Toxicology and Applied Pharmacology 264 (2012) 324–334

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Toxicology and Applied Pharmacology

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Augmentation of aerobic respiration and mitochondrial biogenesis in skeletal muscleby hypoxia preconditioning with cobalt chloride

Saurabh Saxena a, Dhananjay Shukla b, Anju Bansal a,⁎a Experimental Biology Division, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, 110054, Indiab Department of Biotechnology, Gitam University, Gandhi Nagar, Rushikonda, Visakhapatnam-530 045 Andhra Pradesh, India

⁎ Corresponding author. Fax: +91 1123932869.E-mail address: anjubansaldipas@gmail.com (A. Ban

0041-008X/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.taap.2012.08.033

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 June 2012Revised 8 August 2012Accepted 30 August 2012Available online 8 September 2012

Keywords:Hypoxia preconditioningCobalt chlorideExerciseAerobic respiration

High altitude/hypoxia training is known to improve physical performance in athletes. Hypoxia induces hyp-oxia inducible factor-1 (HIF-1) and its downstream genes that facilitate hypoxia adaptation in muscle toincrease physical performance. Cobalt chloride (CoCl2), a hypoxia mimetic, stabilizes HIF-1, which otherwiseis degraded in normoxic conditions. We studied the effects of hypoxia preconditioning by CoCl2 supplemen-tation on physical performance, glucose metabolism, and mitochondrial biogenesis using rodent model. Theresults showed significant increase in physical performance in cobalt supplemented rats without (two times)or with training (3.3 times) as compared to control animals. CoCl2 supplementation in rats augmented thebiological activities of enzymes of TCA cycle, glycolysis and cytochrome c oxidase (COX); and increased theexpression of glucose transporter-1 (Glut-1) in muscle showing increased glucose metabolism by aerobicrespiration. There was also an increase in mitochondrial biogenesis in skeletal muscle observed by increasedmRNA expressions of mitochondrial biogenesis markers which was further confirmed by electron microsco-py. Moreover, nitric oxide production increased in skeletal muscle in cobalt supplemented rats, which seemsto be the major reason for peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) in-duction and mitochondrial biogenesis. Thus, in conclusion, we state that hypoxia preconditioning by CoCl2supplementation in rats increases mitochondrial biogenesis, glucose uptake and metabolism by aerobic res-piration in skeletal muscle, which leads to increased physical performance. The significance of this study liesin understanding the molecular mechanism of hypoxia adaptation and improvement of work performance innormal as well as extreme conditions like hypoxia via hypoxia preconditioning.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Training under hypoxia/high altitude (HA) induces certain biologicalresponses in humans. Living at moderate altitude (2000–2500 m) im-proves the oxygen transport capacity by increase in hematocrit inducedby erythropoietin (Piehl-Aulin et al., 1998;Wehrlin et al., 2006). Previousstudies have reported that increase in the hemoglobin concentration im-proves maximal O2 consumption ( _VO2max) and enhances enduranceperformance (Levine and Stray-Gundersen, 1997; Wehrlin et al., 2006).Moreover, 4 weeks of “living high, training low” has been shown to aug-ment sea-level running performance in practiced runners by increase inhematocrit and _VO2max (Levine and Stray-Gundersen, 1997). However,when only trainingwas performed under hypoxic conditions (living low,training high); increased mitochondrial density, capillary to fiber ratio,and fiber cross-sectional areas in skeletal muscle have been reported(Desplanches and Hoppeler, 1993; Vogt et al., 2001). Other studies thatused similar training protocols demonstrated significant increase in theoxidative enzyme activities and capillary density (Green et al., 1999;

sal).

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Melissa et al., 1997; Terrados et al., 1990). In all of these studies, a signif-icantly higher activity of citrate synthase (CS) was noted after training atthe same level of intensity under hypoxic as compared to normoxic con-ditions, indicating towards an improvement in aerobic performance.Also, in “living low, training high” conditions, enhancements in endur-ance performance and _VO2max have been reported (Melissa et al.,1997; Terrados et al., 1988). These studies suggest that exercise underhypoxia may induce specific muscular and systemic adaptations thatare either absent or exist at low levels at training under normoxic condi-tions. Moderate exposure (2 to 3 weeks) to such altitude sufficientlyelicits such adaptations; therefore, competitive endurance athletesoften live and train at moderate altitudes to improve their endurance ca-pacity (Bailey andDavies, 1997; Boning, 1997; Vogt et al., 2001). Further-more, some studies have reported that training under normobaric and/orintermittent hypoxia also enhances exercise performance (Bonetti andHopkins, 2009; Zoll et al., 2006). At themolecular level, hypoxia trainingactivates hypoxia inducible factor-1 (HIF-1), which is a master regulatorof several hypoxia responsive genes that play a key role in facilitating ad-aptation to hypoxia (Hoppeler and Vogt, 2001; Wenger, 2002; Wilber,2007; Zoll et al., 2006) viz. erythropoietin (Epo), vascular endothelialgrowth factor (VEGF), myoglobin (Mb), glucose transporters-1 and 3

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(Glut-1 and 3), many enzymes of glycolysis, etc. Moreover, exercise in-duces a transient local tissue hypoxia in skeletalmuscle leading to induc-tion of adaptive changes (Koulmann and André-Bigard, 2006). Thus, HIFand its downstream genes play a major role in hypoxia adaptation.

