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Journal of Cosmetic and Laser Therapy
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Ultrasound associated with caffeine increases
basal and beta-adrenoceptor response in
adipocytes isolated from subcutaneous adipose
tissue in pigs
Maria Silvia Mariani Pires-de-Campos, Juliana De Almeida, Valéria Wolf-
Nunes, Elaine Souza-Francesconi & Dora Maria Grassi-Kassisse
To cite this article: Maria Silvia Mariani Pires-de-Campos, Juliana De Almeida, Valéria Wolf-
Nunes, Elaine Souza-Francesconi & Dora Maria Grassi-Kassisse (2016): Ultrasound associated
with caffeine increases basal and beta-adrenoceptor response in adipocytes isolated from
subcutaneous adipose tissue in pigs, Journal of Cosmetic and Laser Therapy
To link to this article: http://dx.doi.org/10.3109/14764172.2015.1063659
Published online: 28 Jan 2016.
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ORIGINAL RESEARCH REPORT 
CONTACT dora maria Grassi-Kassisse doramgk@unicamp.br laboratory of stress study (laBeest), department of structural and functional Biology, institute of 
Biology university of Campinas (uniCamp), Cidade universitária Zeferino Vaz, rua monteiro lobato, 255, Campinas, sp, Brazil, Cep 13083–862.
this study is part of a phd program done by maria silvia mariani pires-de-Campos and supervised by dr. dora maria Grassi-Kassisse.
Color versions of one or more of the figures in the article can be found online at http://www.tandfonline.com/ijcl
© 6 taylor & francis Group, llC
Ultrasound associated with caffeine increases basal and beta-adrenoceptor response 
in adipocytes isolated from subcutaneous adipose tissue in pigs
Maria Silvia Mariani Pires-de-Campos1,2, Juliana De Almeida1, Valéria Wolf-Nunes1, Elaine Souza-Francesconi1, 
and Dora Maria Grassi-Kassisse1
1laboratory of stress study (laBeest), department of structural and functional Biology, Biology institute, university of Campinas (uniCamp), 
Campinas, sp, Brazil; 2physiotherapy, faculty of health sciences (faCis) university methodist of piracicaba (unimep), piracicaba, sp, Brazil
Introduction
Cellulite affects the vast majority of women. It is a change in the 
skin for which there is still no effective treatment defined. New 
forms of treatment are proposed every day, which are physical 
or drug treatments (1, 2). Among the drug proposals, many are 
for topical application; however, their absorption is difficult 
through the skin. This issue leads researchers to investigate the 
use of different chemical substances in the formulation, as well 
as physical methods such as ultrasound (US) to facilitate the 
penetration of drugs through the skin in a process known as 
“phonophoresis” or “sonophoresis” (3).
Due to this demand, the transdermal transport has been 
intensely studied over the past decades. Specifically, since the ini-
tial treatment of polyarthritis with hydrocortisone ointment by 
Fellingher and Schmidt in the 1950s (4), the transdermal delivery 
of therapeutic drugs such as fentanyl, caffeine, heparin, ketopro-
fen, and insulin has become a major concern for clinical medicine 
(5). This technique has several advantages, as it is noninvasive, 
avoids the first-pass effects through the liver, and also prevents the 
degradation of peptides and proteins that can compose the formu-
lation. The downside is that this transport is limited to the outer-
most layer of the epidermis, that is, the stratum corneum (6).
In vitro and in vivo studies (rats, pigs, and humans) using pho-
nophoresis with caffeine were done in skin. Some of them analyz-
ing permeation and others the efficiency of caffeine to localized 
fat treatment. In vitro studies, carried out using diffusion cells 
constructed with pieces of shaved skin, excluding hypodermis, 
extracted from swine dorsal region, demonstrated that US sig-
nificantly accelerated the permeation of caffeine through the skin 
(7). This same group showed in vivo pigs study in which only caf-
feine associated with US reduced the layer of adipose tissue with 
reduction in thickness of hypodermis and adipocytes number (8). 
Borcaud et al. (9) evaluated effects of phonophoresis on transder-
mal transport of fentanyl and caffeine across both hairless rat and 
human skin. Phonophoresis enhanced transdermal drug delivery 
(TDD) of fentanyl (about 35-folds greater than control) and caf-
feine (about 4-folds greater than control) across human and hair-
less rat skin. These experiments were performed using 20-kHz US 
applied at either continuous or pulsed mode and with an average 
intensity of 2.5 W/cm2. The results showed that low-frequency 
US enhanced the transdermal transport of both fentanyl and 
caffeine across human and hairless rat skin. Besides these 
studies, a functional study of adipocytes isolated from areas that 
received topical treatment has not been carried out yet.
