochondrial content is higher in brown compared with white adipose tissue (Xu et al., 2011b). The purpose of this study was to investigate the capacity for lipid oxidation in mitochondria and mitochondrial substrate sensitivity for medium- (octanoyl carnitine) and long-chain (palmitoyl carnitine) fatty acids in skeletal muscle and subcutaneous adipose tissue after 6 weeks of HIT in healthy but overweight subjects. We hypothesized that maximal mitochondrial fat oxidative capacity would be unchanged after the training intervention in both skeletal muscle and subcutaneous adipose tissue. In addition, we hypothesized that mitochondrial substrate sensitivity for medium- and long-chain fatty acids was unchanged after training. Furthermore, we hypothesized that maximal mitochondrial OXPHOS capacity would be increased in both skeletal muscle and subcutaneous adipose tissue after training and that this would be accompanied by an increased mitochondrial content (CS activity in skeletal muscle and mtDNA in adipose tissue).

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Methods Ethical approval The ethics committee of the municipality of Copenhagen and Frederiksberg in Denmark approved the study protocol (journal nr H-3-2012-024). Oral and written consent were obtained from each participant in accordance with the Helsinki Declaration.

Subjects Ten overweight untrained subjects were recruited to participate in the study (two females/eight males). None of the subjects were engaged in structured physical activity of any kind (moderate or high intensity). Subjects were excluded if they had any metabolic disease or used any medications.

Experimental design The study protocol consisted of a baseline test day (pre), 6 weeks of high-intensity training three times a week, and a test day 72 h after the last training session (post). Before the baseline test day, the subjects made a maximal oxygen uptake (VO2max) test so that they were familiarized with the test. One week before the baseline test day and during the fifth week of training, the subjects were asked to register their diet over a period of 4 days (three weekdays and one weekend day). The pre- and post-test day was similar. Subjects met at the laboratory in the fasted state (8:00–9:00 h). Body composition was analyzed using a dual-energy X-ray absorptiometer (Lunar iDXA, GE medical Systems Lunar, Madison, WI, USA), followed by a measurement of blood pressure. A basal blood sample was drawn and this was followed by a muscle biopsy from m. vastus lateralis. The muscle sample was obtained after local anesthesia (lidocaine; 5 mg/mL) of the skin and the superficial muscle fascia using the Bergström needle. The adipose biopsy was obtained from the abdominal subcutaneous adipose tissue. The biopsy (adipose and skeletal muscle) was divided into two portions; one was immediately frozen in liquid nitrogen (within 20 s after sampling) and stored at −80 °C for later analysis, the second part was placed in a relaxing buffer (content described in Boushel et al., 2007) and analyzed for mitochondrial OXPHOS capacity. Thereafter, an electrocardiogram was recorded to exclude subjects with signs of coronary ischemia. Finally, an incremental cycling test to voluntary exhaustion was performed on a stationary bike (Lode, B.V., Groningen, the Netherlands) to determine VO2peak using an online gas exchange system (Cosmed, Rome, Italy). The cycling test commenced at 50 W for 5 min followed by 1 W increases every 4 s (15 W/min) until voluntary exhaustion. Subjects were instructed to keep the pedaling cadence above 60 repetitions per minute during the test. The VO2max was reached if two of the following criteria were reached: (a) heart rate close to the pre-calculated maximum heart rate (220 – age), (b) respiratory exchange ratio above 1.20, (c) A plateau in VO2 despite an increase in workload.

Training program The subjects underwent 6 weeks (three times a week, with approximately 1–2 rest days between training days) of 100% supervised HIT. Each training session lasted for 15 min and consisted of 2 min warm-up (45 W) followed by five 60 s exercise bouts with 90 s cycling at a low intensity in between (25 W). The training load was found at the first training session in week 1. It was determined as the maximal load that the subjects could sustain for 60 s (298 ± 21 W), which corresponds to 128 ± 2% of the maximal load (232 ± 15 W) achieved during the incremental cycling test at the first test day. After the first 2 weeks, the training load was increased by 10%.