The potential benefits of hypoxia preconditioning can be mimickedby cobalt chloride (CoCl2), which is a known hypoxia mimetic (Xi et al.,2004). It produces a chemical hypoxic environment and activates HIF-1under normoxic conditions both in vitro and in vivo (Ciafré et al., 2007;Endoh et al., 2000; Goldberg et al., 1988). The consequences of HIF-1 sta-bilization by Co2+ are the same as in hypoxia viz. induction of Epo, VEGF,Glut-1, heme oxygenase-1 (HO-1) and other hypoxic genes (Endoh et al.,2000; Saxena et al., 2010; Semenza, 2000; Shrivastava et al., 2008b; Xi etal., 2004). Administration of CoCl2 has been shown to provide protectionagainst ischemia/reperfusion injury in brain and kidney (Bergeron et al.,2000;Matsumoto et al., 2003;Miller et al., 2001). Cobalt is also known toinduce erythropoietin production both in vitro (Fisher and Langston,1968) and in vivo (Janda et al., 1965) under normoxic condition. More-over, hypoxia preconditioning by cobalt supplementation has beenreported to induce microvascular remodeling in skeletal muscle andheart (Rakusan et al., 2001; Suzuki, 2002) Studies conducted in our lab-oratory have demonstrated increased hypoxic tolerance, decreased vas-cular leakage, decreased oxidative stress and pro-inflammatory markersin brain and lung of male Sprague–Dawley rats after 7 days supple-mentation of 12.5 mg/kg bw Co2+ (Kalpana et al., 2008; Shrivastava etal., 2008a,b; Shukla et al., 2009, 2011). The effect of cobalt induced hyp-oxia preconditioning on endurance performance has not been studiedmuch. We have earlier reported (Saxena et al., 2010) that hypoxiapreconditioning with cobalt chloride enhances physical performancein rats. It activates cellular oxygen sensing system in rat skeletalmuscle,checks reactive oxygen species (ROS) inducedmuscle fiber damage, im-proves antioxidant status, enhances hematocrit and hemoglobin leveland increases lactate uptake from blood into muscle. It was also foundto enhance the expression of mitochondrial biogenesis markers.

Therefore, the aim of the present study was to evaluate the effectof hypoxic preconditioning by cobalt chloride on cellular aerobic res-piration and mitochondrial biogenesis in skeletal muscle. Further-more, we studied the efficacy of cobalt supplementation in enhancingglucose uptake from blood into the muscle. Also, we tried to decipherthemechanism underlying enhanced peroxisome proliferator activatedreceptor-gamma coactivator-1α (PGC-1α) expression under hypoxiapreconditioning condition.

Materials and methods

Ethical approval. All animal procedures were approved by the Insti-tutional Animal Ethic Committee and the guidelines of UniversitiesFederation for Animal Welfare (UFAW) for animal research werefollowed.

Animals. Male Sprague–Dawley rats (170±10 g) were used for allexperiments. Animals were provided by the experimental animal fa-cility of our institute. Animals were housed in a controlled environ-ment with a 12-h light/dark cycle, access to food (Lipton India Ltd.)and water ad libitum. Body weight, food and water intake were mea-sured daily. Since three rats were housed per cage, average food andwater intake per individual rat was estimated from the total foodand water intake per cage.

Materials. All chemicalswere purchased fromSigma-Aldrich (St. Louis,MO, USA) Antibodies were purchased from Santa Cruz Biotech, SantaCruz, CA, USA.

In our previous study (Saxena et al., 2010), we have found thatoral supplementation of 10 mg/kg body wt. CoCl2∙6H2O for 15 daysincreases the swimming time till exhaustion in rats significantly.Therefore, all the experiments were conducted with the same dosageof cobalt chloride.

Effect of optimum cobalt chloride supplementation on enduranceperformance in sedentary and trained rats. To study the effect ofcobalt supplementation on the physical performance of sedentaryand trained rats, they were randomly divided into 4 groups (n=8 each) according to the treatment and exercise conditions: (i) non-supplemented sedentary (Con); (ii) non-supplemented training (Tr);(iii) Co2+-supplemented sedentary (Cob); and (iv) Co2+- supplementedtraining (CobTr) with the same training protocol. Rats in the non‐supplemented groups were given distilled water with the help of a gas-tric cannula and the rats in cobalt supplemented group were given10 mg/kg CoCl2∙6H2O/day for 15 days. At the end of the 15th day, theirswimming till exhaustion was measured.

