Research Article
Split ViewerEffect of Electroacupuncture Stimulation on Brown Adipose Tissue Thermogenesis
School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing, China
Correspondence to:This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
J Acupunct Meridian Stud 2024; 17(1): 1-11
Published February 29, 2024 https://doi.org/10.51507/j.jams.2024.17.1.1
Copyright © Medical Association of Pharmacopuncture Institute.
Abstract
Objectives: This study aimed to investigate the effects of EA at the GV20 and EX-HN3 acupoints. Specifically, the effects of EA on BAT thermogenesis were analyzed by infrared thermography, western blotting, and real-time polymerase chain reaction (PCR).
Methods: A total of 12 C57BL/6J mice were randomly divided into the EA and control groups. The EA group received EA at GV20 and EX-HN3 for 20 min once daily for 14 days. The control group underwent the same procedure but without EA. The core body temperature was monitored. Infrared thermal images of the back of each mouse in both groups were captured. BAT samples were collected after euthanasia to analyze UCP1 protein and UCP1 mRNA.
Results: The average skin temperature in the scapular region of the EA group was increased by 1.1℃ compared with that of the C group (p < 0.05). Additionally, the average temperature along the governor vessel in the EA group was increased by 1.6℃ (p = 0.045). EA significantly increased the expression of UCP1 protein (p = 0.001) and UCP1 mRNA (p = 0.002) in BAT, suggesting a potential link between EA and BAT thermogenesis.
Conclusion: EA induced BAT thermogenesis, suggesting GV20 and EX-HN3 as potential acupoints for BAT stimulation. The experimental results also highlighted unique meridian characteristics as demonstrated by elevated skin temperature along the governor vessel in mice.
Keywords
INTRODUCTION
Activated brown adipose tissue (BAT), including classic BAT and the newly discovered beige adipose tissue (derived from “browning” of white adipose tissue [WAT]), is crucial for non-shivering heat production in mammals to maintain core body temperature homeostasis [1-4]. Non-shivering heat production is mediated by uncoupling protein 1 (UCP1) located in the inner mitochondrial membranes. UCP1 uncouples mitochondrial ATP synthesis from fuel oxidation and releases energy in the form of heat [5-7]. The heat generated by this mitochondrial uncoupling mechanism is considerable [8,9], which is sufficient to maintain the normal core temperature in cold environments [10]. The results of initial studies suggest that the functional activity of BAT is only present in neonates. However, recent imaging studies have provided evidence of BAT activity in adults, which decreases with age [8]. BAT could not only increase energy consumption but could also absorb many lipid [11] and glucose substrates [12-14] from the circulation as a heat-producing process, thereby reducing plasma triglyceride levels and improving glucose homeostasis. Therefore, promoting the thermogenic function of BAT has become an important method of enhancing energy consumption and improving body weight and insulin resistance. Naturally, BAT thermogenesis has attracted considerable research interest as a target for the prevention and treatment of metabolic disorders, such as obesity and diabetes [15-17].
Methods of activating BAT or inducing the transformation of WAT to beige adipose tissue often require exposure to cold or hormone-induced stressors. However, exposure to cold is not practical in most cases, which cannot be applied in clinical interventions. In addition, it may induce adverse effects, such as elevated blood pressure [18].
Thyroid hormone analogs can be used to induce heat production in patients undergoing thyroidectomy [19]. However, they may affect other organs and tissues in the body and are not suitable for obese and diabetic patients without thyroid problems. Therefore, they are not recommended for human use. Beta3-adrenergic receptor (β3-AR) has been reported to be the most important factor involved in BAT thermogenesis in previous studies [1,3]. However, Jasper et al. suggested that β3-AR is dispensable for the browning of adipose tissue following experiments with β3-AR knockout mice [20]. The role of β3-AR in BAT thermogenesis remains controversial. A specific, effective, convenient, and widely applicable intervention method that can be used in clinical practice to induce BAT thermogenesis should be established. In this study, we planned to examine the effects of electroacupuncture (EA) on BAT thermogenesis to determine whether EA could be established as an effective method for the treatment of BAT-related diseases.
EA is a modern method of acupuncture based on the traditional Chinese medicine (TCM) meridian theory and is widely used in clinical practice. EA involves microbiological stimulation through the administration of small electric currents at select acupoints along meridians for the prevention and treatment of diseases. Several studies have investigated the effects of EA on adipose tissue. Wen et al. found that EA at ST36 could prevent weight gain by modulating HIF-1α-dependent pathways and the inflammatory response in obese adipose tissues [21]. Furthermore, Shen et al. reported that acupuncture at the ST36 and ST44 acupoints could promote white adipose tissue browning by inducing UCP1 expression [22]. Additionally, EA at ST29 and SP6 has been shown to improve metabolism by decreasing white adipose tissue and increasing BAT [23]. These studies were primarily focused on the effects of EA stimulation at lower limb acupoints on inflammation and white adipose tissue browning and did not adequately investigate the potential effects on the function of BAT itself. Therefore, drawing from TCM theory, we aimed to clarify the effects of EA at governor vessel acupoints on the function of BAT itself, which could expand the scope and depth of our understanding.
GV20 and EX-HN3 are specific governor vessel acupoints. According to the TCM meridian theory, the governor vessel is the sea of yang, and it regulates the yang qi (vital energy) of the whole body. The role of yang qi is to warm the whole body, a function that is very similar to the BAT thermogenesis-mediated maintenance of the core body temperature. For body temperature regulation, the hypothalamus occupies a vital integrative position between the sensation of skin and core temperatures and the sympathetic and somatic premotor pathways controlling thermo- effector activation, which includes shivering (“chills”) and BAT thermogenesis [24]. In addition, EA at GV20 and EX-HN3 can induce functional alterations in the neural network centered on the hypothalamus [25]. Therefore, this study was conducted to determine whether EA at GV20 and EX-HN3 could activate BAT thermogenesis considering the close association of GV20 and EX-HN3 with the hypothalamus, as well as their role in vital energy regulation.
MATERIALS AND METHODS
1. Animals
The study was approved by the Animal Ethics Committee of Beijing University of Traditional Chinese Medicine (Ethics number: BUCM 4- 2021102602-4031). C57BL/6J (30 ± 2 g) mice obtained from Beijing HFK Bioscience Co., Ltd. (animal license number: SCXK [Beijing] 2019-0008; Quality testing unit: Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences) were used for this study. All mice were individually housed in cages (temperature, 23-25℃; relative humidity, 50-60%) with a 12/12 h light/dark cycle and
After adaptive feeding for 1 week, the mice were randomly divided into the EA group or the control (C) group. The mice in the EA group (n = 6) were immobilized with a self-made mouse sleeve that did not affect breathing. Two disposable sterile acupuncture needles (size: 0.25 mm × 13 mm) were obliquely inserted at a depth of 0.5 cm into the GV20 and EX-HN3 acupoints. GV20 is located at the midpoint between the auricular apices, whereas EX-HN3 is located midway between the medial ends of the two eyebrows. The locations of the acupoints are shown in Fig. 1. The needle handle was connected to an EA instrument with the following parameters: frequency = 2 Hz and current intensity = 1 mA. The needle handle was slightly vibrated while the mouse was stationary and not struggling. EA was administered for 20 min at the same time each day for 14 days. The mice in the C group (n = 6) did not undergo EA intervention. However, they were immobilized with a self-made mouse sleeve at the same time each day. The core body temperature was monitored at a fixed time every 3 days during the intervention period (a total of 5 recordings). The TH212 Intelligent Digital Rectal Thermometer was used to measure the rectal temperature three times to obtain the average value for the core temperature. After intervention for 14 days, all mice were allowed to move freely above the cage lid. Meanwhile, an infrared camera was placed 0.5 m above the mice to capture thermal images of their backs (the hair on the backs was removed 1 day before imaging). Subsequently, all mice were euthanized using a self-made carbon dioxide euthanasia device, and BAT samples were collected and stored at –80℃ or fixed with paraformaldehyde.
-
Figure 1.Location of the GV20 and EX-HN3 acupoints. (A) Angle and direction of acupuncture points and brain tissue structure at these points. The black circle indicates the hypothalamus. Dots represent the locations of GV20 and EX-HN3 on the human head (B) and mouse head (C).
2. Infrared thermal imaging
Infrared thermal imaging is a technique used to determine the surface temperature based on the principles of radiation transfer. Medical infrared thermal imagers can focus on the invisible infrared radiation emitted by an object and subject it to photoelectric conversion to generate electrical signals. The electrical signal is then stimulated, amplified, and converted into a digital image signal. The image showing the thermal characteristics of the object is presented on the corresponding display device. Warm colors (bright colors) represent high-temperature regions, whereas cool colors (dark colors) represent low-temperature regions. The intensity of the infrared radiation emitted by an object is mainly a function of its temperature. Infrared thermal imaging has been used to study the surface temperature of various mammals [26-28], and some researchers have used it in TCM research including acupuncture meridians and acupoints [29-31].
We used a Testo 865 hand-held infrared thermal imager (Germany) to determine the distribution of the skin surface temperature. The imager was a 160 × 120-pixel infrared pixel detector that can detect objects with a size < 0.3 mm. The imager can detect temperatures ranging from −20℃ to 280℃. The imager has a thermal sensitivity of < 0.12℃. The SUPER infrared super pixel (320 × 240) can be utilized for image enhancement, capturing images through precise displacement, a built-in visible light shooting component with LED lighting, and autofocus (minimum focus, 0.5 m). It can show the isothermal and the low/high/average value of different regions. Moreover, the reflectivity and reflection of the detected temperature can be manually set. The infrared thermal images can be accurately analyzed on a computer using the IRSoft infrared analysis software (Testo, Shanghai, China). The emissivity of mouse skin (e = 0.97), room temperature (23-25℃), relative humidity (50-60%), and detection distance (0.5 m) were the same between the EA and C groups.
