Medical treatment using high-voltage electric potential (HELP) device to generate an electric field (EF) is an alternative therapy commonly used in Japan. However, little is known about the underlying mechanisms of the potential benefits to health. The identification of EF exposure -related biomarkers is key to understanding the beneficial effects of EF therapy. We screened plasma metabolites obtained prior to and immediately after HELP exposure (18 kV, 30 min) in 10 healthy human subjects by via non-targeted plasma metabolomic analysis. Among 161 metabolites, several fatty acid amides containing a signaling molecule oleoylethanolamide (OEA) and fatty acids were significantly upregulated. Under these conditions, HELP exposure had no effect on citric acid and ornithine cycle intermediates. Because OEA is known to induce lipolysis as a putative endogenous ligand of peroxisome proliferator-activated receptor (PPAR)-α, we further confirmed the effect of OEA on gene expression using human subcutaneous cultured adipocytes. Peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) mRNA was upregulated by OEA treatment. OEA-induced ACOX1 mRNA expression was sensitive to a PPAR-α antagonist GW6471. Our findings will provide the new insights into the molecular mechanisms of EF therapy.
A therapeutic device to expose the human body to high-voltage electric potential (HELP) was approved by the Ministry of Health, Labour and Welfare in Japan. High-voltage electric field (EF) therapy is reportedly an effective treatment for stiff shoulders, constipation, insomnia and headache, while the effects of EF exposure on several blood parameters and liver diacylglycerol acyltransferase-2 (DGAT2) mRNA expression have been described [
Endogenous metabolites have been suggested as candidate molecules that may represent an interface between symptoms and therapeutic target proteins. Metabolomics is a relatively recent discipline dedicated to the global study of endogenous metabolites in tissues and biofluids [
The system used for EF exposure has been previously described [6,8,15]. The EF system was equipped with a transformer, a seat, and two insulator-covered electrodes that were placed on a floor plate on which the subject’s feet were located and above the head of the subject. EF generated by the HELP apparatus (Healthtron PRO-18T or Hb9000T; Hakuju Institute for Health Science Co., Ltd., Tokyo, Japan) was created uniformly by transforming 50 Hz alternating current at 18 kV and 9 kV, respectively. The surface EF levels generated by the high voltage (9 kV) electric potential supply were 9.96 kV/m to the neck or 11.6 kV/m to the legs. The safety of this system for human use was established by the Japanese government in 1963.
Ten healthy adults (5 males and 5 females; mean age, 40.2 ± 10.2 years; mean body mass index (BMI), 22.0 ± 2.4 kg/m2) participated in experiment 1 (exposure conditions: 18 kV for 30 min). Ten healthy adults (6 males and 4 females; mean age, 44.4 ± 9.7 years; BMI, 22.3 ± 3.2 kg/m2) participated in experiment 2 (exposure conditions: 9 kV for 30 min). The experiments were performed in the morning and all participants signed an informed consent form after receiving verbal and written information about the study. The experiments were conducted in accordance with the Declaration of Helsinki and the study protocol was approved by the human ethics committee of Hakuju Institute for Health Science Co., Ltd. (Tokyo, Japan).
Blood samples were collected in vacutainer tubes coated with ethylenediaminetetraacetic acid (VP-NA070K; Terumo Corporation, Tokyo, Japan) and immediately centrifuged at 800×g for 5 min using centrifuge to separate plasma from other cellular materials. Subsequently, plasma was transferred to a fresh eppendorf tube and stored at -80°C until processed.
Metabolites were measured as described previously [10,16,17]. In brief, 50 µL of plasma was added to 450 µL of methanol containing internal standards (Solution ID: H3304-1002; Human Metabolome Technologies, Tsuruoka, Japan) at 0°C in order to inactivate enzymes. The extract solution was thoroughly mixed with 500 µL of chloroform and 200 µL of Milli-Q water and centrifuged at 2,300×g and 4°C for 5 min. Then, 350 µL of the upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cut-off filter (Millipore Corporation, Billerica, MA, USA) to remove proteins. The filtrate was centrifugally concentrated and re-suspended in 50 µL of Milli-Q water for CE-MS analysis.
A 500 µL aliquot of plasma was added to 1,500 µL of 1% formic acid/acetonitrile containing internal standard solution (Solution ID: H3304-1002, Human Metabolome Technologies) at 0°C in order to inactivate enzymes. The solution was thoroughly mixed and centrifuged at 2,300×g and 4°C for 5 min. The supernatant was filtrated using a hybrid SPE phospholipid cartridge (55261-U; Supelco, Bellefonte, PA, USA) to remove phospholipids. The filtrate was desiccated and then dissolved in 100 µL of isopropanol/Milli-Q water for LC-MS analysis. LC-TOFMS was performed using an Agilent LC System (Agilent 1200 series RRLC system SL) equipped with an Agilent 6230 TOF mass spectrometer (Agilent Technologies, Waldbronn, Germany). The systems were controlled using Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies). Cationic and anionic compounds were measured using an octadecylsilane column (2×50 mm, 2 µM), as previously described [10,16,17]. Peaks were extracted using the MasterHands automatic integration software (Keio University, Tsuruoka, Japan) to obtain peak information including the m/z ratio, retention time for LC-TOFMS measurement (RT), and peak area. Signal peaks corresponding to isotopomers, adduct ions, and other producted ions of known metabolites were excluded. The remaining peaks were annotated with putative metabolites from the HMT metabolite database based on MT/RT and m/z values as determined by TOFMS. The tolerance range for the peak annotation was configured at ± 0.5 min for MT and ± 10 ppm for m/z. In addition, peak areas were normalized against those of the internal standards and then the resultant relative area values were further normalized by sample amount.
