Journal of Photochemistry and Photobiology B: Biology 149 (2015) 21–26

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

Can low-level laser therapy when associated to exercise decreaseadipocyte area?

http://dx.doi.org/10.1016/j.jphotobiol.2015.04.0331011-1344/� 2015 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Federal University of São Carlos (UFSCar), ViaWashington Luis, km 235, Monjolinho, CEP 13565-905, São Carlos, SP, Brazil.Tel./fax: +55 16 33739810.

E-mail address: antonioaquino@ursa.ifsc.usp.br (A.E. Aquino Jr.).

Antonio E. Aquino Jr. a,b,c,⇑, Marcela Sene-Fiorese b,a, Cynthia A. Castro b,d, Fernanda O. Duarte b,d,Jorge C. Oishi b,d, Gabriela C. Santos b, Karina A. Silva b,d, Fernando Fabrizzi d, Gilberto Moraes d,Selma M.M. Matheus e, Ana Claudia G.O. Duarte b,d, Vanderlei S. Bagnato a,c, Nivaldo A. Parizotto c,f

a Institute of Physics of São Carlos (IFSC), University of São Paulo (USP), São Paulo, Brazilb Laboratory of Nutrition and Metabolism Applied to Exercise, Physical Education and Motor Human Department, Federal University of Sao Carlos (UFSCar), São Paulo, Brazilc Biotechnology Post Graduation Program, Federal University of São Carlos (UFSCar), São Paulo, Brazild Physiological Science Post Graduation Program, Federal University of São Carlos (UFSCar), São Paulo, Brazile Department of Anatomy, Institute of Biosciences (UNESP), Botucatu, São Paulo, Brazilf Electrothermophototherapy Laboratory, Department of Physical Therapy, Federal University of São Carlos (UFSCar), Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 November 2014Accepted 29 April 2015Available online 20 May 2015

Keywords:ObesityExerciseLow-level laser therapyAdipocyte areaCitrate synthase

Obesity affects approximately 20% of the world population, and exercise is the primarynon-pharmacological therapy. The combined use of exercise and low-level laser therapy (LLLT) maypotentiate the effects promoted by exercise. The objective of this study was to investigate the effectsof exercise in combination with phototherapy on adipocyte area, activity of the enzyme citrate synthaseand muscle morphological analysis. We used 64 Wistar rats, which were divided into eight groups with 8rats each: sedentary chow-diet (SC); sedentary chow-diet plus laser therapy (SCL), exercised chow-diet(EC); exercised chow-diet plus laser therapy (ECL); sedentary high-fat diet (SH); sedentary high-fat dietplus laser therapy (SHL); exercised high-fat diet (EH); exercised high-fat diet, laser therapy (EHL). Theanimals were submitted to a program of swimming training for 90 min/5 times per week for 8 weeksand LLLT (GA-Al-AS, 830 nm) at a dose of 4.7 J/point and a total energy of 9.4 J/animal, with durationof 47 s, which was applied to both gastrocnemius muscles after exercise. We conclude that the combineduse of exercise and phototherapy increases the activity of the enzyme citrate synthase and decreases thewhite adipocyte area epididymal, retroperitoneal and visceral in obese rats, enhancing the effects ofexercise.

� 2015 Elsevier B.V. All rights reserved.

1. Introduction

Obesity is a current global concern due to its increasing preva-lence around the world. Furthermore, this chronic, low-gradeinflammatory disease [1] is a precursor of other impairments, suchas dyslipidemias, hypercholesterolemia, an increase in cardiovas-cular risks and diabetes mellitus type II [2,3].

The consequence of increasing serum triglycerides as result ofhypercholesterolemia raises the risk for cardiovascular diseases.Although triglycerides constitute an important component ofenergy metabolism, excess serum triglycerides works as a negativefactor and is redirected to fat deposits in adipose tissue [4]. Thus,

the intracellular deposits of triglycerides are increased, enlargingthe adipocyte area [4]. In general, the main cause of these fatdeposits is a sedentary lifestyle and a poor diet that is usually richin fatty nutrients [5]. An increase in the adipocyte cell area isharmful and is responsible for a chronic inflammatory conditionthat is characterized by abnormal cytokine production and activa-tion of inflammatory signalling pathways, i.e., characteristics ofobesity [6,7].