3. Adipose histology
Adipose tissue was embedded in paraffin, sliced, dewaxed, and stained with routine hematoxylin-eosin (HE) stains. The stained tissue was dehydrated and mounted for observation under a microscope (KF-PRO-020; KFBIO, Ningbo, China).
4. Western blotting
The BAT of mice was extracted for rapid grinding in a precooled mortar with liquid nitrogen. The ground powder and cell lysate were then placed in an Eppendorf tube. After centrifugation at 4℃, BAT protein was extracted, and protein quantification was performed using the bicinchoninic acid method. A 15% SDS-PAGE separation gel solution was prepared, and a 5% gel concentrate was added. The prepared gel was fixed vertically on a glass plate in the gel electrophoresis apparatus. Tissues with 40 μg of protein content were selected from each group of samples, and a 5× sample buffer was added at a volume ratio of 1:4. The protein was denatured after boiling for 5 min in a water bath. The treated samples were placed in the sample holes of the concentrated gel in a predetermined order for electrophoresis. After the protein samples were electrotransferred, the polyvinylidene fluoride (PVDF) membrane (product number: IPVH00010; batch number: R9PA20712; Millipore, Darmstadt, Germany) was sealed with 5% TBS-T skimmed milk powder at room temperature for 60 min. Subsequently, primary anti-UCP1 horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) (number: ZB-2301, batch number: 128964; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) was diluted with a TBS-T rinse solution and sealed at 4℃ for overnight incubation. A large amount of rinse solution was used to remove multiple antibodies. The PVDF membrane was then incubated with the secondary anti-UCP1 horseradish peroxidase-labeled goat anti-mouse IgG (number: ZB-2305, batch number: 129736; Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China), diluted with rinse solution, and shaken at 37℃ for 60 min. Finally, the enhanced chemiluminescence method was used for exposure and imprinting. Image J was used to analyze the gray level of the target band of the scanned image. We quantified the relative expression of UCP1 protein as follows:
UCP1 gray value/glyceraldehyde-3-phosphate dehydrogenase gray value.
5. Real-time polymerase chain reaction (PCR)
BAT samples were placed into a pre-cooling bowl, and liquid nitrogen was added for rapid grinding. TRIzol was added for the extraction of total RNA (TRIzol RNA Purification Kit, Invitrogen, Carlsbad, CA, USA). The optical density (OD) value and concentration of the diluted RNA extract were measured using a nucleic acid ultraviolet spectrophotometer (BioPhotometer, Hamburg, Germany). RNA purity and concentration were evaluated. Subsequently, the extracted RNA was added to a reverse transcription reaction mixture for reverse transcription (M-MLV Reverse Transcriptase; Promega Corporation, Madison, WI, USA), and the obtained complementary DNA and primer mixture were added to the ABI 7500 Fast PCR instrument (Qiagen, Hilden, Germany) for real-time PCR. The real-time PCR program was set at 94℃ for 2 min, followed by 45 cycles at 94℃ for 5 s and 60℃ for 30 s and final maintenance at 72℃ for 10 min. The relative expression of target genes in each sample was then calculated using the 2-ΔΔCT method (Table 1).
-
Table 1 . Target protein primer sequence
Primer sequence Probe Size of PCR product UCP1 forward primer 5’-AGCTTTGCCTCACTCAGGAT-3’ 100 bp UCP1 reverse primer 5’-CGGCTGAGATCTTGTTTCCG-3’ GAPDH forward primer 5’-GGTGAAGGTCGGTGTGAACG-3’ 233 bp GAPDH reverse primer 5’-CTCGCTCCTGGAAGATGGTG-3’ PCR = polymerase chain reaction; UCP1 = uncoupling protein 1; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
6. Statistical analysis
All data were analyzed using SPSS software (version 26.0; SPSS, Inc., Chicago, IL, USA). The result are expressed as the mean ± standard deviation. The measurement data for each group were tested for normality of distribution and homogeneity of variance. If the data showed a normal distribution and homogeneity of variance, the two groups were analyzed by an independent sample t-test; if not, a nonparametric test (rank-sum test) was performed.
RESULTS
1. EA increases the skin temperature in the scapular region of mice
Typical infrared thermography images are shown in Fig. 2. The red, orange, yellow, green, and blue regions represent different temperature regions (from high to low). We used the IRSoft infrared analysis software to assess the skin temperature in the scapular region of mice and selected the lowest, highest, and average temperatures in the scapular region for analysis. The results showed that the skin temperature of the EA group was 1.1℃ higher than that of the C group (
-
Figure 2.Effects of EA on interscapular skin temperature and rectal temperature. (A) Typical infrared thermal images. (B) Comparison of the maximum, minimum, and average scapular skin temperatures of the mice in the EA and C groups. (C) Rectal temperature. (D) Food intake. (E) Body weight. *
p < 0.05, **p < 0.01, ***p < 0.001. C = control; EA = electroacupuncture.
2. EA at specific acupoints (GV20 and EX-HN3) increases the skin temperature along the governor vessel
Unexpectedly, infrared thermography revealed that the EA group had elevated skin temperature along the governor vessel. As shown in Fig. 3A, we gradually concealed low-temperature areas using the IRSoft software. In comparison with the C group, the EA group showed significantly higher skin temperature along the governor vessel (white arrows in Fig. 3A and 3B) on the central line of the body from EX-HN3 (between the two eyebrows) to GV20 (midpoint between the ears). The high temperature along the governor vessel was observed up to the BAT. Although hair was not removed from this body part, the trend in the temperature change was still worthy of attention. We further analyzed the average skin temperature (Fig. 3D) of the mice in the EA group (n = 6) and the C group (n = 6). The average temperature in the EA group was increased by 1.6℃ compared with that in the C group (Z = –2.009,
-
Figure 3.Effects of EA on skin temperature along the governor vessel. (A) Typical infrared thermal images of the EA group. The white arrow indicates the location of the governor vessel acupoints. (B) Infrared thermal images of the C group. The white arrow indicates the location of the governor vessel acupoints. (C) Comparison of the temperature values of a segment of the governor vessel (from EX-HN3 to GV20) between the two groups. (D) Comparison of the average temperature of a segment of the governor vessel between the groups. *
p < 0.05. C = control; EA = electroacupuncture; GV = governor vessel.
3. EA promotes the expression of UCP1 protein and UCP1 mRNA in BAT and activates BAT thermogenesis
We explored the molecular mechanism underlying the effects of EA on skin temperature from the perspective of BAT thermogenesis. HE staining (Fig. 4B) revealed single, small lipid droplets in brown adipocytes and larger lipid droplets in white adipocytes in the C group. Brown adipocytes in the EA group showed a multilocular morphology. The number of white lipid droplets was decreased, which may partially reflect the high level of lipolysis necessary to provide fuel for BAT thermogenesis. We analyzed UCP1 expression after EA by western blotting and UCP1 mRNA expression by real-time PCR. The results showed that EA significantly increased the expression levels of UCP1 protein (
-
Figure 4.Effects of EA on BAT thermogenesis. (A) General images of BAT samples. (B) HE staining of adipose tissue. (C) Representative blots of the levels of UCP1 in BAT determined by western blotting. (D) UCP1 protein expression in the EA group compared with the C group. (E) Detection of UCP1 mRNA expression by real-time PCR. **
p < 0.01. C = control; EA = electroacupuncture; BAT = brown adipose tissue; WAT = white adipose tissue; UCP1 = uncoupling protein 1; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
DISCUSSION
BAT, which plays a significant role in the regulation of energy homeostasis, has been increasingly recognized as a potential target for alleviating metabolic diseases [15,17,20]. However, several existing methods (as mentioned in the Introduction) of inducing BAT activation have limitations and cannot be effectively applied in clinical practice. Notably, acupuncture has a limited side effect profile and is inexpensive, making it an important alternative therapy [32]. EA, which is characterized by parameter objectification, can improve the clinical efficacy of acupuncture and has attracted considerable attention in scientific research [33].
Previous studies have shown that EA at specific acupoints (e.g., ST36) can induce the expression of UCP1 protein and promote browning in white adipose tissue [22]. It has also been shown to reduce obese adipose tissue by inhibiting the inflammatory response [21]. However, these studies were primarily focused on the effects of EA stimulation at lower limb acupoints on white adipose tissue and did not adequately investigate the potential effects on the function of BAT itself. In addition, owing to synergistic effects, clinical outcomes may be better with acupuncture point combinations compared with single-point stimulation. However, the synergistic effects of different acupoint matching methods in the TCM system can vary and should be elucidated [34]. Therefore, this study aimed to explore the potential of EA at GV20 and GV29 in activating BAT thermogenesis. We examined the morphology of interscapular BAT and WAT by HE staining and visualized BAT thermogenesis activation using infrared thermal imaging technology. The novelty of this research lies in the application of TCM theory to acupoint selection and visualization methods with a focus on BAT function.
We used infrared thermography to observe the skin temperature in the scapular region (where BAT is located) of mice to investigate whether the effects of EA on BAT involve temperature changes. The results showed that EA increased the skin temperature in the scapular region of mice at varying degrees. BAT is one of the most vascularized tissues in the body [35]. Thermogenic adipocytes and their progenitors secrete several angiogenic factors to guide vascular cells to expand and remodel depending on the requirements of the adipose tissue microenvironment. Reciprocally, endothelial cells release factors that promote the thermogenic function of brown and beige adipocytes [36]. In mouse BAT, the initiation of angiogenic processes increases thermogenesis and improves whole-body metabolism [37]. The formation and expansion of blood vessels and adipocyte thermogenesis work in synergy. BAT dissipates energy in the form of heat energy to maintain the core body temperature, which is inseparable from a good vascular system [36]. BAT thermogenesis activation will naturally transfer heat energy throughout the vascular system and cause changes in the body surface temperature. Ratko et al. [38] have examined the correlation between the activity of BAT and skin temperature of mice using infrared thermography. Similarly, in this study, we observed the activity of BAT by infrared thermography. Indeed, infrared thermal imaging technology is considered a standard method for quantifying BAT activity [39,40].