The X-ray crystal structure of PPAR-α complexed with agonist AZ242 (1i7g; Protein Data Bank Japan) was used for the molecule docking [
Human white subcutenous preadipocytes (Lonza, Basel, Switzerland) were cultured in preadipocyte basal medium-2 (Lonza) supplemented with 2 mM L-glutamine and 10% fetal bovine serum (Lonza). In brief, 1×104 cells were seeded in wells of 96-well multiwall plates. Differentiation was induced by treating the cells with a differentiation medium containing insulin, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine (all supplied by Lonza) for 6 days. RNA isolated from each sample was processed and hybridized to an Affymetrix GeneChip Human genome U133 Plus 2.0 array according to the protocols described in the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA, USA). Raw data was submitted to the National Center for Biotechnology Information (NCBI) Gene Expression Omunibus (GEO) database (http://www.ncbi.nlm.gov/geo/, platform accession number GSE55539).
qRT-PCR was performed as described [
Oleoylethanolamide (OEA), GW6471 and GW9662 were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
The data was analyzed using Welch’s t-test. A probability (p) value <0.05 was considered statistically significant.
CE- and LC-TOFMS analyses were used to measure the abundance of a total of 161 metabolites in the plasma obtained from healthy participants. The results of hierarchical cluster analysis of metabolic patterns are shown in
Furthermore, the nature of the OEA responsible for EF strength was investigated. The OEA change in relative ratio of after/before was at 1.17 (p=0.079) for 9 kV. We also examined the effect of different processing time (60 min), but found no significant changes caused by HELP (18 kV) exposure (1.09-fold;
OEA is a fatty acid amide known to induce lipolysis as a putative endogenous ligand of PPAR-α [14,20,21]; therefore we hypothesized that increased plasma OEA levels after EF exposure may be linked to changes in the fatty acid content in plasma. We next examined the binding mode study of OEA in active site of PPAR-α using Sybyl-X2.0 software. As shown in
In order to determine whether the docking of OEA on the nuclear receptor PPAR-α reflected a change in the human cellular function, we tested the effect of OEA on gene expression using the Affymetrix GeneChip human genome U133 Plus 2.0 array. The addition of 10 µM OEA to human subcutaneous adipocytes significantly affected the expression of 1013 genes (556 upregulated and 457 downregulated). In particular, genes associated with lipid metabolic process exhibited significant changes in response to OEA treatment (
Metabolite |
Before |
After |
Ratio |
||
---|---|---|---|---|---|
Fatty acid amides | |||||
Oleoylethanolamide | 2.2E-05 ± 5.1E-06 | 2.7E-05 ± 8.4E-06 | 1.24 0.009 ** | ||
Palmitoylethanolamide | 2.1E-05 ± 4.0E-06 | 2.4E-05 ± 5.3E-06 | 1.12 0.017 * | ||
Stearoylethanolamide | 9.2E-06 ± 2.5E-06 | 1.0E-05 ± 2.6E-06 | 1.12 0.013 * | ||
Linoleylethanolamide | 8.4E-06 ± 2.2E-06 | 9.2E-06 ± 3.0E-06 | 1.09 0.265 | ||
Fatty acids and related metabolites | |||||
Oleic acid | 1.2E-03 ± 7.9E-04 | 1.8E-03 ± 1.1E-03 | 1.52 0.009 ** | ||
