Moxibustion-Simulating Bipolar Radiofrequency Suppresses...

Research ArticleMoxibustion-Simulating Bipolar RadiofrequencySuppresses Weight Gain and Induces Adipose TissueBrowning via Activation of UCP1 and FGF21 ina Mouse Model of Diet-Induced Obesity

Young Jun Koh Ju-Hee Lee and Sung Yun Park

College of Korean Medicine Dongguk University 32 Dongguk-ro Ilsandong-gu Goyang-si Gyeonggi-do 10326 Republic of Korea

Correspondence should be addressed to Sung Yun Park bmeparkdonggukackr

Received 4 June 2018 Accepted 29 August 2018 Published 10 September 2018

Academic Editor Gihyun Lee

Copyright copy 2018 Young Jun Koh et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Obesity is a pathological condition associatedwith various diseases including diabetes stroke arthritis infertility andheart disease Moxibustion is widely used to prevent and manage obesity in traditional Asian medicine We tested our hypothesisthat moxibustion-simulating bipolar radiofrequency (M-RF) can suppress total body and white adipose tissue (WAT) weight gainvia induction of WAT browning in a mouse model of diet-induced obesity (DIO) Methods We designed an M-RF device thatcould accurately adjust the depth and temperature at which heat stimulation was administered into the abdomen of DIO miceHigh-fat-fed male C57BL6 mice were treated with the M-RF device every two or three days for three weeks We then harvestedWAT and serum from the mice and measured total body and WAT weight size of adipocytes mitochondrial contents featuresof the dead adipocyte environment and levels of uncoupling protein 1 (UCP1) and fibroblast growth factor 21 (FGF21) ResultsHeat stimulation by M-RF in DIO mice resulted in precise temperature adjustment in the mice abdomen with variance less than1∘C Additionally M-RF stimulation inhibited body and WAT weight gain resulting in increased formation of beige adipocytesincreased mitochondrial content and decreased formation of dead adipocytes in WAT Moreover treatment of M-RF inducedexpression of UCP1 and FGF21 in serum andor epididymal WATs in DIO mice Conclusion Heat stimulation by M-RF treatmentinduced upregulation of UCP1 and FGF21 expression in serum andor WATs which was correlated with reduced total body andWAT weight gain in DIO mice

1 Introduction

Moxibustion based on natural materials such as herbs iswidely used in traditional Asian medicine clinics to pre-vent and manage various diseases such as polycystic ovarysyndrome ulcerative colitis heart disease irritable bowelsyndrome diabetic limbs and obesity [1ndash6] Previous animalstudies showed that preventive moxibustion can also be usedto adjust the levels of fat accumulation blood lipids andfemale sex hormones in rats and mice [6 7] Some researcherhas proved that moxibustion leads to significant decreaseof 5-hydroxytryptamine and adrenocorticotrophic hormonewhich play a role in decreasing blood flow into fat tissuein mouse [8] However moxibustion is limited in that itdirectly stimulates the outer layer of the skin with relatively

high temperatures that may be difficult to adjust and itgenerates smoke during use [9ndash11] Thus development of aneffective easy-to-use and safe moxibustion system is need-ed

Application of radiofrequency (RF)-induced thermaleffects based on electrical systems is a minimally invasivetreatment method used in various medical fields Radiofre-quencies are commonly used to treat tumors in the liverlung pancreas and kidney as well as to induce fat reductionand cellulite improvement [12ndash16] Because RF is used toincrease the temperature of a target point from 42∘C to 46∘Cfor hyperthermia therapy [17 18] or for ablation therapy (inwhich temperature changes ranging from 50∘C to 102∘C areapplied) skin can be simulated using RF devices to increasethe temperature [10 19]

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 4737515 12 pageshttpsdoiorg10115520184737515

2 Evidence-Based Complementary and Alternative Medicine

Obesity accompanied by WAT growth poses seriousconcerns because it is closely related to the onset of severalmetabolic diseases including type 2 diabetes fatty liverdisease and cardiovascular diseases [20ndash22] Recent studieshave spurred interest in the antiobesity effects of WATbrowning which induces the characteristics of brown adi-pose tissue (BAT) in WATs outside of typical BAT locations[23] Browning of adipocytes in WAT which has geneticdifferences because of its distinct developmental origin fromBAT [24] can be induced by various stimulations such assmall molecules and environmental cues [25] WAT brown-ing occurs via several transcriptional factors coregulatorslipid droplet-associated proteins microRNAs and growthfactors aswell asmitochondrial uncoupling proteins [25ndash27]

Obesity is associated with chronic inflammation whichleads to various diseases such as diabetes hypertensionand cardiovascular diseases [28] Chronic inflammation inobesity is mainly caused by recruitment of inflammatorymacrophages into the WATs as well as by conversion of resi-dent macrophages in the WATs [29 30] In the adipose tissueof obese individuals macrophages with phagocytic activitiessurround and remove dead adipocytes in the WATs [29]Therefore it is important to characterize the adipose tissuein terms of macrophage infiltration and conversion status

Obese patients who try losing weight via lifestyle inter-ventions such as adjusting food intake and increasing theamount of physical exercise often fail to see significantimprovements in their condition moreover pharmacothera-peutics aimed at managing obesity are often accompanied byside effects such as metabolic and psychologic diseases [31]Thus a novel mode of intervention that safely manages obe-sity and its related conditions is needed In light of this unmetneed several trials have used not only medications butalso noninvasive medical devices to treat obesity [32] Herewe describe the effects of moxibustion-simulating bipolarradiofrequency (M-RF) treatment on body weight reductionand adipocyte browning induction in a mouse model of diet-induced obesity (DIO)

2 Materials and Methods

21 Equipment To improve upon the method of classicalmoxibustion we designed a bipolar RF device that is able toprecisely control the depth and temperature at which stimula-tion is given thereby allowing quantitative intervention Themain equipment consisted of two primary devices an energygenerator and a temperature measuring device (Figures 1(a)and 1(b)) A bipolar RF device mainly consisted of a powercontrol board display panel and six bipolar probes Thepower control board could generate a maximum power of446 W with 50 Ω 676 W with 200 Ω and 508 W with500 Ω which was delivered to the subject through bipolarprobesThe bipolar probes were circular (diameters of 5 mm18 mm and 3 mm for the inner circle outer circle andwidth respectively) and coated with Ag as a skin protectant(Figure 1(c)) The process was monitored and controlled by adisplay panel consisting of a 10110158401015840 thin film transistor-liquidcrystal display that had high resolution (1280lowast800) whichis standard for 4-wire touch screens K-type temperature

detecting needles were used to measure the temperaturesat the surface of the abdomen and approximately 1 mmbelow the surface The temperature data were sampled at20 Hz and transmitted to the LabVIEW system (NationalInstruments Austin TX USA) The heating performance ofthe M-RF Stimulator was measured with skin samples ofYucatan pig and mice as shown in Figures 1(d) and 1(e) Eachexperimental procedure was conducted over a course of threeminutes The subjects were contained in plastic cube boxes(350 mm times 450 mm times 350 mm) maintained at 23∘C ambienttemperature and 50ndash60 humidity

22 Mouse and Treatment All animal experiments wereapproved by the Institutional Animal Care and Use Com-mittee of Dongguk University (approval No IACUC-2017-017-1) Eight-week-old male C57BL6 mice were purchasedfrom Central Lab Animal Inc (Seoul Korea) The micewere provided with ad libitum access to water and high-fatdiet (HFD) The HFD (60 Kcal fat Research Diets NewBrunswick NJ) was provided for four weeks to induce diet-induced obesity (DIO) Afterwards the mice were randomlydistributed into the following four treatment groups (1)no heat application (control n = 10) (2) low-temperaturestimulation (RF-L 367∘C n = 10) (3) middle-temperaturestimulation (RF-M 379∘C n = 10) and (4) high-temperaturestimulation (RF-H 388∘C n = 10) We applied M-RF ontothe abdominal skin of DIO mice at the three differenttemperatures for one minute per day every two days overthree weeks while maintaining the HFD At the end ofthe three-week application of M-RF mice were anesthetizedby intramuscular injection of a combination of anesthetics(mixture of tiletamine zolazepam and xylazine each 10mgkg) weighed and sacrificed Blood was collected bycardiac puncture and centrifuged at 848 g for 15 minutes at4∘C after which the resulting serumwas harvested TheWATspecimens were immediately weighed and fixed in neutralbuffered 4 formaldehyde for histochemical studies Theserum and WAT were stored at -80∘C until further use