BAT adaptive thermogenesis is driven by proton leakage across the mitochondrial membrane of brown adipocytes, which is facilitated by the high expression of UCP1 in the inner mitochondrial membrane of BAT [1,5]. Considering that UCP1 is the driving force of BAT thermogenesis pathway activation, we evaluated UCP1 expression in BAT by western blotting to clarify the mechanism underlying the increase in skin temperature. The results showed that EA induced UCP1 expression. Moreover, we measured the expression level of UCP1 mRNA by real-time PCR to determine whether EA could affect UCP1 at the gene level. The results showed that the expression level of UCP1 mRNA in the EA group was significantly increased. Therefore, the infrared thermal imaging and molecular biology results of the present study indicated that EA at the GV20 and EX-HN3 acupoints could activate BAT thermogenesis (Fig. 5).
-
Figure 5.Schematic of the effects of EA on the thermogenesis pathway of brown adipocytes in mice.
Previous studies have demonstrated that EA at GV20 could induce neural activity in specific areas of the brain [41]. Shortly after the EA needle is removed, the neural activity of the brain network formed by 15 functional brain regions, such as the frontal lobe, para-hippocampal gyrus, and hypothalamus, can still be observed [24]. A functional magnetic resonance imaging-based study by Salazar et al. on humans and rats showed that EA could activate hypothalamic neurons and enhance sympathetic activity [42]. From an anatomical perspective, GV20 is located at the top of the brain and in the cap-shaped fascia. The acupoints are surrounded by the left and right superficial temporal arteries and veins, occipital arteries and veins, and branches of the occipital and frontal nerves. GV20 is a significant acupoint for regulating brain function and is a key acupoint on the governor vessel.
Mice are considered the most suitable experimental animals for medical scientific research due to their high similarity to humans in terms of genes, anatomical structures, and even physiological processes, including metabolism and the nervous system [43]. There has been some progress in studying the meridian system in mice, and previous findings have provided a foundation for our research [44,45]. However, it is important to acknowledge that mice cannot fully represent humans, and we must take into account the anatomical and meridian system differences between mice and humans as different species. Therefore, further research and validation are necessary to ensure that our knowledge of the meridian system can be accurately applied to humans.
TCM is an ancient discipline practiced over the course of thousands of years by Chinese people in the struggle against diseases. According to “Inner Canon of Huangdi”, the oldest TCM classic [46], “people are closely connected with the natural world”. Aligning with the natural rhythm by regulating yin and yang is the basic principle of TCM. In TCM theory, the governor vessel is believed to be the sea of yang, regulating the yang qi of a body with 12 meridians, with the head being the point where the yang qi of various meridians converges. GV20 is the point formed after the convergence, which plays an important role in regulating the yin and yang balance (energy metabolism) of the body. Based on the meridian principle [47] and acupoint specificity [48,49], stimulating acupoints can lead to responses in corresponding meridians. In this study, we observed that EA at GV20 and EX-HN3 increased the skin temperature along the governor vessel. However, the results of the present study do not explain the meridian phenomenon, which is also a limitation of this study.
In conclusion, this study visualized the activation of BAT thermogenesis using infrared thermal imaging technology. The findings were obtained by conducting acupoint selection based on TCM theory combined with modern neuroanatomy research. This study is the first to associate the effects of GV20 stimulation on yang qi with the thermogenesis of BAT, an energy-producing tissue. Although the methods and theoretical basis of EA in TCM are different from those of Western medicine, the main therapeutic effects of both of them are similar.
LIMITATIONS AND FUTURE DIRECTIONS
1. Limitations
Our study provides important insights into the effects of EA on BAT thermogenesis; however, several limitations should be considered.
A limitation of our study is the small sample size. We only included 12 mice, dividing them into the EA group and the control group. This limited sample size raises concerns regarding the generalizability of our findings and the statistical power of our analysis. Future studies with larger sample sizes are needed to validate our results and increase the reliability of the findings.
Our research utilized C57BL/6J mice as an animal model, which is a commonly employed model in scientific studies. However, it is crucial to recognize that there may be inherent differences in physiology between mice and humans. Consequently, there could be limitations in the extent to which findings from the meridian system of mice can be extrapolated to humans.
Additionally, although our study observed an increase in skin temperature and UCP1 levels in the EA group, the exact mechanisms underlying these effects remain unclear. The changes in skin temperature measured by infrared thermography may be related to EA-induced metabolic changes; however, the effects of EA on metabolism were not specifically elucidated in our study. Therefore, further experiments are necessary to investigate and clarify the specific molecular pathways and signaling cascades involved in the EA-induced activation of BAT thermogenesis. These future experiments would contribute to a deeper understanding of the underlying mechanisms.
2. Future directions
Based on the limitations identified in our study, there are several areas that warrant further investigation.
Future research should focus on deciphering the precise mechanisms through which EA activates BAT thermogenesis. Advanced techniques such as transcriptomic analysis, proteomics, and metabolomics can offer valuable insights into the specific molecular pathways and signaling components implicated in the effects of EA. Furthermore, examining the effects of EA on metabolic parameters, body composition, and insulin sensitivity is crucial to establish its therapeutic potential for metabolic diseases. By monitoring the changes induced by EA, we can gain a better understanding of how EA influences the body’s metabolism and its potential benefits in treating metabolic disorders.
Additionally, translating the findings from animal studies to clinical practice should be prioritized. Well-designed clinical trials should be conducted to investigate the effects of EA on BAT thermogenesis in humans. Such trials could assess the effects of EA on metabolic disorders, body composition, and other relevant clinical outcomes.
By addressing limitations and exploring future research directions, we can deepen our understanding of the effects of EA on BAT thermogenesis and its potential therapeutic applications in metabolic disorders. These endeavors will contribute to the advancement of evidence-based acupuncture and its integration into clinical practice.
ACKNOWLEDGEMENTS
We would like to thank the equipment providers and Editage for English language editing.
FUNDING
This work was funded by the National Natural Science Foundation of China (Award Number: 81973938 and 82274654).
AUTHORS’ CONTRIBUTIONS
Ting Li, Meng Wu, and Zhigang Li designed and performed experiments. Junjian Tian and Yitong Li analyzed the results and participated in the revision of the manuscript. Ting Li and Meng Wu wrote the paper. All authors approved the final version of the manuscript.
DATA AVAILABILITY
The data that support the findings of this study are available from the corresponding author upon request.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
References
- Oelkrug R, Polymeropoulos ET, Jastroch M. Brown adipose tissue: physiological function and evolutionary significance. J Comp Physiol B 2015;185:587-606.
- Contreras C, Gonzalez F, Fernø J, Diéguez C, Rahmouni K, Nogueiras R, et al. The brain and brown fat. Ann Med 2015;47:150-68. https://doi.org/10.3109/07853890.2014.919727.
- Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev 2004;84:277-359.
- Zhang Z, Yang D, Xiang J, Zhou J, Cao H, Che Q, et al. Non-shivering thermogenesis signalling regulation and potential therapeutic applications of brown adipose tissue. Int J Biol Sci 2021;17:2853-70. https://doi.org/10.7150/ijbs.60354.
- Fedorenko A, Lishko PV, Kirichok Y. Mechanism of fatty-acid-dependent UCP1 uncoupling in brown fat mitochondria. Cell 2012;151:400-13. https://doi.org/10.1016/j.cell.2012.09.010.
- Calderon-Dominguez M, Mir JF, Fucho R, Weber M, Serra D, Herrero L. Fatty acid metabolism and the basis of brown adipose tissue function. Adipocyte 2015;5:98-118.
- Heaton GM, Wagenvoord RJ, Kemp A Jr, Nicholls DG. Brown-adipose-tissue mitochondria: photoaffinity labelling of the regulatory site of energy dissipation. Eur J Biochem 1978;82:515-21. https://doi.org/10.1111/j.1432-1033.1978.tb12045.x.
- Leitner BP, Huang S, Brychta RJ, Duckworth CJ, Baskin AS, McGehee S, et al. Mapping of human brown adipose tissue in lean and obese young men. Proc Natl Acad Sci U S A 2017;114:8649-54. https://doi.org/10.1073/pnas.1705287114.
- Fernández-Verdejo R, Marlatt KL, Ravussin E, Galgani JE. Contribution of brown adipose tissue to human energy metabolism. Mol Aspects Med 2019;68:82-9.
- Gomolin IH, Aung MM, Wolf-Klein G, Auerbach C. Older is colder: temperature range and variation in older people. J Am Geriatr Soc 2005;53:2170-2.
- Bartelt A, Bruns OT, Reimer R, Hohenberg H, Ittrich H, Peldschus K, et al. Brown adipose tissue activity controls triglyceride clearance. Nat Med 2011;17:200-5.
- Guerra C, Navarro P, Valverde AM, Arribas M, Brüning J, Kozak LP, et al. Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance. J Clin Invest 2001;108:1205-13. https://doi.org/10.1172/JCI13103.
- Gunawardana SC, Piston DW. Reversal of type 1 diabetes in mice by brown adipose tissue transplant. Diabetes 2012;61:674-82. https://doi.org/10.2337/db11-0510.
