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Acetic acid enhances endurance capacity of exercise-trained mice by increasing skeletal muscle oxidative properties a

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Jeong Hoon Pan , Jun Ho Kim , Hyung Min Kim , Eui Seop Lee , Dong-Hoon Shin , b

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Seongpil Kim , Minkyeong Shin , Sang Ho Kim , Jin Hyup Lee & Young Jun Kim

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Department of Food and Biotechnology, Korea University, Sejong, Republic of Korea

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R&D Center, Daesang Corp., Icheon, Republic of Korea

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School of Global Sport Studies, Korea University, Sejong, Republic of Korea Published online: 22 May 2015.

Click for updates To cite this article: Jeong Hoon Pan, Jun Ho Kim, Hyung Min Kim, Eui Seop Lee, Dong-Hoon Shin, Seongpil Kim, Minkyeong Shin, Sang Ho Kim, Jin Hyup Lee & Young Jun Kim (2015): Acetic acid enhances endurance capacity of exercisetrained mice by increasing skeletal muscle oxidative properties, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1034652 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1034652

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Bioscience, Biotechnology, and Biochemistry, 2015

Acetic acid enhances endurance capacity of exercise-trained mice by increasing skeletal muscle oxidative properties Jeong Hoon Pan1, Jun Ho Kim1, Hyung Min Kim1, Eui Seop Lee1, Dong-Hoon Shin1, Seongpil Kim2, Minkyeong Shin2, Sang Ho Kim3, Jin Hyup Lee1 and Young Jun Kim1,* 1

Department of Food and Biotechnology, Korea University, Sejong, Republic of Korea; 2R&D Center, Daesang Corp., Icheon, Republic of Korea; 3School of Global Sport Studies, Korea University, Sejong, Republic of Korea

Received February 2, 2015; accepted March 17, 2015

Downloaded by [New York University] at 13:51 25 May 2015

http://dx.doi.org/10.1080/09168451.2015.1034652

Acetic acid has been shown to promote glycogen replenishment in skeletal muscle during exercise training. In this study, we investigated the effects of acetic acid on endurance capacity and muscle oxidative metabolism in the exercise training using in vivo mice model. In exercised mice, acetic acid induced a significant increase in endurance capacity accompanying a reduction in visceral adipose depots. Serum levels of non-esterified fatty acid and urea nitrogen were significantly lower in acetic acid-fed mice in the exercised mice. Importantly, in the mice, acetic acid significantly increased the muscle expression of key enzymes involved in fatty acid oxidation and glycolytic-to-oxidative fiber-type transformation. Taken together, these findings suggest that acetic acid improves endurance exercise capacity by promoting muscle oxidative properties, in part through the AMPK-mediated fatty acid oxidation and provide an important basis for the application of acetic acid as a major component of novel ergogenic aids. Key words:

acetic acid; muscle protein expression; oxidative properties; exercise training; mouse

One of the major determinants of endurance capacity is increased fat oxidation, which leads to the sparing of glycogen consumption in muscle and the liver during exercise.1,2) β-oxidation in muscular mitochondria followed by aerobic respiration is required to generate adequate ATP for muscular energy during exercise3); thus, regulation of energy metabolism by increasing fat oxidation and decreasing carbohydrate consumption enhances endurance capacity during prolonged exercise. AMP-activated protein kinase (AMPK) is a key regulator in mediating the acute and prolonged effects of exercise on fatty acid metabolism in skeletal muscle.4,5) Importantly, AMPK is activated by depletion of ATP and increased AMP, such as diet restriction/hypoglycemia, exercise, and muscular contraction. Activated AMPK is known to stimulate an increase in muscle *Corresponding author. Email: [email protected] © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

glucose transport6) and fatty acid oxidation7) and to inhibit ATP-consuming process to restore the energy balance.8,9) Mouse skeletal muscle fibers are classified into four types based on their contractile and metabolic properties: type I (oxidative); type IIa (mixed oxidative-glycolytic); type IIx (glycolytic); and type IIb (glycolytic) myofibers. These are characterized based on their energy source during exercise or physical activity: type I fibers preferentially metabolize fatty acids as an energy substrate and express the slow isoforms of contractile proteins, whereas type II fibers preferentially use carbohydrates and express the fast isoforms of contractile proteins.10,11) Exercise training stimulates a remodeling program in skeletal muscle; increased expression of genes involved in the oxidative slowtwitch contractile apparatus, mitochondrial respiration, and fatty acid oxidation.12–14) In addition, skeletal muscles rich in oxidative fibers are resistant to muscle wasting15) and may contribute to enhanced endurance performance. Acetic acid, a major organic acid in vinegar, has been shown to promote fatty acid oxidation and muscle glycogen repletion during exercise in rodents.16,17) Recently, it is reported that administration of acetic acid increased energy expenditure and suppressed body fat mass,18) implying that acetic acid could enhance endurance capacity by reprogramming muscle fiber type and energy utilization in muscle during exercise. Therefore, we investigated the effects of acetic acid on endurance capacity and related physiological metabolism in mice, and this finding may provide an important basis for using acetic acid as a novel source for ergogenic aid.