Mitochondria, high-intensity training Analytical procedures Blood analysis Blood glucose concentrations were measured directly in full blood using the YSI analyzer (2300 STAT plus; Yellow Springs, OH, USA). Glycosylated hemoglobin (HbA1c) was analyzed using a device (DCA2000+; Bayer Healthcare, Elkhart, IN, USA) based on the latex immunoagglutination inhibition method.

Skeletal muscle analysis Enzyme activities. For both CS activity and HAD activity, approximately 2 mg of the dissected tissue was homogenized in 600 μL 0.3 M K2 HPO4, 0.05% bovine serum albumin (BSA), pH 7.7 for 2 min on a Tissuelyzer (Qiagen, Venlo, Limburg, the Netherlands). 6 μL of 10% triton was added and the samples were left on ice for 15 min before they were stored at −80 °C for later analysis. For CS, the homogenate was diluted 50 times in a solution containing 0.33 mM acetyl-CoA, 0.6 mM oxaloacetate, 0.157 mM DTNB, 39 mM Tris-HCl (pH 8.0). The change in 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB) to TNB at 37 °C was measured spectrophotometrically at 415 nm (Srere, 1969) on an automatic analyzer, Cobas 6000, C 501 (Roche Diagnostics, Mannheim, Germany). For HAD, the homogenate was diluted 70 times in a solution containing 0.33 mM acetoacetyl–CoA, 180 μM nicotinamide adenine dinucleotide (NADH), 41.7 μM ethylenediaminetetraacetic acid (EDTA), 27.1 mM imidazole (pH 7.0). The changes in NADH at 37 °C were measured spectrophotometrically at 340 nm (Bergmeyer, 1974) on an automatic analyzer, Cobas 6000, C 501 (Roche Diagnostics). Enzyme activities are expressed as micromoles substrate per minute per gram dry weight of muscle tissue. Protein content. Approximately 15 mg of wet tissue was freeze dried and dissected free of all visible blood, connective tissue, and fat tissue. Approximately 3 mg (dry weight) of the biopsies were homogenized in 270 μL cold RIPA buffer added protease and phosphates inhibitors [50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2.5 mM phenylmethylsulfonyl fluoride, 20 mM β-glycerophosphate, 10 mM pyrophosphate, 2 mM sodium ortovanadate, including mini EDTA-free protease inhibitor tablet according to the instructions of the manufacturer (Roche Diagnostics)]. Homogenization was done at 30 Hz for 2 × 2 min at −20 °C in a TissueLyser (Qiagen Retsch, Haan, Germany), or until the sample was completely dissolved. Thereafter, the homogenate was sonicated for 1 min. Protein concentration was measured by bicinchoninic acid assay (Pierce, Rockford, IL, USA) in triplicate, and a maximal coefficient of variation of 5% between replicates was accepted. An equal amount of protein (12 μg), diluted in Laemmli sample buffer was heated to 95 °C for 10 min and separated on 12% Criterion TGX Stain-Free polyacrylamide SDS gels (Criterion, Bio-Rad, Copenhagen, Denmark). After SDS-electrophoresis, the gels were activated with ultraviolet (UV) light for 5 min, followed by a 1-s image in a LAS 4000 image analyzer (GE Healthcare, Little Chalfont, UK). The activated gel was transferred to a polyvinylidene fluoride membrane (0.2 μm pores, Bio-Rad) in 7 min using the Trans-Blot Turbo Transfer System (Bio-Rad) with Trans-Blot Turbo Midi Transfer Packs. After transfer, another 1-s image was taken of membrane and gel with UV light to visualize protein transfer. The membranes were blocked for 1 h at room temperature with either skimmed milk or BSA diluted in Tris-buffered saline (10 mM Tris Base, 150 mM NaCl, pH 7.4) + 0.05% Tween 20. The membranes were then incubated with the primary antibody overnight at 4 °C. The primary antibodies were anti-adipose triglyceride lipase (ATGL), anti-CD36, antiFABPpm, diacylglycerol acyl transferase 2 (DGAT2; Abcam,