- Wang J, Wu Q, Zhou Y, Yu L, Yu L, Deng Y, et al. The mechanisms underlying olanzapine-induced insulin resistance via the brown adipose tissue and the therapy in rats. Adipocyte 2022;11:84-98. https://doi.org/10.1080/21623945.2022.2026590.
- Cheng L, Wang J, Dai H, Duan Y, An Y, Shi L, et al. Brown and beige adipose tissue: a novel therapeutic strategy for obesity and type 2 diabetes mellitus. Adipocyte 2021;10:48-65.
- Tournissac M, Leclerc M, Valentin-Escalera J, Vandal M, Bosoi CR, Planel E, et al. Metabolic determinants of Alzheimer's disease: a focus on thermoregulation. Ageing Res Rev 2021;72:101462. https://doi.org/10.1016/j.arr.2021.101462.
- Poher AL, Altirriba J, Veyrat-Durebex C, Rohner-Jeanrenaud F. Brown adipose tissue activity as a target for the treatment of obesity/insulin resistance. Front Physiol 2015;6:4.
- van der Lans AA, Hoeks J, Brans B, Vijgen GH, Visser MG, Vosselman MJ, et al. Cold acclimation recruits human brown fat and increases nonshivering thermogenesis. J Clin Invest 2013;123:3395-403. https://doi.org/10.1172/JCI68993.
- Broeders EP, Vijgen GH, Havekes B, Bouvy ND, Mottaghy FM, Kars M, et al. Thyroid hormone activates brown adipose tissue and increases non-shivering thermogenesis--a cohort study in a group of thyroid carcinoma patients. PLoS One 2016;11:e0145049. Erratum in: PLoS One 2018;13:e0209225.
- de Jong JMA, Wouters RTF, Boulet N, Cannon B, Nedergaard J, Petrovic N. The β3-adrenergic receptor is dispensable for browning of adipose tissues. Am J Physiol Endocrinol Metab 2017;312:E508-18. https://doi.org/10.1152/ajpendo.00437.2016.
- Wen CK, Lee TY. Electroacupuncture prevents white adipose tissue inflammation through modulation of hypoxia-inducible factors-1α-dependent pathway in obese mice. BMC Complement Altern Med 2015;15:452.
- Shen W, Wang Y, Lu SF, Hong H, Fu S, He S, et al. Acupuncture promotes white adipose tissue browning by inducing UCP1 expression on DIO mice. BMC Complement Altern Med 2014;14:501. https://doi.org/10.1186/1472-6882-14-501.
- Zhang F, Ma T, Tong X, Liu Y, Cui P, Xu X, et al. Electroacupuncture improves metabolic and ovarian function in a rat model of polycystic ovary syndrome by decreasing white adipose tissue, increasing brown adipose tissue, and modulating the gut microbiota. Acupunct Med 2022;40:347-59.
- Morrison SF. Central neural control of thermoregulation and brown adipose tissue. Auton Neurosci 2016;196:14-24.
- Zhang G, Qu S, Zheng Y, Chen J, Deng G, Yang C, et al. Key regions of the cerebral network are altered after electroacupuncture at the Baihui (GV20) and Yintang acupuncture points in healthy volunteers: an analysis based on resting fcMRI. Acupunct Med 2013;31:383-8.
- Usamentiaga R, Venegas P, Guerediaga J, Vega L, Molleda J, Bulnes FG. Infrared thermography for temperature measurement and non-destructive testing. Sensors (Basel) 2014;14:12305-48. https://doi.org/10.3390/s140712305.
- McCafferty DJ, Gilbert C, Paterson W, Pomeroy PP, Thompson D, Currie JI, et al. Estimating metabolic heat loss in birds and mammals by combining infrared thermography with biophysical modelling. Comp Biochem Physiol A Mol Integr Physiol 2011;158:337-45.
- Franco NH, Gerós A, Oliveira L, Olsson IAS, Aguiar P. ThermoLabAnimal - a high-throughput analysis software for non-invasive thermal assessment of laboratory mice. Physiol Behav 2019;207:113-21. https://doi.org/10.1016/j.physbeh.2019.05.004.
- Fu Y, Ni JX, Marmori F, Zhu Q, Tan C, Zhao JP. Infrared thermal imaging-based research on the intermediate structures of the lung and large intestine exterior-interior relationship in asthma patients. Chin J Integr Med 2016;22:855-60.
- Yang HQ, Xie SS, Hu XL, Chen L, Li H. Appearance of human meridian-like structure and acupoints and its time correlation by infrared thermal imaging. Am J Chin Med 2007;35:231-40. https://doi.org/10.1142/S0192415X07004771.
- de Souza RC, Pansini M, Arruda G, Valente C, Brioschi ML. Laser acupuncture causes thermal changes in small intestine meridian pathway. Lasers Med Sci 2016;31:1645-9.
- Van Hal M, Dydyk AM, Green MS. Acupuncture. In: Aboubakr S, Abu-Ghosh A, Ackley WB, Adolphe TS, Aeby TC, Aeddula NR, et al, editors. StatPearls [Internet]. Treasure Island: StatPearls Publishing, 2023. Available from: https://pubmed.ncbi.nlm.nih.gov/30335320/.
- Zhu J, Li J, Yang L, Liu S. Acupuncture, from the ancient to the current. Anat Rec (Hoboken) 2021;304:2365-71.
- Jun Z, Xia LI, Hui Z, Kun YE, Xin W, Xuefei W, et al. Effects of acupuncture on functional gastrointestinal disorders: special effects, coeffects, synergistic effects in terms of single or compatible acupoints. J Tradit Chin Med 2023;43:397-408.
- Brakenhielm E, Cao Y. Angiogenesis in adipose tissue. Methods Mol Biol 2008;456:65-81.
- Shamsi F, Wang CH, Tseng YH. The evolving view of thermogenic adipocytes - ontogeny, niche and function. Nat Rev Endocrinol 2021;17:726-44.
- Sun K, Kusminski CM, Luby-Phelps K, Spurgin SB, An YA, Wang QA, et al. Brown adipose tissue derived VEGF-A modulates cold tolerance and energy expenditure. Mol Metab 2014;3:474-83. https://doi.org/10.1016/j.molmet.2014.03.010.
- Ratko M, Habek N, Kordić M, Dugandžić A. The use of infrared technology as a novel approach for studies with female laboratory animals. Croat Med J 2020;61:346-53.
- Habek N, Kordić M, Jurenec F, Dugandžić A. Infrared thermography, a new method for detection of brown adipose tissue activity after a meal in humans. Infrared Phys Technol 2018;89:271-6. https://doi.org/10.1016/j.infrared.2018.01.020.
- Ang QY, Goh HJ, Cao Y, Li Y, Chan SP, Swain JL, et al. A new method of infrared thermography for quantification of brown adipose tissue activation in healthy adults (TACTICAL): a randomized trial. J Physiol Sci 2017;67:395-406.
- Deng D, Duan G, Liao H, Liu Y, Wang G, Liu H, et al. Changes in regional brain homogeneity induced by electro-acupuncture stimulation at the Baihui acupoint in healthy subjects: a functional magnetic resonance imaging study. J Altern Complement Med 2016;22:794-9. https://doi.org/10.1089/acm.2015.0286.
- Salazar TE, Richardson MR, Beli E, Ripsch MS, George J, Kim Y, et al. Electroacupuncture promotes central nervous system-dependent release of mesenchymal stem cells. Stem Cells 2017;35:1303-15. https://doi.org/10.1002/stem.2613.
- Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, et al. Initial sequencing and comparative analysis of the mouse genome. Nature 2002;420:520-62.
- Vieira JS, Toreti JA, de Carvalho RC, de Araújo JE, Silva ML, Silva JRT. Analgesic effects elicited by neuroactive mediators injected into the ST 36 acupuncture point on inflammatory and neuropathic pain in mice. J Acupunct Meridian Stud 2018;11:280-9. https://doi.org/10.1016/j.jams.2018.05.006.
- Chang S. The meridian system and mechanism of acupuncture-a comparative review. Part 1: the meridian system. Taiwan J Obstet Gynecol 2012;51:506-14.
- Zou Q, Wang Y, Shu Z, Yang K, Wang J, Lu K, et al. Topological analysis of the language networks of ancient traditional Chinese medicine books. Evid Based Complement Alternat Med 2020;2020:8810016. https://doi.org/10.1155/2020/8810016.
- Zhou W, Benharash P. Effects and mechanisms of acupuncture based on the principle of meridians. J Acupunct Meridian Stud 2014;7:190-3. https://doi.org/10.1016/j.jams.2014.02.007.
- Qiu K, Yin T, Hong X, Sun R, He Z, Liu X, et al. Does the acupoint specificity exist? Evidence from functional neuroimaging studies. Curr Med Imaging 2020;16:629-38.
- Lee DY, Jiu YR, Hsieh CL. Metabolism modulation in rat tissues in response to point specificity of electroacupuncture. Sci Rep 2022;12:210.
Related articles in JAMS
Article
Research Article
J Acupunct Meridian Stud 2024; 17(1): 1-11
Published online February 29, 2024 https://doi.org/10.51507/j.jams.2024.17.1.1
Copyright © Medical Association of Pharmacopuncture Institute.
Effect of Electroacupuncture Stimulation on Brown Adipose Tissue Thermogenesis
Ting Li† , Meng Wu† , Junjian Tian , Yitong Li , Zhigang Li*
School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing, China
Correspondence to:Zhigang Li
School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing, China
E-mail lizhigang620@126.com
†These authors contributed equally to this work and should be considered co-first authors.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Brown adipose tissue (BAT) is a unique thermogenic tissue in mammals mediated by uncoupling protein 1 (UCP1). The energy generated by glucose and triglyceride metabolism is released and transmitted throughout the body as heat. Understanding the factors influencing BAT function is crucial to determine its metabolic significance and effects on overall health. Although studies have shown that electroacupuncture (EA) at specific acupoints (e.g., ST36) can stimulate BAT, its effects at other acupoints are not well understood. Further research is needed to investigate the potential effects of EA at these acupoints and their association with BAT activation.
