Eur J Appl Physiol DOI 10.1007/s00421-014-2879-9

Original Article

Metabolic adaptations in skeletal muscle, adipose tissue, and whole‑body oxidative capacity in response to resistance training Malin Alvehus · Niklas Boman · Karin Söderlund · Michael B. Svensson · Jonas Burén 

Received: 18 October 2013 / Accepted: 22 March 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Purpose The effects of resistance training on mitochondrial biogenesis and oxidative capacity in skeletal muscle are not fully characterized, and even less is known about alterations in adipose tissue. We aimed to investigate adaptations in oxidative metabolism in skeletal muscle and adipose tissue after 8 weeks of heavy resistance training in apparently healthy young men. Methods  Expression of genes linked to oxidative metabolism in the skeletal muscle and adipose tissue was assessed before and after the training program. Body composition, peak oxygen uptake (VO2 peak), fat oxidation, activity of mitochondrial enzyme in muscle, and serum adiponectin levels were also determined before and after resistance training. Results  In muscle, the expression of the genes AdipoR1 and COX4 increased after resistance training (9 and 13 %, respectively), whereas the expression levels of the genes PGC-1α, SIRT1, TFAM, CPT1b, and FNDC5 did not change. In adipose tissue, the expression of the genes SIRT1 and CPT1b decreased after training (20 and 23 %, respectively). There was an increase in lean mass (from Communicated by Martin Flueck. M. Alvehus (*) · J. Burén  Department of Public Health and Clinical Medicine, Medicine, Umeå University, Umeå, Sweden e-mail: [email protected]

59.7 ± 6.1 to 61.9 ± 6.2 kg), VO2 peak (from 49.7 ± 5.5 to 56.3 ± 5.0 ml/kg/min), and fat oxidation (from 6.8 ± 2.1 to 9.1 ± 2.7 mg/kg fat-free mass/min) after training, whereas serum adiponectin levels decreased significantly and enzyme activity of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase did not change. Conclusion  Despite significant increases in VO2 peak, fat oxidation, and lean mass following resistance training, the total effect on gene expression and enzyme activity linked to oxidative metabolism was moderate. Keywords Resistance training · Oxidative capacity · Skeletal muscle · Adipose tissue · Gene expression · Enzyme activity Abbreviations AdipoR1 Adiponectin receptor 1 COX4 Cytochrome c oxidase subunit 4 CPT1 Carnitine palmitoyltransferase 1 CS Citrate synthase FFM Fat-free mass HAD 3-Hydroxyacyl-CoA dehydrogenase MFO Maximal fat oxidation PGC-1α  Peroxisome proliferator-activated receptor γ co-activator-1α SIRT1 Sirtuin 1 Tfam Mitochondrial transcription factor A VO2 peak Peak oxygen uptake

N. Boman · M. B. Svensson  Department of Surgical and Perioperative Science, Sports Medicine, Umeå University, Umeå, Sweden


K. Söderlund  The Swedish School of Sport and Health Sciences (GIH), Stockholm, Sweden

Exercise training induces favorable metabolic alterations involving increased oxidative capacity and metabolic flexibility. A majority of studies investigating oxidative capacity


and mitochondrial biogenesis in human skeletal muscle have studied the effects of endurance training, whereas resistance training has received less attention. Recent reports have shown that compared to endurance training alone, a combination of endurance and resistance training triggers greater induction of gene expression and protein activity in oxidative metabolic pathways (Ruas et al. 2012; Wang et al. 2011). Adiponectin and its receptor, AdipoR1, have a central role in oxidative metabolism and mitochondrial biogenesis (Civitarese et al. 2006; Iwabu et al. 2010); in response to endurance training, the expression of the gene encoding AdipoR1 increases in muscle (Bluher et al. 2006). AdipoR1 regulates the expression and activation of peroxisome proliferator-activated receptor γ co-activator-1α (PGC1α) (Iwabu et al. 2010), which is induced by exercise and increases fatty acid oxidation and mitochondrial biogenesis in skeletal muscle (Arany 2008). Following activation of the PGC-1α pathway, mitochondrial transcription factor A (Tfam) is the potent final effector activating the duplication of mitochondrial DNA molecules (Kang and Li Ji 2012). Sirtuin 1 (SIRT1), also regulated by AdipoR1 (Iwabu et al. 2010), is a deacetylating protein involved in the activation of PGC-1α that acts as a crucial modulator of fatty acid oxidation (Serra et al. 2013). To be oxidized, long chain fatty acids must be converted to their acylcarnitine form before entering the mitochondrial matrix. This reaction is catalyzed by the enzyme carnitine palmitoyltransferase (CPT) 1 and is the rate-limiting step in fatty acid oxidation (Jeppesen and Kiens 2012). CPT1 gene expression and protein activity increase following endurance training in human muscle tissue (Berthon et al. 1998; Tunstall et al. 2002). An additional enzyme, essential for fatty acid oxidation, is 3-hydroxyacyl-CoA dehydrogenase (HAD) that catalyzes the third step in the mitochondrial β-oxidation (Yang et al. 2005). The end product of β-oxidation, acetyl-CoA, then enters the citric acid cycle via the activity of citrate synthase (CS), a strong biomarker for mitochondrial content in muscle (Larsen et al. 2012). As a final step in the oxidation of nutrients, ATP is generated in the respiratory chain by oxidative reactions coupled with phosphorylation. A marker for muscle oxidative capacity is the mitochondrial enzyme cytochrome c oxidase subunit 4 (COX4), which mediates the final reaction in the electron transport chain. Protein levels of COX4 increase quickly in muscle after repeated high-intensity training (Burgomaster et al. 2007). Adipose tissue is a highly active metabolic and endocrine organ. The endocrine function of adipose tissue involves the production and secretion of hundreds of substances that act in a local or systemic manner (Harwood 2012). Adipokines, including leptin and adiponectin, are almost exclusively produced by adipocytes. In addition,