Muscle samples. Another experiment with 32 rats was set, again di-vided into four groups with similar dose and training protocol asmentioned above. On the 16th day, the rats were sacrificed underketamin (80 mg/kg i.p.) as anesthesia and the whole gastrocnemiusmuscle was collected from the calf region of the hind limb of therats. The red gastrocnemius muscle (100–150 mg) was then separat-ed from the whole muscle, washed in phosphate-buffered saline(PBS) twice, snap frozen after removal of hair and fatty tissues, andstored at −80 °C for subsequent biochemical and protein expressionstudies. The red gastrocnemius muscle was chosen because it is ac-tively recruited during swimming and is highly oxidative in nature(Delp and Duan, 1996; Gonchar, 2005). Before killing, blood sampleswere collected by retro-orbital puncture.

Determination of biochemical activities of enzymes. Biochemical ac-tivities of enzymes of cellular respiration in skeletal muscle weremeasured spectrophotometrically. Citrate synthase (CS, EC 2.3.3.1), suc-cinate dehydrogenase (SDH, EC 1.3.99.1), hexokinase (EC 2.7.1.1), andphosphofructokinase (PFK, EC 2.7.1.11) were determined in muscle bythe methods of Srere (1963), Veeger et al. (1969), Easterby and Qadri(1982) and Ling et al. (1966), respectively. Activity of cytochrome c ox-idase (COX, 1.9.3.1) was measured using commercially available kits(Sigma-Aldrich, St Louis, MO, USA) as per the manufacturer's instruc-tions. Lactate dehydrogenase (LDH, EC 1.1.1.2) activity was determinedusing commercially available kits (Randox Laboratories Ltd, Crumlin,UK) as per the manufacturer's instructions.

ATP level measurement. ATP is called as energy currency in the cell.ATP level in muscle was measured by using commercially availablekit (Sigma-Aldrich, St Louis, MO, USA) as per manufacturer's protocol.

Nitric oxide (NO) measurement. NO levels in muscle were deter-mined indirectly by the quantification of its oxidized products of deg-radation (NO2

−) using Griess reagent by the method of Barrias et al.(2002). Briefly, 50 mg of muscle tissue was homogenized in 500 μLPBS and centrifuged at 2500 g for 10 min. Equal volumes of superna-tant and Griess reagent were mixed and incubated for 15 min. A chro-mophoric azo-derivative thus formed was measured at 540 nm in amicroplate reader (Molecular Devices, CA, USA).

Protein expression studies. Western blot techniquewas used to studythe protein expressions of HIF-1α, nuclear respiratory factor-1 (NRF-1),PGC-1α, Glut-1, Glut-4, endothelial nitric oxide synthase (eNOS), neu-ronal nitric oxide synthase (nNOS) pyruvate dehydrogenase kinase-1and 4 (PDK-1 and 4) and prolyl hydroxylase domain (PHD) containingproteins: PHD-1 and PHD-2. Muscle samples were homogenized andcytosolic and nuclear fractions were isolated by using the Nuclear/Cytosolic Fractionation Kit (Biovision, Mountain View, CA, USA) asper themanufacturer's instructions. HIF-1α, PGC-1α and NRF-1 expres-sions were studied in the nuclear fraction; and Glut-1, Glut-4, PDK-1,PHD-1, PHD-2, eNOS and nNOS expressions were examined in thecytosolic fraction. Protein concentrations were quantified by usingBradford's reagent. Fifty micrograms of protein was electrophoresed

Table 1Details of primers used for reverse transcription-PCR.

Gene Primer sequence Productsize (bp)

Annealingtemperature (°C)

COX-1 F5′-GAGCAGGAATAGTAGGGACA-3′R5′-GGAGTAGAAATGATGGAGGA-3′

261 57

COX-4A F5′-TGGGACTACAACAAGAATGA-3′R5′-AGTGAAGCCGATGAAGAAC-3′

134 55

COX-4B F5′-CCCCTATGTTGACTGCTATG-3′R5′-TTACTGTCTTCCATTCGTTG-3′

212 56

CS F5′-TATTTTGGCTGCTGGTAACT-3′R5′-GTTCGGTTTATTCCCTCTG-3′

220 55

PFKm F5′-GATGTCTGGGAAAATCAAAG-3′R5′-GTGCTCAAAATCTGTCTGGT-3′

145 55

TFAm F5′-GGTGTATGAAGCGGATTTT-3′R5′-CGAGGTCTTTTTGGTTTTC-3′

186 53

α-Tubulin F5′-ATCACAGGCAAGGAAGATGC-3′R5′-GGGCTGGGTAAATGGAGAAC-3′

247 59

Fig. 1. Effect of hypoxia preconditioning with cobalt chloride supplementation(10 mg/kg bw CoCl2∙6H2O for 15 days) on endurance performance in both sedentaryand trained rats. Values are mean±SD. n=8 in each group. aSignificantly differentfrom control. bSignificantly different from training. †Significantly different (pb0.01).