Objectives: This study aimed to investigate the effects of EA at the GV20 and EX-HN3 acupoints. Specifically, the effects of EA on BAT thermogenesis were analyzed by infrared thermography, western blotting, and real-time polymerase chain reaction (PCR).
Methods: A total of 12 C57BL/6J mice were randomly divided into the EA and control groups. The EA group received EA at GV20 and EX-HN3 for 20 min once daily for 14 days. The control group underwent the same procedure but without EA. The core body temperature was monitored. Infrared thermal images of the back of each mouse in both groups were captured. BAT samples were collected after euthanasia to analyze UCP1 protein and UCP1 mRNA.
Results: The average skin temperature in the scapular region of the EA group was increased by 1.1℃ compared with that of the C group (p < 0.05). Additionally, the average temperature along the governor vessel in the EA group was increased by 1.6℃ (p = 0.045). EA significantly increased the expression of UCP1 protein (p = 0.001) and UCP1 mRNA (p = 0.002) in BAT, suggesting a potential link between EA and BAT thermogenesis.
Conclusion: EA induced BAT thermogenesis, suggesting GV20 and EX-HN3 as potential acupoints for BAT stimulation. The experimental results also highlighted unique meridian characteristics as demonstrated by elevated skin temperature along the governor vessel in mice.
Keywords: Electroacupuncture, Brown adipose tissue, Thermogenesis, Infrared thermography, Uncoupling protein
INTRODUCTION
Activated brown adipose tissue (BAT), including classic BAT and the newly discovered beige adipose tissue (derived from “browning” of white adipose tissue [WAT]), is crucial for non-shivering heat production in mammals to maintain core body temperature homeostasis [1-4]. Non-shivering heat production is mediated by uncoupling protein 1 (UCP1) located in the inner mitochondrial membranes. UCP1 uncouples mitochondrial ATP synthesis from fuel oxidation and releases energy in the form of heat [5-7]. The heat generated by this mitochondrial uncoupling mechanism is considerable [8,9], which is sufficient to maintain the normal core temperature in cold environments [10]. The results of initial studies suggest that the functional activity of BAT is only present in neonates. However, recent imaging studies have provided evidence of BAT activity in adults, which decreases with age [8]. BAT could not only increase energy consumption but could also absorb many lipid [11] and glucose substrates [12-14] from the circulation as a heat-producing process, thereby reducing plasma triglyceride levels and improving glucose homeostasis. Therefore, promoting the thermogenic function of BAT has become an important method of enhancing energy consumption and improving body weight and insulin resistance. Naturally, BAT thermogenesis has attracted considerable research interest as a target for the prevention and treatment of metabolic disorders, such as obesity and diabetes [15-17].
Methods of activating BAT or inducing the transformation of WAT to beige adipose tissue often require exposure to cold or hormone-induced stressors. However, exposure to cold is not practical in most cases, which cannot be applied in clinical interventions. In addition, it may induce adverse effects, such as elevated blood pressure [18].
Thyroid hormone analogs can be used to induce heat production in patients undergoing thyroidectomy [19]. However, they may affect other organs and tissues in the body and are not suitable for obese and diabetic patients without thyroid problems. Therefore, they are not recommended for human use. Beta3-adrenergic receptor (β3-AR) has been reported to be the most important factor involved in BAT thermogenesis in previous studies [1,3]. However, Jasper et al. suggested that β3-AR is dispensable for the browning of adipose tissue following experiments with β3-AR knockout mice [20]. The role of β3-AR in BAT thermogenesis remains controversial. A specific, effective, convenient, and widely applicable intervention method that can be used in clinical practice to induce BAT thermogenesis should be established. In this study, we planned to examine the effects of electroacupuncture (EA) on BAT thermogenesis to determine whether EA could be established as an effective method for the treatment of BAT-related diseases.
EA is a modern method of acupuncture based on the traditional Chinese medicine (TCM) meridian theory and is widely used in clinical practice. EA involves microbiological stimulation through the administration of small electric currents at select acupoints along meridians for the prevention and treatment of diseases. Several studies have investigated the effects of EA on adipose tissue. Wen et al. found that EA at ST36 could prevent weight gain by modulating HIF-1α-dependent pathways and the inflammatory response in obese adipose tissues [21]. Furthermore, Shen et al. reported that acupuncture at the ST36 and ST44 acupoints could promote white adipose tissue browning by inducing UCP1 expression [22]. Additionally, EA at ST29 and SP6 has been shown to improve metabolism by decreasing white adipose tissue and increasing BAT [23]. These studies were primarily focused on the effects of EA stimulation at lower limb acupoints on inflammation and white adipose tissue browning and did not adequately investigate the potential effects on the function of BAT itself. Therefore, drawing from TCM theory, we aimed to clarify the effects of EA at governor vessel acupoints on the function of BAT itself, which could expand the scope and depth of our understanding.
GV20 and EX-HN3 are specific governor vessel acupoints. According to the TCM meridian theory, the governor vessel is the sea of yang, and it regulates the yang qi (vital energy) of the whole body. The role of yang qi is to warm the whole body, a function that is very similar to the BAT thermogenesis-mediated maintenance of the core body temperature. For body temperature regulation, the hypothalamus occupies a vital integrative position between the sensation of skin and core temperatures and the sympathetic and somatic premotor pathways controlling thermo- effector activation, which includes shivering (“chills”) and BAT thermogenesis [24]. In addition, EA at GV20 and EX-HN3 can induce functional alterations in the neural network centered on the hypothalamus [25]. Therefore, this study was conducted to determine whether EA at GV20 and EX-HN3 could activate BAT thermogenesis considering the close association of GV20 and EX-HN3 with the hypothalamus, as well as their role in vital energy regulation.
MATERIALS AND METHODS
1. Animals
The study was approved by the Animal Ethics Committee of Beijing University of Traditional Chinese Medicine (Ethics number: BUCM 4- 2021102602-4031). C57BL/6J (30 ± 2 g) mice obtained from Beijing HFK Bioscience Co., Ltd. (animal license number: SCXK [Beijing] 2019-0008; Quality testing unit: Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences) were used for this study. All mice were individually housed in cages (temperature, 23-25℃; relative humidity, 50-60%) with a 12/12 h light/dark cycle and
After adaptive feeding for 1 week, the mice were randomly divided into the EA group or the control (C) group. The mice in the EA group (n = 6) were immobilized with a self-made mouse sleeve that did not affect breathing. Two disposable sterile acupuncture needles (size: 0.25 mm × 13 mm) were obliquely inserted at a depth of 0.5 cm into the GV20 and EX-HN3 acupoints. GV20 is located at the midpoint between the auricular apices, whereas EX-HN3 is located midway between the medial ends of the two eyebrows. The locations of the acupoints are shown in Fig. 1. The needle handle was connected to an EA instrument with the following parameters: frequency = 2 Hz and current intensity = 1 mA. The needle handle was slightly vibrated while the mouse was stationary and not struggling. EA was administered for 20 min at the same time each day for 14 days. The mice in the C group (n = 6) did not undergo EA intervention. However, they were immobilized with a self-made mouse sleeve at the same time each day. The core body temperature was monitored at a fixed time every 3 days during the intervention period (a total of 5 recordings). The TH212 Intelligent Digital Rectal Thermometer was used to measure the rectal temperature three times to obtain the average value for the core temperature. After intervention for 14 days, all mice were allowed to move freely above the cage lid. Meanwhile, an infrared camera was placed 0.5 m above the mice to capture thermal images of their backs (the hair on the backs was removed 1 day before imaging). Subsequently, all mice were euthanized using a self-made carbon dioxide euthanasia device, and BAT samples were collected and stored at –80℃ or fixed with paraformaldehyde.
-
Figure 1. Location of the GV20 and EX-HN3 acupoints. (A) Angle and direction of acupuncture points and brain tissue structure at these points. The black circle indicates the hypothalamus. Dots represent the locations of GV20 and EX-HN3 on the human head (B) and mouse head (C).
2. Infrared thermal imaging
Infrared thermal imaging is a technique used to determine the surface temperature based on the principles of radiation transfer. Medical infrared thermal imagers can focus on the invisible infrared radiation emitted by an object and subject it to photoelectric conversion to generate electrical signals. The electrical signal is then stimulated, amplified, and converted into a digital image signal. The image showing the thermal characteristics of the object is presented on the corresponding display device. Warm colors (bright colors) represent high-temperature regions, whereas cool colors (dark colors) represent low-temperature regions. The intensity of the infrared radiation emitted by an object is mainly a function of its temperature. Infrared thermal imaging has been used to study the surface temperature of various mammals [26-28], and some researchers have used it in TCM research including acupuncture meridians and acupoints [29-31].
We used a Testo 865 hand-held infrared thermal imager (Germany) to determine the distribution of the skin surface temperature. The imager was a 160 × 120-pixel infrared pixel detector that can detect objects with a size < 0.3 mm. The imager can detect temperatures ranging from −20℃ to 280℃. The imager has a thermal sensitivity of < 0.12℃. The SUPER infrared super pixel (320 × 240) can be utilized for image enhancement, capturing images through precise displacement, a built-in visible light shooting component with LED lighting, and autofocus (minimum focus, 0.5 m). It can show the isothermal and the low/high/average value of different regions. Moreover, the reflectivity and reflection of the detected temperature can be manually set. The infrared thermal images can be accurately analyzed on a computer using the IRSoft infrared analysis software (Testo, Shanghai, China). The emissivity of mouse skin (e = 0.97), room temperature (23-25℃), relative humidity (50-60%), and detection distance (0.5 m) were the same between the EA and C groups.