23 Anesthesia and Euthanasia Mice were anesthetized andeuthanized by intramuscular injection of the combination ofanesthetics (tiletamine-zolazepam 50mgkg and xylazine 12mgkg) before RF treatment and histologic analysis

24 Histologic and Morphometric Analysis Mice were anes-thetized by intramuscular injection of anesthetics and WATswere fixed by cardiac perfusion of 1 paraformaldehyde inPBS and whole-mounted To visualize the adipocytes andmitochondrial content in WATs the tissues were incubatedfor four hours at room temperature with the following chem-icals 44-difluoro-4-bora-3a4a-diaza-s-indacene (BODIPY)for adipocytes (1 120583gml in PBS Invitrogen Carlsbad CAUSA) and MitoTracker Red CMXRos (MitoTracker) formitochondrial contents (100 nM in PBS Invitrogen) Toverify the dead adipocytes immunohistochemistry was per-formed as previously described [29 33] Briefly the WATswere incubated for an hour at room temperature with block-ing solution containing 5 whole donkey serum (JacksonImmunoResearch Laboratories IncWest Grove PAUSA) in

Evidence-Based Complementary and Alternative Medicine 3

(a)

(b)

(c)

(d) (e)

RF Stimulator

Mouse(abdominal side)

LabVIEW

40

38

36

34

0

32

30

28

26

ControlRF-L

RF-MRF-H

Tem

pera

ture

(∘C)

RF-LRF-MRF-H

1 2 30Minutes after application

0

20

25

30

35

40

Tem

pera

ture

(∘C)

1 2 3 4 50Minutes after application

Figure 1 Development of the M-RF Stimulator (a) Block diagram of the M-RF Stimulator and temperature measurement system (b)Photo for front side of the M-RF Stimulator (c) Bipolar electrode plate (5 mm 18 mm and 3 mm for diameters of the inner circle outercircle and width respectively) in the M-RF Stimulator for use in mice (d) Temperature change (maximum temperatures of 399∘C 384∘C372∘C and 372∘C for RF-H RF-M RF-L and moxibustion (Moxibustion) respectively) in Yucatan pigs (e) Temperature change (maximumtemperatures of 388∘C 379∘C and 367∘C for moxibustion RF-H RF-M and RF-L respectively) in mice

PBS-T (03 Triton X-100 in PBS) After blocking the tissueswere incubated overnight at room temperature while shakingwith rat anti-mouse F480 antibody (clone ClA3-1 diluted11000 Serotec) in blocking solution to visualize infiltrationof phagocytic macrophages inWATs which is a characteristicof apoptotic adipocytes [29] After five washes in PBS-Twhole-mounted tissues were incubated for four hours atroom temperature with Cy3-conjugated anti-rat antibody(diluted 1500 Jackson ImmunoResearch Laboratories Inc)in blocking solution For negative control experimentsprimary or secondary antibodies were omitted during theimmunohistochemistry process All images were capturedusing a Nikon Eclipse Ts2 inverted fluorescent microscope(Nikon Japan) equipped with high-definition color camera(DS-Qi2 Nikon) and then analyzed with the NIS Elements

Imaging Software (version 430 Nikon) To calculate theadipocyte size mitochondrial content and UCP1 expressionstained images for BODIPY MitoTracker and UCP1 werecaptured and measured To determine the adipocyte size inWATs the diameters of adipocytes were measured in fiverandomly selected regions (sim100 adipocytes per each region)perWATThemitochondrial content and expression of UCP1weremeasured by analyzing the densities of theMitoTracker-or UCP1-positive areas based on the pixels in the regions ofinterest During analysis only pixels with an intensity ofmorethan 50were chosen to exclude background fluorescenceThenumber of beige adipocytes in five randomly selected regions(sim100 adipocytes per each region) per WAT was countedDead adipocytes were detected using double-stained colorimages for BODIPY and F480 We counted the number of


BODIPYminusclustered macrophage+ clustered regions for deadadipocytes in 10 randomly selected regions (sim100 adipocytesper each region) per WAT in a blind manner

25Western Blotting Total protein fromWATswas extractedusing a tissue homogenizer and lysis buffer (50 mM Tris-Cl150 mM NaCl 1 4-nonylphenyl-polyethylene glycol 5 mMethylenediaminetetraacetic acid 1 M threo-14-Dimercapto-23-butanediol) containing protease inhibitor cocktail (Gen-DEPOT Barker TX USA) The homogenates were cen-trifuged for 15min at 15928 g and 4∘CThe supernatants werecollected and centrifuged again and the second supernatantswere used for Western blotting analysis To detect mouseFGF21 protein protein-loaded membranes were first reactedwith anti-FGF21 antibody (diluted 11000 Abcam Cam-bridge MA USA) and then incubated with HRP-conjugatedsecondary antibody (diluted 15000Thermo Scientific Rock-ford IL USA) Finally the FGF21 protein was detected withHRP substrate (Amersham Bioscience BuckinghamshireUK) using a Fusion FX7 chemiluminescence imaging system(Vilber Lourmat France)

26 ELISA for Serum FGF21 To quantify FGF21 in mouseserum an enzyme-linked immunosorbent assay (ELISA) wasperformed using the mouse FGF21 ELISA kit (RampD SystemsMinneapolis MN USA) according to the manufacturerrsquosinstructions Briefly mice were anesthetized by intramus-cular injection of a combination of anesthetics and 1 mlof whole blood was collected via cardiac puncture with 28-gauge syringe The blood samples were then centrifuged for15 minutes at 848 g and 4∘C after which the resulting serumsamples were stored at -80∘C until analysis

27 Statistics Values are presented as the means plusmn standarddeviation (SD) Statistical analyses consisted of one-wayanalysis of variance (ANOVA) followed by Tukeyrsquos multiplecomparison or a Studentrsquos t-test P values lt 005 were consid-ered statistically significant All experiments were performedindependently at least three times and the data were analyzedusing the Prism 50 software (GraphPad Software Inc SanDiego CA USA)

3 Results

31 Thermal Stimulation Using an M-RF Stimulator To eval-uate the performance of the M-RF Stimulator we measuredthe temperature of Yucatan pigs and mice The temperaturecurves of the M-RF Stimulator (399 plusmn 012∘C 384 plusmn 012∘Cand 372 plusmn 013∘C (mean plusmn SD) for maximum temperatures ofRF-HRF-M and RF-L respectively) showed patterns similarto those of moxibustion (372 plusmn 021∘C maximum tempera-ture) at 2 mm from the skin of Yucatan pigs (Figure 1(d)) andat 1 mm from the skin of mice (388∘C 379∘C and 367∘Cfor RF-H RF-M and RF-L respectively) (Figure 1(e)) Theaverage times required for the temperature to increase fromapproximately 30∘C to the maximum temperature for eachtemperature curve in the Yucatan pig experiment were 174sec 168 sec 189 sec and 133 sec for RF-H RF-M RF-L andmoxibustion respectively (Figure 1(d))