Linoleic acid | 1.2E-03 ± 7.9E-04 | 1.8E-03 ± 1.1E-03 | 1.47 0.017 * | ||
Linolenic acid | 4.6E-05 ± 2.6E-05 | 6.2E-05 ± 3.2E-05 | 1.35 0.044 * | ||
Palmitoleic acid | 6.0E-05 ± 5.1E-05 | 8.0E-05 ± 5.6E-05 | 1.34 0.054 | ||
3.8E-04 ± 2.9E-04 | 5.8E-04 ± 4.1E-04 | 1.51 0.003 ** | |||
3.2E-05 ± 2.2E-05 | 4.4E-05 ± 3.0E-05 | 1.35 0.004 ** | |||
1.1E-05 ± 3.8E-06 | 1.5E-05 ± 6.0E-06 | 1.38 0.002 ** | |||
8.9E-06 ± 5.4E-06 | 1.3E-05 ± 8.1E-06 | 1.46 0.007 ** | |||
1.5E-05 ± 9.5E-06 | 2.1E-05 ± 1.5E-05 | 1.44 0.014 * | |||
Arachidonic acid | 1.0E-04 ± 4.4E-05 | 1.4E-04 ± 7.1E-05 | 1.41 0.004 ** | ||
Ethyl arachidonate | 2.5E-05 ± 2.1E-05 | 3.5E-05 ± 2.8E-05 | 1.39 0.020 * | ||
Nervonic acid | 2.0E-06 ± 9.5E-07 | 2.6E-06 ± 8.1E-07 | 1.31 0.001 ** | ||
Myristoleic acid | 2.3E-04 ± 1.4E-04 | 2.1E-04 ± 1.1E-04 | 0.90 0.299 | ||
Ricinoleic acid | 7.7E-06 ± 5.5E-06 | 1.0E-05 ± 8.0E-06 | 1.31 0.054 | ||
FA(22:5) | 2.6E-05 ± 1.6E-05 | 3.8E-05 ± 2.6E-05 | 1.46 0.013 * | ||
FA(22:4) | 1.4E-05 ± 1.3E-05 | 2.1E-05 ± 1.8E-05 | 1.46 0.014 * | ||
FA(20:3) | 4.1E-06 ± 5.0E-06 | 5.6E-06 ± 7.3E-06 | 1.38 0.084 | ||
FA(19:1) | 3.3E-06 ± 1.6E-06 | 4.4E-06 ± 2.5E-06 | 1.35 0.030 * | ||
FA(17:1) | 4.8E-06 ± 3.1E-06 | 6.2E-06 ± 3.7E-06 | 1.30 0.067 | ||
FA(14:3) | 5.3E-06 ± 2.4E-06 | 5.3E-06 ± 2.6E-06 | 0.99 0.967 | ||
FA(17:0) | 3.2E-06 ± 1.4E-06 | 4.4E-06 ± 2.0E-06 | 1.37 0.004 ** | ||
FA(14:0) | 1.0E-05 ± 7.6E-06 | 1.3E-06 ± 9.1E-06 | 1.28 0.032 ** | ||
Palmitic acid | 2.5E-04 ± 1.3E-04 | 3.4E-04 ± 1.8E-04 | 1.36 0.019 * | ||
Stearic acid | 1.3E-04 ± 4.9E-05 | 1.8E-04 ± 8.0E-05 | 1.45 0.003 ** | ||
Heptadecanoic acid | 4.3E-06 ± 1.7E-06 | 6.0E-06 ± 3.0E-06 | 1.41 0.008 ** | ||
Lauric acid | 1.9E-03 ± 5.5E-04 | 1.6E-03 ± 4.0E-04 | 0.84 0.008 ** | ||
Decanoic acid | 7.2E-04 ± 4.0E-04 | 5.1E-04 ± 2.3E-04 | 0.71 0.005 ** | ||
Pelargonic acid | 9.2E-04 ± 1.3E-04 | 6.4E-04 ± 1.4E-04 | 0.70 0.001 ** | ||
Octanoic acid | 4.6E-04 ± 1.1E-04 | 3.2E-04 ± 5.9E-05 | 0.70 0.001 ** | ||
Heptanoic acid | 7.2E-04 ± 1.2E-04 | 4.0E-04 ± 7.9E-05 | 0.57 0.001 ** | ||
Hexanoic acid | 9.0E-04 ± 1.7E-04 | 5.6E-04 ± 1.2E-04 | 0.63 0.002 ** | ||
Valeric acid | 1.2E-03 ± 3.6E-04 | 6.4E-04 ± 2.0E-04 | 0.54 0.008 ** | ||
2-Hydroxyvaleric acid | 1.8E-03 ± 1.9E-03 | 1.9E-03 ± 2.2E-03 | 1.05 0.361 | ||
11-Aminoundecanoic acid | 1.7E-04 ± 5.1E-05 | 1.7E-04 ± 5.4E-05 | 1.00 0.997 | ||
Acylcarnitines | |||||
AC(20:1) | 9.4E-06 ± 4.3E-06 | 9.1E-06 ± 5.1E-06 | 0.97 0.767 | ||
AC(20:0) | 5.4E-06 ± 2.1E-06 | 4.8E-06 ± 1.2E-06 | 0.89 0.366 | ||
AC(18:2) | 8.0E-05 ± 3.0E-05 | 5.9E-05 ± 9.2E-06 | 0.74 0.039 * | ||
AC(18:1) | 1.7E-04 ± 6.9E-05 | 1.5E-04 ± 5.6E-05 | 0.85 0.178 | ||
AC(18:0) | 4.9E-05 ± 1.9E-05 | 4.3E-05 ± 1.3E-05 | 0.87 0.270 | ||
AC(16:2) | 7.5E-06 ± 3.6E-06 | 4.3E-06 ± 2.0E-06 | 0.60 0.003 ** | ||
AC(16:1) | 2.8E-05 ± 1.3E-05 | 1.9E-05 ± 1.1E-05 | 0.69 0.008 ** | ||
Palmitoylcarnitine | 1.1E-04 ± 4.5E-05 | 8.5E-05 ± 1.5E-05 | 0.76 0.112 | ||
AC(14:2) | 3.0E-05 ± 1.9E-05 | 1.3E-05 ± 5.3E-06 | 0.44 0.008 ** | ||
AC(14:0) | 1.8E-05 ± 8.6E-06 | 1.1E-05 ± 4.9E-06 | 0.63 0.019 * | ||