3. Adipose histology
Adipose tissue was embedded in paraffin, sliced, dewaxed, and stained with routine hematoxylin-eosin (HE) stains. The stained tissue was dehydrated and mounted for observation under a microscope (KF-PRO-020; KFBIO, Ningbo, China).
4. Western blotting
The BAT of mice was extracted for rapid grinding in a precooled mortar with liquid nitrogen. The ground powder and cell lysate were then placed in an Eppendorf tube. After centrifugation at 4℃, BAT protein was extracted, and protein quantification was performed using the bicinchoninic acid method. A 15% SDS-PAGE separation gel solution was prepared, and a 5% gel concentrate was added. The prepared gel was fixed vertically on a glass plate in the gel electrophoresis apparatus. Tissues with 40 μg of protein content were selected from each group of samples, and a 5× sample buffer was added at a volume ratio of 1:4. The protein was denatured after boiling for 5 min in a water bath. The treated samples were placed in the sample holes of the concentrated gel in a predetermined order for electrophoresis. After the protein samples were electrotransferred, the polyvinylidene fluoride (PVDF) membrane (product number: IPVH00010; batch number: R9PA20712; Millipore, Darmstadt, Germany) was sealed with 5% TBS-T skimmed milk powder at room temperature for 60 min. Subsequently, primary anti-UCP1 horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) (number: ZB-2301, batch number: 128964; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) was diluted with a TBS-T rinse solution and sealed at 4℃ for overnight incubation. A large amount of rinse solution was used to remove multiple antibodies. The PVDF membrane was then incubated with the secondary anti-UCP1 horseradish peroxidase-labeled goat anti-mouse IgG (number: ZB-2305, batch number: 129736; Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China), diluted with rinse solution, and shaken at 37℃ for 60 min. Finally, the enhanced chemiluminescence method was used for exposure and imprinting. Image J was used to analyze the gray level of the target band of the scanned image. We quantified the relative expression of UCP1 protein as follows:
UCP1 gray value/glyceraldehyde-3-phosphate dehydrogenase gray value.
5. Real-time polymerase chain reaction (PCR)
BAT samples were placed into a pre-cooling bowl, and liquid nitrogen was added for rapid grinding. TRIzol was added for the extraction of total RNA (TRIzol RNA Purification Kit, Invitrogen, Carlsbad, CA, USA). The optical density (OD) value and concentration of the diluted RNA extract were measured using a nucleic acid ultraviolet spectrophotometer (BioPhotometer, Hamburg, Germany). RNA purity and concentration were evaluated. Subsequently, the extracted RNA was added to a reverse transcription reaction mixture for reverse transcription (M-MLV Reverse Transcriptase; Promega Corporation, Madison, WI, USA), and the obtained complementary DNA and primer mixture were added to the ABI 7500 Fast PCR instrument (Qiagen, Hilden, Germany) for real-time PCR. The real-time PCR program was set at 94℃ for 2 min, followed by 45 cycles at 94℃ for 5 s and 60℃ for 30 s and final maintenance at 72℃ for 10 min. The relative expression of target genes in each sample was then calculated using the 2-ΔΔCT method (Table 1).
-
&md=tbl&idx=1' data-target="#file-modal"">Table 1
Target protein primer sequence.
Primer sequence Probe Size of PCR product UCP1 forward primer 5’-AGCTTTGCCTCACTCAGGAT-3’ 100 bp UCP1 reverse primer 5’-CGGCTGAGATCTTGTTTCCG-3’ GAPDH forward primer 5’-GGTGAAGGTCGGTGTGAACG-3’ 233 bp GAPDH reverse primer 5’-CTCGCTCCTGGAAGATGGTG-3’ PCR = polymerase chain reaction; UCP1 = uncoupling protein 1; GAPDH = glyceraldehyde-3-phosphate dehydrogenase..
6. Statistical analysis
All data were analyzed using SPSS software (version 26.0; SPSS, Inc., Chicago, IL, USA). The result are expressed as the mean ± standard deviation. The measurement data for each group were tested for normality of distribution and homogeneity of variance. If the data showed a normal distribution and homogeneity of variance, the two groups were analyzed by an independent sample t-test; if not, a nonparametric test (rank-sum test) was performed.
RESULTS
1. EA increases the skin temperature in the scapular region of mice
Typical infrared thermography images are shown in Fig. 2. The red, orange, yellow, green, and blue regions represent different temperature regions (from high to low). We used the IRSoft infrared analysis software to assess the skin temperature in the scapular region of mice and selected the lowest, highest, and average temperatures in the scapular region for analysis. The results showed that the skin temperature of the EA group was 1.1℃ higher than that of the C group (
-
Figure 2. Effects of EA on interscapular skin temperature and rectal temperature. (A) Typical infrared thermal images. (B) Comparison of the maximum, minimum, and average scapular skin temperatures of the mice in the EA and C groups. (C) Rectal temperature. (D) Food intake. (E) Body weight. *
p < 0.05, **p < 0.01, ***p < 0.001. C = control; EA = electroacupuncture.
2. EA at specific acupoints (GV20 and EX-HN3) increases the skin temperature along the governor vessel
Unexpectedly, infrared thermography revealed that the EA group had elevated skin temperature along the governor vessel. As shown in Fig. 3A, we gradually concealed low-temperature areas using the IRSoft software. In comparison with the C group, the EA group showed significantly higher skin temperature along the governor vessel (white arrows in Fig. 3A and 3B) on the central line of the body from EX-HN3 (between the two eyebrows) to GV20 (midpoint between the ears). The high temperature along the governor vessel was observed up to the BAT. Although hair was not removed from this body part, the trend in the temperature change was still worthy of attention. We further analyzed the average skin temperature (Fig. 3D) of the mice in the EA group (n = 6) and the C group (n = 6). The average temperature in the EA group was increased by 1.6℃ compared with that in the C group (Z = –2.009,
-
Figure 3. Effects of EA on skin temperature along the governor vessel. (A) Typical infrared thermal images of the EA group. The white arrow indicates the location of the governor vessel acupoints. (B) Infrared thermal images of the C group. The white arrow indicates the location of the governor vessel acupoints. (C) Comparison of the temperature values of a segment of the governor vessel (from EX-HN3 to GV20) between the two groups. (D) Comparison of the average temperature of a segment of the governor vessel between the groups. *
p < 0.05. C = control; EA = electroacupuncture; GV = governor vessel.
3. EA promotes the expression of UCP1 protein and UCP1 mRNA in BAT and activates BAT thermogenesis
We explored the molecular mechanism underlying the effects of EA on skin temperature from the perspective of BAT thermogenesis. HE staining (Fig. 4B) revealed single, small lipid droplets in brown adipocytes and larger lipid droplets in white adipocytes in the C group. Brown adipocytes in the EA group showed a multilocular morphology. The number of white lipid droplets was decreased, which may partially reflect the high level of lipolysis necessary to provide fuel for BAT thermogenesis. We analyzed UCP1 expression after EA by western blotting and UCP1 mRNA expression by real-time PCR. The results showed that EA significantly increased the expression levels of UCP1 protein (
-
Figure 4. Effects of EA on BAT thermogenesis. (A) General images of BAT samples. (B) HE staining of adipose tissue. (C) Representative blots of the levels of UCP1 in BAT determined by western blotting. (D) UCP1 protein expression in the EA group compared with the C group. (E) Detection of UCP1 mRNA expression by real-time PCR. **
p < 0.01. C = control; EA = electroacupuncture; BAT = brown adipose tissue; WAT = white adipose tissue; UCP1 = uncoupling protein 1; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
DISCUSSION
BAT, which plays a significant role in the regulation of energy homeostasis, has been increasingly recognized as a potential target for alleviating metabolic diseases [15,17,20]. However, several existing methods (as mentioned in the Introduction) of inducing BAT activation have limitations and cannot be effectively applied in clinical practice. Notably, acupuncture has a limited side effect profile and is inexpensive, making it an important alternative therapy [32]. EA, which is characterized by parameter objectification, can improve the clinical efficacy of acupuncture and has attracted considerable attention in scientific research [33].
Previous studies have shown that EA at specific acupoints (e.g., ST36) can induce the expression of UCP1 protein and promote browning in white adipose tissue [22]. It has also been shown to reduce obese adipose tissue by inhibiting the inflammatory response [21]. However, these studies were primarily focused on the effects of EA stimulation at lower limb acupoints on white adipose tissue and did not adequately investigate the potential effects on the function of BAT itself. In addition, owing to synergistic effects, clinical outcomes may be better with acupuncture point combinations compared with single-point stimulation. However, the synergistic effects of different acupoint matching methods in the TCM system can vary and should be elucidated [34]. Therefore, this study aimed to explore the potential of EA at GV20 and GV29 in activating BAT thermogenesis. We examined the morphology of interscapular BAT and WAT by HE staining and visualized BAT thermogenesis activation using infrared thermal imaging technology. The novelty of this research lies in the application of TCM theory to acupoint selection and visualization methods with a focus on BAT function.