ABSTRACT
Background: The topical use of caffeine has been indicated for the lipodystrophies treatment as it pro-
motes increased lipolysis. Ultrasound (US) is often used in cutaneous diseases, esthetic conditions, and as 
a skin permeation enhancer. Objective: We investigate the lipolytic response of adipocytes isolated from 
subcutaneous adipose pigs tissue subjected to treatment with topical application of phonophoresis asso-
ciated with caffeine. Method: We treated dorsal regions of pigs (Landrace  Large White, 35 days, 15 kg, 
n  6) daily for 15 days with gel, gel  US [3 MHz, continuous, 0.2 Wcm2, 1 min/cm2, in total 2 min], gel  
caffeine (5%w/w), and gel  caffeine  US. We used a fifth untreated region as control. Twenty-four hours 
after the last application, we isolated the adipocytes of each treated area and quantified the basal and 
stimulated lipolytic responses to isoprenaline. The results, in mmol glycerol/106cells/60 min, were analyzed 
with analysis of variance or ANOVA followed by Newman–Keuls test. The value of p  0.05 was indicative 
of statistical difference. Results: Only the adipocytes isolated from the area treated with caffeine  US 
showed increased basal lipolysis (0.76  0.26; p  0.0276) and maximal isoprenaline stimulation (0.38  0.15, 
p  0.0029) compared with the other areas. Conclusion: The results demonstrate that increased lipolysis of 
caffeine  US is due to an increase in basal and beta-adrenoceptor response by caffeine, and caffeine’s 
effect is local, avoiding unwanted effects.
ARTICLE HISTORY
received 10 october 2014
accepted 8 June 2015
KEYWORDS
Glycerol; isoproterenol; 
phonophoresis; topical 
agents
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2 M. S. M. PIRES-DE-CAMPOS ET AL.
lipolysis of adipocytes isolated from this treated area will be 
increased when compared with other treatment areas, acting as 
local effect.
Materials and methodsAnimals
We used five uncastrated male hybrid pigs (Landrace  Large 
White), 35 days old (∼ 15 kg), which were obtained from a com-
mercial breeder. The animals were kept in pigpen, divided into 
individual boxes. Twice a day, the stalls were stocked with water 
and feed specific for pigs, composed of a mixture of ground corn, 
soybean meal, meat meal, calcium salts added of Fapec polynu-
cleotides (8). As a prophylactic measure against ectoparasites, 
pigs were pretreated with Ivomecâ. One day before beginning 
the treatments, the dorsal region was carefully trichotomized 
with a shearing machine, avoiding damage to the corneal layer. 
During the experiments, the pigs were cared for in accordance 
with the principles set out by Olfert et al. (23) for the use of ani-
mals in research and education, and the experimental protocols 
we used were approved by the Animal Research Ethics Commit-
tee (EEC/UNICAMP, Protocol 614-2).
Topical treatments
The dorsal region of each pig, trichotomized, was divided into 
five areas (8 cm2 each), one for each of the five treatments: appli-
cation of gel (GEL; see text for composition); application of gel 
and caffeine (CAF; see text for composition); application of gel 
and US; application of gel, caffeine, and US (CAF  US); and a 
control area which did not receive treatment (Figure 1).
The amount of gel applied in each area was 3 g. All treat-
ments were performed once a day, always at the same time, for 
The adipocytes are cells that primarily compose adipose tis-
sue, and can be isolated so their lipolytic function is evaluated. 
In adipocytes there are basal and stimulate lipolysis. Lipolysis in 
these cells is predominantly stimulated by adrenergic agonists 
that interact with beta-adrenoceptors (b-ARs) stimulating the 
Gs protein (Gs), with subsequent activation of adenylyl cyclase, 
which increases the synthesis of cyclic adenosine monophos-
phate (cAMP). This product, in turn, activates the protein 
kinase A or PKA that phosphorylates the hormone-sensitive 
lipase (HSL) and the perilipins. This signaling cascade starts 
the lipolysis, which releases fatty acids and glycerol from the 
adipose cell (10).
