Korean J Physiol Pharmacol 2021; 25(2): 167-175
Published online March 1, 2021 https://doi.org/10.4196/kjpp.2021.25.2.167
Copyright © Korean J Physiol Pharmacol.
Yelim Seo1, Young-Won Kim1, Donghee Lee1, Donghyeon Kim2, Kyoungseo Kim2, Taewoo Kim2, Changyeob Baek2, Yerim Lee2, Junhyeok Lee2, Hosung Lee2, Geonwoo Jang2, Wonyeong Jeong2, Junho Choi2, Doegeun Hwang2, Jung Soo Suh2, Sun-Woo Kim3 , Hyoung Kyu Kim3, Jin Han3, Hyoweon Bang1, Jung-Ha Kim4, Tong Zhou5,*, and Jae-Hong Ko1,*
Departments of 1Physiology and 2Medicine, College of Medicine, Chung-Ang University, Seoul 06974, 3Cardiovascular and Metabolic Disease Center, SMART Marine Therapeutics Center, Inje University, Busan 47392, 4Department of Family Medicine, College of Medicine, Chung-Ang University Hospital, Seoul 06973, Korea, 5Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV 89557, USA
Correspondence to:Jae-Hong Ko
E-mail: akdongyi01@cau.ac.kr
Tong Zhou
E-mail: tongz@med.unr.edu
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Far-infrared rays (FIR) are known to have various effects on atoms and molecular structures within cells owing to their radiation and vibration frequencies. The present study examined the effects of FIR on gene expression related to glucose transport through microarray analysis in rat skeletal muscle cells, as well as on mitochondrial biogenesis, at high and low glucose conditions. FIR were emitted from a bio-active material coated fabric (BMCF). L6 cells were treated with 30% BMCF for 24 h in medium containing 25 or 5.5 mM glucose, and changes in the expression of glucose transporter genes were determined. The expression of GLUT3 (Slc2a3) increased 2.0-fold (p < 0.05) under 5.5 mM glucose and 30% BMCF. In addition, mitochondrial oxygen consumption and membrane potential (ΔΨm) increased 1.5- and 3.4-fold (p < 0.05 and p < 0.001), respectively, but no significant change in expression of Pgc-1a, a regulator of mitochondrial biogenesis, was observed in 24 h. To analyze the relationship between GLUT3 expression and mitochondrial biogenesis under FIR, GLUT3 was down-modulated by siRNA for 72 h. As a result, the ΔΨm of the GLUT3 siRNA-treated cells increased 3.0-fold (p < 0.001), whereas that of the control group increased 4.6-fold (p < 0.001). Moreover, Pgc-1a expression increased upon 30% BMCF treatment for 72 h; an effect that was more pronounced in the presence of GLUT3. These results suggest that FIR may hold therapeutic potential for improving glucose metabolism and mitochondrial function in metabolic diseases associated with insufficient glucose supply, such as type 2 diabetes.
Keywords: Glucose, Glucose transporter type 3, Infrared rays, Mitochondrial biogenesis, Radiation
Far-infrared rays (FIR) are electromagnetic waves ranging between 3 and 100 μm that hold the ability to penetrate up to 2.5 cm into the skin, affecting muscles, as well as blood and lymph vessels, owing to their radiation and vibration frequency. FIR have a vibrational frequency of 3–100 THz that transfers to atoms and molecular structures [1] and, importantly, can induce a muscle wound healing effect [2,3]. A previous work confirmed that FIR can enhance mitochondrial copy number, oxygen consumption, as well as genes related to apoptosis and mitophagy in rat muscle tissues [4]. These results suggest that FIR activates the PINK1-mediated mitochondrial quality control pathway and mitochondrial biogenesis, which consequently increases the efficiency of oxidative respiration in skeletal muscle. However, the effects of FIR on mitochondrial morphology and membrane potential remain to be validated, and the precise mechanism of FIR-mediated upregulation of mitochondrial function is unclear.
Mitochondria are cellular organelles present in high numbers in the brain, liver, heart, and muscles. They use glucose and fatty acids to synthesize ATP as an energy source for cellular function [5]. Mitochondrial dysfunction is implicated in cardiovascular disease, aging, metabolic disease, cancer, and degenerative diseases [6,7]. Indeed, diabetes is closely related to increased reactive oxygen species production, which is directly responsible for mitochondrial dysfunction, and is one of the key factors underlying the pathological mechanism of diabetic synovial stenosis [8]. Type 2 diabetes mellitus is a condition characterized by metabolic imbalance associated with impaired mitochondrial ATP levels [9]. Due to the close relationship with energy-consuming diseases, research on the development of targeted therapeutics that can directly control mitochondrial function is being actively conducted [10,11].