32 Thermal Stimulation by M-RF Suppresses Body and WATWeight Gain in DIOMice To determine the effects of M-RF-induced heat stimulation on body weight gain in DIO mice(control) we applied M-RF on DIO mice at three levels oftemperature low (367∘C F-L) middle (379∘C RF-M) andhigh (388∘C RF-H) for three weeks (three min per day everytwo days) At three weeks after the start of M-RF treatmenttotal body weight measurements of the control group thatreceived noheat stimulationhad significantly increased (3913plusmn 3736 g n = 10) whereas those of RF-L (3322 plusmn 2371 gn = 10 P lt 001 versus control) RF-M ( 3292 plusmn 08585 g n= 10 P lt 001 versus control) and RF-H (3404 plusmn 3505 g n= 10 P lt 001 versus control) had not increased significantly(Figure 2(a)) Moreover in contrast with the control (at endof the experiment 4367 plusmn 1268 g n = 10) all three levelsof M-RF stimulation groups showed changes in body weightrelative to the day of first application (RF-L -046 plusmn 0826 gn = 10 P lt 001 versus control RF-M -108 plusmn 0858 g n =10 P lt 001 versus control RF-H -042 plusmn 185 g n = 10 Plt 001 versus control) (Figure 2(b)) Consistently weights ofepididymal andmesenteric WATs inM-RF-treated DIOmicedid not significantly increase in all three levels (RF-L 0895 plusmn00284 g n = 10 P lt 001 eWAT versus control and 0538 plusmn01039 g n = 10 P lt 001 mWAT versus control RF-M 08866plusmn 00286 g n =10 P lt 001 eWAT versus control and 0565 plusmn01373 g n = 10 P lt 001 mWAT versus control RF-H 1359plusmn 00427 g n = 10 P lt 001 eWAT versus control and 0641 plusmn01664 g n = 10 P lt 001 mWAT versus control) comparedto the control (2630 plusmn 00610 gram n = 10 for eWAT and1194 plusmn 01673 g n = 10 for mWAT) (Figures 2(c) and 2(e))Moreover ratios of WAT-to-body weight were lower in RF-L (269 plusmn 0085 n = 10 P lt 001 eWAT versus control and162 plusmn 0313 n = 10 P lt 001 mWAT versus control) RF-M(269 plusmn 0087 n = 10 P lt 001 eWAT versus control and 172plusmn 0417 n = 10 P lt 001 mWAT versus control) and RF-Hgroups (399 plusmn 0125 n = 10 P lt 001 eWAT versus controland 188 plusmn 0489 n = 10 P lt 00101 mWAT versus control)than in the control (672 plusmn 0156 n = 10 for eWAT and 305plusmn 0428 n = 10 for mWAT) (Figures 2(d) and 2(f))

33 Thermal Stimulation by M-RF Decreases the Size ofAdipocytes and Increases Mitochondrial Contents in the WATsof DIO Mice To describe the cellular changes in WATsinduced by M-RF stimulation we harvested the epididymaland mesenteric WATs from the M-RF-treated DIO mice andobserved the size andmitochondrial contents of adipocytes inboth WATs using whole-mounted immunostaining (Figures3(a) and 3(d)) In the control group the average diameter ofthe adipocytes was 1243 plusmn 2214 120583m in epididymal (n = 5000cells) and 9721 plusmn 1789 120583m in mesenteric WATs (n = 5000cells) (Figures 3(b) and 3(e)) In M-RF-treated groups thesizes of adipocytes in both epididymal and mesenteric WATswere significantly smaller (7319 plusmn 1356 120583m n = 5000 cells Plt 001 eWAT versus control and 5860 plusmn 9446 120583m n = 5000cells P lt 001 mWAT versus control) RF-M (7503 plusmn 1525120583m n = 5000 cells P lt 001 eWAT versus control and 5057plusmn 622 120583m n = 5000 cells P lt 001 mWAT versus control)and RF-H (8461 plusmn 1848 120583m n = 5000 cells P lt 001 eWATversus control and 6108 plusmn 1578 120583m n = 5000 cells P lt 001


Body

wei

ght (

g)45

40

35

30

0

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

2 3 4 5 6 71

Number of applications

RF-MRF-H

ControlRF-L

(a)

6

4

2

0

minus2

minus4

Body

wei

ght c

hang

e (g)

relat

ive t

o ba

selin

e

lowastlowastlowastlowastlowastlowast lowastlowast

lowastlowastlowastlowast



lowastlowastlowastlowast lowastlowast


2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(b)

Control RF-L RF-M RF-H0

1

2

3

Epid

idym

al W

AT w

eigh

t (g)

lowastlowast


(c)Control RF-L RF-M RF-H

0

2

4

6

8

Ratio

eWAT

to B

W (

)

lowastlowast


(d)


05

10

15

Mes

ente

ric W

AT w

eigh

t (g)


lowastlowast

(e)Control RF-L RF-M RF-H

0

1

2

3

4

Ratio

mW

AT to

BW

()

lowastlowast


(f)Figure 2 M-RF stimulation suppresses total body andWATweight gain in DIO mice (a) Body weights (g) of control (n = 10) RF-L (n = 10)RF-M (n = 10) or RF-H (n = 10) groups measured before M-RF stimulation (b) Body weight changes (g) relative to the base level (the dayof first application) in each group (n = 10) (c e) Weights (g) of WATs in each group (n = 10) (d f) Ratio of eWAT (epididymal WAT) weight(d) and mWAT (mesentericWAT) (f) to body weight at the end-points of the experiment in each group (n = 10) Results are presented as themeans plusmn SD lowast P lt 005 versus control lowastlowast P lt 001 versus control

mWAT versus control) (Figures 3(b) and 3(e)) Furthermorecontents ofmitochondria in the adipocytes of epididymal andmesenteric WATs were increased by all three levels of M-RFstimulation (RF-L 1959 plusmn 01447 AU n = 5000 cells P lt

001 eWAT versus control and 2275 plusmn 01275 AU n = 5000cells P lt 001 mWAT versus control RF-M 2077 plusmn 00999AU n = 5000 cells P lt 001 eWAT versus control and 1983 plusmn02004 AU n = 5000 cells P lt 001 mWAT versus control


BODIPY MitoTracker Merged


Control

RF-L

RF-M

RF-H

Control

RF-L

RF-M

RF-H

Epid

idym

al ad

ipos

e tiss

ueM

esen

teric

adip

ose t

issue

(d)

(a)

(b)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40

0


Control RF-L RF-M RF-H

(c)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05

0Control RF-L RF-M RF-H


(e)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40


lowastlowast


(f)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05

0

25


lowastlowast lowastlowast lowastlowast

Figure 3 M-RF stimulation reduces the size of adipocytes and increases mitochondrial contents in the WATs of DIO mice (a d) Whole-mounted WATs stained with BODIPY (green) and MitoTracker (red) Note that all three levels of M-RF stimulation resulted in decreasesin the size of adipocytes and increases in the mitochondrial contents compared to the control Scale bar 200 120583m (b c e and f) Diameterof adipocytes and density of MitoTracker-positive area were measured in five randomly selected regions (sim100 adipocytes per region) perWAT in control (n = 10) RF-L (n = 10) RF-M (n = 10) and RF-H (n = 10) and presented as micrometers for diameter of adipocytes and asan arbitrary unit (AU) for the ratio of the pixel densities compared to the control for intensity of MitoTracker Results are presented as themeans plusmn SD lowastlowast P lt 001 versus control

RF-H 1983 plusmn 02004 AU n = 5000 cells P lt 001eWAT versus control and 2141 plusmn 02071 AU n = 5000cells P lt 001 mWAT versus control) (Figures 3(c) and3(f))

34Thermal Stimulation byM-RF Induces Expression of UCP1and Formation of Beige Adipocytes in the WATs of DIOMice To identify the underlying mechanism of increasedmitochondrial contents induced by M-RF treatment in DIO


Control

Imm

unoh

istoc

hem

istry

for U

CP1

RF-L

RF-M RF-H

(a) (b)30

20

10

0

UCP

1-po

sitiv

e are

a rat

io (A

U)



(c)

Num

ber o

f bei

ge ad

ipoc

ytes

(n)

20

15

5

0

10


lowastlowast

lowastlowast

lowastlowast

(d)