Cambridge, UK), anti-HSL, anti-lipoprotein lipase (LPL; Santa Cruz Biotechnology, Inc., Heidelberg, Germany) and DGAT1 (Novus Biologicals, Littleton, CO, USA). Secondary antibodies were goat anti-rabbit or goat anti-mouse horseradish peroxidase conjugated (Dako, Glostrup, Denmark). After primary and secondary antibody incubations, the membranes were washed 3 × 10 min in Tris-buffered saline ± 0.05% Tween 20. Blots were developed in ECL detection reagents (GE Healthcare) and the chemiluminescence emitted from immune complexes was visualized with a LAS 4000 image analyzer (GE Healthcare). The images of the membranes and stain-free gels were quantified by ImageQuant TL software version 7.0 (GE Healthcare). Analysis included the determination of intensities of the band of interest relative to total Stain-Free fluorescence. Representative blots are provided in Fig. 1. Adipose tissue. mtDNA was analyzed in the subcutaneous adipose tissue as previously described (Kraunsoe et al., 2010). Mitochondrial respiration. Mitochondrial OXPHOS capacity was measured in permeabilized skeletal muscle fibers (Pfi). The details of the procedure have been described previously (Boushel et al., 2007). Briefly, skeletal muscle fibers were gently dissected free of fat and connective tissue, using two sharp needles. The dissection procedure was followed by a chemical permeabilization using saponin (50 μg/mL), and washed twice in a respiration medium (MiR05 (Boushel et al., 2007)). All respiratory measurements on Pfi were carried out in duplicate after hyperoxygenation to avoid any potential oxygen limitation to respiration. Mitochondrial OXPHOS capacity was also measured in adipose tissue (from the abdominal region). The details of this procedure have been described previously (Kraunsoe et al., 2010). Briefly, adipose tissue was placed in cold BIOPS and carefully dissected on ice with forceps under a magnifying glass and this was done to remove capillaries and connective tissue. The respiratory measurements were carried out in duplicate at air saturation.

Mitochondrial respiratory protocols The following three protocols were applied on both adipose and skeletal muscle tissue (blebbistatin; a myosin II ATPase inhibitor was added to the Pfi analysis (Perry et al., 2011)). The myosin II ATPase inhibitor ensures relaxation of the Pfi thereby improving diffusion. Protocol 1 (evaluating palmitoyl carnitine sensitivity): malate (2 mM), ADP (5 mM) and palmitoyl carnitine (5 – 10 – 25 – 50 – 100 μM). Protocol 2 (evaluating complex I and II OXPHOS capacity): state 2 respiration (LEAK) was assessed with malate (2 mM), followed by octanoyl carnitine (1.5 mM), state 3 respiration was reached with ADP (5 mM; ETFP). Subsequently, glutamate (10 mM; CIP) was added followed by succinate (10 mM; CI + IIP), this state is referred to as maximal coupled state 3 respiration, cytochrome c (10 μM) was added to control for outer mitochondrial membrane integrity. Oligomycin (2 μg/mL; state 4o) was added to inhibit the ATP synthase followed by antimycin A (5.0 mM; ROX), which inhibit complex III. Finally TMPD (0.2 M) and ascorbate (0.8 M) was added for complex IV (flux through COX). Protocol 3 (evaluating octanoyl carnitine sensitivity; only in skeletal muscle): malate (2 mM; LEAK), ADP (5 mM) and octanoyl carnitine (10 – 25 – 50 – 100 – 250 – 500 – 1000 μM). It could be expected that oxaloacetate would accumulate in protocol 3, thereby inhibiting respiration, but this was not the case in the present study. When comparing ETFP flux rates from protocol 2 with flux rates from protocol 3 after 1000 μM octanoyl carnitine, no differences (pre or post) were present.

