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

Abstract

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.

Graphical abstract

A proposed signaling pathway regulated by acetic acid administration in skeletal muscle of exercise-trained mice.

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 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 slow-twitch 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 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 ΔC_t and ΔΔC_t values.

Immunoblot analysis

Antibodies were obtained from the following sources: AMPKα and phospho-AMPKα 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 one-way or two-way ANOVA using the SAS software for Windows release 9.2 (SAS Institute Inc., Cary, NC, 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 <0.05 are reported as statistically significant.

Results

Acetic acid prevents diet-induced visceral fat accumulation

Acetic acid administration significantly reduced weight gain and visceral fat depot in both sedentary and exercised mice compared to the corresponding control groups (p = 0.0011 and p < 0.0001 for overall acetic acid effect). Mesenteric fat depot was significantly decreased in exercised control mice compared to that of sedentary mice, while no significant differences were observed in weight gain and total visceral fat between sedentary and exercised control mice (Fig. 1). There were no significant differences in food intake among all treatment groups (data not shown).

Fig. 1. Effects of dietary acetic acid on body weight of sedentary and trained groups (A and B, respectively), body weight gain (C), and visceral fat depot (D) of exercise-trained mice.

Notes: Animals were subjected to the exercise training program for 8 weeks. Visceral fat depot includes epididymal, mesenteric, and retroperitoneal fat. Values represent mean ± SE (n = 6). *Significantly different from respective control (*p < 0.05, **p < 0.01). ^†S-control vs. Ex-Control (p < 0.05).

Acetic acid promotes endurance capacity by increasing fatty acid oxidation in muscle

The endurance capacity of mice was determined by the maximum running time on the treadmill. The maximum running time was significantly increased in acetic acid group compared to the control group at Week 6 and Week 8 (p < 0.05, Fig. 2). Exercise training of control group induced an average 1.4-fold increase in maximum running time of mice at Week 8 compared to their baseline, while acetic acid administration with exercise induced an average 1.8-fold increase compared to their baseline. The serum levels of major metabolic substrates after treadmill running showed that acetic acid administration significantly decreased serum levels of glucose (p = 0.005 for overall acetic acid effect), non-esterified fatty acid (NEFA) (p = 0.028 for overall acetic acid effect), and urea nitrogen (p = 0.010 for overall acetic acid effect) (Table 1).

Fig. 2. Effects of dietary acetic acid on endurance running time.

Notes: Endurance capacity was evaluated with treadmill exercise every 2 weeks. Values represent mean ± SE (n = 6). *Significantly different from Ex-Control (p < 0.05).

Table 1. Serum parameters.

	S-Control	S-Acetic acid	Ex-Control	Ex-Acetic acid	Effects (p value)^a
					E	A	E × A
Glucose, mg/dL	38.6 ± 1.54	40.2 ± 0.99	40.0 ± 0.78	35.17 ± 1.47*	0.286	0.005	0.634
Cholesterol, mg/dL	14.8 ± 0.44	15.0 ± 1.16	13.5 ± 0.39	9.63 ± 1.43	0.034	0.264	0.205
TG, mg/dL	0.64 ± 0.18	0.46 ± 0.13	0.42 ± 0.12	0.57 ± 0.14	0.905	0.905	0.915
NEFA, μEq/L	107.4 ± 12.89	89.4 ± 3.34	110.8 ± 3.06	87.25 ± 8.71*	0.945	0.028	0.749
Urea nitrogen, mg/dL	6.5 ± 0.27	6.0 ± 0.04	6.15 ± 0.22	4.18 ± 0.84*	0.020	0.010	0.109
Creatine kinase, IU/L	36.5 ± 5.55	36.6 ± 1.31	50.67 ± 13.68	62.0 ± 3.02	0.040	0.559	0.573
Lactate, mM	1.12 ± 0.11	1.21 ± 0.12	1.07 ± 0.12	1.07 ± 0.11	0.810	0.383	0.267

At the end of study, the animals ran for 30 min according to the training protocol and were sacrificed by Avertin (2,2,2-tribromoethanol) asphyxiation. Values represent means ± SE (n = 6).

^a E, Exercise; A, Acetic acid.

^* Significantly different from control (p < 0.05).

To further explore the impact of acetic acid on fatty acid oxidation in muscle, we analyzed the mRNA expressions of key regulators in muscle oxidative metabolism. Since the serum parameters showed no effect of acetic acid in sedentary group, we focused on the exercised group for further metabolic analyses. Acetic acid significantly up-regulated muscle mRNA expressions of CPT1β, UCP2, UCP3, and HSL, and down-regulated ACC2 mRNA compared to those of the control group (Fig. 3(A)). In addition, acetic acid administration increased the expressions of proteins involved in regulation of muscle oxidative properties such as LKB1, pAMPK, and PGC-1α (Fig. 3(C)).