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on SDS-PAGE and electroblotted onto a nitrocellulose membrane. Themembrane was blocked with 5% non-fat dry milk for 1 h, washed andprobed with respective rabbit polyclonal antibodies. The mem-brane was washed with PBS-Tween-20 (0.1%) and incubated withanti-rabbit-IgG-HRP conjugate (1:40,000) for 2 h. The membranewas then incubated with chemiluminescent substrate (Sigma-Aldrich,St Louis, MO, USA) and the bands were developed using X-ray films(Kodak). All antibodies were purchased from Santa Cruz Biotech andthe chemiluminescence peroxidase substrate kit was purchased fromSigma. The intensities of the bands were quantified by using ImageJsoftware (National Institutes of Health; freely available at http://rsb.info.nih.gov/ij/).

mRNA expression studies. Reverse transcription PCR (RT-PCR) tech-nique was used to study mRNA expression in the muscle. Total RNAwas extracted from muscle homogenates by using Trizol method. TheRNA quality and quantity were checked by both spectrophotometryand agarose gel electrophoresis. cDNA was prepared using first strandc-DNA synthesis kit (Fermentas) as per manufacturer's instructions.PCR primers for various genes were purchased from Integrated DNATechnologies, Inc., USA. The details of the sequences, annealing tem-perature, and amplicon size are represented in Table 1. PCR wasconducted using a thermal cycler (MJ Research) with the followingconditions: Initial denaturation was carried out at 94 °C for 2 minfollowed by denaturation at 94 °C for 1 min, annealing for 1 min andextension at 68 °C for 1 min for a total of 30 cycles followed by finalextension at 68 °C for 10 min. The PCR products were electrophoresedon 2% agarose gel and visualized by UV transillumination. The imagesof the PCR products were acquired and the intensities of the bandswere quantified by using ImageJ software (National Institutes ofHealth; freely available at http://rsb.info.nih.gov/ij/).

DNA binding activity. Nuclear extracts were prepared from redgastrocnemius using a nuclear‐cytoplasmic extraction kit (Biovision,Mountainview, CA, USA), following the manufacturer's instructions,and stored at −80 °C until further analysis. To assess nuclear HIF-1binding activity, gel mobility shift assays were carried out. The bindingmixture (25 μL) containing 5 μg protein of nuclear extract and 1 μg ofpoly dI–dC was incubated in a binding buffer (10 mmol/L Tris–HClpH 7.4, 50 mmol/L NaCl, 50 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/LEDTA, 5 mmol/L DTT) on ice for 10 min. Later 10 pmol of biotinylatedHIF-1 probe (Operon, Germany) with the following sequences wasadded and incubated at room temperature for an additional 30 min:HIF-1 F 5′-GCCCTACGTGCTGTCTCA-3′, HIF-1 R 5′TGAGACAGCAC-GTAGGGC-3′, HIF-1 mutant F 5′CCTAAAAGCTGTCTCA-3′, HIF-1 mutantR 5′TGAGACAGCTTTTAGG 3′. The samples were separated on a 6% na-tive polyacrylamide DNA retardation gel and then electroblotted ontopositively charged nylon membranes. Biotinylated DNA–protein com-plex was detected with peroxidase-conjugated streptavidin and achemiluminescent substrate kit (Peirce, Rockford, IL, USA).

Electron microscopy. Mitochondrial density in skeletal muscle wasdetermined by electron microscopy. Freshly removed red gastrocne-mius muscle from three rats of each group were taken, cut into piecesof ~1 mm3 size and processed as mentioned by Nisoli et al. (2004).For morphometric studies of mitochondria, 20 randomly selectedareas per animal were photographed at 25,000× magnification andcounted. For morphometric analysis of images, the University of Texasimage analysis software (http://ddsdx.uthscsa.edu/dig/download.html)was used.

Data analysis. All the experiments were performed on three differ-ent occasions and data are presented as mean±SD. One‐way analysisof variance with post-hoc Bonferroni analysis was used to determinestatistical significance between groups and pb0.05 was consideredas significant.

Results

Endurance performance

The endurance performancewas evaluated by calculating the swim-ming time until exhaustion in rats (Fig. 1). There was a two-fold(pb0.01) increase in endurance performance in cobalt supplementedgroup as observed by increased swimming time relative to controlgroup. Cobalt supplementation along with training further increasedthe swimming time relative to both control (3.3 fold; pb0.01) andtraining only group (1.4 fold; pb0.01).

Metabolic adaptations

To evaluate whether cobalt supplementation affects the glucose up-take inmuscles, wemeasured the expression ofmain glucose transport-er proteins Glut-1 and Glut-4 in gastrocnemius muscles (Fig. 2). Theexpression of Glut-1 increased significantly after cobalt supplementa-tion as compared to saline treated control but no change was observedin Glut-4 expression level. Swimming training with or without cobaltsupplementation for 14 days resulted in a marked increase expressionof Glut-1 and Glut-4 relative to control. However, Glut-1 expressionwas further enhanced by cobalt supplementation along with training(1.3 fold, pb0.01) as compared to training alone. A significant decrease(pb0.01) in blood glucose level in cobalt supplemented only and train-ing only groups relative to saline treated control further confirms the in-crease of glucose uptake from the blood. We also observed decreasedblood glucose level in cobalt supplementation along with traininggroup relative to training only group (Table 2).