Endogenously, the b-ARs are activated by norepinephrine 
and epinephrine, which are mostly known as lipolytic agents 
(10). In contrast, adenosine A1 receptors (A1Rs) inhibit adeny-
lyl cyclase and reduce the levels of intracellular cAMP, so that 
the stimulation of A1R opposes the lipolytic action of b-AR 
(11). Lipolysis can also be decreased by increasing the activity 
of cAMP phosphodiesterase, which hydrolyzes cAMP to AMP 
and adenosine (11). In contrast, insulin induces phosphodi-
esterase enzyme activation causing a decrease in lipolysis effect 
of b-lipolytic agents (10).
Basal lipolysis suffers influence mainly by adenosine, which 
can act in different receptor subtypes. There are four subtypes of 
adenosine receptors: A1, A2a, A2b, and A3 that signal primar-
ily through the stimulation (A2a and A2b) and inhibition (A1 
and A3) of cAMP synthesis. A2 receptors are also known to be 
expressed in preadipocytes, whereas A1Rs are predominantly 
expressed in mature adipocytes. Adenosine, acting through 
A1R, has been implicated in many of the physiological func-
tions of adipocytes. It has been associated, in particular, with an 
inhibition of lipolysis (12).
Caffeine is the most common stimulant used for treatment 
of lipodystrophy (5, 13, 14). It is a derivative of xanthine, which 
inhibits phosphodiesterase of cAMP, increasing and prolonging 
its dependent adrenergic responses, and blocks the adenosine 
receptors A1 and A2A (15, 16). For this reason, caffeine is widely 
used to potentiate the lipolytic response and is found in a large 
number of cosmetic preparations for topical application (16). 
Despite some controversy (17), studies suggest that caffeine 
can break down the stored fat through stimulation of lipolysis, 
reducing fat cell deposits (18, 19). Due to unwanted side effects 
of caffeine ingestion, like nervous excitation, sleep disturbances, 
heart rate increase, and diuresis, its application in cellulite treat-
ment is preferably topical (8).
The population of adrenoceptors in adipocytes may vary, 
depending on the adipose tissue location and the species stud-
ied. The adipocytes isolated from hypodermis of pigs have rel-
evant similarities with those in the human hypodermis (20). 
Moreover, the responses of b-adrenergic stimulation and inhi-
bition in pigs is similar to those of humans, which suggests that 
the proportion of adrenoceptor subtypes in the adipose tissue of 
the two species is similar: 70–80%, 20%, and less than 10% for 
b1, b2, and b3, respectively (21, 22).
Thus, the aim of this study was to investigate the lipolytic 
response of adipocytes isolated from subcutaneous adipose tis-
sues of pigs subjected to treatment of topical caffeine applica-
tion and phonophoresis. As US promotes caffeine permeation, 
the hypothesis of this study is that basal and beta-stimulated 
Figure 1. the dorsal region with different shaved areas of one of the pigs from 
this study.
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JOURNAL OF COSMETIC AND LASER THERAPy 3
performed exactly as previously described by Pires-de-Campos 
et al. (8). Briefly, intensities for those in vitro (7) and in vivo (8) 
US studies were calculated as suggested by Watson (24). Watson 
shows that to induce therapeutic effects the target tissue should 
be kept at 40 and 45°C for 5 min, so it is necessary to have a 
variation of 5°C. For this US, frequency of 3 MHz, intensity of 
0.2 W/cm2 (SATA), power of 0.8 W, effective radiation area or 
ERA of 4 cm2 with continuous emission, and application time 
of 1 min/cm2 were used; it means a total of 2 min, 24 J (7, 8) 
(Figure 3).
Isolation of adipocyte and lipolysis measurement
The pigs were sacrificed by deepening of anesthesia (xylazine 
 ketamine—at initial doses of 2 mg/kg  20 mg/kg, respec-
tively) after 12 h fasting and 24 h after the last day of treatment. 