Figure 4 M-RF stimulation increases UCP1 expression and formation of beige adipocytes in the WATs of DIO mice (a) Whole-mountedWATs immunostained for UCP1 (red) Scale bar 100 120583m (b) Higher magnification image of RF-L in (a) showing characteristic beigeadipocytes Scale bar 50 120583m Note that all three levels of M-RF stimulation increased UCP1 expression and formation of beige adipocytescompared to control Scale bar 50 120583m (c d) Ratio of UCP1-positive areas compared to control (c) and numbers of beige adipocytes (d)calculated in five randomly selected regions (sim100 adipocytes per each region) per WAT in control (n = 10) RF-L (n = 10) RF-M (n = 10)and RF-H (n = 10) Results are presented as means plusmn SD lowastlowast P lt 001 versus control

mice we analyzed UCP-1 expression in epididymal WATsbecause activation of UCP-1 is a component of mitochondrialactivation [34] that is responsible for browning of WATs [25]Similar to the changes in mitochondrial contents UCP-1 washighly expressed in the epididymal WATs following RF-L(2438 plusmn 01440 AU n = 5000 cells P lt 001 versus control)RF-M (2558 plusmn 00646 AU n = 5000 cells P lt 001 versuscontrol) and RF-H (2189 plusmn 03965 AU n = 5000 cells Plt 001 versus control) (Figures 4(a) and 4(c)) Consistentwith previous reports [33] while beige adipocytes were rarelyobserved in the WATs of control DIO mice (0200 plusmn 0421n = 5000 cells) large numbers of beige adipocytes withsmall droplets were observed in the eWATs of DIO mice thatreceived RF-L (134 plusmn 284 n = 5000 cells P lt 001 versus

control) RF-M (141 plusmn 448 n = 5000 cells P lt 001 versuscontrol) and RF-H (127plusmn 416 n= 5000 cells Plt 001 versuscontrol) stimulation (Figures 4(b) and 4(d))

35 Thermal Stimulation by M-RF Inhibits Infiltration ofMacrophages into Dead Adipocytes in the WATs of DIOMice To investigate the presence of dead adipocytes andmacrophage infiltration into WATs whole-mounted WATswere costained with BODIPY (neutral lipid binding chemi-cal) and F480 (phagocytic macrophage marker) In controlmice we observed dead adipocytes with macrophage clumps(BODIPYminusF480+ cells) in their epididymal WATs (19 plusmn088 n = 5000 cells P lt 001) but these were not foundin their mesenteric WATs (data not shown) (Figure 5(a))


BODIPY F480 Merged

Control

RF-L

RF-M

RF-H

(a)

Num

ber o

f dea

d ad

ipoc

ytes

(n)

3

2

0

1lowastlowast

lowastlowast

lowastlowast

Control RF-L RF-M RF-H(b)

Figure 5 M-RF stimulation suppresses dead adipocyte formation in theWATs of DIO mice (a) Whole-mountedWATs double-stained withBODIPY (green) and F480 (red) Note that all three levels of M-RF stimulation resulted in decreased dead adipocyte formation comparedto control Scale bar 100 120583m (b) Number of BODIPYminusclustered F480+ dead adipocytes counted in 10 randomly selected regions (sim100adipocytes per region) per WAT in control (n = 10) RF-L (n = 10) RF-M (n = 10) and RF-H (n = 10) Results are presented as the means plusmnSD lowastlowast P lt 001 versus control


Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)

Figure 6 M-RF stimulation increases the level of FGF21 in serum and WAT of DIO mice (a) ELISA for serum FGF21 in control (n = 5)RF-L (n = 5) RF-M (n = 5) and RF-H (n = 5) (b) RepresentativeWestern blotting for FGF21 inWATs of DIOmice in control (n = 5) RF-L (n= 5) RF-M (n = 5) and RF-H (n = 5) (c) Quantification of (b) and calculated in terms of ratio relative to control Note that all three levels ofM-RF stimulation significantly increased the level of FGF21 in serum and WAT compared to the control Results are presented as the meanplusmn SD lowast P lt 005 versus control lowastlowast P lt 001 versus control

In contrast we rarely detected BODIPYminusF480+ deadadipocytes in the epididymal WATs of RF-L (020 plusmn 042 n= 5000 cells P lt 001 versus control) RF-M (010 plusmn 032 n =5000 cells P lt 001 versus control) and RF-H (030 plusmn 048 n= 5000 cells P lt 001 versus control) groups (Figure 5(b))

36 Thermal Stimulation by M-RF Increases the Expressionof FGF21 in Both Serum and WATs of DIO Mice FGF21 hasfavorable effects in several metabolic diseases including type2 diabetes dyslipidemia and obesity We analyzed the level ofFGF21 from serumand epididymalWATs fromDIOmice andfound that M-RF stimulation increased serum FGF21 levelsin RF-L (3215 plusmn 2839 n = 5 P lt 005 versus control) RF-M(1590plusmn 1743 n = 5 P lt 001 versus control) and RF-H (6681plusmn 1125 n = 5 P lt 001 versus control) groups whereas thecontrol group showed a significantly lower amount of serumFGF21 (1314 plusmn 1092 n = 5) (Figure 6(a)) Similarly FGF21protein expression was increased in the epididymal WATs inresponse to all three levels of M-RF stimulation (RF-L 1245plusmn 2730AUn= 5 Plt 005 versus control RF-M 4878plusmn 1454

AU n = 5 P lt 005 versus control RF-H 3794 plusmn 1115 AU n= 5 P lt 005 versus control) (Figures 6(b) and 6(c))

4 Discussion

Wemanufactured amoxibustion-simulating heat stimulationdevice using bipolar RF and showed that our bipolar RF waseffective for precise adjustment of depth and temperature ofthermal stimulation targeting abdominal WAT which pro-vides an advantage over monopolar RF devices Our resultsdemonstrated that thermal stimulation by M-RF treatmentinduced profound reductions in total body and WATweightwhich were accompanied by adipose tissue browning andelevation of UCP1 and FGF21 expression in DIO mice

Tissue heating by RF stimulation occurs in response tothe induction of ion currents into target tissue by applyingan RF wave field between an electrode plate and a groundplate While the two plates of monopolar RF are located onopposing sides the two plates of bipolar RF are positionedclosely on the same surface Studies have shown that by


changing the power of an RF generator makes it easy toprecisely increase the temperature by depth in bipolar RFcompared with monopolar RF [35 36] Because of theseadvantages bipolar RF is used in various medical fields fortumor treatment and fat reduction [12 13 37]

Our findings show that heat stimulation by RF treatmentleads to beige adipocyte induction in DIO mice which isinteresting because beige adipocyte induction in WATs is akey feature of cold exposure [25] Beige adipocyte inductioncan be promoted by several external stimuli including 120573

3-

adrenergic receptor agonist A2A agonist thiazolidinedione

miRNA 155 and cold exposure [25 38ndash40] which exerttheir effects by activating peroxisome proliferator-activatedreceptor gamma peroxisome proliferator-activated receptorgamma coactivator-1 alpha (PGC-1120572) 120573

3-adrenergic recep-

tor and adenosine receptor in WAT and other tissues [2538ndash40] Specifically cold exposure is a strong inducer ofbeige adipocyte induction that functions by increasing mito-chondrial biogenesis [25] however the detailed mechanismresponsible for beige adipocyte induction by heat stimulationhas yet to be clearly identified A previous study indicatedthat heat stimulation by RF may result in fat reduction andcellulite improvement and that heat stimulation may alsocause minor inflammation and promote collagen formation[15] Another report suggested a unique effect of bipolar RF inenhancing fat metabolism which may contribute to treatingcellulite Because bipolar RF can penetrate to depths of morethan 3 mm in depth heat stimulation by bipolar RF can alterthe surrounding adipose tissue by inducing thermal damage[41] Thus it is possible that heat stimulation by the M-RFdevice that we designed is strong enough to cause browningof WAT and lead to reduced total body weight

The present findings also show that treatment with RFinhibits the infiltration of macrophages into dead adipocytesin WATs which is a characteristic inflammatory pheno-type in obesity Obesity can induce chronic inflammationthereby leading to onset of various diseases such as diabeteshypertension and cardiovascular diseases [28] In obeseindividuals phagocytic macrophages can be recruited intoWATs from the bone marrow or resident macrophages inWATs may be converted into phagocytic macrophages [2930] Such changes in the macrophage environment lead toproduction of inflammatory cytokines such as interleukin-1120573 monocyte chemoattractant protein-1 and tumor necrosisfactor-120572 [28] Therefore it is possible that the decrease inthe number of dead adipocytes and inhibition of macrophageinfiltration in WATs are responsible for the weight reductioncaused by RF stimulation