Glucose transport across the plasma membrane is fundamental to mammalian energy metabolism and is mediated by tissue-specific glucose transporter proteins in the cell membrane [12]. Several studies have highlighted the expression of multiple glucose transporters in the same tissue for regulation of glucose and energy homeostasis [13]. Two main types of glucose carriers exist, namely sodium-glucose linked transporters and glucose transporters (GLUTs). Based on sequence similarity, GLUT proteins are classified into Class 1, comprising the well-characterized glucose transporters (GLUT1–4, 14), Class 2 (GLUT5, 7, 9, 11), and Class 3 (GLUT6, 8, 10, 12, 13) [14,15]. Of the many glucose transporters, GLUT1, GLUT3, and GLUT4 are expressed in rat L6 skeletal muscle cells [16], a cell line that retains many properties of skeletal muscle [17,18]. In cells such as muscle, the transport of glucose across the plasma membrane limits its availability [19,20]. Recent research showed that reduced mitochondrial ATP production due to insulin resistance in skeletal muscle is accompanied by a functional reduction in Class 1 GLUTs [14]. Thus, it has been suggested that GLUTs are closely related to changes in mitochondrial function.
The purpose of this study was to demonstrate whether FIR could increase intracellular transport of glucose and promote mitochondrial energy metabolism, thereby improving intracellular glucose deficiency conditions such as occurs in type 2 diabetes, resulting in therapeutic effects.
The FIR fabric samples were provided by Ventex Co. (Seoul, Korea). The bio-active materials coated fabric (BMCF) contained over 30 kinds of minerals, including SiO2, Mg, Al2O3, Na, Ca, and Fe2O3. The fabric was irradiated with FIR and the Korea Far-Infrared Association (Seoul, Korea) confirmed that the emission rate between 5 and 20 µm was 89.5%. The emission energy was 3.45 × 102 W/m2 · µm (issue number KFI-789) at 37°C. In the current study, we used BMCF that emitted 0% and 30% FIR. No abnormal skin reactions of any kind were observed during the safety evaluation of the BMCF.
Rat skeletal muscle L6 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 25 or 5.5 mM glucose (L001-05 or L001-02; WELGENE, Gyeongsan, Korea), and containing 10% fetal bovine serum and 1% penicillin/streptomycin [21,22]. The cells were used between passage 29 and 33. For FIR radiation conditions, 0% and 30% BMCF was attached inside the culture dish at a distance of 1 cm from the cells [23], followed by incubation at 37°C in 5% CO2 for 24 and 72 h. The experimental design is shown in Supplementary Fig. 1.
L6 cells were cultured and left to stabilize in DMEM plus 25 mM glucose for 24 h, before sub-culturing in 5.5 mM glucose medium.
mRNA expression and mitochondrial DNA copy number (mtDNA-CN) were measured using the Fast Start DNA Master SYBR Green I kit and the LightCycler 2.0 system (Roche, Basel, Switzerland). All primers used for qPCR analysis are described in Supplementary Table 1.
Mitochondrial oxygen consumption was measured according to the method described by Frezza
ΔΨm was measured using tetramethylrhodamine ethyl ester (TMRE; excitation/emission = 549/574 nm; Abcam, Cambridge, UK). L6 cells were treated with TMRE at a final concentration of 100 nM and allowed to react at 37°C for 30 min to induce TMER accumulation in the mitochondria [27]. To measure mitochondrial mass, cells were stained with acridine orange 10-nonyl bromide (NAO; Invitrogen, Waltham, MA, USA) at a final concentration of 2.5 µM in phosphate-buffered saline (PBS) solution [28], incubated in the dark at 37°C for 30 min, and then washed twice with PBS. TMRE and NAO fluorescence for each group were observed under an LSM 700 confocal microscope (Carl-Zeiss, Oberkochen, Germany). Mitochondrial mass measurements were performed at an excitation wavelength of 488 nm, and the emission of NAO was measured beyond 585 nm. The TMRE and NAO mean intensity of the region of interest (ROI) were measured in each cell and analyzed by Image J software (NIH, Bethesda, MD, USA) [29].
All collected data were analyzed using IBM SPSS Statistics v. 20.0 (IBM Corp., Armonk, NY, USA). Statistical significance was determined using the Student's t-test and one-way and two-way ANOVA. A p-value < 0.05 was considered statistically significant. The Student's t-test was used to evaluate mRNA relative expression, mtDNA-CN relative ratio, and RCR/mtDNA-CN ratio data. The one-way and two-way ANOVA were used for assessing TMRE and NAO fluorescence intensity per cell area ratio data.