Methods Animal and diet. The care and treatment of experimental animals conformed to a protocol approved by the Institutional Animal Care and Use Committee of Korea University (Seoul, Korea). Sixty female C57BL/ 6 mice (5-week-old) were purchased from Samtako Co. Ltd (Osan, Korea) and were housed in individual cages

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in a windowless room with a 12 h light–dark cycle. During a 2-week adaptation period, all mice in the exercised group were subjected to running exercise 3 times (velocity of 10 m/min on 0° inclination for 15 min with shock grid OFF, followed by 10 min with shock grid ON) to acclimate to the treadmill. At the end of the adaptation period, all animals were subjected to an endurance test for the measurement of their baselines on the running time to exhaustion. To minimize individual variations on endurance capacity baseline, we selected 12 mice with the closest value to the mean baseline from the original 30 mice. Finally, selected mice were divided into two groups (Ex-control and Ex-acetic acid). Food grade acetic acid was dissolved in normal saline and administered orally to mice at 10 mL/kg body weight once daily for 8 weeks with vehicle. Body weight and food intake were recorded weekly. At the end of study, the mice were fasted for 4 h, ran for 30 min according to the endurance protocol, and then immediately euthanized by Avertin (2,2,2-Tribromoethanol, Sigma-Aldrich). Blood was collected by cardiac puncture and internal organs (visceral fat and skeletal muscle) were also weighed. Exercise training and endurance protocol. During the experimental period, all animals were trained on a motorized treadmill (YS-03-2; Mirae-ST Corp., Daejeon, Korea) three times a week. Training was performed for 15 min (5 min at 10 m/min, then an increase of 1 m/min every minute for 10 min) on a 10° incline with a shock grid (0.97 mA, 1 Hz) to encourage the mice to run. Endurance capacity was determined every other week by placing animals on an individual treadmill at room temperature. The exercise regimen was started with shock grid ON and 10° inclination at 10 m/min for 5 min, speed was increased by 1 m/min up to 20 m/min (10 min with increase speed), and then held at 20 m/min until exhaustion. Based on previous studies that measured treadmill endurance capacity,19– 21) the mice were defined as exhausted if they were willing to sustain on the shock grid five times for more than 2 s or remain on the shock grid for five consecutive seconds. At the moment of exhaustion, the mouse was removed from the treadmill. The total running time until exhaustion was recorded and used as the index of endurance capacity. Biochemical parameter analysis in serum. The serum was separated from whole blood samples by centrifugation at 2000 × g for 20 min at 4 °C. Serum levels of glucose, urea nitrogen, creatine kinase, triglyceride (TG), total cholesterol, and lactate were measured with an ARCO PC (Biotecnica Instruments SpA, Rome, Italy) using commercial kits as specified by the manufacturer. Total RNA extraction and determination of mRNA level by quantitative PCR. Soleus muscle tissues were homogenized in 1 mL of Easyblue reagent, and then total RNA was isolated according to the manufacturer’s protocol (iNtRON Biotechnology, Sungnam, Kyunggi,

Korea). RNA was quantified by spectroscopy (NanoDrop, Wilmington, NC, USA). Total RNA (2 μg) was reverse transcribed to cDNA with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster, CA, USA) using random and oligo (dT) primers in a 20 μL reaction according to the manufacturer’s protocol. The resulting cDNA was used in the qPCR along with specific primers for the following target genes: carnitine palmitoyltransferase 1β (CPT1β; NM_013200.1), uncoupling protein 2 (UCP2; NM_062227.2), UCP3 (NM_013167.2), hormone-sensitive lipase (HSL; NM_012859.1), acetyl-CoA carboxylase 2 (ACC2; NM_053922.1), AMP-activated protein kinase (AMPK; NM_023991.1), peroxisome proliferator-activated receptor δ (PPARδ; NM_013141.2), myosin heavy chain I (MHC I; NM_001135158.1), and MHC IIb (NM_019325.1). Each 20 μL reaction contained 100 ng of cDNA, 10× power SYBR Green Mastermix (mBiotech, Hanam, Korea), and forward and reverse primers. All reactions were carried out in an ABI 7500 thermocycler (Applied Biosystems) using the following thermal cycling program: 50 °C for 2 min, 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The results were normalized to β-actin (NM_031144.2) as an internal standard, and the relative quantity of each gene is presented in terms of 2–ΔΔCt, which was calculated using the ΔCt and ΔΔCt values. Immunoblot analysis. Antibodies were obtained from the following sources: AMPKα and phosphoAMPKα Thr172 (Cell Signaling Technology, Danvers, MA, USA), LKB1, PGC-1α, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), UCP (Abcam, Gyeonggi, Korea), MHC I, IIa and IIb (BA-F8, SC-71, and BF-F3, respectively, Developmental Studies Hybridoma Bank, Iowa city, IA). Conventional immunoblotting procedures were used to detect the target proteins. Soleus muscle tissues were collected to extract protein using RIPA buffer (Cell Signaling Technology). Lysates were then cleared by centrifugation at 15,000 × g for 20 min. Total protein concentration was determined by Bradford assay. Equal amounts of protein were separated on 12% SDS/PAGE and the proteins were transferred to polyvinylidene difluoride membranes. The membranes were then blocked for 30 min in a PBS solution containing 5% BSA and 0.1% Tween-20, and then probed with primary antibody overnight in 5% BSA and 0.1% Tween-20 in PBS. After washing, membranes were incubated for 1 h with horseradish peroxidase-linked secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) in PBS solution containing 5% nonfat milk powder and 0.1% Tween-20. Finally, after three 5 min washes in 0.1% PBS/Tween-20, proteins were visualized by ImageQuant LAS 4000 (General Electric, Pittsburgh, PA, USA). Band intensities were quantified with imageJ software (Rasband W S, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/). Statistical analysis. Data were analyzed by oneway or two-way ANOVA using the SAS software for Windows release 9.2 (SAS Institute Inc., Cary, NC,

Acetic acid regulates muscle oxidative genes

USA) on the W64_VSHOME platform. Two-way ANOVA with repeated measures was performed to assess mean differences between groups for body weight, adipose depot, and serum analysis. One-way ANOVA with repeated measures was performed to assess mean differences between groups for gene expression and endurance capacity over time. The least squares means option using a Tukey–Kramer adjustment was used for multiple comparisons among the experimental groups. Data are shown as the mean ± SE. p values of