We removed the tissues and the adipocytes were isolated from 
different areas. The isolation followed the modification of the 
procedure described by Grassi-Kassisse et al. (25). Briefly, we 
weighted the adipose tissue of each area, divided it into five sam-
ples of 2–3 g, and each sample was then chopped and digested 
in 20-mL polyethylene bottle containing 6 mL of Krebs Ringer 
bicarbonate buffer (KRB), 25 mM HEPES, and 6 mM glucose, 
at pH 7.4, with 1 mg/mL of collagenase (type 2, Clostridium 
histolyticum) and 3% bovine serum albumin (BSA, fraction V, 
free fatty acid) (referred to as KRBA). The bottles were shaken 
(60 cycles/min) at 37°C for 45 min, after which the resulting 
cell suspension was filtered through a nylon mesh (200 mm) 
and washed three times with 10 mL of KRBA. Cell suspensions 
of five bottles were combined and washed 3 times with 3-min 
intervals between the washes. Before incubation, an aliquot of 
the final suspension was used to count cells in Malassez cham-
ber (26). Aliquots of the cell suspension (100,000 cells) were dis-
tributed in 2-mL polyethylene bottles containing 1 mL of fresh 
KRBA, and then we added 10 mL of isoprenaline (final concen-
tration: 10 nM to 10 mM) to the bottles, followed by 60 min of 
incubation with agitation at 60 cycles/min, at 37°C. The reac-
tion was stopped in an ice bath. After approximately 30 min, 
15 consecutive days, exactly as described previously by Pires-
de-Campos et al. (8).
Composition and gel application
The gel, consisting of 1% carboxyvinyl acid, 10% propylene 
glycol, sodium acetate buffer (0.1 M; pH 7.1), 25% absolute 
ethyl alcohol, and triethanolamine (pH 7.0), was applied to the 
skin in circular friction by only one physiotherapist trained 
to perform massage until accomplishing hyperemia and 
increased skin temperature. Thisgel served as a vehicle for 
topical application of caffeine and was prepared as described by 
Pires-de-Campos et al. (8).
Treatment with gel and caffeine
The gel with caffeine was prepared as described by Pires-de-
Campos et al. (8), adding 5% of anhydrous caffeine (Sigmaâ 
Chemical CO., St Louis, MO, USA) to the gel described above. 
Caffeine was dissolved in 0.1 M sodium acetate buffer (pH 7.1) 
containing 25% of absolute ethyl alcohol and propylene glycol 
at 10%; the final solution (pH 7.0–7.5) was incorporated into 
the gel (8).
Caffeine gel was applied to the skin on a daily basis, for 15 
days, using circular friction (therapy massage) by a single thera-
pist trained to perform massage until accomplishing hyperemia 
and increased skin temperature, which takes 2 min (Figure 2).
Application of ultrasound
Ultrasound (US; Sonomaster Microcontrolado, KWâ Indús-
tria Nacional de Tecnologia Ltda, Amparo, SP, Brazil) was 
applied daily, for 15 days, in areas of skin covered by gel (US) or 
caffeine gel (CAF  US), during 2 min with parameters 
described as follows: first, the frequency of US was set at 3 
MHz and was calibrated with a balance of US from OHMIC 
CS Instruments Co. (Easton, MD, USA) and the attenuation 
evaluation of the ultrasonic wave to the gel with caffeine was 
Figure 2. Caffeine gel application. Figure 3. ultrasound (us) application.
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4 M. S. M. PIRES-DE-CAMPOS ET AL.
None of the treatments caused a change in the adrenocep-
tors’ sensitivity evaluated by the pD2 value (Table 1). As the 
same pig received all the treatments (Figures 1–3), we discarded 
the anesthetic effects at lipolysis.
Discussion
Esthetics has become the focus of intense commercial activ-
ity, with different product offerings, especially for localized fat 
reduction (1). However, the use and efficiency of such products 
are still controversial and there are few scientific studies that 
validate these practices (1, 16).
The focus of this work is to study lipolysis induced by 
b-agonists with a local effect. The functional effects and the 
ligand-binding patterns for a variety of bAR agonists and antag-
onists are rather atypical in porcine compared with rodent adi-
pocytes (29). The porcine adipocyte has approximately 70–80% 
b1AR, 20% b2AR, and 10% b3AR, as indicated by of the subtype 
transcripts concentration (21) and by binding of ligands found 
to be specific for the cloned porcine b1AR and b2AR (30). Pig 
skin model is the most indicated model, because according to 
the literature (31, 32), it is the one that most closely resembles 
the human skin in its morphological and functional aspects, 
and these similarities explain why this model is widely used in 
cosmetic and dermatological studies (20, 33). In these tissues, 
there is also an important similarity in the adipocyte response 
to b-adrenergic agonists, since the ratio of adrenoceptor sub-
types b1, b2, and b3 is similar in both species, and this subtype 
distribution is totally different from that in rodent adipocytes, 
which have  90% b3AR (20, 21, 34, 35).