We used infrared thermography to observe the skin temperature in the scapular region (where BAT is located) of mice to investigate whether the effects of EA on BAT involve temperature changes. The results showed that EA increased the skin temperature in the scapular region of mice at varying degrees. BAT is one of the most vascularized tissues in the body [35]. Thermogenic adipocytes and their progenitors secrete several angiogenic factors to guide vascular cells to expand and remodel depending on the requirements of the adipose tissue microenvironment. Reciprocally, endothelial cells release factors that promote the thermogenic function of brown and beige adipocytes [36]. In mouse BAT, the initiation of angiogenic processes increases thermogenesis and improves whole-body metabolism [37]. The formation and expansion of blood vessels and adipocyte thermogenesis work in synergy. BAT dissipates energy in the form of heat energy to maintain the core body temperature, which is inseparable from a good vascular system [36]. BAT thermogenesis activation will naturally transfer heat energy throughout the vascular system and cause changes in the body surface temperature. Ratko et al. [38] have examined the correlation between the activity of BAT and skin temperature of mice using infrared thermography. Similarly, in this study, we observed the activity of BAT by infrared thermography. Indeed, infrared thermal imaging technology is considered a standard method for quantifying BAT activity [39,40].
BAT adaptive thermogenesis is driven by proton leakage across the mitochondrial membrane of brown adipocytes, which is facilitated by the high expression of UCP1 in the inner mitochondrial membrane of BAT [1,5]. Considering that UCP1 is the driving force of BAT thermogenesis pathway activation, we evaluated UCP1 expression in BAT by western blotting to clarify the mechanism underlying the increase in skin temperature. The results showed that EA induced UCP1 expression. Moreover, we measured the expression level of UCP1 mRNA by real-time PCR to determine whether EA could affect UCP1 at the gene level. The results showed that the expression level of UCP1 mRNA in the EA group was significantly increased. Therefore, the infrared thermal imaging and molecular biology results of the present study indicated that EA at the GV20 and EX-HN3 acupoints could activate BAT thermogenesis (Fig. 5).
-
Figure 5. Schematic of the effects of EA on the thermogenesis pathway of brown adipocytes in mice.
Previous studies have demonstrated that EA at GV20 could induce neural activity in specific areas of the brain [41]. Shortly after the EA needle is removed, the neural activity of the brain network formed by 15 functional brain regions, such as the frontal lobe, para-hippocampal gyrus, and hypothalamus, can still be observed [24]. A functional magnetic resonance imaging-based study by Salazar et al. on humans and rats showed that EA could activate hypothalamic neurons and enhance sympathetic activity [42]. From an anatomical perspective, GV20 is located at the top of the brain and in the cap-shaped fascia. The acupoints are surrounded by the left and right superficial temporal arteries and veins, occipital arteries and veins, and branches of the occipital and frontal nerves. GV20 is a significant acupoint for regulating brain function and is a key acupoint on the governor vessel.
Mice are considered the most suitable experimental animals for medical scientific research due to their high similarity to humans in terms of genes, anatomical structures, and even physiological processes, including metabolism and the nervous system [43]. There has been some progress in studying the meridian system in mice, and previous findings have provided a foundation for our research [44,45]. However, it is important to acknowledge that mice cannot fully represent humans, and we must take into account the anatomical and meridian system differences between mice and humans as different species. Therefore, further research and validation are necessary to ensure that our knowledge of the meridian system can be accurately applied to humans.
TCM is an ancient discipline practiced over the course of thousands of years by Chinese people in the struggle against diseases. According to “Inner Canon of Huangdi”, the oldest TCM classic [46], “people are closely connected with the natural world”. Aligning with the natural rhythm by regulating yin and yang is the basic principle of TCM. In TCM theory, the governor vessel is believed to be the sea of yang, regulating the yang qi of a body with 12 meridians, with the head being the point where the yang qi of various meridians converges. GV20 is the point formed after the convergence, which plays an important role in regulating the yin and yang balance (energy metabolism) of the body. Based on the meridian principle [47] and acupoint specificity [48,49], stimulating acupoints can lead to responses in corresponding meridians. In this study, we observed that EA at GV20 and EX-HN3 increased the skin temperature along the governor vessel. However, the results of the present study do not explain the meridian phenomenon, which is also a limitation of this study.
In conclusion, this study visualized the activation of BAT thermogenesis using infrared thermal imaging technology. The findings were obtained by conducting acupoint selection based on TCM theory combined with modern neuroanatomy research. This study is the first to associate the effects of GV20 stimulation on yang qi with the thermogenesis of BAT, an energy-producing tissue. Although the methods and theoretical basis of EA in TCM are different from those of Western medicine, the main therapeutic effects of both of them are similar.
LIMITATIONS AND FUTURE DIRECTIONS
1. Limitations
Our study provides important insights into the effects of EA on BAT thermogenesis; however, several limitations should be considered.
A limitation of our study is the small sample size. We only included 12 mice, dividing them into the EA group and the control group. This limited sample size raises concerns regarding the generalizability of our findings and the statistical power of our analysis. Future studies with larger sample sizes are needed to validate our results and increase the reliability of the findings.
Our research utilized C57BL/6J mice as an animal model, which is a commonly employed model in scientific studies. However, it is crucial to recognize that there may be inherent differences in physiology between mice and humans. Consequently, there could be limitations in the extent to which findings from the meridian system of mice can be extrapolated to humans.
Additionally, although our study observed an increase in skin temperature and UCP1 levels in the EA group, the exact mechanisms underlying these effects remain unclear. The changes in skin temperature measured by infrared thermography may be related to EA-induced metabolic changes; however, the effects of EA on metabolism were not specifically elucidated in our study. Therefore, further experiments are necessary to investigate and clarify the specific molecular pathways and signaling cascades involved in the EA-induced activation of BAT thermogenesis. These future experiments would contribute to a deeper understanding of the underlying mechanisms.
2. Future directions
Based on the limitations identified in our study, there are several areas that warrant further investigation.
Future research should focus on deciphering the precise mechanisms through which EA activates BAT thermogenesis. Advanced techniques such as transcriptomic analysis, proteomics, and metabolomics can offer valuable insights into the specific molecular pathways and signaling components implicated in the effects of EA. Furthermore, examining the effects of EA on metabolic parameters, body composition, and insulin sensitivity is crucial to establish its therapeutic potential for metabolic diseases. By monitoring the changes induced by EA, we can gain a better understanding of how EA influences the body’s metabolism and its potential benefits in treating metabolic disorders.
Additionally, translating the findings from animal studies to clinical practice should be prioritized. Well-designed clinical trials should be conducted to investigate the effects of EA on BAT thermogenesis in humans. Such trials could assess the effects of EA on metabolic disorders, body composition, and other relevant clinical outcomes.
By addressing limitations and exploring future research directions, we can deepen our understanding of the effects of EA on BAT thermogenesis and its potential therapeutic applications in metabolic disorders. These endeavors will contribute to the advancement of evidence-based acupuncture and its integration into clinical practice.
ACKNOWLEDGEMENTS
We would like to thank the equipment providers and Editage for English language editing.
FUNDING
This work was funded by the National Natural Science Foundation of China (Award Number: 81973938 and 82274654).
AUTHORS’ CONTRIBUTIONS
Ting Li, Meng Wu, and Zhigang Li designed and performed experiments. Junjian Tian and Yitong Li analyzed the results and participated in the revision of the manuscript. Ting Li and Meng Wu wrote the paper. All authors approved the final version of the manuscript.
DATA AVAILABILITY
The data that support the findings of this study are available from the corresponding author upon request.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Fig 1.
Fig 2.
Fig 3.
Fig 4.
Fig 5.
-
Table 1 . Target protein primer sequence.
Primer sequence Probe Size of PCR product UCP1 forward primer 5’-AGCTTTGCCTCACTCAGGAT-3’ 100 bp UCP1 reverse primer 5’-CGGCTGAGATCTTGTTTCCG-3’ GAPDH forward primer 5’-GGTGAAGGTCGGTGTGAACG-3’ 233 bp GAPDH reverse primer 5’-CTCGCTCCTGGAAGATGGTG-3’ PCR = polymerase chain reaction; UCP1 = uncoupling protein 1; GAPDH = glyceraldehyde-3-phosphate dehydrogenase..
References
- Oelkrug R, Polymeropoulos ET, Jastroch M. Brown adipose tissue: physiological function and evolutionary significance. J Comp Physiol B 2015;185:587-606.
- Contreras C, Gonzalez F, Fernø J, Diéguez C, Rahmouni K, Nogueiras R, et al. The brain and brown fat. Ann Med 2015;47:150-68. https://doi.org/10.3109/07853890.2014.919727.
- Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev 2004;84:277-359.
- Zhang Z, Yang D, Xiang J, Zhou J, Cao H, Che Q, et al. Non-shivering thermogenesis signalling regulation and potential therapeutic applications of brown adipose tissue. Int J Biol Sci 2021;17:2853-70. https://doi.org/10.7150/ijbs.60354.
- Fedorenko A, Lishko PV, Kirichok Y. Mechanism of fatty-acid-dependent UCP1 uncoupling in brown fat mitochondria. Cell 2012;151:400-13. https://doi.org/10.1016/j.cell.2012.09.010.
- Calderon-Dominguez M, Mir JF, Fucho R, Weber M, Serra D, Herrero L. Fatty acid metabolism and the basis of brown adipose tissue function. Adipocyte 2015;5:98-118.
- Heaton GM, Wagenvoord RJ, Kemp A Jr, Nicholls DG. Brown-adipose-tissue mitochondria: photoaffinity labelling of the regulatory site of energy dissipation. Eur J Biochem 1978;82:515-21. https://doi.org/10.1111/j.1432-1033.1978.tb12045.x.
- Leitner BP, Huang S, Brychta RJ, Duckworth CJ, Baskin AS, McGehee S, et al. Mapping of human brown adipose tissue in lean and obese young men. Proc Natl Acad Sci U S A 2017;114:8649-54. https://doi.org/10.1073/pnas.1705287114.
- Fernández-Verdejo R, Marlatt KL, Ravussin E, Galgani JE. Contribution of brown adipose tissue to human energy metabolism. Mol Aspects Med 2019;68:82-9.