Table 2 represents the biochemical activities of key enzymes ofglycolysis (PFK and hexokinase), TCA cycle (CS and SDH) and electron

Fig. 2. Effect of cobalt supplementation on the protein expression of glucose transporters Glut-1 and Glut-4. Representative immunoblots with their mean band intensities normal-ized with α‐tubulin. Data represents the mean±SD of three independent experiments. Data analyzed by one-way ANOVA with post‐hoc Bonferroni analysis. aSignificantly differentfrom control. bSignificantly different from training. †Significantly different (pb0.01).

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transport chain (COX). There was a significant increase in CS activityin cobalt supplemented (65%, pb0.01) as well as trained rats (93%,pb0.01) as compared to control. Cobalt supplementation along withtraining further enhanced CS activity significantly (pb0.01) as com-pared to training without cobalt supplementation. Similarly, a signif-icant increase in SDH activity was noted after cobalt supplementation(26%, pb0.01) and training (38%, pb0.01) relative to control. Cobaltsupplementation along with training further promoted SDH activity(16%, pb0.05) as compared to training only. Also, cobalt supplemen-tation and training enhanced PFKm activity significantly as comparedto control. Cobalt supplementation along with training further in-creased PFKm activity as compared to training. Furthermore, therewas a significant rise in hexokinase activity in cobalt supplementedand trained rats as compared to control but no significant changewas observed in cobalt supplemented along with training rats ascompared to training. Moreover, cobalt supplementation as well astraining enhanced COX activity significantly as compared to control.Cobalt supplementation along with training further enhanced COXactivity relative to training alone. There was no change in LDH activityin skeletal muscle in cobalt supplemented animals but training re-duced it significantly (Table 2).

Table 2Key metabolic enzyme activities, ATP level and nitrite level in skeletal muscle following hypoxBonferroni analysis. aSignificantly different from control. bSignificantly different from training.

Con Co

CS (μmol/g protein/min) 0.164±0.005 0SDH (μmol/g protein/min) 0.98±0.02 1Hexokinase (μmol/g protein/min) 36.2±2.3 4PFK (μmol/g protein/min) 0.122±0.007 0.1COX (μmol/g protein/min) 189±9 2LDH activity (U/g protein) 8614±425 84ATP (μmol/g protein/min) 0.032±0.002 0.0Nitrite level (mol/mg protein) 18.3±1.3 2Blood glucose (g/100 mL) 85.3±2.9 7

A significant enhancement in ATP level was observed in cobaltsupplemented (31%, pb0.01) and trained (56%, pb0.01) rats relativeto control. Cobalt supplementation along with training further increasedATP level (16%, pb0.01) as compared to training only (Table 2). A highermuscle ATP level augments muscle endurance.

Protein expressions of PDK-1 and PDK-4

Pyruvate dehydrogenase kinases (PDKs) are the enzymes, which in-hibit the entry of pyruvate into TCA cycle by checking the activity of thepyruvate dehydrogenase (PDH) enzyme that converts pyruvate to ace-tyl coenzyme A (Ac-CoA). The protein expression levels of PDK-1 andPDK-4 were found unaffected across the groups (data not shown).

mRNA and protein expressions of mitochondrial biogenesis markers

Semiquantitativemeasurement ofmRNA expression of variousmet-abolic enzymes as markers of mitochondrial biogenesis was performedand is summarized in Fig. 3. The expression of COX‐1, COX-4a, COX-4b,CS and PFKm increased after cobalt supplementation relative to control.Training only increased the mRNA expressions of these enzymes as

ia preconditioning. n=8 for each group. Data analyzed by one-way ANOVA with post‐hoc*Significantly different (pb0.05). †Significantly different (pb0.01).

b Tr CobTr

.27±0.013†a 0.316±0.014†a 0.376±0.017†a, b

.23±0.05†a 1.35±0.05†a 1.56±0.06†a, b

2.3±3.1†a 45.8±3.1†a 48.2±3.4†a

58±0.006†a 0.170±0.007†a 0.187±0.007†a ⁎b

37±14†a 255±11†a 285±13†a, b

51±391 4898±265†a 5021±268†a

42±0.003†a 0.050±0.003†a 0.058±0.003†a, b

9.2±1.6†a 24.6±1.6†a 29.2±1.8†a, b

4.1±3.0†a 75.2±3.0†a 71.1±2.1†a⁎b

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compared to control however training along with cobalt supplementa-tion further elevated the expression as compared to training only group.CS expression level remained unaltered in cobalt supplemented alongwith training group as compared to training. Furthermore, mitochon-drial transcription factor A (TFAm) mRNA expression level was also in-duced by cobalt supplementation and training.

PGC-1α is a key regulator of mitochondrial biogenesis and NRF-1induces the expression of nuclear coded mitochondrial proteins(Garnier et al., 2005; Liang and Ward, 2006). In our study, cobalt sup-plementation increased the protein expressions of PGC-1α and NRF-1as compared to control. Training also increased the protein levels ofPGC-1α and NRF-1 compared with control, which were further in-creased by cobalt along with training (Fig. 4).

Mitochondrial biogenesis. There was a significant increase in themitochondrial density in skeletalmuscle in cobalt supplemented seden-tary rats as compared to control (Fig. 5),which clearly demonstrates theenhancement of mitochondrial biogenesis in skeletal muscle of hypoxiapreconditioned rats.