Diet-induced obesity is generally treated through controlledexercise [8], which is pivotal for improving the oxidative capacityof the mitochondria. Exercise results in the use of triglycerides asthe main energy source to produce ATP [9]. This metabolic prefer-ence is dependent on mitochondrial activity. Several studies haveshown that one of the effects of low-level laser therapy (LLLT) ismitochondrial alteration, increasing its size and oxidative capacity[10,11]. We hypothesised that the morphological and functionalenhancements of mitochondria are associated with alterations in

Table 2Low-level laser therapy (LLLT) parameters.

Type Ga-Al-As Energy density 1.66 J/cm2

Wavelength 830 nm (infrared) Treatment time 47 sFrequency Continuous wave

(CW)Total energy delivered 9.4 J

Optical output 100 mW Power density 35.36 W/cm2

Spot diameter 0.6 mm Energy per point 4.7 J/point

22 A.E. Aquino Jr. et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 21–26

restricted mitochondrial enzymes, such as citrate synthase. Thisenzyme plays a central role in the oxidative capacity of mitochon-dria, and an increase of its activity after LLLT has been reported[12]. For this reason, we analysed the influence of LLLT on the adi-pocyte area in different white adipose tissues and the citrate syn-thase activity using obese rats in sedentary and exercisedconditions. We expected that LLLT would increase both the citratesynthase activity and promote change in adipocyte area.

2. Materials and methods

2.1. Animals

All animal procedures were performed according to the Guidefor the Care and Use of Laboratory Animals published by the USNational Institutes of Health (NIH publication no. 85-23, revised1996), and protocols were approved by the ethics committee ofUniversidade Federal de São Carlos-UFSCar (no. 067/2010). In thisstudy, 64 male Wistar rats (90 days old and weighing303.31 ± 19.83 g) were used. The animals were divided into eightgroups with eight rats per group (n = 8) and assigned as: sedentarychow-diet (SC); sedentary chow-diet plus laser therapy (SCL), exer-cised chow-diet (EC); exercised chow-diet plus laser therapy (ECL);sedentary high-fat diet (SH); sedentary high-fat diet plus lasertherapy (SHL); exercised high-fat diet (EH); exercised high-fat diet,laser therapy (EHL). Except for the SC, SCL, EC and ECL groups, all ofthe other groups were fed ad libitum with the high-fat diet(3 weeks) before experimental period for the development ofexogenous obesity. The rats were kept in individual cages withfood and water ad libitum for 8 weeks under a 12:12-h light–darkcycle at 23 ± 1 �C.

2.2. Diet

The rats were fed either a normocaloric diet (N), MP-77 stan-dard rat chow diet provided in pellet form (Primor�, São Paulo,Brazil), which contained 23 g of protein, 49 g of carbohydrates,4 g of total fat, 5 g of fibre, 7 g of ash, and 6 g of vitamins per100 g of diet; or a high-fat diet, which consisted of the same com-mercial rat chow plus peanuts, milk chocolate and sweet biscuitsin a ratio of 3:2:2:1. [13]. The quantities of caloric and total fatin diets are showed in Table 1.

2.3. Exercise and LLLT protocols

The exercise program consisted of swimming in individualtanks filled with water, which was maintained at 28–32 �C. Theanimals in the trained groups swam for 30, 60, and 90 min/dayon the first, second, and third days, respectively, to becomeadapted to the program. The swimming period was then set to90 min/day for 5 days/week for 8 weeks. All of the rats swam withan attached load of 3–5% of their body mass. This load was weeklyadjusted and recorded. The values of loads were used to calculateof mean supported load per group. This exercise program is consid-ered to be of moderate intensity [14].

The LLLT parameters are shown in Table 2. All applications wereperformed by the same person using Thera Laser (DMC Equipment,São Carlos, SP, Brazil). The probe was held stationary and contacted

Table 1Difference between chow-diet and high fat diet in calories and total fat.

Chow diet High fat diet

Calories 4.07 kcal/g 5.12 kcal/gFat 4 g 35 g

the skin at a 90� angle and with slight pressure. The laser was usedfor the transcutaneous irradiation of the muscle gastrocnemius inboth legs (1 point in each). We decided not to use a sham groupbecause we included the appropriate positive and negative con-trols to with respect to LLLT (positive, high-fat diet with LLLT;and negative, high-fat diet without LLLT) and diet (positive,high-fat; and negative, chow-diet; both with or without LLLT).The laser was applied after the daily exercise regimen becausethe stress induced from exercise results in the maximal absorptionand metabolic effects. The same protocol was previously used byour laboratory [11,15]. All animals were handled similarly, pro-moting the same routine.