AC(13:1) | 4.5E-05 ± 4.6E-05 | 3.1E-05 ± 2.9E-05 | 0.69 0.054 | ||
AC(12:1) | 2.5E-05 ± 2.1E-05 | 9.1E-06 ± 4.8E-06 | 0.36 0.015 * | ||
AC(12:0) | 3.2E-05 ± 2.3E-05 | 1.1E-05 ± 5.7E-06 | 0.35 0.007 ** | ||
Carnitine | 2.7E-02 ± 5.5E-03 | 2.7E-02 ± 5.5E-03 | 1.01 0.614 | ||
8.4E-03 ± 1.5E-03 | 8.2E-03 ± 1.8E-03 | 0.98 0.744 | |||
Phospholipids and relative metabolites | |||||
Sphingosine | 2.9E-05 ± 8.5E-06 | 3.7E-05 ± 2.0E-05 | 1.26 0.168 | ||
Ethanolamine phosphate | 4.9E-04 ± 1.8E-04 | 4.8E-04 ± 2.3E-04 | 0.98 0.848 | ||
Ethanolamine | 8.5E-04 ± 1.7E-04 | 8.3E-04 ± 1.7E-04 | 0.98 0.586 | ||
Glycerol | 9.6E-02 ± 3.7E-02 | 9.6E-02 ± 2.3E-02 | 1.01 0.962 | ||
Nucleic acids and related metabolites | |||||
ATP | 6.7E-04 ± 3.0E-04 | 7.2E-04 ± 3.0E-04 | 1.07 0.716 | ||
ADP | 8.8E-04 ± 3.5E-04 | 1.0E-03 ± 4.2E-04 | 1.17 0.316 | ||
AMP | 2.2E-04 ± 1.9E-04 | 2.6E-04 ± 1.4E-04 | 1.15 0.618 | ||
GDP | 1.0E-04 ± 3.9E-05 | 1.2E-04 ± 5.4E-05 | 1.14 0.341 | ||
UDP | 5.2E-05 ± 2.0E-05 | 6.7E-05 ± 2.8E-05 | 1.30 0.040 * | ||
IDP | 6.1E-03 ± 4.6E-04 | 6.2E-03 ± 6.2E-04 | 1.01 0.788 | ||
Uric acid | 2.8E-02 ± 5.9E-03 | 2.9E-02 ± 7.2E-03 | 1.02 0.185 | ||
Uridine | 8.6E-04 ± 1.7E-04 | 8.0E-04 ± 1.7E-04 | 0.93 0.310 | ||
8-Hydroxy-2’-deoxyguanine | 5.7E-04 ± 3.6E-04 | 6.6E-04 ± 4.1E-04 | 1.15 0.541 | ||
Pyrophosphate | 9.8E-04 ± 1.7E-04 | 1.1E-03 ± 1.1E-04 | 1.10 0.096 | ||
1-Methylnicotinamide | 1.2E-04 ± 5.9E-05 | 1.2E-04 ± 7.0E-05 | 1.02 0.793 | ||
Tricarboxylic acid cycle intermediates and relative metabolites | |||||
Citric acid | 1.9E-02 ± 2.9E-03 | 2.0E-02 ± 2.9E-03 | 1.07 0.066 | ||
Isocitric acid | 1.0E-03 ± 2.9E-04 | 1.0E-03 ± 2.8E-04 | 1.03 0.414 | ||
Lactic acid | 9.9E-02 ± 5.4E-03 | 1.0E-01 ± 3.8E-02 | 1.05 0.716 | ||
Succinic acid | 5.4E-04 ± 1.4E-04 | 5.3E-04 ± 8.1E-05 | 0.98 0.731 | ||
Pyruvic acid | 2.8E-03 ± 1.1E-03 | 2.7E-03 ± 9.8E-04 | 0.97 0.738 | ||
Malic acid | 7.8E-04 ± 2.0E-04 | 7.4E-02 ± 2.1E-04 | 0.95 0.482 | ||
1.1E-03 ± 2.9E-04 | 1.1E-03 ± 2.9E-04 | 0.99 0.811 | |||
Urea cycle | |||||
Ornithine | 1.1E-02 ± 4.2E-03 | 9.9E-03 ± 3.5E-03 | 0.93 0.234 | ||
Citrulline | 5.7E-03 ± 1.2E-03 | 5.8E-03 ± 1.3E-03 | 1.01 0.450 | ||
Urea | 4.3E-01 ± 6.9E-02 | 4.2E-01 ± 8.2E-02 | 0.99 0.717 | ||
Arg | 2.9E-02 ± 8.7E-03 | 2.8E-02 ± 8.1E-03 | 0.97 0.605 | ||
Guanidoacetic acid | 8.4E-04 ± 3.6E-04 | 7.8E-04 ± 2.9E-04 | 0.93 0.089 | ||
Glyceric acid | 1.2E-03 ± 2.5E-04 | 1.1E-03 ± 2.7E-04 | 0.89 0.056 | ||
Amino acids and related metabolites | |||||
Ala | 8.6E-02 ± 3.1E-02 | 7.7E-02 ± 2.5E-02 | 0.90 0.016 * | ||
b-Ala | 5.8E-04 ± 2.2E-04 | 5.5E-04 ± 1.6E-04 | 0.94 0.331 | ||
Asn | 7.4E-03 ± 1.4E-03 | 7.2E-03 ± 1.3E-03 | 0.97 0.307 | ||
Asp | 1.6E-03 ± 4.5E-04 | 1.5E-03 ± 4.7E-04 | 0.99 0.866 | ||
Tyr | 1.9E-02 ± 3.8E-03 | 1.8E-02 ± 4.2E-03 | 0.92 0.033 * | ||
Phe | 3.1E-02 ± 4.1E-03 | 2.9E-02 ± 3.4E-03 | 0.91 0.013 ** | ||
Trp | 2.1E-02 ± 2.9E-03 | 1.9E-02 ± 2.6E-03 | 0.90 0.002 ** | ||
Lys | 5.6E-02 ± 1.1E-02 | 5.5E-02 ± 1.1E-02 | 0.98 0.531 | ||
Ile | 4.4E-02 ± 1.4E-02 | 4.0E-02 ± 9.9E-03 | 0.90 0.090 | ||
Met | 6.2E-03 ± 1.6E-03 | 5.1E-03 ± 1.4E-03 | 0.82 0.023 * | ||
Cystine | 2.3E-03 ± 7.2E-04 | 1.7E-03 ± 6.5E-04 | 0.77 0.038 * | ||