Statistics and calculations Data are presented as means ± standard error of the mean in the text and in the tables and figures. P < 0.05 was considered

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Fig. 1. Protein content measured by Western blots. Representative blots from four subjects’ pre-and post-training are presented to the right of the figure. Data are means ± SE. *P < 0.05. ATGL, adipose triglyceride lipase; CD36, cluster of differentiation 36; DGAT, diacylglycerol acyl transferase; FABPpm, fatty acid binding protein; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; PLIN5, perilipin 5.

significant. No gender differences existed in any of the mitochondrial measurements; therefore, the group was considered as one. Statistical analysis of differences in mitochondrial OXPHOS capacity between the groups was carried out with a two-way analysis of variance for repeated measures. The following restrictive assumptions, normality and equal variance, was checked before the statistical analysis was conducted. Significant main effects or interactions were further analyzed by the Holm–Sidak post-hoc test. If the normality and equal variance test failed, data were transformed and reanalyzed and this is noted in the figure legend. Differences between pre- and post-training were evaluated using a paired t-test. All statistical analysis was performed using the software program SigmaPlot 12.5 (Systat Software, San Jose, CA, USA). The calculation of mitochondrial sensitivity for palmitoyl carnitine (Kmapp) and mitochondrial respiratory capacity (Vmax) has been described previously (Larsen et al., 2012a). Mitochondrial sensitivity and mitochondrial respiratory capacity was also calculated using the Lineweaver–Burk plot. Intrinsic mitochondrial function was calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS activity; skeletal muscle or mtDNA; adipose tissue).

Results Body weight tended (P = 0.075) to increase with training (Table 1). Fat percentage was unchanged, but lean body mass was increased (P < 0.05) after training. No difference was seen in fasting blood glucose concentration, but a decreased HbA1c was observed (P < 0.05). Maximal oxygen uptake increased significantly (P < 0.05) after HIT (2.9 ± 0.2 vs 3.1 ± 0.2 L/min, respectively). No difference was seen in blood pressure (diastolic or systolic) after the training period. Subject characteristics are given in Table 1.

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Table 1. Subject characteristics

Age (year) Weight (kg) Fat percentage (%) LBM (kg) VO2peak (L/min) VO2peak (mL/min/kg) VO2peak (mL/min/kg [LBM]) Wmax (W) Glucose (mM) HbA1c (%) BP Dia (mmHg) BP Sys (mmHg)

Pre-HIT

Post-HIT

P-value

38 ± 3 100.1 ± 5.0 37.9 ± 2.6 59.9 ± 3.1 2.9 ± 0.2 29 ± 2 48 ± 1 232 ± 15 4.5 ± 0.1 5.5 ± 0.1 121 ± 3 83 ± 2

– 101.1 ± 5.3 37.7 ± 2.8 60.5 ± 3.2 3.1 ± 0.2 31 ± 2 51 ± 2 255 ± 16 4.4 ± 0.2 5.3 ± 0.1 121 ± 4 81 ± 2

0.075 0.372 0.010 0.002 0.006 0.004 0.001 0.645 0.040 0.856 0.216

Data are means ± SE. BP, blood pressure; Dia, diastolic; HbA1c, glycated hemoglobin; HIT, highintensity interval training; LBM, lean body mass; Sys, systolic; VO2peak, maximal oxygen uptake.

Energy intake, macro nutrition composition, and protein intake per body weight did not change with training (Table 2). Muscle characteristics CS activity was significantly (P < 0.05) increased after HIT, with no difference in HAD activity (Table 3). The ratio between HAD and CS activities was significantly (P < 0.05) different after HIT (Table 3). HSL expression was significantly (P < 0.05) increased after training and a tendency (P = 0.098) toward an increase was seen in FABPm expression (Table 3). No differences were seen

Mitochondria, high-intensity training Table 2. Dietary intake

Energy intake (MJ) Carbohydrate (%) Fat (%) Protein (%) Alcohol (%) Protein (g/kg bw)

Pre-HIT

During HIT

P-value

9.5 ± 0.5 44 ± 2 35 ± 1 18 ± 1 3±1 1.1 ± 0.1

9.7 ± 0.6 47 ± 2 32 ± 2 17 ± 1 3±1 1.1 ± 0.1

0.773 0.618 0.270 0.457 0.430 0.607

Data are means ± SE. bw, body weight; HIT, high-intensity interval training.