Fig. 3. Effects of dietary acetic acid on selected gene expressions for fat oxidation (A, B), and protein expressions (C) from soleus muscle of exercise-trained mice.

Notes: ACC2, acetyl CoA carboxylase; CPT1β, carnitine palmitoyltransferase 1 beta; UCP2, uncoupling protein 3; UCP3, uncoupling protein 3; HSL, hormone-sensitive lipase; AMPK, AMP-activated protein kinase; pAMPK, phosphorylated AMPK; PPARδ, peroxisome proliferator-activated receptor δ; MHC I, myosin heavy chain I; MHCIIa, myosin heavy chain IIa; MHC IIb, myosin heavy chain IIb; LKB1, Liver kinase B1; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator-1α. Relative quantities of each gene were presented in terms of 2^–ΔΔCt, calculated using the ΔC_t and ΔΔC_t values. Values represent means ± SE (n = 3). *Significantly different from Ex-Control (*p < 0.05, **p < 0.01, ***p < 0.001).

Acetic acid enhances exercise-mediated responses in the skeletal muscle

AMPK is known to be the main regulator of exercise-mediated responses in the skeletal muscle, and PPARδ, a key enzyme for such muscle fiber transformation, is one of the primary downstream targets of AMPK.^22–24) The gene expressions of AMPK and PPARδ were significantly up-regulated by acetic acid, resulting in increased MHCI (oxidative type I-isoform) and MHCIIa (mixed oxidative-glycolytic type IIa-isoform), and decreased MHCIIb (glycolytic type IIb-isoform) expressions in the soleus muscle of mice (Fig. 3(B)). The twitch speed of type IIa fiber is fast and it contains high glycogen content, but mitochondrial density and oxidative capacity are high. Thus, type IIa fiber is more sensitive to the mode of exercise. Consistent with the changes of MHCI and MHCIIb mRNA levels, their protein ratio was significantly increased by acetic acid treatment in exercised mice (Fig. 3(C)).

Discussion

There is strong evidence both in vitro and in vivo that acetic acid reduces body fat by increasing hepatic fatty acid oxidation and energy expenditure.^18,25,26) Consistently, our current study confirmed that supplementation with acetic acid prevented visceral fat accumulation without change in food intake in exercise-trained mice. Our results also showed that acetic acid administration during treadmill exercise of mice is associated with the enhanced muscle oxidative properties through AMPK-mediated signaling pathway.

It is reported that the beneficial effects of acetic acid on hepatic lipid metabolism are mediated by AMPK.^26,27) These effects include the both down-regulation of lipogenic genes and up-regulation of fat oxidative genes in the liver by AMPK signaling pathway. However, there is no evidence that the effects of acetic acid on fat oxidative properties in exercise-trained mice, especially in muscle. We showed that acetic acid suppressed the expression of the lipogenic gene ACC2 and increased the expression of several oxidative genes, including CPT1β, UCP2, UCP3, and HSL, through enhanced AMPK activity in skeletal muscle. In addition to oxidative mRNA expression, we also found the up-regulated protein expression involved in fat oxidation such as LKB1, pAMPK/AMPK, and PGC-1α, indicating that acetic acid controls fuel utilization by enhancing fatty acid oxidation in muscle during exercise. In fact, it was suggested that LKB1-AMPK-PGC-1 signaling is critical to the beneficial effects of training.²⁸⁾ AMPK can be activated by increased AMP-to-ATP ratios and by phosphorylation through upstream AMPK kinases such as LKB1.^29,30) Accordingly, LKB1 phosphorylates and thus activates AMPK at site Thr172 of the α-subunit.^31,32) Moreover, AMPK plays major role in the regulation of PGC-1α expression.³³⁾ Suwa et al.³⁴⁾ reported that the 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-induced activation of AMPK in muscle caused up-regulated expression of PGC-1α, and Terada et al.³⁵⁾ reported the increase in mRNA expression of PGC-1α after endurance training.³⁵⁾

Meanwhile, phosphorylation/inhibition of muscle ACC2 mediated by activation of AMPK is the principal pathway regulating fatty acid oxidation in muscle during exercise.^36,37) Phosphorylation of ACC2 suppresses the malonyl-CoA-mediated inhibition of CPT-1, which is a rate-limiting enzyme in mitochondrial fatty acid beta-oxidation.³⁶⁾ The importance of ACC2 in the regulation of muscle lipid metabolism is shown by the increased rates of fatty acid oxidation, energy expenditure, and reduced malonyl-CoA levels in mice followed by disruption of the ACC2 gene.³⁸⁾ Taken together, the present findings strongly suggest that acetic acid could promote exercise-based adaptations in muscle oxidative metabolism.