Leu | 8.3E-02 ± 2.1E-02 | 7.6E-02 ± 1.4E-02 | 0.92 0.131 | ||
His | 2.2E-02 ± 2.8E-03 | 2.2E-02 ± 2.6E-03 | 0.99 0.799 | ||
Thr | 2.8E-02 ± 9.8E-03 | 2.8E-02 ± 9.9E-03 | 0.98 0.529 | ||
Val | 1.1E-01 ± 2.1E-02 | 1.1E-01 ± 1.8E-02 | 0.96 0.282 | ||
Ser | 1.6E-02 ± 5.4E-03 | 1.6E-02 ± 5.2E-03 | 1.02 0.571 | ||
Glu | 3.4E-02 ± 2.1E-02 | 3.6E-02 ± 2.0E-02 | 1.05 0.343 | ||
Gln | 1.1E-01 ± 3.2E-02 | 1.1E-01 ± 3.0E-02 | 1.02 0.602 | ||
Gly | 3.1E-02 ± 8.5E-03 | 3.1E-02 ± 9.1E-03 | 1.00 0.952 | ||
Pro | 6.8E-02 ± 2.4E-02 | 6.5E-02 ± 2.2E-02 | 0.95 0.115 | ||
Hydroxyproline | 2.9E-03 ± 1.7E-03 | 2.7E-03 ± 1.7E-03 | 0.95 0.042 * | ||
5-Oxoproline | 1.8E-03 ± 4.1E-04 | 2.9E-03 ± 3.9E-03 | 1.62 0.390 | ||
Taurine | 5.6E-03 ± 1.7E-03 | 5.3E-03 ± 1.5E-03 | 0.95 0.524 | ||
Hypotaurine | 4.2E-04 ± 1.3E-04 | 4.0E-04 ± 9.6E-05 | 0.94 0.527 | ||
Isethionic acid | 1.8E-04 ± 2.2E-05 | 1.9E-04 ± 2.8E-05 | 1.08 0.040 * | ||
Kynurenine | 4.3E-04 ± 1.2E-04 | 3.9E-04 ± 8.9E-05 | 0.92 0.109 | ||
Sarcosine ( |
4.4E-04 ± 1.2E-04 | 4.0E-04 ± 1.3E-04 | 0.90 0.036 * | ||
1.0E-03 ± 1.7E-04 | 9.8E-04 ± 2.3E-04 | 0.97 0.289 | |||
1.6E-04 ± 3.4E-05 | 1.8E-04 ± 4.6E-05 | 1.14 0.026 * | |||
1.7E-04 ± 1.6E-05 | 1.9E-04 ± 2.7E-05 | 1.14 0.027 * | |||
8.3E-04 ± 4.7E-04 | 8.3E-04 ± 5.3E-04 | 1.00 0.901 | |||
9.2E-04 ± 3.1E-04 | 8.7E-04 ± 2.9E-04 | 0.94 0.117 | |||
3-Metylhistidine | 2.9E-03 ± 2.2E-03 | 2.6E-03 ± 2.0E-03 | 0.93 0.091 | ||
2-Aminobutyric acid | 5.9E-03 ± 2.0E-03 | 6.0E-03 ± 2.1E-03 | 1.02 0.518 | ||
3-Aminobutyric acid | 1.2E-04 ± 3.4E-05 | 1.2E-04 ± 2.7E-05 | 0.99 0.928 | ||
3-Aminoisobutyric acid | 5.7E-04 ± 3.6E-04 | 5.9E-04 ± 3.7E-04 | 1.03 0.242 | ||
4-Methyl-2-oxovaleric acid | 1.1E-02 ± 3.8E-03 | 1.1E-02 ± 3.1E-03 | 0.96 0.424 | ||
5-Methoxyindoleacetic acid | 1.3E-04 ± 4.2E-05 | 1.2E-04 ± 5.2E-05 | 0.94 0.204 | ||
Indole-3-acetic acid | 1.6E-04 ± 6.5E-05 | 1.5E-04 ± 5.0E-05 | 0.91 0.104 | ||
2-Oxoisovaleric acid | 1.6E-03 ± 3.8E-04 | 1.6E-03 ± 3.2E-04 | 1.00 0.999 | ||
2-Aminoadipic acid | 1.8E-04 ± 4.1E-05 | 1.7E-04 ± 3.3E-05 | 0.96 0.304 | ||
Homovanillic acid | 1.7E-04 ± 7.0E-05 | 1.8E-04 ± 8.0E-05 | 1.06 0.251 | ||
Pipecolic acid | 8.1E-04 ± 4.0E-04 | 7.7E-04 ± 3.8E-04 | 0.96 0.276 | ||
Hippuric acid | 5.4E-04 ± 3.7E-04 | 4.6E-04 ± 3.3E-04 | 0.85 0.022 * | ||
Methionine sulfoxide | 5.4E-04 ± 1.8E-04 | 5.4E-04 ± 1.6E-04 | 0.99 0.893 | ||
Sufotyrosine | 7.4E-05 ± 2.3E-05 | 6.8E-05 ± 1.8E-05 | 0.92 0.565 | ||
Creatine | 1.3E-02 ± 7.0E-03 | 1.2E-02 ± 5.6E-03 | 0.93 0.218 | ||
Creatinine | 2.1E-02 ± 4.5E-03 | 2.1E-02 ± 4.3E-03 | 0.99 0.671 | ||
Phosphocreatine | 8.7E-05 ± 2.6E-05 | 1.0E-04 ± 2.3E-05 | 1.16 0.047 * | ||
Phosporylcholine | 23.0E-04 ± 7.7E-05 | 3.0E-04 ± 8.1E-05 | 1.00 0.999 | ||
Glycerophosphocholine | 3.3E-04 ± 1.2E-04 | 3.0E-04 ± 8.9E-05 | 0.89 0.362 | ||
Choline | 6.5E-03 ± 1.3E-03 | 6.4E-03 ± 1.3E-03 | 0.99 0.776 | ||
Betaine | 2.3E-02 ± 4.6E-03 | 2.3E-02 ± 4.8E-03 | 0.99 0.642 | ||
g-Butyrobetaine | 8.2E-04 ± 1.4E-04 | 7.8E-04 ± 1.5E-04 | 0.95 0.046 * | ||
2-Hydroxyglutaric acid | 1.5E-04 ± 5.3E-05 | 1.7E-04 ± 4.2E-05 | 1.10 0.365 | ||
Bile acids | |||||
Deoxycholic acid | 5.9E-05 ± 4.4E-05 | 5.5E-05 ± 4.1E-05 | 0.93 0.413 | ||