Table 3. Muscle enzyme activities and mitochondrial and genomic DNA in adipose tissue

Pre-HIT

Post-HIT

Enzyme activities (skeletal muscle) CS activity (μmol/g/min) 107 ± 8 145 ± 7 HAD activity (μmol/g/min) 100 ± 7 117 ± 5 HAD/CS ratio 0.94 ± 0.04 0.81 ± 0.03 COX flux (pmol/s/mg) 92 ± 8 133 ± 11 Mitochondrial and genomic DNA (adipose tissue) mtDNA (ds mtDNA/mg 5.9 ± 0.6 6.5 ± 0.5 tissue) 106 Cells per mg tissue ([ds 5609 ± 752 5539 ± 372 gDNA/2]/mg tissue) mtDNA per cell (ds mtDNA/ 1131 ± 87 1168 ± 44 [ds gDNA/2]) COX flux (pmol/s/mg) 1.22 ± 0.17 1.40 ± 0.19

P-value 0.012 0.236 0.020 0.026 0.352 0.921 0.703 0.434

Data are means ± SE. COX, cytochrome C oxidase; CS, citrate synthase; ds, double stranded; g, genomic; HAD, β-hydroxy-acyl-CoA-dehydrogenase; HIT, high-intensity interval training; mtDNA, mitochondrial DNA.

in CD36, DGAT1, DGAT2, LPL, PLIN5, and ATGL expression after training (Fig. 1).

Mitochondrial OXPHOS capacity (skeletal muscle) Mitochondrial lipid OXPHOS capacity (ETFP) was not different after training (Fig. 2a). Complex I-linked OXPHOS capacity (CIP) was not different after training (Fig. 2b), but complex I- and II-linked OXPHOS capacity (CI+IIP) was (P < 0.05) increased after training (Fig. 2c). COX flux was (P < 0.05) increased after training (Table 3). Intrinsic mitochondrial function was similar after training with all substrate combinations (data not shown). COX flux and CS activity showed a significant correlation (P = 0.01; data not shown). Substrate control ratio (ETFP/CIP) was comparable after training (Table 4), whereas (CIP/CI+IIP) was decreased (P < 0.05) after training (Table 4). Mitochondrial substrate sensitivity and maximal respiratory capacity with octanoyl carnitine and palmitoyl carnitine were similar before and after training (Fig. 3a,b). The same result was obtained when mitochondrial sensitivity and respiratory capacity was calculated using Michaelis–Menten kinetics or the Lineweaver–Burk plot.

Fig. 2. Mitochondrial OXPHOS capacity in human skeletal muscle. White bars are pre-training and black bars are posttraining. (a) Maximal mitochondrial lipid OXPHOS capacity (ETFp). (b) Mitochondrial OXPHOS capacity with complex I linked substrates (CIp). (c) Maximal mitochondrial OXPHOS capacity with complex I- and II-linked substrates (CI + IIp). Data are mean ± SE. *P < 0.05.

Adipose tissue mtDNA per mg of adipose tissue or mtDNA per cell was not different after training (Table 3).

Mitochondrial respiratory capacity (adipose tissue) HIT did not change mitochondrial lipid OXPHOS capacity, complex I (CIP) and complex I + II (CI + IIP)linked OXPHOS capacity or COX flux (Table 3) in

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Larsen et al. Table 4. Mitochondrial ratios in human skeletal muscle and subcutaneous adipose tissue

Skeletal muscle SCR (CIP/CI + IIP) SCR (ETFP/CIP) Adipose tissue SCR (CIP/CI + IIP) SCR (ETFP/CIP)

Pre-HIT

Post-HIT

P-value

0.42 ± 0.03* 0.66 ± 0.08*

0.33 ± 0.02* 0.66 ± 0.05*

0.029 0.939

0.60 ± 0.02 0.89 ± 0.02

0.57 ± 0.02 0.90 ± 0.02

0.570 0.643

Data are means ± SE. *P < 0.05. Different from subcutaneous adipose tissue. HIT, high-intensity interval training; SCR, substrate control ratio.