- Gomolin IH, Aung MM, Wolf-Klein G, Auerbach C. Older is colder: temperature range and variation in older people. J Am Geriatr Soc 2005;53:2170-2.
- Bartelt A, Bruns OT, Reimer R, Hohenberg H, Ittrich H, Peldschus K, et al. Brown adipose tissue activity controls triglyceride clearance. Nat Med 2011;17:200-5.
- Guerra C, Navarro P, Valverde AM, Arribas M, Brüning J, Kozak LP, et al. Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance. J Clin Invest 2001;108:1205-13. https://doi.org/10.1172/JCI13103.
- Gunawardana SC, Piston DW. Reversal of type 1 diabetes in mice by brown adipose tissue transplant. Diabetes 2012;61:674-82. https://doi.org/10.2337/db11-0510.
- Wang J, Wu Q, Zhou Y, Yu L, Yu L, Deng Y, et al. The mechanisms underlying olanzapine-induced insulin resistance via the brown adipose tissue and the therapy in rats. Adipocyte 2022;11:84-98. https://doi.org/10.1080/21623945.2022.2026590.
- Cheng L, Wang J, Dai H, Duan Y, An Y, Shi L, et al. Brown and beige adipose tissue: a novel therapeutic strategy for obesity and type 2 diabetes mellitus. Adipocyte 2021;10:48-65.
- Tournissac M, Leclerc M, Valentin-Escalera J, Vandal M, Bosoi CR, Planel E, et al. Metabolic determinants of Alzheimer's disease: a focus on thermoregulation. Ageing Res Rev 2021;72:101462. https://doi.org/10.1016/j.arr.2021.101462.
- Poher AL, Altirriba J, Veyrat-Durebex C, Rohner-Jeanrenaud F. Brown adipose tissue activity as a target for the treatment of obesity/insulin resistance. Front Physiol 2015;6:4.
- van der Lans AA, Hoeks J, Brans B, Vijgen GH, Visser MG, Vosselman MJ, et al. Cold acclimation recruits human brown fat and increases nonshivering thermogenesis. J Clin Invest 2013;123:3395-403. https://doi.org/10.1172/JCI68993.
- Broeders EP, Vijgen GH, Havekes B, Bouvy ND, Mottaghy FM, Kars M, et al. Thyroid hormone activates brown adipose tissue and increases non-shivering thermogenesis--a cohort study in a group of thyroid carcinoma patients. PLoS One 2016;11:e0145049. Erratum in: PLoS One 2018;13:e0209225.
- de Jong JMA, Wouters RTF, Boulet N, Cannon B, Nedergaard J, Petrovic N. The β3-adrenergic receptor is dispensable for browning of adipose tissues. Am J Physiol Endocrinol Metab 2017;312:E508-18. https://doi.org/10.1152/ajpendo.00437.2016.
- Wen CK, Lee TY. Electroacupuncture prevents white adipose tissue inflammation through modulation of hypoxia-inducible factors-1α-dependent pathway in obese mice. BMC Complement Altern Med 2015;15:452.
- Shen W, Wang Y, Lu SF, Hong H, Fu S, He S, et al. Acupuncture promotes white adipose tissue browning by inducing UCP1 expression on DIO mice. BMC Complement Altern Med 2014;14:501. https://doi.org/10.1186/1472-6882-14-501.
- Zhang F, Ma T, Tong X, Liu Y, Cui P, Xu X, et al. Electroacupuncture improves metabolic and ovarian function in a rat model of polycystic ovary syndrome by decreasing white adipose tissue, increasing brown adipose tissue, and modulating the gut microbiota. Acupunct Med 2022;40:347-59.
- Morrison SF. Central neural control of thermoregulation and brown adipose tissue. Auton Neurosci 2016;196:14-24.
- Zhang G, Qu S, Zheng Y, Chen J, Deng G, Yang C, et al. Key regions of the cerebral network are altered after electroacupuncture at the Baihui (GV20) and Yintang acupuncture points in healthy volunteers: an analysis based on resting fcMRI. Acupunct Med 2013;31:383-8.
- Usamentiaga R, Venegas P, Guerediaga J, Vega L, Molleda J, Bulnes FG. Infrared thermography for temperature measurement and non-destructive testing. Sensors (Basel) 2014;14:12305-48. https://doi.org/10.3390/s140712305.
- McCafferty DJ, Gilbert C, Paterson W, Pomeroy PP, Thompson D, Currie JI, et al. Estimating metabolic heat loss in birds and mammals by combining infrared thermography with biophysical modelling. Comp Biochem Physiol A Mol Integr Physiol 2011;158:337-45.
- Franco NH, Gerós A, Oliveira L, Olsson IAS, Aguiar P. ThermoLabAnimal - a high-throughput analysis software for non-invasive thermal assessment of laboratory mice. Physiol Behav 2019;207:113-21. https://doi.org/10.1016/j.physbeh.2019.05.004.
- Fu Y, Ni JX, Marmori F, Zhu Q, Tan C, Zhao JP. Infrared thermal imaging-based research on the intermediate structures of the lung and large intestine exterior-interior relationship in asthma patients. Chin J Integr Med 2016;22:855-60.
- Yang HQ, Xie SS, Hu XL, Chen L, Li H. Appearance of human meridian-like structure and acupoints and its time correlation by infrared thermal imaging. Am J Chin Med 2007;35:231-40. https://doi.org/10.1142/S0192415X07004771.
- de Souza RC, Pansini M, Arruda G, Valente C, Brioschi ML. Laser acupuncture causes thermal changes in small intestine meridian pathway. Lasers Med Sci 2016;31:1645-9.
- Van Hal M, Dydyk AM, Green MS. Acupuncture. In: Aboubakr S, Abu-Ghosh A, Ackley WB, Adolphe TS, Aeby TC, Aeddula NR, et al, editors. StatPearls [Internet]. Treasure Island: StatPearls Publishing, 2023. Available from: https://pubmed.ncbi.nlm.nih.gov/30335320/.
- Zhu J, Li J, Yang L, Liu S. Acupuncture, from the ancient to the current. Anat Rec (Hoboken) 2021;304:2365-71.
- Jun Z, Xia LI, Hui Z, Kun YE, Xin W, Xuefei W, et al. Effects of acupuncture on functional gastrointestinal disorders: special effects, coeffects, synergistic effects in terms of single or compatible acupoints. J Tradit Chin Med 2023;43:397-408.
- Brakenhielm E, Cao Y. Angiogenesis in adipose tissue. Methods Mol Biol 2008;456:65-81.
- Shamsi F, Wang CH, Tseng YH. The evolving view of thermogenic adipocytes - ontogeny, niche and function. Nat Rev Endocrinol 2021;17:726-44.
- Sun K, Kusminski CM, Luby-Phelps K, Spurgin SB, An YA, Wang QA, et al. Brown adipose tissue derived VEGF-A modulates cold tolerance and energy expenditure. Mol Metab 2014;3:474-83. https://doi.org/10.1016/j.molmet.2014.03.010.
- Ratko M, Habek N, Kordić M, Dugandžić A. The use of infrared technology as a novel approach for studies with female laboratory animals. Croat Med J 2020;61:346-53.
- Habek N, Kordić M, Jurenec F, Dugandžić A. Infrared thermography, a new method for detection of brown adipose tissue activity after a meal in humans. Infrared Phys Technol 2018;89:271-6. https://doi.org/10.1016/j.infrared.2018.01.020.
- Ang QY, Goh HJ, Cao Y, Li Y, Chan SP, Swain JL, et al. A new method of infrared thermography for quantification of brown adipose tissue activation in healthy adults (TACTICAL): a randomized trial. J Physiol Sci 2017;67:395-406.
- Deng D, Duan G, Liao H, Liu Y, Wang G, Liu H, et al. Changes in regional brain homogeneity induced by electro-acupuncture stimulation at the Baihui acupoint in healthy subjects: a functional magnetic resonance imaging study. J Altern Complement Med 2016;22:794-9. https://doi.org/10.1089/acm.2015.0286.
- Salazar TE, Richardson MR, Beli E, Ripsch MS, George J, Kim Y, et al. Electroacupuncture promotes central nervous system-dependent release of mesenchymal stem cells. Stem Cells 2017;35:1303-15. https://doi.org/10.1002/stem.2613.
- Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, et al. Initial sequencing and comparative analysis of the mouse genome. Nature 2002;420:520-62.
- Vieira JS, Toreti JA, de Carvalho RC, de Araújo JE, Silva ML, Silva JRT. Analgesic effects elicited by neuroactive mediators injected into the ST 36 acupuncture point on inflammatory and neuropathic pain in mice. J Acupunct Meridian Stud 2018;11:280-9. https://doi.org/10.1016/j.jams.2018.05.006.
- Chang S. The meridian system and mechanism of acupuncture-a comparative review. Part 1: the meridian system. Taiwan J Obstet Gynecol 2012;51:506-14.
- Zou Q, Wang Y, Shu Z, Yang K, Wang J, Lu K, et al. Topological analysis of the language networks of ancient traditional Chinese medicine books. Evid Based Complement Alternat Med 2020;2020:8810016. https://doi.org/10.1155/2020/8810016.
- Zhou W, Benharash P. Effects and mechanisms of acupuncture based on the principle of meridians. J Acupunct Meridian Stud 2014;7:190-3. https://doi.org/10.1016/j.jams.2014.02.007.
- Qiu K, Yin T, Hong X, Sun R, He Z, Liu X, et al. Does the acupoint specificity exist? Evidence from functional neuroimaging studies. Curr Med Imaging 2020;16:629-38.
- Lee DY, Jiu YR, Hsieh CL. Metabolism modulation in rat tissues in response to point specificity of electroacupuncture. Sci Rep 2022;12:210.