Acetic acid has been shown to affect hepatic and muscular glycogen metabolism by facilitating glycogen replenishment in rodents with or without exercise.^17,39,40) This effect of acetic acid suggests the preferential use of fat (over glucose) as an energy source in the liver and muscle during exercise, resulting in improved endurance performance. Therefore, we measured serum levels of major metabolic substrates after exercise to test the effect of acetic acid on fuel utilization. Serum levels of NEFA, which is one of the major fuels available when glycogen and glucose are used for oxidative metabolism during exercise,⁴¹⁾ were significantly lower in the acetic acid group. Consistent with the reduced NEFA levels, animals that were fed acetic acid exhibited reduced serum urea nitrogen levels. When the body cannot obtain energy from carbohydrate and fat sources, proteins are catabolized to a greater degree. Accordingly, urea nitrogen concentration was dramatically increased following sustained exercise, indicating that urea nitrogen is a very sensitive index of endurance ability.^42,43) Therefore, our results, along with those of previous studies on glycogen replenishment, suggest that acetic acid could stimulate fatty acid oxidation and reduce carbohydrate and protein catabolism following exercise. However, further studies are required to confirm the effects of acetic acid on endurance performance with respiratory measurements.

Exercise-based adaptations in muscle are linked to increased expression of genes involved in fatty acid oxidation and oxidative fiber-type transformation,^13,44,45) and these adaptations may contribute to improved endurance performance.^46,47)

In the present study, acetic acid supplementation along with training enhanced exercise-based adaptations in muscle, as reflected by the increased expression of genes involved in energy homeostasis such as LKB1, AMPK, PPARδ, and PGC-1α. Indeed, PPARδ, the primary downstream target of AMPK, is a key regulator of lipid and carbohydrate metabolism in muscle,⁴⁸⁾ and targeted activation of PPARδ in muscle leads to increased oxidative muscle fibers and enhanced running endurance.^11,49) Narkar et al.¹⁰⁾ showed that simultaneous treatment with AMPK and PPARδ agonists in mice induced the expression of muscle oxidative genes in a synergistic fashion, suggesting that the interaction between AMPK and PPARδ contributes to skeletal muscle transcriptome reprogramming following exercise. In addition, the MHC isoforms I, IIa, and IIb have been widely used as the primary biomarkers to determine fiber-type composition, and it has been shown that the expression of MHCI and IIb is up-regulated and down-regulated, respectively, in muscle after endurance exercise training.⁵⁰⁾ Interestingly, muscle-specific deletion of PPARδ decreased MHCI mRNA levels and increased MHCIIb mRNA levels in muscle.⁵¹⁾ In addition, increased expression of PGC-1α in muscle caused the fast-to-slow fiber-type transformation accompanied suggesting that PGC-1α solely converts all fibers, but other signals also influence the fiber phenotype such as calcineurin-Mef2 pathway.⁵²⁾ Indeed, PGC-1α is downstream of the calcium/calcineurin/CaM kinase pathway. Although the exact mechanism of PGC-1α on the fiber-type transformation has not been fully elucidated, it is quite obvious that the PGC-1α is one of key factors influencing the fiber-type transformation. Our current results, showing that acetic acid promotes exercise-based alterations in MHC isoform expression through up-regulated mRNA expression of AMPK, PPARδ, and protein expression of pAMPK/AMPK and PGC-1α in muscle, is consistent with these previous findings. There is strong possibility for acetic acid along with exercise having a combinatorial effect on the biological response during exercise.

The present study demonstrated that acetic acid administration promotes the expression of genes and proteins that enhance muscle oxidative properties following sustained exercise training. In addition, the reduced serum levels of NEFA and urea nitrogen indicate increased fatty acid oxidation during exercise in mice given acetic acid. These observations provide an important basis for the application of acetic acid as a major component of novel ergogenic aids and warrant further clinical evaluation.

Author Contributions

JH Pan and JH Kim designed the study and drafted the manuscript. YJ Kim, D-H Shin, S Kim, and M Shin reviewed the manuscript. HM Kim and ES Lee participated in a part of treadmill experiment and animal care. SH Kim and JH Lee consulted about the design of the study and troubleshooting of western blot. All authors reviewed and approved the final manuscript.

Acknowledgments

We thank “Carcinogenesis laboratory” members of Kyungbook National University for technical advice.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by High Value-added Food Technology Development Program, funded by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET) [grant number 112114-2].