Chenodeoxycholic acid | 4.1E-05 ± 4.9E-05 | 3.6E-05 ± 4.7E-05 | 0.87 0.101 | ||
Taurodeoxycholic acid | 8.7E-06 ± 9.4E-06 | 5.5E-06 ± 6.2E-06 | 0.63 0.110 | ||
Taurochenodeoxycholic acid | 1.4E-05 ± 1.1E-05 | 8.3E-06 ± 6.5E-06 | 0.60 0.066 | ||
Glycochenodeoxycholic acid | 8.8E-05 ± 6.0E-05 | 5.7E-05 ± 3.8E-05 | 0.65 0.046 * | ||
Glycodeoxycholic acid | 2.9E-05 ± 2.2E-05 | 2.1E-05 ± 1.6E-05 | 0.73 0.120 | ||
Glycoursodeoxycholic acid | 1.2E-05 ± 1.1E-05 | 9.3E-06 ± 7.4E-06 | 0.78 0.313 | ||
Steroids and related metabolites | |||||
Cortisol | 8.0E-05 ± 3.3E-05 | 7.3E-05 ± 2.3E-05 | 0.91 0.371 | ||
Cortisone | 1.9E-05 ± 6.8E-06 | 1.9E-05 ± 5.7E-06 | 1.00 0.963 | ||
Pregnenolone sulfate | 3.1E-05 ± 1.1E-05 | 2.9E-05 ± 8.8E-06 | 0.94 0.386 | ||
Cholesterol sulfate | 5.9E-05 ± 1.5E-05 | 5.4E-05 ± 1.5E-05 | 0.93 0.023 * | ||
Cholesterol | 1.2E-03 ± 2.8E-04 | 1.1E-03 ± 3.3E-04 | 0.87 0.290 | ||
7-Dehydrocholesterol | 9.5E-06 ± 2.9E-06 | 8.8E-06 ± 2.2E-06 | 0.93 0.578 | ||
Dehydroisoandrosterone 3-sulfate | 1.0E-03 ± 3.0E-04 | 1.1E-03 ± 2.8E-04 | 1.08 0.004 ** | ||
Etiocholan-3a-ol-17-one | 7.1E-06 ± 3.7E-06 | 7.9E-06 ± 3.8E-06 | 1.12 0.058 | ||
Etiocholan-3a-ol-17-one sulfate | 5.0E-04 ± 2.4E-04 | 5.3E-06 ± 2.5E-04 | 1.07 0.034 * | ||
Other metabolites | |||||
Gluconic acid | 3.4E-04 ± 7.1E-05 | 3.6E-04 ± 6.6E-05 | 1.05 0.348 | ||
Mucic acid | 1.2E-04 ± 3.8E-05 | 1.2E-04 ± 3.2E-05 | 1.02 0.635 | ||
Threonic acid | 1.9E-03 ± 1.2E-03 | 2.2E-03 ± 1.3E-03 | 1.15 0.014 * | ||
Trimethylamine N-oxide | 1.3E-03 ± 5.8E-04 | 1.2E-03 ± 5.5E-04 | 0.91 0.097 | ||
Indole-3-carboxaldehyde | 1.9E-05 ± 6.1E-06 | 2.0E-05 ± 5.1E-06 | 1.06 0.295 | ||
3-Indoxysulfuric acid | 5.7E-04 ± 2.9E-04 | 5.8E-04 ± 3.3E-04 | 1.03 0.631 | ||
a-Tocopherol | 2.6E-04 ± 1.0E-04 | 2.4E-04 ± 8.2E-05 | 0.94 0.405 | ||
2-Hydroxybutyric acid | 3.2E-03 ± 1.4E-03 | 4.4E-03 ± 2.1E-03 | 1.40 0.002 ** | ||
3-Hydroxybutyric acid | 4.8E-03 ± 7.2E-03 | 6.4E-03 ± 8.3E-03 | 1.32 0.099 | ||
* Indicates a significant difference (* |
Because FAT/CD36, acyl-CoA synthetase long-chain family member 3 (ACSL3), and ACOX1 are PPARα-responsive genes, we next confirmed the effect of OEA (10 µM) on the target proteins at the mRNA level by qRT-PCR. qRT-PCR showed increased expression of about 1.65-fold, 1.51-fold, 1.54-fold and 1.88-fold for FAT/CD36, ACSL3, HDLBP, and ACOX1, respectively (
The results of the present study clearly indicate that plasma fatty acids in healthy human subjects are sensitive to acute EF exposure. Interestingly, 60% or more of significant increase following EF exposure was fatty acid family in plasma. In contrast, about 30% of significant decrease following EF exposure was acylcarnitine family in plasma. It is notable that EF exposure did not seem to adversely alter physiological parameters of the healthy volunteers, at least in terms of citric acid or ornithine cycles. Moreover, a stress-responsive hormone cortisol was not affected by EF exposure. Our findings also clarified that EF exposure elicits an increase in plasma OEA, a fatty acid ethanolamide. In contrast, LoVerme et al. reported that cold exposure increased the OEA content in white adipose tissue, but not plasma [