Fig. 3. Induction of keymetabolic enzymemRNA expression asmitochondrial biogenesismarke(COX-1), cytochrome c oxidase-IVA (COX-IVA), cytochrome c oxidase-IVB (COX-IVB), citrate sfactor A (TFAm) with their mean band intensities normalized with α‐tubulin. Data represenwith post‐hoc Bonferroni analysis. aSignificantly different from control. bSignificantly different

HIF-1α protein expression and DNA-protein binding studies. West-ern blotting experiments showed enhanced expression of HIF-1α incobalt supplemented animals with or without training as comparedto respective controls (Fig. 4). To further assess the DNA bind-ing activity of HIF-1 to hypoxia response element (HRE), electro-phoretic mobility shift assay (EMSA) was carried out using highlyspecific oligonucleotide probe consisting of an enhancer region ofEPO gene. A very low DNA binding activity was detected in controlanimals. Training increased in DNA binding activity of HIF-1, however,much stronger DNA binding activity was observed in animalssupplemented with cobalt both in sedentary and trained conditions(Fig. 6).

Protein expression of PHD-1 and PHD-2. PHDs are the proteins re-sponsible for the degradation of HIF-1α in the presence of oxygen.Fig. 7 represents the protein expression levels of PHD-1 and PHD-2.The protein expression level of PHD-1 decreased after cobalt supple-mentation but increased significantly in trained rats compared withcontrol. Cobalt supplementation along with the training group had

rs by cobalt supplementation. RepresentativemRNA expression of cytochrome c oxidase-1ynthase (CS), phosphofructokinase muscle type (PFKm) and mitochondrial transcriptionts the mean±SD of three independent experiments. Data analyzed by one-way ANOVAfrom training. †Significantly different (pb0.01).

Fig. 4. Effect of cobalt supplementation on protein expression levels of HIF-1α, NRF-1 and PGC-1α. Representative immunoblots of HIF-1α, NRF-1 and PGC-1αwith their mean band in-tensities normalizedwithα-tubulin. Data represents themean±SDof three independent experiments. Data analyzed by one-wayANOVAwith post‐hocBonferroni analysis. aSignificantlydifferent from control. bSignificantly different from training. †Significantly different (pb0.01).

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lesser PHD-1 expression as compared to training only group. The pro-tein expression of PHD-2 increased in cobalt only and training onlygroups as compared to control; cobalt supplementation along withtraining group had more PHD-2 expression compared with trainingonly.

NOS expressions and NO production in muscle. NO level, as mea-sured by nitrite formation was significantly increased in skeletalmuscle of rats supplementedwith CoCl2 (sedentary as well as trained)as compared to control (Table 2). Fig. 8 represents the protein expres-sions of nNOS and eNOS in skeletal muscle of rats. The protein expres-sion of eNOS increased in skeletal muscle of CoCl2 supplemented,trained, and trained as well as supplemented rats; whereas nNOSexpression increased after cobalt supplementation but reduced intraining groups.

Discussion

In the present study, we report that hypoxia preconditioning withcobalt chloride enhances metabolic status by increasing glucoseuptake, aerobic respiration and mitochondrial biogenesis in muscle.Furthermore, we observed that hypoxia preconditioning with cobaltchloride enhances physical performance in sedentary as well as intrained rats. Glucose uptake is a regulatory step in muscle glucosemetabolism (Kern et al., 1990). Increase in glucose transporter ex-pression (Glut-1) in cobalt supplemented groups shows that glucoseuptake is up-regulated in skeletal muscle after cobalt supplementa-tion. Glut-4 is a major facilitative glucose transporter in skeletal

muscles and is very important in contraction induced glucose uptakeinto the tissue (Zoll et al., 2006). Glut-4 level in skeletal muscle in-creases readily in response to exercise training (Kraniou et al.,2004), and decreases with detraining (McCoy et al., 1994). In agree-ment with this, training with or without cobalt supplementation en-hanced Glut-4 expression in skeletal muscle but cobaltsupplementation with or without training did not affect Glut-4 ex-pression as compared to their respective controls. Moreover, the bio-chemical activities of various TCA cycle and glycolytic enzymes likeCS, SDH, hexokinase, and PFKm were found to be enhanced after co-balt supplementation. COX activity also increased in cobalt treatedrats. Previous studies have shown that enzyme activities of CS, SDH,PFK and COX in skeletal muscle increase after training in hypoxia con-ditions (Desplanches et al., 1996; Melissa et al., 1997; Ponsot et al.,2006) suggesting an increase in aerobic metabolism in skeletal mus-cles. Our results therefore indicate that cobalt supplementation in-creases glucose uptake and metabolism through aerobic respiration.Furthermore, PDKs are the enzymes that suppress TCA cycle by phos-phorylating and inactivating pyruvate dehydrogenase (PDH) that co-verts pyruvate to Ac-CoA under low oxygen conditions. Unable toenter the citric acid cycle, pyruvate converts into lactate by LDH activ-ity. Among the currently known four isoforms of PDK; PDK-1, 2, and 4are expressed in rat skeletal muscle (Peters et al., 2001). Among these,PDK-1 is transcriptionally regulated by HIF-1 (Kim et al., 2006). Westudied the protein expression of PDK-1 and 4 and found that PDK1expression did not change with either cobalt supplementation ortraining despite high HIF-1 expression (data not shown). Further-more, high LDH activity is a marker of anaerobic performance

Fig. 5. Hypoxia preconditioning with cobalt chloride enhances mitochondrial density in skeletal muscle. The representative micrographs of red gastrocnemius myofibers show theincrease in mitochondrial number in subsarcolemmal region. Arrows indicate the mitochondria (magnification, ×25,000).