2.4. Sample preparation

At the end of the experimental period (8 weeks), the animalswere euthanized by decapitation. The blood was immediately col-lected and centrifuged, and aliquots of the plasma were frozen at�80 �C for subsequent analyses. White adipose tissues (epididy-mal, retroperitoneal and visceral) were immediately excised,weighed and frozen at �20 �C. The gastrocnemius muscles werealso removed and frozen at �80 �C.

2.5. Citrate synthase activity

The citrate synthase activity in samples of gastrocnemius mus-cle was assayed using the colorimetric method [16] and the CSAssay Kit (Sigma, St. Louis, Missouri, USA). The reaction protocolwas performed using: 0.2 mM acetyl-CoA, 0.5 mM OAA, and0.1 mM DTNB in 100 mM Tris–HCl (pH 7.5). The reaction was incu-bated for 1.5 min at 25 �C, and the absorbance was recorded at412 nm.

2.6. Adipocyte area

Samples (100 mg) of epididymal, retroperitoneal and visceraladipose tissue were fixed in 0.2 M collidine buffer (pH 7.4) contain-ing 2% osmium tetroxide at 37 �C. After 24 h, the samples werewashed with warmed saline [17]. Histological preparations of theadipose tissue were photographed with a CCD-Iris camera (SonyCorp., Tokyo, Japan) interfaced with an Olympus BX60 optic micro-scope (Olympus Corp., New York, NY, USA) and a computer. Theadipocyte area being 400 cells from each animal, totalling 2000cells per group. It was measured using image analysis software(Image-Pro Plus 3.0; Media Cybernetics, Silver Spring, MD, USA)and was expressed in �10�3 lm2.

2.7. Morphological analysis

Animals (one per group, totalling 8 animals) were euthanized,and their right gastrocnemius muscles were immediately removedand perfused with Karnovsky’s fixative solution (2.0% glutaralde-hyde and 4.0% paraformaldehyde at a 1:1 ratio in 0.1 M sodiumphosphate buffer [pH 7.4]). The samples were routinely processedfor transmission electron microscopy and photographed using aPhilips CM100 FEI. The index of the junctional measure was calcu-lated as the product of the maximum length of the fifth junction in

A.E. Aquino Jr. et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 21–26 23

the long axis of the muscle fibre from each animal. This wasaccomplished using an image analysis system (KS300 Zeiss) andAxiovision 4.0 software [18].

2.8. Statistical analysis

The Kolmogorov–Smirnov test was used to analyse the normal-ity of the data. The metabolic parameters were evaluated by atwo-way ANOVA test with post hoc analysis (Tukey–Kramer mul-tiple comparisons) to compare the groups. All data are expressed asthe means ± SD. The Statistical software 7.0 (StatSoft, Tulsa, OK,USA) was used for the statistical analysis, and significant differ-ences were accepted at p < 0.05.

3. Results

The citrate synthase activity in the gastrocnemius muscle wascompared between the rats treated without and with LLLT(Table 3). For SC vs. SCL and SH vs. SHL, the citrate synthase activitytended to increase with LLLT treatment. However, for EC vs. ECLand EH vs. EHL, this activity was remarkably increased with LLLT

Table 3The effects of LLLT on the citrate synthase activity.

SC SCL SH SHL

2.00 ± 0.43 2.89 ± 0.24 2.47 ± 0.37 3.02 ± 0.58

Data are expressed as mean ± SD. Sedentary Chow-diet (SC n = 8); Sedentary Chow-diet p(ECL n = 8); Sedentary High-fat diet (SH n = 8); Sedentary High-fat diet plus laser (SHL n =The letter superscript represents the comparison between groups: aSC vc. SCL, bSH vs. S

c EC vs. ECL.d EH vs. EHL (P < 0.05).e Ec v EH groups.

Fig. 1. Histological analysis of the adipocyte area for epididymal, retroperitoneal and visChow-diet (SC n = 8); Sedentary Chow-diet plus laser (SCL n = 8); Exercised Chow-dietn = 8); Sedentary High-fat diet plus laser (SHL n = 8); Exercised High-fat diet (EH n = 8); ESCL groups (a), SH vs. SHL groups (b), EC vs. ECL groups (c) and EH vs. EHL groups (dsignificance (p < 0.001) was determinate by ANOVA followed by Tukeys post hoc analys

(p < 0.001). A comparison of the sedentary and trained groups(SH vs. EH) also showed a significant difference in the activity ofthis enzyme (p < 0.001).