Our results showed that heat stimulation by RF treatmentinduces profound mitochondrial biogenesis and expressionof UCP1 in WATs of DIO mice Browning of WATs isaccompanied by generation of mitochondria and expres-sion of related genes in the WATs [42] During browningthrough mitochondrial biogenesis in WATs activation ofPGC-1120572 peroxisome proliferator-activated receptor gammaPR domain containing 16 and other coactivators induce theexpression of mitochondria-related genes including UCP1[23] UCP1 is mitochondrial carrier protein expressed brownadipocytes that are responsible for generating heat through

proton transport via ATP synthesis [43 44] ParticularlyUCP1 plays crucial roles in the development of BAT and theproduction of beige adipocytes from white adipocytes evenoutside typical BAT locations [23] Expression of adipocyte-specific UCP1 leads to prevention of obesity by modulatingmitochondrial membrane potential [45] Conversely dele-tion of UCP1 induces obesity by abolishing diet-inducedthermogenesis in mice kept at thermoneutral temperatureregardless of the type of diet (normal chow high-fat)[46] Although the mechanism underlying RF stimulation-induced UCP1 expression and adipocyte browning in WATremains unclear our results demonstrated that browning canoccur in the WATs of DIO mice upon RF stimulation

A clinical study of twelve healthy males using a tem-perature-controlled water bath showed that levels of serumFGF21 and free fatty acids increased after immersing theirlower legs in hot water at 39∘C 42∘C or 43∘C [47] Moreoverheat treatment and activation of heat shock proteins havebeen shown to improve insulin sensitivity of skeletal muscleas well as to reduce plasma triglyceride and free fatty acidsof genetically obese mice in conjunction with a decreasein WAT mass [48] Traditionally only cold exposure wasassociated with formation of beige adipocytes [24 25 42]however previous reports have shown that heat stimulationcan also induce beige adipocyte formation [15 47] whichled us to test the effects of RF heat stimulation in WATbrowning Consistent with the results of a previous report[47] our results demonstrated that heat stimulation by RFtreatment can induce elevation of FGF21 in the serum andWATs FGF21 has diverse metabolic effects that are beneficialfor managing obesity increasing fatty acid oxidation in theliver and improving insulin sensitivity in obese individuals[49 50] Recent studies have shown that elevation of adipose-derived and serum FGF21 levels lead to upregulation ofUCP1 transcription in BAT and WAT [51ndash53] Moreoverseveral in vitro and in vivo studies demonstrated that highconcentrations of serum FGF21 cause browning ofWATs andare positively correlated with nonshivering thermogenesisFurthermore FGF21 increases the expression of cell deathactivator CIDE-A carnitine palmitoyltransferase-1 beta andUCP1 in both brown and white adipocytes in a PGC-1120572-dependent manner [51] FGF21 also plays central roles inactivation of the sympathetic nerve system and increases inenergy expenditure which are correlated with weight loss[54] Moreover FGF21 has been found to increase CCL11expression to recruit eosinophils into WAT where CCL11leads to infiltration of macrophages and generation of beigeadipocytes from adipocyte precursors [55] Thus we assumethat M-RF treatment in DIO mice induced the expressionof FGF21 and UCP1 which in turn influenced adipocytebrowning by activation of the sympathetic nerve and changesin the commitment of adipocyte precursors

5 Conclusions

Building upon the idea of moxibustion we manufacturedan M-RF heat stimulation device and demonstrated itsantiobesity effects in DIO mice Heat stimulation using M-RF effectively suppressed body and WAT weight gain while


increasing the formation of beige adipocytes mitochondrialcontent and higher expression of UCP1 and FGF21 in DIOmice

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declared that no conflicts of interest exist

Authorsrsquo Contributions

Young Jun Koh Ju-Hee Lee and Sung Yun Park participatedin the study design conducted the experiments and statisticalanalysis and wrote the manuscript All authors have read andapproved the manuscript

Acknowledgments

This work was supported by the National Research Founda-tion of Korea (NRF-2017R1C1B2005982 to YJ Koh)

References

[1] Y-J Liao Y Shi L-Q Yu and J-Q Fang ldquoConsiderationsabout study on the underlying mechanism of acu-moxibustionin the treatment of obesity type polycystic ovary syndrome byregulating adiponectinrdquoZhen Ci Yan Jiu vol 37 no 1 pp 72ndash762012

[2] J Sun H Zhang C Wang M Yang et al ldquoRegulating theBalance ofTh17Treg via Electroacupuncture andMoxibustionAnUlcerative Colitis MiceModel Based Studyrdquo Evidence-BasedComplementary and Alternative Medicine vol 2017 Article ID7296353 13 pages 2017

[3] C F Tan C Wang L Du et al ldquoEffect of Electroacupunctureand Moxibustion Pretreatment on Expression of AutophagyRelated Proteins LC 3 and Beclin 1 in Rats with MyocardialIschemia-reperfusion Injuryrdquo Zhen Ci Yan Jiu vol 43 no 1 pp1ndash7 2018

[4] H Zhang F Xie H Gong et al ldquoEffects of heat-sensitive mox-ibustion on HPA axis in rats with irritable bowel syndromerdquoZhongguo Zhen Jiu vol 37 no 12 pp 1315ndash1321 2017

[5] P Liang Z Gu and A Wei ldquoMoxibustion at Geshu (BL 17) fordiabetic limb arterial obliteration at early stagerdquo Zhen Jiu vol37 no 12 pp 1280ndash1284 2017

[6] S-P Zhu Y-W He H Chen et al ldquoEffects of PreventiveAcupuncture and Moxibustion on Fat Accumulation BloodLipid and Uterus E

2of Menopause Ratsrdquo Evidence-Based Com-

plementary and Alternative Medicine vol 2014 Article ID621975 9 pages 2014

[7] L Duan G Zhao B Ji Y Cao and X Chen ldquoEffect of crude-herb moxibustion on blood lipids in rats with dyslipidemiardquoJournal of Traditional Chinese Medical Sciences vol 1 no 2 pp140ndash147 2014

[8] H B Wang X H Li Y W He et al ldquoEffect of preventiveacupuncture and preventive moxibustion in Guanyuan (CV4)on contents of ACTH NE and 5-HT in menopausal ratrdquo

Journal of BeijingUniversity of Traditional ChineseMedicine vol31 no 12 pp 45ndash52 2008

[9] A Bensoussan S P Myers and A-L Carlton ldquoRisks associatedwith the practice of traditional Chinese medicinerdquo Archives ofFamily Medicine vol 9 no 10 pp 1071ndash1078 2000

[10] H-S Myoung J-S Park S-P Cho J Lee H-S Choi and K-JLee ldquoA design of RF stimulator which is similar to temperaturedistribution by moxibustion (preliminary study)rdquo in Proceed-ings of the 32nd Annual International Conference of the IEEEEngineering in Medicine and Biology Society (EMBC rsquo10) pp1238ndash1241 2010

[11] H Deng and X Shen ldquoThe Mechanism of MoxibustionAncient Theory and Modern Researchrdquo Evidence-Based Com-plementary and Alternative Medicine vol 2013 Article ID379291 7 pages 2013

[12] F Burdıo A Guemes JM Burdıo et al ldquoLarge hepatic ablationwith bipolar saline-enhanced radiofrequency An experimentalstudy in in Vivo porcine liver with a novel approachrdquo Journal ofSurgical Research vol 110 no 1 pp 193ndash201 2003

[13] J M Lee J K Han S H Kim et al ldquoA comparative exper-imental study of the in-vitro efficiency of hypertonic saline-enhanced hepatic bipolar and monopolar radiofrequency abla-tionrdquo Korean Journal of Radiology vol 4 no 3 pp 163ndash1692003

[14] D Duncan ldquoMegasessions Efficacy of fewer longer treatmentsessions for fat reduction in noninvasive body contouring usinga radiofrequency based devicerdquo Journal ofDrugs inDermatology(JDD) vol 16 no 5 pp 478ndash480 2017

[15] R A Weiss ldquoNoninvasive radio frequency for skin tighteningand body contouringrdquo Seminars in Cutaneous Medicine andSurgery vol 32 no 1 pp 9ndash17 2013

[16] Z Alizadeh F Halabchi R Mazaheri M Abolhasani and MTabesh ldquoReview of the mechanisms and effects of noninvasivebody contouring devices on cellulite and subcutaneous fatrdquoInternational Journal of Endocrinology and Metabolism vol 14no 4 p e36727 2016