Microarray data extracted from a previous study [23] were assembled for GO map analysis. We confirmed that pathways related to glucose transport and regulation were stimulated by FIR in rat skeletal muscle (Fig. 1). Response to glucose (GO:0009749, p < 0.001), regulation of glucose transport (GO:0010827, p < 0.01), glucose transport (GO:0015758, p < 0.05), positive regulation of glucose transport (GO:0010828, p < 0.05), regulation of glucose import (GO:0046324, p < 0.05), glucose import (GO:0046323, p = 0.057), cellular response to glucose stimulus (GO:0071333), cellular glucose homeostasis (GO:0001678), positive regulation of glucose import (GO:0046326), glucose metabolic process (GO:0006006), glucose homeostasis (GO:0042593), and regulation of glucose metabolic process (GO:0010906) were all activated by FIR. These results confirm that FIR hold potential to affect glucose metabolism.
To assess the effect of FIR on glucose metabolism, L6 cells were exposed to BMCF for 24 h under 25 and 5.5 mM glucose conditions. Interestingly, genes related to glucose metabolism identified in the microarray data (Table 1) were found to be altered in the
Table 1 . List of glucose transport-related genes from microarray data of BMCF-worn rat skeletal muscle.
Gene symbol | Gene accession | Fold change | p-value | Gene description |
---|---|---|---|---|
NM_017006 | 1.717 | 0.865 | Glucose-6-phosphate dehydrogenase | |
NM_031325 | 1.518 | 1.000 | UDP-glucose 6-dehydrogenase | |
XM_006252469 | 1.359 | 1.000 | UDP-glucose glycoprotein glucosyltransferase 2 | |
NM_031741 | 1.212 | 1.000 | Solute carrier family 2 (facilitated glucose/fructose transporter), member 5 | |
NM_001108963 | 1.199 | 1.000 | Solute carrier family 2 (facilitated glucose transporter), member 10 | |
NM_001106098 | 1.138 | 1.000 | Solute carrier family 35 (UDP-GlcNAc/UDP-glucose transporter), member D2 | |
NM_001191994 | 1.138 | 1.000 | Solute carrier family 37 (glucose-6-phosphate transporter), member 2 | |
NM_017102 | 1.110 | 1.000 | Solute carrier family 2 (facilitated glucose transporter), member 3 | |
XM_006223919 | 1.089 | 1.000 | Similar to Na+ dependent glucose transporter 1 | |
NM_133611 | 1.082 | 1.000 | Solute carrier family 2 (facilitated glucose transporter), member 13 | |
XM_001067659 | 1.059 | 1.000 | Suppressor of glucose, autophagy associated 1 | |
NM_001107421 | 1.055 | 1.000 | Glucose-fructose oxidoreductase domain containing 2 | |
XM_001066043 | 1.047 | 1.000 | GDP-D-glucose phosphorylase 1 | |
NM_001191551 | 1.024 | 1.000 | Solute carrier family 2 (facilitated glucose transporter), member 9 | |
NM_001107451 | 1.005 | 1.000 | Solute carrier family 2 (facilitated glucose transporter), member 12 | |
NM_001107286 | 1.001 | 1.000 | TDP-glucose 4,6-dehydratase | |
NM_031795 | –1.002 | 1.000 | UDP-glucose ceramide glucosyltransferase | |
NM_207592 | –1.005 | 1.000 | Glucose-6-phosphate isomerase | |
NM_176080 | –1.011 | 1.000 | Na+ dependent glucose transporter 1 | |
NM_133596 | –1.016 | 1.000 | UDP-glucose glycoprotein glucosyltransferase 1 | |
NM_053494 | –1.029 | 1.000 | Solute carrier family 2, (facilitated glucose transporter) member 8 | |
NM_001134547 | –1.042 | 1.000 | Similar to Na+ dependent glucose transporter 1 | |
XM_006225659 | –1.050 | 1.000 | Solute carrier family 2 (facilitated glucose transporter), member 7 | |
NM_001024743 | –1.058 | 1.000 | UDP-glucose pyrophosphorylase 2 | |
NM_001106698 | –1.058 | 1.000 | Hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) | |
NM_031589 | –1.080 | 1.000 | Solute carrier family 37 (glucose-6-phosphate transporter), member 4 | |
NM_022590 | –1.084 | 1.000 | Solute carrier family 5 (sodium/glucose cotransporter), member 2 | |
NM_013098 | –1.085 | 1.000 | Glucose-6-phosphatase, catalytic subunit | |
NM_001127556 | –1.114 | 1.000 | Similar to sodium-glucose cotransporter-like 1 | |
XM_008773019 | –1.141 | 1.000 | Similar to Na+ dependent glucose transporter 1 | |
NM_001106562 | –1.191 | 1.000 | Solute carrier family 2 (facilitated glucose transporter), member 6 | |
NM_176077 | –1.221 | 1.000 | Glucose 6 phosphatase, catalytic, 3 | |
NM_013033 | –1.252 | 1.000 | Solute carrier family 5 (sodium/glucose cotransporter), member 1 | |
NM_001106383 | –1.253 | 1.000 | Solute carrier family 5 (glucose activated ion channel), member 4 | |
NM_001011944 | –1.295 | 1.000 | Solute carrier family 37 (glucose-6-phosphate transporter), member 1 | |
NM_001170334 | –1.462 | 1.000 | Glucose-fructose oxidoreductase domain containing 1 |
BMCF, bio-active materials coated fabric.