When comparing SC vs. SCL, a significant increase (p < 0.001)was found in the adipocyte area of the three types of white adiposetissue analysed in this study (Fig. 1). When comparing the high-fatsedentary groups (SH vs. SHL), the area of the retroperitoneal whiteadipose tissue was significantly increased in SHL group. Therefore,the visceral white adipose tissue was decreased in this group(p < 0.001). The exercise groups (EC vs. ECL) showed a significantlyreduced in adipocyte area only in the visceral white adipose tissuein ECL group (p < 0.001). In the exercise groups feed with high-fatdiet (EH vs. EHL), there was a reduction in the adipocyte area for allthree types of white adipose tissue in EHL group (p < 0.001).

From our ultrastructural analysis of the muscle fibres of the gas-trocnemius muscle, normal, intact, organised myofibrils thatformed well-defined sarcomeres were observed in all groups(Figs. 2 and 3). The only morphological differences detected wererelated to the different types of muscle fibres present in the musclesamples. Several mitochondria were present, either isolated or inintermyofibrillar chains, which highlights the preservation of their

EC ECL EH EHL

2.78 ± 0.34 4.71 ± 1.46c 5.58 ± 1.09e 7.44 ± 0.88d

lus laser (SCL n = 8); Exercised Chow-diet (EC n = 8); Exercised Chow-diet plus laser8); Exercised High-fat diet (EH n = 8); Exercised High-fat diet plus laser (EHL n = 8).

HL.

ceral white adipose tissue were expressed in means ± standard deviation. Sedentary(EC n = 8); Exercised Chow-diet plus laser (ECL n = 8); Sedentary High-fat diet (SHxercised High-fat diet plus laser (EHL n = 8). The comparison were expressed: SC vc.). The comparison between the sedentary and exercise groups (e). The statisticalis.

Fig. 2. Electron micrographs of gastrocnemius muscle in the chow-diet groups (one animal each group). Sedentary chow-diet (SC), sedentary chow-diet plus laser therapy(SCL), Exercised chow-diet (EC) and exercised chow-diet plus laser therapy (ECL). Sarcomere (black line), Z line (black arrow), mitochondria (M).

Fig. 3. Electron micrographs of gastrocnemius muscle in the high-fat diet groups (one animal each group). Sedentary high-fat diet (SH), sedentary high-fat diet plus lasertherapy (SHL), Exercised high-fat diet (EH) and exercised high-fat diet plus laser therapy (EHL). Sarcomere (black line), Z line (black arrow), mitochondria (M).

24 A.E. Aquino Jr. et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 21–26

crests. Well-developed triads were also noted (Fig. 3A), and lipiddroplets were not present. As shown in Fig. 2B, the size and num-ber of the mitochondria were increased.

4. Discussion and conclusions

The process of mitochondrial biogenesis is triggered by factorssuch as environmental stress, exercise, temperature, caloric

restriction, obesity induced by diet and oxidative stress in additionto changes in cell division, differentiation and renewal [19,20]. Thisprocess is also accompanied by changes in the size, weight andnumber of the mitochondrial units. As a major factor in this pro-cess, the PGC1-a (peroxisome proliferator activated receptorgamma, coactivator 1 alpha) is modulated by exercise [21].

PGC1-a is closely linked with PPARa (peroxisomeproliferator-activated receptor alpha) in the activation of fatty acid

A.E. Aquino Jr. et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 21–26 25

oxidation, the activity of ERRs (estrogen-related receptors) in thetricarboxylic acid cycle, the activity of NRF-1 (nuclear respiratoryfactor 1) and NRF-2 (nuclear respiratory factor 2) in the electrontransport chain, the activity of cytochrome C oxidase and mito-chondrial DNA [22–24].

With this in mind, phototherapy is based on concepts proposedby Tiina Karu, whose principles assert that lasers promote changesand adaptations in cellular metabolism. These changes and adapta-tions occur through the absorption of light by the enzyme cyto-chrome C oxidase (an enzyme in the terminal electron transportchain), which functions as a proton acceptor, altering cellularmetabolism [25]. This process results in chemical activation orchanges in the redox state of mitochondrial respiratory chain com-ponents, mainly cytochrome C oxidase and NADH dehydrogenase[26]. Furthermore, structural changes, such as the formation ofgiant mitochondria, as well as increased energy synthesis resultingfrom changes in the metabolic process have also been observed inprevious studies from our laboratory and others [10,11,27]. Theseeffects on mitochondrial enzyme activity were also observed inthe present study. The combination of exercise and laser therapyincreased the citrate synthase activity regardless of diet type, sug-gesting increased oxidative capacity in this organelle. Other studieshave shown similar effects, including the increased activity of cyto-chrome C oxidase upon laser exposure [11,25,27–29].