[17] D Fuentes R Cardan R J Stafford J Yung G D Dodd IIIand Y Feng ldquoHigh-fidelity computer models for prospectivetreatment planning of radiofrequency ablation with in vitroexperimental correlationrdquo Journal of Vascular and Interven-tional Radiology vol 21 no 11 pp 1725ndash1732 2010

[18] Y Feng and D Fuentes ldquoModel-based planning and real-timepredictive control for laser-induced thermal therapyrdquo Interna-tional Journal of Hyperthermia vol 27 no 8 pp 751ndash761 2011

[19] H-S Myoung and K-J Lee ldquoA Unique Electrical ThermalStimulation System Comparable to Moxibustion of Subcuta-neous Tissuerdquo Evidence-Based Complementary and AlternativeMedicine vol 2014 Article ID 518313 6 pages 2014

[20] N Esser S Legrand-Poels J Piette A J Scheen andN PaquotldquoInflammation as a link between obesity metabolic syndromeand type 2 diabetesrdquoDiabetes Research andClinical Practice vol14 pp 187ndash189 2014

[21] S Milic D Lulic and D Stimac ldquoNon-alcoholic fatty liverdisease and obesity biochemicalmetabolic and clinical presen-tationsrdquo World Journal of Gastroenterology vol 20 no 28 pp9330ndash9337 2014

[22] F B Ortega C J Lavie and S N Blair ldquoObesity and cardio-vascular diseaserdquo Circulation Research vol 118 no 11 pp 1752ndash1770 2016


[23] L Sidossis and S Kajimura ldquoBrown and beige fat in humansthermogenic adipocytes that control energy and glucose home-ostasisrdquo The Journal of Clinical Investigation vol 125 no 2 pp478ndash486 2015

[24] K A Lo and L Sun ldquoTurningWAT intoBATA reviewon regu-lators controlling the browning of white adipocytesrdquo BioscienceReports vol 33 no 5 pp 711ndash719 2013

[25] M Kissig S N Shapira and P Seale ldquoSnapShot Brown andBeige Adipose Thermogenesisrdquo Cell vol 166 no 1 pp 258ndash258e1 2016

[26] M Rosell M Kaforou A Frontini et al ldquoBrown and whiteadipose tissues intrinsic differences in gene expression andresponse to cold exposure in micerdquo American Journal of Physi-ology-Endocrinology and Metabolism vol 306 no 8 pp E945ndashE964 2014

[27] A Smorlesi A Frontini A Giordano and S Cinti ldquoTheadipose organWhite-brownadipocyte plasticity andmetabolicinflammationrdquo Obesity Reviews vol 13 no 2 pp 83ndash96 2012

[28] K Ohashi R Shibata TMurohara andNOuchi ldquoRole of anti-inflammatory adipokines in obesity-related diseasesrdquo Trends inEndocrinology amp Metabolism vol 25 no 7 pp 348ndash355 2014

[29] J K Young S Kang J L Hyuek et al ldquoBone marrow-derived circulating progenitor cells fail to transdifferentiate intoadipocytes in adult adipose tissues in micerdquo The Journal ofClinical Investigation vol 117 no 12 pp 3684ndash3695 2007

[30] A Castoldi C N de Souza N O Saraiva Camara and P MMoraes-Vieira ldquoThe macrophage switch in obesity develop-mentrdquo Frontiers in Immunology vol 6 p 637 2015

[31] C M Apovian L J Aronne D H Bessesen et al ldquoPharma-cological management of obesity an endocrine society clinicalpractice guidelinerdquo The Journal of Clinical Endocrinology ampMetabolism vol 100 no 2 pp 342ndash362 2015

[32] R Nassab ldquoThe evidence behind noninvasive body contouringdevicesrdquo Aesthetic Surgery Journal vol 35 no 3 pp 279ndash2932015

[33] Y J Koh B Park J Park et al ldquoActivation of PPAR120574 inducesprofound multilocularization of adipocytes in adult mousewhite adipose tissuesrdquo Experimental ampMolecularMedicine vol41 no 12 pp 880ndash895 2009

[34] C Porter DNHerndonMChondronikola et al ldquoHuman andMouse Brown Adipose Tissue Mitochondria Have ComparableUCP1 Functionrdquo Cell Metabolism vol 24 no 2 pp 246ndash2552016

[35] X Buy A Basile G Bierry J Cupelli and A Gangi ldquoSaline-infused bipolar radiofrequency ablation of high-risk spinal andparaspinal neoplasmsrdquo American Journal of Roentgenology vol186 no 5 pp S322ndashS326 2006

[36] A Gazis O Beuing B Jollenbeck J Franke and M SkalejldquoBipolar radio frequency ablation of spinal neoplasms in latestage cancer disease A report of three casesrdquoThe Spine Journalvol 37 no 1 pp E64ndashE68 2012

[37] SNGoldberg ldquoRadiofrequency tumor ablation principles andtechniquesrdquo European Journal of Ultrasound vol 13 no 2 pp129ndash147 2001

[38] A M Cypess L S Weiner C Roberts-Toler et al ldquoActivationof human brown adipose tissue by a 1205733-adrenergic receptoragonistrdquo Cell Metabolism vol 21 no 1 pp 33ndash38 2015

[39] TGnad S Scheibler I vonKugelgen et al ldquoAdenosine activatesbrown adipose tissue and recruits beige adipocytes via A

2119860

receptorsrdquo Nature vol 516 no 7531 pp 395ndash399 2014

[40] Y Chen F Siegel S Kipschull et al ldquoMiR-155 regulates differ-entiation of brown and beige adipocytes via a bistable circuitrdquoNature Communications vol 4 article 1769 2013

[41] M H Khan F Victor B Rao and N S Sadick ldquoTreatmentof cellulite Part II Advances and controversiesrdquo Journal of theAmerican Academy of Dermatology vol 62 no 3 pp 373ndash3842010

[42] I ShabalinaN Petrovic J A deJong A Kalinovich B Cannonand J Nedergaard ldquoUCP1 in BriteBeige adipose tissue mito-chondria is functionally thermogenicrdquo Cell Reports vol 5 no5 pp 1196ndash1203 2013

[43] D Ricquier and F Bouillaud ldquoMitochondrial uncoupling pro-teins from mitochondria to the regulation of energy balancerdquoThe Journal of Physiology vol 529 no 1 pp 3ndash10 2000

[44] B Cannon and J Nedergaard ldquoBrown adipose tissue Functionand physiological significancerdquo Physiological Reviews vol 84no 1 pp 277ndash359 2004

[45] F Baumruk P Flachs M Horakova D Floryk and J KopeckyldquoTransgenic UCP1 in white adipocytes modulates mitochon-drial membrane potentialrdquo FEBS Letters vol 444 no 2-3 pp206ndash210 1999

[46] H M Feldmann V Golozoubova B Cannon and J Neder-gaard ldquoUCP1 Ablation Induces Obesity and Abolishes Diet-Induced Thermogenesis in Mice Exempt from Thermal Stressby Living at Thermoneutralityrdquo Cell Metabolism vol 9 no 2pp 203ndash209 2009

[47] J-B Lee and T-WKim ldquoPassive heat loading links lipolysis andregulation of fibroblast growth factor-21 in humansrdquo Journal ofThermal Biology vol 45 pp 163ndash167 2014

[48] R S Rogers M-S Beaudoin J L Wheatley D C Wrightand P C Geiger ldquoHeat shock proteins In vivo heat treatmentsreveal adipose tissue depot-specific effectsrdquo Journal of AppliedPhysiology vol 118 no 1 pp 98ndash106 2015

[49] F M Fisher and E Maratos-Flier ldquoUnderstanding the Physiol-ogy of FGF21rdquoAnnual Review of Physiology vol 78 pp 223ndash2412016

[50] M Giralt A Gavalda-Navarro and F Villarroya ldquoFibroblastgrowth factor-21 energy balance and obesityrdquo Molecular andCellular Endocrinology vol 418 part 1 pp 66ndash73 2015