Accession no. | Gene symbol | Gene description | Fold change | |
Lipid metabolic process | ||||
---|---|---|---|---|
Up-regulated gene | ||||
NM_002732 | PRKACG | protein kinase, cAMP-dependent, catalytic, gamma | 8.04 | |
NM_001243900 | HDLBP | high density lipoprotein binding protein | 6.00 | |
NM_000949 | PRLR | prolactin receptor | 5.43 | |
NM_001204051 | SLC25A27 | solute carrier family 25, member 27 | 5.21 | |
NM_022898 | BCL11B | B-cell CLL/lymphoma 11B | 5.09 | |
NM_000198 | HSD3B2 | hydroxy-delta-5-sterold dehydrogenase, 3 beta- and steroid delta-isomerase | 4.88 | |
NM_002649 | PIK3CG | phosphoinositide-3-kinase, catalytic, gamma polypeptide | 4.60 | |
NM_004457 | ACSL3 | acyl-CoA synthetase long-chain family member 3 | 4.29 | |
NM_018327 | SPTLC3 | serine palmitoyltransferase, long chain base subunit 3 | 3.89 | |
NM_002011 | FGFR4 | fibroblast growth factor receptor 4 | 3.84 | |
NM_001098537 | PNPLA7 | patatin-like phospholipase domain containing 7 | 3.82 | |
NM_001127178 | PIGG | phosphatidylinositol glycan anchor biosynthesis, class G | 3.73 | |
NM_030791 | SGPP1 | sphingosine-1-phosphate phosphatase 1 | 3.55 | |
NM_007253 | CYP4F8 | cytochrome P 450, family 4, subfamily F, polypeptide 8 | 3.45 | |
NM_024419 | PGS1 | phosphatidylglycerophosphate synthase 1 | 3.10 | |
NM_203347 | LCN15 | lipocalin 15 | 3.04 | |
NM_001040442 | FABP6 | fatty acid binding protein 6, ileal | 2.79 | |
NM_000778 | CYP4A11 | cytochrome P 450, family 4, subfamily A, polypeptide 11 | 2.75 | |
NM_001185039 | ACOX1 | acyl-CoA oxidase 1, palmitoyl | 2.70 | |
NM_018667 | SMPD3 | sphingomyelin phosphodiesterase 3, neutral membrane | 2.64 | |
NM_014602 | PIK3R4 | phosphoinositide-3-kinase regulatory subunit 4 | 2.51 | |
NM_015879 | ST8SIA3 | ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 3 | 2.40 | |
NM_024870 | PREX2 |
phosphatidylinositol-3,4,5-triphosphate-dependent Raexchange | 2.16 | |
NM_012400 | PLA2G2D | phospholipase A2, group IID | 2.16 | |
NM_005050 | ABCD4 | ATP-binding cassette sub-family D member 4 | 2.08 | |
NM_001127458 | CRLS1 | cardiolipin synthase 1 | 1.74 | |
NM_198531 | ATP9B | ATPase, class II, type 9B | 1.70 | |
NM_001144772 | NSMAF | neutral sphingomyelinase activation associated factor | 1.69 | |
NM_001144382 | PLCL2 | phospholipase C-like 2 | 1.61 | |
NM_000072 | CD36 | fatty acid translocase/CD36 | 1.58 | |
Down-regulated genes | ||||
NM_001756 | SERPINA6 | serpin peptidase inhibitor, clade A, member 6 | 0.14 | |
NM_000545 | HNF1A | HNF1 homeobox A | 0.15 | |
NM_024586 | OSBPL9 | oxysterol binding protein-like 9 | 0.16 | |
NM_001831 | CLU | clusterin | 0.16 | |
NM_022060 | ABHD4 | abhydrolase domain containing 4 | 0.19 | |
NM_032047 | B3GNT5 | UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 5 | 0.19 | |
NM_153343 | ENPP6 | ectonucleotide pyrophosphatase/phosphodiesterase 6 | 0.25 | |
NM_015974 | CRYL1 | crystallin, lamda 1 | 0.25 | |
NM_006330 | LYPLA1 | lysophospholipase I | 0.28 | |
NM_001160147 | DDHD1 | DDHD domain containing 1 | 0.39 | |
NM_153240 | NPHP3 | nephronophthisis 3 (adolescent) | 0.44 | |