subcutaneous adipose tissue (Fig. 4a–c); with this and the unchanged mtDNA, it follows that intrinsic mitochondrial function was similar after training (data not shown). COX flux and mtDNA showed a significant correlation (P = 0.03; data not shown). No differences were seen in any of the ratios calculated (ETFP/CIP; CIP/CI + IIP) from the subcutaneous adipose tissue after training (Table 4). Mitochondrial substrate sensitivity with palmitoyl carnitine or maximal respiratory capacity was not different after training (Fig. 5a). The same result was obtained when mitochondrial sensitivity and

Fig. 3. White circles are pre-training and black circles are post-training. (a) Fatty acid kinetics in human skeletal muscle with octanoyl carnitine as the fatty acid substrate. (b) Fatty acid kinetics in human skeletal muscle with palmitoyl carnitine as the fatty acid substrate. The respiratory rates reported in these figures are respiratory rates exclusively corresponding to the addition of different octanoyl carnitine or palmitoyl carnitine concentrations. The inserted figure is showing that the data are fitting to a straight line according to the Lineweaver–Burk plot (only PRE training data, mean of the whole group). Data are mean ± SE.

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Fig. 4. Mitochondrial OXPHOS capacity in human adipose tissue. White bars are pre-training and black bars are posttraining. (a) Maximal mitochondrial lipid OXPHOS capacity (ETFp). (b) Mitochondrial OXPHOS capacity with complex I linked substrates (CIp). (c) Maximal mitochondrial OXPHOS capacity with complex I- and II-linked substrates (CI + IIp). Data are mean ± SE.

respiratory capacity was calculated using Michaelis– Menten kinetics or the Lineweaver–Burk plot. The L/P ratio was significantly lower in adipose tissue compared with skeletal muscle tissue, whereas the two different substrate control ratios (ETFP/CIP; CIP/CI + IIP) were significantly higher in adipose tissue compared with skeletal muscle tissue (Table 4). Discussion The novel finding in the present study was that mitochondrial fat oxidation in skeletal muscle and abdominal

adipose tissue was similar after 6 weeks of high-intensity training in healthy overweight subjects. Maximal mitochondrial OXPHOS capacity (CI + IIP) was increased after training in skeletal muscle but not in subcutaneous adipose tissue. In addition and supporting the above observations, a similar activity of HAD but an increased CS activity was observed after training in skeletal muscle. No difference was found in mtDNA content from subcutaneous adipose tissue after training. An increased absolute maximal oxygen uptake was found in the present study; this is in agreement with most (Perry et al., 2007, 2008), but not all studies (McKay et al., 2009) investigating HIT. An increase was seen in CS activity in the present study, indicating an increase mitochondrial volume (Larsen et al., 2012b), which confirms results from previous studies (Perry et al., 2007, 2008; Burgomaster et al., 2008; Jacobs et al., 2013). An increased capacity for lipid oxidation by the mitochondria in skeletal muscle has previously been reported after HIT (Jacobs et al., 2013) and a combination of endurance and high-intensity training (Pesta et al., 2011). The present study found a similar mitochondrial lipid OXPHOS capacity after training, accompanied by an unchanged HAD activity in skeletal muscle. In the study by Jacobs and colleagues, subjects trained at 100% of peak power, whereas the subjects in the present study trained at 128% of peak power and this difference in training intensity and possibly substrate utilization pattern during exercise and recovery may explain why different results were obtained. The energy expenditure during each training session could also serve as an explanation for the difference seen in mitochondrial lipid OXPHOS capacity between the present study and the study by Jacobs and colleagues. It has previously been reported that whole-body fat oxidation increases after HIT at 90% of VO2peak (10 bouts of 4 min separated by 2-min rest) (Talanian et al., 2007). Considering the modest increase in VO2max and lean body mass and the lack of increase in mitochondrial fat oxidation, we do not think that whole-body fat oxidation is increased, but this is only speculation. Helge and colleagues reported that subjects consuming a high amount of lipid in their diet over 7 weeks increased HAD activity probably because of an increased flux through the ß-oxidation pathway (Helge & Kiens, 1997). In the present study, we also focused on some of the key proteins regulating lipid transport, storage, and lipolysis in skeletal muscle. HSL was increased after training, which is in contrast to another study, where HSL was not changed after HIT training (Perry et al., 2008). It has been suggested previously, that ATGL is the primary lipase responsible for lipolysis and therefore oxidation (Haemmerle et al., 2006; Alsted et al., 2009), and in the present study, ATGL protein content was unchanged after training as was mitochondrial fatty acid oxidation. A tendency (P < 0.1) for an