[51] F M Fisher S Kleiner N Douris et al ldquoFGF21 regulatesPGC-1120572 and browning of white adipose tissues in adaptivethermogenesisrdquo Genes amp Development vol 26 no 3 pp 271ndash281 2012

[52] M J W Hanssen E Broeders R J Samms et al ldquoSerumFGF21 levels are associated with brown adipose tissue activityin humansrdquo Scientific Reports vol 5 2015

[53] B Ni J S Farrar J A Vaitkus and F S Celi ldquoMetabolicEffects of FGF-21 Thermoregulation and Beyondrdquo Frontiers inEndocrinology vol 6 p 148 2015

[54] BM Owen X Ding D AMorgan et al ldquoFGF21 acts centrallyto induce sympathetic nerve activity energy expenditure andweight lossrdquo Cell Metabolism vol 20 no 4 pp 670ndash677 2014

[55] Z Huang L Zhong J T H Lee et al ldquoThe FGF21-CCL11 AxisMediates Beiging of White Adipose Tissues by Coupling Sym-pathetic Nervous System to Type 2 Immunityrdquo Cell Metabolismvol 26 no 3 pp 493ndash508e4 2017

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


Obesity accompanied by WAT growth poses seriousconcerns because it is closely related to the onset of severalmetabolic diseases including type 2 diabetes fatty liverdisease and cardiovascular diseases [20ndash22] Recent studieshave spurred interest in the antiobesity effects of WATbrowning which induces the characteristics of brown adi-pose tissue (BAT) in WATs outside of typical BAT locations[23] Browning of adipocytes in WAT which has geneticdifferences because of its distinct developmental origin fromBAT [24] can be induced by various stimulations such assmall molecules and environmental cues [25] WAT brown-ing occurs via several transcriptional factors coregulatorslipid droplet-associated proteins microRNAs and growthfactors aswell asmitochondrial uncoupling proteins [25ndash27]

Obesity is associated with chronic inflammation whichleads to various diseases such as diabetes hypertensionand cardiovascular diseases [28] Chronic inflammation inobesity is mainly caused by recruitment of inflammatorymacrophages into the WATs as well as by conversion of resi-dent macrophages in the WATs [29 30] In the adipose tissueof obese individuals macrophages with phagocytic activitiessurround and remove dead adipocytes in the WATs [29]Therefore it is important to characterize the adipose tissuein terms of macrophage infiltration and conversion status

Obese patients who try losing weight via lifestyle inter-ventions such as adjusting food intake and increasing theamount of physical exercise often fail to see significantimprovements in their condition moreover pharmacothera-peutics aimed at managing obesity are often accompanied byside effects such as metabolic and psychologic diseases [31]Thus a novel mode of intervention that safely manages obe-sity and its related conditions is needed In light of this unmetneed several trials have used not only medications butalso noninvasive medical devices to treat obesity [32] Herewe describe the effects of moxibustion-simulating bipolarradiofrequency (M-RF) treatment on body weight reductionand adipocyte browning induction in a mouse model of diet-induced obesity (DIO)

2 Materials and Methods

21 Equipment To improve upon the method of classicalmoxibustion we designed a bipolar RF device that is able toprecisely control the depth and temperature at which stimula-tion is given thereby allowing quantitative intervention Themain equipment consisted of two primary devices an energygenerator and a temperature measuring device (Figures 1(a)and 1(b)) A bipolar RF device mainly consisted of a powercontrol board display panel and six bipolar probes Thepower control board could generate a maximum power of446 W with 50 Ω 676 W with 200 Ω and 508 W with500 Ω which was delivered to the subject through bipolarprobesThe bipolar probes were circular (diameters of 5 mm18 mm and 3 mm for the inner circle outer circle andwidth respectively) and coated with Ag as a skin protectant(Figure 1(c)) The process was monitored and controlled by adisplay panel consisting of a 10110158401015840 thin film transistor-liquidcrystal display that had high resolution (1280lowast800) whichis standard for 4-wire touch screens K-type temperature

detecting needles were used to measure the temperaturesat the surface of the abdomen and approximately 1 mmbelow the surface The temperature data were sampled at20 Hz and transmitted to the LabVIEW system (NationalInstruments Austin TX USA) The heating performance ofthe M-RF Stimulator was measured with skin samples ofYucatan pig and mice as shown in Figures 1(d) and 1(e) Eachexperimental procedure was conducted over a course of threeminutes The subjects were contained in plastic cube boxes(350 mm times 450 mm times 350 mm) maintained at 23∘C ambienttemperature and 50ndash60 humidity

22 Mouse and Treatment All animal experiments wereapproved by the Institutional Animal Care and Use Com-mittee of Dongguk University (approval No IACUC-2017-017-1) Eight-week-old male C57BL6 mice were purchasedfrom Central Lab Animal Inc (Seoul Korea) The micewere provided with ad libitum access to water and high-fatdiet (HFD) The HFD (60 Kcal fat Research Diets NewBrunswick NJ) was provided for four weeks to induce diet-induced obesity (DIO) Afterwards the mice were randomlydistributed into the following four treatment groups (1)no heat application (control n = 10) (2) low-temperaturestimulation (RF-L 367∘C n = 10) (3) middle-temperaturestimulation (RF-M 379∘C n = 10) and (4) high-temperaturestimulation (RF-H 388∘C n = 10) We applied M-RF ontothe abdominal skin of DIO mice at the three differenttemperatures for one minute per day every two days overthree weeks while maintaining the HFD At the end ofthe three-week application of M-RF mice were anesthetizedby intramuscular injection of a combination of anesthetics(mixture of tiletamine zolazepam and xylazine each 10mgkg) weighed and sacrificed Blood was collected bycardiac puncture and centrifuged at 848 g for 15 minutes at4∘C after which the resulting serumwas harvested TheWATspecimens were immediately weighed and fixed in neutralbuffered 4 formaldehyde for histochemical studies Theserum and WAT were stored at -80∘C until further use

23 Anesthesia and Euthanasia Mice were anesthetized andeuthanized by intramuscular injection of the combination ofanesthetics (tiletamine-zolazepam 50mgkg and xylazine 12mgkg) before RF treatment and histologic analysis

24 Histologic and Morphometric Analysis Mice were anes-thetized by intramuscular injection of anesthetics and WATswere fixed by cardiac perfusion of 1 paraformaldehyde inPBS and whole-mounted To visualize the adipocytes andmitochondrial content in WATs the tissues were incubatedfor four hours at room temperature with the following chem-icals 44-difluoro-4-bora-3a4a-diaza-s-indacene (BODIPY)for adipocytes (1 120583gml in PBS Invitrogen Carlsbad CAUSA) and MitoTracker Red CMXRos (MitoTracker) formitochondrial contents (100 nM in PBS Invitrogen) Toverify the dead adipocytes immunohistochemistry was per-formed as previously described [29 33] Briefly the WATswere incubated for an hour at room temperature with block-ing solution containing 5 whole donkey serum (JacksonImmunoResearch Laboratories IncWest Grove PAUSA) in


(a)

(b)

(c)

(d) (e)

RF Stimulator


LabVIEW

40

38

36

34

0

32

30

28

26

ControlRF-L

RF-MRF-H

Tem

pera

ture

(∘C)

RF-LRF-MRF-H


0

20

25

30

35

40

Tem

pera

ture

(∘C)










3 Results





Body

wei

ght (

g)45

40

35

30

0


lowastlowastlowast

2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(a)

6

4

2

0

minus2

minus4

Body

wei

ght c

hang

e (g)

relat

ive t

o ba

selin

e







2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(b)


1

2

3

Epid

idym

al W

AT w

eigh

t (g)

lowastlowast



0

2

4

6

8

Ratio

eWAT

to B

W (

)

lowastlowast


(d)


05

10

15

Mes

ente

ric W

AT w

eigh

t (g)


lowastlowast


0

1

2

3

4

Ratio

mW

AT to

BW

()

lowastlowast








Control

RF-L

RF-M

RF-H

Control

RF-L

RF-M

RF-H

Epid

idym

al ad

ipos

e tiss

ueM

esen

teric

adip

ose t

issue

(d)

(a)