According to Azeemi et al. (2008), the use of a specific spectrumof light causes enzymatic changes, especially with respect tolipases [30]. Some factors, such as the light spectrum, provide con-ditions for increasing the oxidative capacity of the cells, allowingthe increase of hydrolysis of triglycerides and, consequently, agreater amount of substrate that is available for energy production;the use of this substrate depends on the energy demand.

In fact, we observed an increase in the adipocyte area of all tis-sues studied for the sedentary groups (chow-diet and high-fat)submitted to laser therapy (except in visceral adipose tissue inSHL). Moreover, the reduction in the adipocyte area epididymal,retroperitoneal and visceral tissue in the exercised groups thatwere also treated with laser therapy showed that the energydemand is the main reason for this variation. Another interestingresult observed in this study is that the adipocyte area in the vis-ceral adipose tissue was significantly reduced in the rats fed ahigh-fat diet plus laser therapy compared to those without lasertherapy (SH vs. SHL), suggesting that important metabolic differ-ences exist between the types of adipose tissue studied. The differ-ences in adipocyte area between the fat deposit regions indicatethat the visceral adipose tissue has a more pronounced lipolyticcharacteristic than the other regions [31–33]. Thus, when com-bined with exercise, laser therapy promotes a decrease in the fatdeposition in this tissue, regardless of the type of diet consumed,which may be beneficial because the accumulation of visceral adi-pose tissue is strongly related to the development of metabolicsyndrome [34,35]. The results obtained in this study suggest thatthe use of a low-intensity laser (830 nm) is more effective whencombined with exercise. On the other hand, this change in adipo-cyte area has been suggested to occur by both increased mitochon-drial structure [10,11,27] and function [15]. Furthermore, theincrease citrate synthase activity in ECL and EHL groups highlightsthe influence of these treatments (exercise and LLLT) on the pro-cess of mitochondrial biogenesis and, most importantly, on theactive biogenesis of mitochondria.

Thus, through a signalling cascade that is potentiated by actionof PGC1-a, several pathways are activated, increasing the amountof available ATP due to the increased energy demand resultingfrom exercise in combination with laser therapy. Additionally,the amount of available triglycerides, which were greatest in ourexperimental model that consumed the high-fat diet, is also a fac-tor that influences the action of PGC1-a [24]. Thus, we believe that

LLLT acts on PPARa and PGC1-a, causing an increase in their activ-ity in beta-oxidation, which when exacerbated by the amount oftriglycerides from a high-fat diet, results in a large amount of finalproduct (acetyl-CoA).

In this context, through the activity of PGC1-a and the increasedactivity of the receptors NRF-1 and NRF-2, the activity of the tricar-boxylic acid and the electron transport chain are increased withinthe mitochondria, promoting the excess acetyl-CoA to be degradedand generating greater amounts of ATP at the end of the process[23,24].

Moreover, according to Karu et al. (2005), there is an increase inthe rate of DNA synthesis upon the activation of PGC1-a and itssubsequent effects on the activity of the receptors NRF-1 andNRF-2, causing a phenomenon known as photobiomodulation.This increase may also be related with influence of activatingreceptor and increase the level of mitochondrial DNA.

In addition to this system, the activation of the adipocyte mem-brane by raise cyclic adenosine monophosphate (cAMP) levelsstimulate the triggers of hydrolysis of triglycerides into fatty acidsand glycerol through lipase cytoplamatic modulation [36,37]. Thismodulation promotes a decrease in the amount of circulatingtriglycerides, and the triglycerides pool is also hydrolysed uponcombined treatment with exercise and laser therapy, previouslypublished [38].

In the present study, the association between phototherapy andmoderate-intensity exercise in response to experimental obesitywas effective in decreasing the adipocyte area. This occurred as adirect consequence of the increase in energy demand caused byexercise, which increased the mitochondrial oxidative capacity.

Conflict of interest statement

The authors have neither financial nor personal conflicts ofinterest.

Acknowledgement

The authors contributed equally to this work. This article wassupported by Grants Institute of Physics of São Carlos (IFSC),University of São Paulo (USP, São Paulo, Brazil).

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