Eur J Appl Physiol

adipose tissue is the major storage site for excess energy and provides the muscles with fatty acids when required (Harwood 2012). Only a limited number of studies have investigated possible adaptive changes in metabolic enzymes and mitochondrial biogenesis in response to exercise training in healthy human adipose tissue. It has been reported that PGC-1α and AdipoR1 gene expression in adipose tissue increases with 4 weeks of endurance training (Bluher et al. 2007; Ruschke et al. 2010). To date, the influence of resistance training on oxidative metabolism in adipose tissue has not been well characterized. The aim of the present study was to examine the impact of resistance training, designed to induce muscle hypertrophy, on the expression of genes and activity of enzymes linked to oxidative metabolism and mitochondrial biogenesis in skeletal muscle and adipose tissue. We hypothesized that 8 weeks of resistance training in young healthy men would increase the expression of genes and activity of enzymes associated with oxidative capacity.

Materials and methods Subjects Apparently healthy young men with slight to moderate experience with resistance training were recruited for this study via advertisement on campus. Exclusion criteria included smoking, lactose intolerance, and past or present intake of steroid hormone substances. After initial screening, 29 subjects underwent baseline testing. In the present study, only subjects who underwent muscle or adipose tissue biopsy both at baseline and after completing the training program were included (a total of 17 subjects). One subject was excluded from the analysis of skeletal muscle gene expression due to experimental error. Ethics statement This study was approved by the Regional Ethical Review Board at Umeå University. All subjects provided written informed consent before entering the study. Experimental protocol Subject characterization, blood sampling, and tissue sampling were performed at baseline and at the end of the study. Subject characterization and blood sampling were performed after overnight fasting and after refraining from exercise for 48 h. Body composition measures and biopsy collection were performed on separate days. Body weight was measured in light clothing to the nearest 0.1 kg. Height was measured to the nearest 0.1 cm.

Eur J Appl Physiol

Body composition was determined by dual energy X-ray absorptiometry (Lunar iDXA, GE Healthcare, Waukesha, WI, USA). The coefficient of variation was 0.5 % for total lean mass and 0.8 % for total body fat. In the fasting state, subjects consumed a volume of energy drink (Gainomax Recovery, Norrmejerier, Umeå, Sweden) corresponding to 1 g carbohydrate/kg body weight. Following 1 h of rest, VO2 peak and fat oxidation were determined using a graded incremental exercise test on a cycle ergometer (Ergomedic 839E, Monark, Vansbro, Sweden). After a warm-up, the subjects started cycling at a work rate of 80 W followed by 40 W increments every 3 min until voluntary exhaustion. Respiratory gas exchange measurements of oxygen uptake (O2) and carbon dioxide production (CO2) were performed using an Oxycon Pro analysis system (Jaeger, Wuerzberg, Germany). Average values for VO2 and VCO2 (expressed as L × min−1) were calculated over the last minute of each work stage. Fat oxidation rates were calculated using stoichiometric equations by Jeukendrup and Wallis (2005). Maximal fat oxidation (g/min) was determined as the highest value of fat oxidation rate during the incremental exercise test. Biopsies were obtained in the afternoon between 1 and 2 PM and 48–56 h after the last training session. Biopsies from skeletal muscle (vastus lateralis) were collected using a conchotome; visible blood, fat, and connective tissue were removed. Subcutaneous adipose tissue biopsies were obtained from the periumbilical region using needle aspiration. The fat tissue was washed with saline and blood clots were removed. Biopsies were immediately snap-frozen in liquid nitrogen and stored at −80 °C until analysis. Biopsy procedures were performed under local anesthesia [Carbocain® (5 mg/mL) containing adrenaline (5 μg/mL); AstraZeneca, Södertälje, Sweden]. The 8-week intervention also included an investigation of the possible effects on steroid metabolism after consumption of a beverage containing proteins and carbohydrates or only carbohydrates during the exercise sessions. Since intake of these two beverages was not of interest for the present study and did not differentially affect the relevant parameters, all subjects were considered as one group. Training program The included men performed a resistance training program, designed to attain muscle hypertrophy, for 8 weeks. The training program was designed using guidelines from the American College of Sports Medicine position stand “Progression models in resistance training for healthy adults” (Ratamess et al. 2009). The concept of the training program was to train every major muscle group two times per week for 8 weeks by performing 8–15 repetitions at a 2/2 s eccentric/concentric tempo for 2–4 sets with 60–120 s of