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Fig. 5. White circles are pre-training and black circles are post-training. (a) Fatty acid kinetics in human subcutaneous adipose tissue with palmitoyl carnitine as the fatty acid substrate. The respiratory rates reported in these figures are respiratory rates exclusively corresponding to the addition of different octanoyl carnitine or palmitoyl carnitine concentrations. The inserted figure is showing that the data are fitting to a straight line according to the Lineweaver–Burk plot (only PRE training data, mean of the whole group). Data are mean ± SE.

increased protein expression was seen for FABPm content, which is in agreement with data from Perry and colleagues (Perry et al., 2008), but contradictory to another study (Burgomaster et al., 2007). It has previously been reported that PLIN5 protein expression is high in tissues with a high oxidative capacity for lipids (Vigelsø et al., 2013). In the present study, PLIN5 expression was similar after training, which is contradictory to a study by Shepherd and colleagues where an increase in PLIN5 was found after both endurance and sprint interval training (Shepherd et al., 2013). However, although Shepherd and colleagues observed a higher PLIN5 protein expression after training, they did not find an increased whole-body fat oxidation (Shepherd et al., 2013). Shepherd and colleagues also suggested that PLIN2 and 5 protein expression correlates with insulin sensitivity (Shepherd et al., 2013). Vigelsø and colleagues investigated the perilipin proteins in subjects that differed in insulin sensitivity and found no difference in PLIN5 expression between subjects with impaired glucose tolerance and control subjects, but a tendency toward a reduced PLIN5 protein content was observed in patients with type 2 diabetes compared with healthy control subjects (Vigelsø et al., 2013). In the present study, a decreased HbA1c was seen after training, which indicates improved insulin sensitivity and given the unchanged muscle PLIN5 protein content, it implies that PLIN5 does probably not play a key role in regard to insulin sensitivity. In the present study, complex I-linked OXPHOS capacity was similar after HIT training, and this is in

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contrast to the higher complex I-linked OXPHOS capacity reported by Jacobs and colleagues (Jacobs et al., 2013). However, in the present study, glutamate and malate was used as complex I-linked substrates, whereas Jacobs and colleagues also added pyruvate (Jacobs et al., 2013) and since a former study found that addition of pyruvate and malate increased complex I-linked OXPHOS capacity after training (a combination of endurance and high-intensity training; Walsh et al., 2001), this may explain the observed difference. Maximal OXPHOS capacity (with complex I- and II-linked substrates) was increased after training in the present study, which is in agreement with other studies (Pesta et al., 2011; Jacobs et al., 2013). Intrinsic mitochondrial function was evaluated and no differences were found after training in the present study, which is supported by the results of another HIT study (Jacobs et al., 2013). It has previously been reported that elite athletes have a higher intrinsic mitochondrial function compared with active subjects (Jacobs & Lundby, 2013) and given the lack of a training effect, this may suggest that elite athletes are selected with an inherent genetical higher intrinsic mitochondrial function. Palmitoyl carnitine sensitivity in skeletal muscle was similar after training. The sensitivity for palmitoyl carnitine in the present study is in the same range as previously reported in rats (Smith et al., 2012). It has been suggested that carnitine palmitoyltransferase I (CPT-I) is rate limiting in long-chain fatty acid oxidation (Kim et al., 2000), indicated by a lower sensitivity for palmitoyl CoA (higher Kmapp) compared with palmitoyl