Fig. 6. Identification of HIF-1 DNA binding activity by EMSA at the Epo enhancer region.Nuclear extracts from red gastrocnemius muscle were prepared from different groups.50 μg of protein extract was used for the EMSA. The gel picture shown is representativeof three independent experiments.

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(Kaczor et al., 2005). Simultaneously, LDH expression is highly regu-lated by HIF-1 (Firth et al., 1994). In our study, muscle LDH activitywas found to be unaltered in cobalt supplemented rats. Training re-duced LDH activity, which is in agreementwith Revan and Erol (2011)and Burneiko et al. (2006). Therefore, in view of the above,we infer thathypoxia preconditioning with cobalt supplementation increases cellu-lar aerobic respiration in skeletal muscle.

Skeletal muscles adapt to endurance exercise with an increase inmitochondria content and ATP level (Hood and Saleem, 2007;Joyner and Coyle, 2008). Our results of electronmicroscopy of skeletalmuscle sections confirmed that mitochondria density increases inskeletal muscle as a result of hypoxia preconditioning by cobalt.Furthermore, it has been demonstrated by Garnier et al. (2005) thatmitochondrial adaptation to endurance training in humans isassociated with activation of PGC-1α, as well as its downstreamtranscription factors (NRF-1, TFAm, etc.), which induce coordinatedexpression of mitochondrial transcripts. Also, it promotes the remod-eling of muscle tissue to a fiber-type composition that is metabolicallymore oxidative and less glycolytic in nature (Liang and Ward, 2006).We observed an increase in the mRNA or protein expressions of theabove mentioned genes and mitochondria enzymes (CS, COX-I,COX-IV A and B) following cobalt induced hypoxia preconditioningwith or without training, indicating an increased mitochondrialdensity in muscles to sustain from fatigue for a longer period. Further-more, the ATP content of skeletal muscle was found to be increased as aresult of both exercise and Co2+ administration. ATP content directlyinfluences exercise economy and higher levels of ATP after cobalt

Fig. 7. Effect of cobalt supplementation on protein expressions of PHD-1 and PHD-2. Representative immunoblots of PHD-1 and PHD-2 with their mean band intensities normalizedwith α-tubulin. Data represents the mean±SD of three independent experiments. Data analyzed by one-way ANOVAwith post‐hoc Bonferroni analysis. aSignificantly different fromcontrol. bSignificantly different from training. †Significantly different (pb0.01).

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supplementation provide direct evidences of increased muscle endur-ance. To the best of our knowledge, our study is the first one to showthe effect of cobalt supplementation on mitochondrial biogenesis.

Fig. 8. Cobalt supplementation enhances eNOS and nNOS expressions in skeletal muscle innormalized with α-tubulin. Data represents the mean±SD of three independent experimendifferent from control. bSignificantly different from training. †Significantly different (pb0.01

The NO level elevated in muscles of cobalt supplemented animals.It has been shown that NO plays an important role in mitochondrialbiogenesis in skeletal muscles (Nisoli and Carruba, 2006). Because of

rats. Representative immunoblots of eNOS and nNOS with their mean band intensitiests. Data analyzed by one-way ANOVA with post‐hoc Bonferroni analysis. aSignificantly).

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its vasodilation property, it regulates blood flow to tissues and thus ithelps in supplying respiratory substrates tomitochondria and redistrib-utes heat generated by respiring mitochondria. It also regulates thebinding to and release of O2 from hemoglobin (Wolzt et al., 1999) andthus supplies O2 to mitochondria. eNOS is predominant in oxidativeskeletal muscles and seems to be the source of NO production that is in-volved in mitochondrial biogenesis (Bossy-Wetzel and Lipton, 2003;Nisoli et al., 2004). In the present study, the NO level was found to besignificantly high in the skeletal muscles of supplemented as well astrained rats (Table 2). The expression of eNOS and nNOS also increasedafter cobalt supplementation (Fig. 8). Training enhanced the expressionof eNOS and nNOS but the effect on nNOS was not very profound per-haps because the major source of NO in aerobic muscle is eNOS (Learyand Shoubridge, 2003; Nisoli and Carruba, 2006; Nisoli et al., 2003)not nNOS. eNOS is an HIF-1 regulated protein (Coulet et al., 2003) andin some reports, the expression of nNOS is also found to be regulatedby hypoxia (Prabhakar et al., 1996; Ward et al., 2005). Therefore, oneof the major reasons for the increase in performance by cobalt supple-mentation may be NO mediated mitochondrial biogenesis. To furtherinvestigate this, we studied the protein–protein interaction of HIF-1with PGC-1α and NRF-1. First, we immunoprecipitated HIF-1 alongwith its interacting proteins in solution and then using the Westernblotting technique, we checked whether the above mentioned proteinsinteract with HIF-1 or not. We did not find any direct interaction be-tween HIF-1 and the above mentioned proteins, which indicates thatHIF-1 does not directly influence their expression (data not shown).Previous studies by Nisoli et al. (2003, 2004) have reported thatupregulation of eNOS expression and NO production enhances the ex-pression of PGC-1α. Our study also indicates towards a similar mecha-nism of PGC-1α and mitochondrial biogenesis induction by hypoxia;cobalt supplementation activates HIF-1 that increases expression ofeNOS and NO production in skeletal muscle, which further leads tothe increase in the expression of PGC-1α that orchestrates genes re-sponsible for mitochondrial biogenesis.