rest between sets of each exercise. The progression of the program used an undulating model where even-numbered weeks (2nd, 4th, 6th, and 8th weeks) meant performing each exercise to concentric failure, while exercises were performed with a slight strength reserve during odd-numbered weeks. The number of sessions (2–4 per week) was based on the participants’ previous experience of training; fewer sessions per week led to more exercises per session and slightly more repetitions per set. The participants were instructed to desist from strenuous endurance training but to maintain general lifestyle during the intervention period. Each training session started with 10 min of warm-up cycling at approximately 75 W, which was the only type of endurance training included in the program. Prior to the training program, the participants underwent a familiarization session to determine initial loads. Participants were instructed in correct technique and were individually coached in weight selection and progression during the familiarization and full study periods. Training loads and repetitions were recorded throughout the training program. Training was performed in the gym at the unit for Sports Medicine at Umeå University or at an adjacent training facility (IKSU Sport Center, Umeå). Serum analysis Circulating total adiponectin levels were measured using a radioimmunoassay according to the manufacturer’s instructions (Linco Research, St. Louis, MO, USA) (intra-assay coefficient of variation 13 %). RNA extraction and qRT‑PCR Total RNA was extracted from adipose tissue and skeletal muscle biopsies using the RNeasy® Lipid Tissue Mini Kit according to the manufacturer’s instructions (Qiagen Nordic, Qiagen House, West Sussex, UK). RNA yield and purity were determined using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity was evaluated by 1 % agarose gel electrophoresis with GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA) under ultraviolet light. cDNA synthesis was performed by reverse transcription of RNA using TaqMan® reverse transcription reagents (Applied Biosystems, Foster City, CA, USA). Relative quantification real-time PCR was carried out on an ABI Prism® 7000 Sequence Detection System (Applied Biosystems) using Universal PCR Master Mix 2X (Applied Biosystems) and TaqMan gene expression assays (Applied Biosystems) for adiponectin (Hs00605917_m1), ADIPOR1 (Hs01114951_m1), PGC1-α (Hs01016719_m1), COX4I1 (Hs00971639_m1), TFAM (mitochondrial transcription


factor A; Hs00273372_s1), SIRT-1 (Hs01009005_m1), CPT1b (Hs03046298_s1), FNDC5 (Hs00401006_m1), and RPLPO (Hs 99999902_m1). Relative expression levels were calculated according to the 2−ΔΔCt method. Housekeeping gene expression was evaluated by comparing expression levels of β-actin, GAPDH, and RPLP0 before and after the intervention period. Target genes were normalized to RPLP0, as this gene was most stable before and after the intervention period and had the lowest coefficient of variation. Extraction of muscle for enzyme activity measurements Samples were freeze-dried, dissected free of blood and connective tissue, powdered, weighed and homogenized with a buffer containing 50 mM Na2HPO4, 1 mM EDTA, 0.05 % v/v Triton X-100 pH 7.4. To 1 mg of freeze-dried muscle, 150 μl ice-cold buffer was added. The homogenization was carried out using a Bullet Blender 1.5 (Next Advance, New York, USA) with zirconium oxide beads 0.5 and 1.0 mm, approximately 50 mg added to each sample. The samples, weighing between 0.7 and 2.5 mg, were homogenized for 2 min at speed 4 and then centrifuged at, 10,000 rpm for 1 min. The supernatant was used for enzyme activity measurements. CS was assayed at 412 nm and HAD at 340 nm using Beckman Coulter DU 800 spectrophotometer. The method used for CS determination was modified from Alp et al. (1976) and HAD activity was determined using a modified method from Essén et al. (1975). The analysis was carried out at 25 °C.

Eur J Appl Physiol Table 1  Subject characteristics Baseline Age (years) Body weight (kg) Lean mass (kg) Fat mass (kg) VO2 peak (ml/kg/min)

After exercise

25.3 ± 2.8 78.6 ± 7.4 59.7 ± 6.1 15.4 ± 3.3 49.7 ± 5.5

81.2 ± 8.4*** 61.9 ± 6.2*** 15.7 ± 3.6 56.3 ± 5.0***

6.8 ± 2.1

9.1 ± 2.7**

MFO/FFM (mg/kg FFM/min) Values are mean ± standard deviation

N  = 17, except for body weight after exercise, where N  = 16, VO2 and MFO/FFM N = 15


VO2 peak peak oxygen uptake, MFO maximal fat oxidation, FFM fatfree mass *** P 

Metabolic adaptations in skeletal muscle, adipose tissue, and whole-body oxidative capacity in response to resistance training.

The effects of resistance training on mitochondrial biogenesis and oxidative capacity in skeletal muscle are not fully characterized, and even less is...
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