(b)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40

0



(c)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05



(e)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40


lowastlowast


(f)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05

0

25







Control

Imm

unoh

istoc

hem

istry

for U

CP1

RF-L

RF-M RF-H

(a) (b)30

20

10

0

UCP

1-po

sitiv

e are

a rat

io (A

U)



(c)

Num

ber o

f bei

ge ad

ipoc

ytes

(n)

20

15

5

0

10


lowastlowast

lowastlowast

lowastlowast

(d)






BODIPY F480 Merged

Control

RF-L

RF-M

RF-H

(a)

Num

ber o

f dea

d ad

ipoc

ytes

(n)

3

2

0

1lowastlowast

lowastlowast

lowastlowast




Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)





4 Discussion






3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

(b)

(c)

(d) (e)

RF Stimulator


LabVIEW

40

38

36

34

0

32

30

28

26

ControlRF-L

RF-MRF-H

Tem

pera

ture

(∘C)

RF-LRF-MRF-H


0

20

25

30

35

40

Tem

pera

ture

(∘C)










3 Results





Body

wei

ght (

g)45

40

35

30

0


lowastlowastlowast

2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(a)

6

4

2

0

minus2

minus4

Body

wei

ght c

hang

e (g)

relat

ive t

o ba

selin

e







2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(b)


1

2

3

Epid

idym

al W

AT w

eigh

t (g)

lowastlowast



0

2

4

6

8

Ratio

eWAT

to B

W (

)

lowastlowast


(d)


05

10

15

Mes

ente

ric W

AT w

eigh

t (g)


lowastlowast


0

1

2

3

4

Ratio

mW

AT to

BW

()

lowastlowast








Control

RF-L

RF-M

RF-H

Control

RF-L

RF-M

RF-H

Epid

idym

al ad

ipos

e tiss

ueM

esen

teric

adip

ose t

issue

(d)

(a)

(b)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40

0



(c)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05



(e)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40


lowastlowast


(f)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05

0

25







Control

Imm

unoh

istoc

hem

istry

for U

CP1

RF-L

RF-M RF-H

(a) (b)30

20

10

0

UCP

1-po

sitiv

e are

a rat

io (A

U)



(c)

Num

ber o

f bei

ge ad

ipoc

ytes

(n)

20

15

5

0

10


lowastlowast

lowastlowast

lowastlowast

(d)






BODIPY F480 Merged

Control

RF-L

RF-M

RF-H

(a)

Num

ber o

f dea

d ad

ipoc

ytes

(n)

3

2

0

1lowastlowast

lowastlowast

lowastlowast




Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)





4 Discussion






3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























3 Results





Body

wei

ght (

g)45

40

35

30

0


lowastlowastlowast

2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(a)

6

4

2

0

minus2

minus4

Body

wei

ght c

hang

e (g)

relat

ive t

o ba

selin

e







2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(b)


1

2

3

Epid

idym

al W

AT w

eigh

t (g)

lowastlowast



0

2

4

6

8

Ratio

eWAT

to B

W (

)

lowastlowast


(d)


05

10

15

Mes

ente

ric W

AT w

eigh

t (g)


lowastlowast


0

1

2

3

4

Ratio

mW

AT to

BW

()

lowastlowast








Control

RF-L

RF-M

RF-H

Control

RF-L

RF-M

RF-H

Epid

idym

al ad

ipos

e tiss

ueM

esen

teric

adip

ose t

issue

(d)

(a)

(b)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40

0



(c)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05



(e)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40


lowastlowast


(f)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05

0

25







Control

Imm

unoh

istoc

hem

istry

for U

CP1

RF-L

RF-M RF-H

(a) (b)30

20

10

0

UCP

1-po

sitiv

e are

a rat

io (A

U)



(c)

Num

ber o

f bei

ge ad

ipoc

ytes

(n)

20

15

5

0

10


lowastlowast

lowastlowast

lowastlowast

(d)






BODIPY F480 Merged

Control

RF-L

RF-M

RF-H

(a)

Num

ber o

f dea

d ad

ipoc

ytes

(n)

3

2

0

1lowastlowast

lowastlowast

lowastlowast




Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)





4 Discussion






3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Body

wei

ght (

g)45

40

35

30

0


lowastlowastlowast

2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(a)

6

4

2

0

minus2

minus4

Body

wei

ght c

hang

e (g)

relat

ive t

o ba

selin

e







2 3 4 5 6 71


RF-MRF-H

ControlRF-L

(b)


1

2

3

Epid

idym

al W

AT w

eigh

t (g)

lowastlowast



0

2

4

6

8

Ratio

eWAT

to B

W (

)

lowastlowast


(d)


05

10

15

Mes

ente

ric W

AT w

eigh

t (g)


lowastlowast


0

1

2

3

4

Ratio

mW

AT to

BW

()

lowastlowast








Control

RF-L

RF-M

RF-H

Control

RF-L

RF-M

RF-H

Epid

idym

al ad

ipos

e tiss

ueM

esen

teric

adip

ose t

issue

(d)

(a)

(b)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40

0



(c)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05



(e)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40


lowastlowast


(f)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05

0

25







Control

Imm

unoh

istoc

hem

istry

for U

CP1

RF-L

RF-M RF-H

(a) (b)30

20

10

0

UCP

1-po

sitiv

e are

a rat

io (A

U)



(c)

Num

ber o

f bei

ge ad

ipoc

ytes

(n)

20

15

5

0

10


lowastlowast

lowastlowast

lowastlowast

(d)






BODIPY F480 Merged

Control

RF-L

RF-M

RF-H

(a)

Num

ber o

f dea

d ad

ipoc

ytes

(n)

3

2

0

1lowastlowast

lowastlowast

lowastlowast




Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)





4 Discussion






3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Control

RF-L

RF-M

RF-H

Control

RF-L

RF-M

RF-H

Epid

idym

al ad

ipos

e tiss

ueM

esen

teric

adip

ose t

issue

(d)

(a)

(b)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40

0



(c)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05



(e)

Dia

met

er o

f adi

pocy

tes (

m

) 160

120

80

40


lowastlowast


(f)

Inte

nsity

of M

itoTr

acke

r(A

U)

20

15

10

05

0

25







Control

Imm

unoh

istoc

hem

istry

for U

CP1

RF-L

RF-M RF-H

(a) (b)30

20

10

0

UCP

1-po

sitiv

e are

a rat

io (A

U)



(c)

Num

ber o

f bei

ge ad

ipoc

ytes

(n)

20

15

5

0

10


lowastlowast

lowastlowast

lowastlowast

(d)






BODIPY F480 Merged

Control

RF-L

RF-M

RF-H

(a)

Num

ber o

f dea

d ad

ipoc

ytes

(n)

3

2

0

1lowastlowast

lowastlowast

lowastlowast




Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)





4 Discussion






3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Control

Imm

unoh

istoc

hem

istry

for U

CP1

RF-L

RF-M RF-H

(a) (b)30

20

10

0

UCP

1-po

sitiv

e are

a rat

io (A

U)



(c)

Num

ber o

f bei

ge ad

ipoc

ytes

(n)

20

15

5

0

10


lowastlowast

lowastlowast

lowastlowast

(d)






BODIPY F480 Merged

Control

RF-L

RF-M

RF-H

(a)

Num

ber o

f dea

d ad

ipoc

ytes

(n)

3

2

0

1lowastlowast

lowastlowast

lowastlowast




Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)





4 Discussion






3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















BODIPY F480 Merged

Control

RF-L

RF-M

RF-H

(a)

Num

ber o

f dea

d ad

ipoc

ytes

(n)

3

2

0

1lowastlowast

lowastlowast

lowastlowast




Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)





4 Discussion






3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Seru

m F

GF2

1 (p

gm

l)200

150

0

100

50


lowastlowast

lowastlowast

lowast

(a)

Control

FGF21

-actin

RF-L RF-M RF-H

(b)

Leve

l of F

GF2

1 in

WAT

(AU

)

0

60

40

20lowast


lowast

lowast

(c)





4 Discussion






3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















3-



3-adrenergic recep-






5 Conclusions




Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Data Availability






Acknowledgments


References












































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





































2119860






















of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Moxibustion-Simulating Bipolar Radiofrequency Suppresses...

Documents

Transcript of Moxibustion-Simulating Bipolar Radiofrequency Suppresses...