HIF-prolyl hydroxylases (HPHs), also referred as prolyl hydroxylasedomain (PHD) proteins play a critical role in regulating HIF abundanceand oxygen homeostasis (Webb et al., 2009). PHDs are an evolutionarilyconserved subfamily of dioxygenases that uses iron and ascorbate ascofactors and oxygen and 2-oxoglutarate as co-substrates. These pro-teins are responsible for the oxygen-dependent hydroxylation of theα-subunit of HIF at proline 402 and 564 residues. This hydroxylation fur-ther signals HIF-α polyubiquitination and proteasomal degradation.Among the four isoforms (PHD1, PHD2, PHD3 and P4H-TM), PHD2 isthe most abundant and active form that hydroxylates the α-subunit ofHIF (Appelhoff et al., 2004). It has been shown that PHD2 and PHD3have a hypoxia responsive element (HRE) sequence (Metzen et al.,2005; Pescador et al., 2005) and are transcriptionally induced by hypoxiain an HIF-1 dependent manner (Berra et al., 2006; D'Angelo et al., 2003;Marxsen et al., 2004), which promotes a negative feedback loop.Prolonged hypoxia or HIF-1 expression can unexpectedly enhance theexpression of these PHDs (Berra et al., 2006). In agreement with this,we found an increase in PHD2 expression under cobalt supplementationand training. In contrast, PHD-1 expression was lowered in cobaltsupplemented rats with or without as compared to respective controlswhereas it increased in training alone group as compared to control.Erez et al. (2004) have also shown that PHD-1 expression decreasesunder hypoxic conditions and aryl hydrocarbon nuclear transporter(ARNT/HIF-1β) has a role in the regulation of PHD-1 under hypoxic con-ditions though it requires further studies.

In conclusion, our main findings are: (i) Hypoxia preconditioningwith cobalt enhances the physical performance in sedentary as wellas trained rats. (ii) It increases aerobic respiration and ATP productionin skeletal muscle. (iii) It augments metabolic status in skeletal mus-cles by enhancing glucose uptake. (iv) Hypoxic preconditioning alsoimproves mitochondria biogenesis in skeletal muscle. (v) There wasno direct interaction found between HIF-1 and PGC-1α, and NRF-1

proteins but an increase in the expression of eNOS and NO productionin skeletal muscle indicates toward NO induced PGC-1α expression inskeletal muscle.

AbbreviationsARNT Aryl hydrocarbon nuclear receptorATP Adenosine triphosphateCoCl2 cobalt chlorideCOX cytochrome c oxidaseCo2+ cobaltous ionCS Citrate synthaseCoIP Co-immunoprecipitationeNOS Endothelial nitric oxide synthaseEMSA electrophoretic mobility shift assayEpo ErythropoietinGlut Glucose transporterHA High AltitudeHIF Hypoxia inducible factorHO-1 Heme oxygenase-1HRE Hypoxia response elementLDH Lactate dehydrogenaseMb MyoglobinnNOS Neuronal nitric oxide synthaseNO Nitric oxideNO2

- Nitrite ionNRF-1 Nuclear respiratory factor-1PBS Phosphate buffer salinePDH Pyruvate dehydrogenasePDK Pyruvate dehydrogenase kinasePFKm Phosphate fructokinase muscle typePGC-1α peroxisomeproliferator activated receptor gamma coactivator-

1αPHD Prolyl hydroxylase domain containing proteinROS Reactive oxygen speciesSDH Succinate dehydrogenaseTCA Tri carboxylic acid_VO2max maximal oxygen consumptionVEGF vascular endothelial growth factorTFAm Mitochondrial transcription factors A

Conflict of interest statement

The authors have no conflict of interest for this study.

Acknowledgment

Financial assistance for this work was provided by the Defense Re-search and Development Organization, Government of India. The au-thors acknowledge the technical support and encouragement providedby Dr. Rameshwar Singh and Mr. Bhagwat Singh. Saurabh Saxena wasa recipient of the Senior Research Fellow (SRF) scholarship from theCouncil for Scientific and Industrial Research (CSIR), Govt. of India andDhananjay Shukla was a recipient of the Senior Research Fellowshipfrom University Grants Commission (UGC), Govt. of India.

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