Given the observed increase in
Changes in the mtDNA-CN in L6 cells following incubation with 25 or 5.5 mM glucose plus BMCF treatment for 24 h were assessed. The mtDNA-CN increased 1.7- and 1.3-fold in the 25 and 5.5 mM glucose conditions (p < 0.05), respectively, compared to the non-treated control (Fig. 3A). Next, changes in mitochondrial oxygen consumption in relation to the change in mtDNA-CN were determined to evaluate mitochondrial ATP production ability. The results indicated a 1.2- and 1.5-fold increase in ATP production ability of mitochondria under 30% BMCF and 25 and 5.5 mM glucose conditions (p < 0.05 and p < 0.01), respectively, compared to the non-treated control (Fig. 3B). In addition, TMRE fluorescence tended to increase upon treatment with 30% BMCF (Fig. 3C). In particular, by analyzing the TMRE fluorescence intensity ROI, the ΔΨm was found to be significantly increased by 3.4-fold following 30% BMCF compared to the non-treated control (p < 0.001, Fig. 3D). However, no significant differences in
The correlation between increased
Here, we aimed to assess changes in glucose transporter expression and mitochondrial biogenesis following exposure to FIR under high and low glucose levels. In fact, 5.5 mM glucose is usually considered a normal glucose serum concentration and corresponds with a low glucose culture medium
In our previous study, microarray analysis revealed that mRNA expression in rat muscle cells was stimulated by FIR radiation in a dose-dependent manner at 10% and 30% BMCF [23]. Additional analysis of genetic data through GO analysis confirmed the possibility that FIR could affect glucose metabolism (Fig. 1). The present study explored further the impact of FIR on the expression of genes associated with glucose metabolism under high and low glucose conditions in rat skeletal muscle cells (L6). The expression of
Our previous study demonstrated that FIR activate mitophagy and improve mitochondrial oxidative respiration based on mtDNA-CN and RCR [4]. Herein, it was confirmed that both parameters increased under high and low glucose conditions following FIR treatment compared to non-treated controls (Fig. 3A, B). Determining mitochondrial ATP production and the ΔΨm allows for an assessment of the functional state of mitochondria [26,38,39]. The RCR per mtDNA-CN ratio increased significantly (p < 0.05), as well as the ΔΨm, following 30% BMCF treatment under 5.5 mM glucose when compared with that of 25 mM glucose (Fig. 3B–D). The change in ΔΨm and mass is related to mitochondrial energy metabolism activity and/or mitochondrial biogenesis. Biogenesis of mitochondria, particularly under stress conditions, is often reflected by an increase in their mass [40]. Genetic down-modulation of GLUT3 expression through siRNA confirmed that TMRE and NAO decreased when
Altogether, this study highlights the therapeutic potential of FIR for the treatment of cellular insulin resistance, which leads to glucose deficiency and conditions such as degenerative disease or type 2 diabetes, potentially owing to decreased mitochondrial function. Additional studies are warranted to explore the exact transport pathway and the stimulatory mechanism of glucose involved in FIR-mediated improvement of mitochondrial biogenesis at low glucose conditions.
Supplementary data including one table and one figure can be found with this article online at https://doi.org/10.4196/kjpp.2021.25.2.167.
kjpp-25-2-167-supple.pdfThis research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (grant number: 2017R1D1A1B06035273) and Chung-Ang University Research Grants in 2016.
Y.S., Y-W.K., and D.L. designed the experiments and interpreted the data. D.H., J.C., D.K., K.K., J.L., C.B., W.J., H.L., G.J., T.K., Y.L., S-W.K., J.S.S., and Y.S. performed the experiments. H.K.K., J.H., and H.B. analyzed the data. Y.S. and J.K. wrote the manuscript. T.Z. and J-H.K. supervised the study.
The authors declare no conflicts of interest.
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