Mitochondria, high-intensity training carnitine, the latter being dependent on carnitine palmitoyltransferase II (CPT-II; Smith et al., 2012). Unpublished results from our laboratory in healthy middle-aged males have shown that the sensitivity for both palmitoyl CoA and carnitine (CPT-I and CPT-II) was increased after 2 weeks of HIT training (authors’ unpublished observations). This could indicate that CPT-I and -II sensitivity are affected in the same direction with training. The lack of difference in the present study in regard to palmitoyl carnitine sensitivity could again be due to the difference in training intensity between the two studies. It has previously been reported that CPT-I sensitivity was increased after 8 weeks of endurance training in obese subjects (Bruce et al., 2006), but it is possible that the HIT training in the present study may have no effect on CPT-I and -II activity and thus explain the similar mitochondrial sensitivity for palmitoyl carnitine observed in the present study. Mitochondrial substrate sensitivity or mitochondrial OXPHOS capacity with octanoyl carnitine (medium-chain fatty acid) was not influenced by training, which is in line with the observation of similar octanoyl carnitine sensitivity in lean and obese subjects with different physical fitness levels (Larsen et al., 2011). A novel observation was the similar mitochondrial OXPHOS capacity or palmitoyl carnitine substrate sensitivity in subcutaneous adipose tissue after training, which has not been investigated in human subcutaneous adipose tissue before. It has previously been reported that mitochondrial content increases after 4–10 weeks of training in rats (Stallknecht et al., 1991; Sutherland et al., 2009); whether the finding by Stallknecht and colleagues was due to the exercise training or a result of the exposure to cold water is unknown, whereas 10 days of combined endurance and HIT had no effect on mitochondrial content (CS activity) in human subcutaneous adipose tissue (Camera et al., 2010). This is in agreement with the functional measurements from the present study, where no differences were found in mitochondrial OXPHOS capacity from subcutaneous adipose tissue, but also the fact that no difference was found in mitochondrial content (mtDNA) in the present study after training. Camera and colleagues reported no difference in palmitate oxidation after 10 days of training in adipose tissue (Camera et al., 2010), which is in line with our results. Six weeks of HIT does not seem to induce a transition from white to brown adipose tissue in subcutaneous adipose tissue from humans. Camera and colleagues speculated that the adaptive capacity and

response may vary between different adipose tissue depots and that this may explain the lack of a difference in fat oxidative capacity after training (Camera et al., 2010). It has been reported that adipocytes in humans are replaced at a rate of approximately 10% per year (Spalding et al., 2008) and we speculate that the duration of the training intervention was too short to actually see an improvement in mitochondrial function in the subcutaneous adipose tissue in the present study. More longterm training studies are therefore needed to clarify a possible effect on mitochondrial function in human adipose tissue. In summary, no differences were seen in mitochondrial lipid oxidation in either skeletal muscle or subcutaneous adipose tissue after 6 weeks of high-intensity training and this was accompanied by similar HAD activity in skeletal muscle. An increased mitochondrial OXPHOS capacity and content (CS activity) was found in the skeletal muscle, with no differences present in subcutaneous adipose tissue in these parameters. Furthermore, an increased mitochondrial sensitivity for long-chain fatty acid (palmitoyl carnitine) was found in skeletal muscle, with no difference in subcutaneous adipose tissue. Perspectives Results from the present study indicate that this specific modality of high-intensity training does not improve the mitochondria’s ability to oxidize fatty acids in either skeletal muscle or adipose tissue, which is contradictory to the effect of regular endurance training. An increase in mitochondrial content was found in the trained skeletal muscle, whereas no difference was found in mitochondrial content in adipose tissue. This could indicate that a longer intervention is necessary to see improvements in adipose tissue in regard to mitochondrial function. Key words: Mitochondrial content, exercise adaptations.

Acknowledgements Tine L. Dohlmann and Morten Hindsø are thanked for helping with recruitment and training of the subjects. Regitze Kraunsøe, Kathrine Qvist, Christina N. Hansen, and Jeppe Bach are thanked for their skilled technical assistance. The samples for this project are generated from an EU-funded project “Metapredict” 7th Framework.

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The effect of high-intensity training on mitochondrial fat oxidation in skeletal muscle and subcutaneous adipose tissue.

High-intensity interval training (HIT) is known to increase mitochondrial content in a similar way as endurance training [60-90% of maximal oxygen upt...
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