NM_003080 | SMPD2 | sphingomyelin phosphodiesterase 2, neutral membrane | 0.45 | |
NM_016466 | ANKRD23 | ankyrin repeat domain 23 | 0.54 | |
NM_001039667 | ANGPTL4 | angiopoietin-like 4 | 0.55 | |
NM_016174 | CERCAM | cerebral endothelial cell adhesion molecule | 0.56 | |
NM_001101667 | ACOX3 | acyl-CoA oxidase 3, pristanoyl | 0.58 | |
NM_003702 | PPAP2A | phosphatidic acid phosphatase type 2A | 0.59 | |
NM_001143835 | NFRKB | nuclear factor related to kappa B binding protein | 0.62 | |
NM_003105 | SORL1 | sortilin-related receptor, L (DLR class) A repeats containing | 0.64 |
In the present study, OEA binding to PPAR-α was stabilized through the formation of hydrogen bouds with Ser280, Tyr314, and Phe273, whereas a previous study reported hydrogen bounding with Ser280 and His440 [
Another goal of the present study was to gain insights into the genetic components affected by OEA through a large-scale analysis of gene expression analysis. In human white subcutenous adipocytes, FAT/CD36, ACSL3, HDLBP, ACOX1, and PPAR-α were upregulated in response to OEA. In contrast to PPAR-α, PPAR-γ had no effect. ACOX1, CYP4A11, ACSL3, and FAT/CD36 are reportedly involved in lipid metabolism genes induced by a PPAR-α agonist [
An experimental pretest-posttest design study by Sirikulchayanonta
OEA might have additional functions as an agonist or activator against the transient receptor potential vanilloid type-1 (TRPV1), GPR119, or GPR55 [
In conclusions, acute EF exposure induced notable effects on plasma OEA levels in healthy subjects. In human subcutaneous adipocytes, ACOX1 gene expression induced by OEA was sensitive to the PPAR-α antagonist GW6471. Our findings not only provide a clear example to understand the molecular mechanisms of health merit induced by EF therapy, but might also be important to the development of alternative medicine therapies and electroceuticals.
We are grateful to Dr. Makoto Kikuchi (Professor Emeritus, National Defence Medical College, Japan) for encouragement. We thank Katherine Nixon who provided medical writing services on behalf of Enago Crimson Interactive Pvt. Ltd.
Peroxisomal Acyl-coenzyme A Oxidase 1
Acyl-CoA Synthetase Long-chain Family Member 3
Capillary Electrophoresis-time-of-flight Mass Spectrometry Electron Ionization-mass Spectrometry
Cytochrome P450, Family 4, Subfamily A, Polypeptide 11
Diacylglycerol Acyltransferase-2; EF: Electric Field; ELISA: Enzyme Linked Immunosorbent Assay
Fatty Acid Translocase/ Cluster of Differentiation 36
Glyceraldehyde 3-phosphate Dehydrogenase
G Protein-coupled Receptor
N-((2S)-2-(((1Z)-1-methyl-3-oxo-3-(4-(trifluoromethyl)phenyl) prop-1-enyl)amino)-3-(4-(2-(5-methyl-2-phenyl-1,3-oxazol-4-yl) ethoxy)phenyl)propyl)propanamide
2-Chloro-5-nitroN-phenylbenzamide
High Density Lipoprotein Binding Protein
High Density Lipoprotein-cholesterol
Highvoltage Electric Potentialc
Liquid Chromatography-timeof-flight Mass Spectrometry Electron Ionization-Mass Spectrometry
Oleoylethanolamide; PPAR-α: Peroxisome Proliferator-activated Receptor-alpha
Quantitative Reverse Transcription Polymerase Chain Reaction
Transient Receptor Potential Vanilloid 1