In this study we demonstrate that adipocytes isolated from 
skin areas exposed to caffeine and associated with US initially 
showed a significantly higher basal production of glycerol. The 
definition of the mechanisms involved with basal lipolysis is still 
incomplete; it is known that insulin is an important antilipoly-
tic agent; however, it is efficient for the inhibition of stimulated 
lipolysis and has almost no effect on basal lipolysis (10). Some 
clarifications appear with studies on the role of lipases in adipo-
cytes that have advanced after the cloning of the adipose triacyl-
glycerol lipase enzyme. Studies indicate that the HSL is the main 
the floating adipocytes were discarded and the infranatant was 
stored for glycerol analysis.
Lipolysis was then quantified following the glycerol release in 
the incubation medium. Aliquots of infranatant (100 mL) were 
used to quantify the glycerol. We analyzed each test in tripli-
cate, and the results represent the average of the experiments 
performed in different areas of each of the animals. Glycerol 
production was measured after action of the enzymes: glycerol 
kinase, glycerol-3-phosphate oxidase, and peroxidase. Glyc-
erol is phosphorylated by ATP to obtain glycerol-1-phosphate 
and ADP, in a reaction catalyzed by glycerol kinase. Glycerol-
1-phosphate is then oxidized by glycerol phosphate oxidase 
into dihydroxyacetone phosphate and hydrogen peroxide. A 
quinoneimine dye is produced by the coupling of peroxidase-
catalyzed 4-aminoantipyrine (4-AAP) and sodium N-ethyl-N-
(3-sulfopropyl)-m-anisidine (ESPA) with hydrogen peroxide, 
which presents maximum absorbance at 540 nm. The increase 
in absorbance at 540 nm is directly proportional to the glycerol 
contained in the samples. We performed all tests using com-
mercial kits (catalog number 02700, Labor Labâ AS, Guarulhos, 
SP, Brazil [27]).
Concentration–effect curves for isoprenaline were built and 
the values of the concentration that cause 50% of the maximum 
response were calculated (pD2-log EC50). The results were 
expressed in glycerol/106cells/60 min. Concentration–response 
curves were built using the maximum response of glycerol 
release induced by isoprenaline in control adipocytes as 100%.
Statistical analysis
The results are presented as mean  standard deviations. In 
order to analyze data normal distribution we used Shapiro–
Wilk test. Since the normality supposition was verified we used 
one-way analysis of variance (ANOVA) followed by Newman–
Keuls multiple comparison test (28), considering statistical 
significance at 5%. Analyses were done using GraphPad Prism 
software (Prism 5 for Windows, version 5.01).
Results
Adipocytes isolated from skin areas exposed to caffeine treat-
ment associated with US showed a significant increase in basal 
release of glycerol of 55% more than the values obtained by adi-
pocytes isolated from the control area (Table 1, in mmol of glyc-
erol/106 cells/60 min, Control: 0.49  0.05; Gel: 0.61  0.12; US: 
0.48  0.2; Caffeine: 0.51  0.13; Caffeine  US: 0.76  0.26, 
p  0.0276). Adipocytes isolated from this area also showed 
a significant increase in lipolysis stimulated by isoprenaline 
(10 mM; Figure 4, and Table 1, 10 mM: Control: 0.25  0.03; 
Gel: 0.19  0.07; US: 0.20  0.07; Caffeine: 0.18  0.05; Caffeine 
 US: 0.38  0.15, p  0.0029). The CAF  US treatment 
promoted an increase of 52% in the production of glycerol at 
the maximum concentration of isoprenaline, considering the 
glycerol produced by adipocytes isolated from the control area.
Adipocytes isolated from areas treated only with the applica-
tion of Gel, or US, or CAF did not show any significant change 
in basal lipolysis or lipolysis stimulated by isoprenaline (1 and 
10 mM; Figure 4, Table 1).
Table 1. Basal lipolysis, lipolytic potency, and maximal lipolytic response to 
isoprenaline in isolated subcutaneous adipocytes from pigs treated with caffeine 
by phonophoresis.
isoprenaline
treatment Basal pd2 emax
Control 0.49  0.05 7.74  0.92 0.25  0.03
Gel 0.61  0.12 7.39  0.93 0.19  0.07
ultrasound 0.48  0.20 7.95  0.60 0.20  0.07
Caffeine 0.51  0.13 7.62  0.69 0.18  0.05
Caffeine  ultrasound 0.76  0.26* 7.35  0.51 0.38  0.15#
the values are the mean  sd of five experiments and are expressed as mmol 
glycerol/106cells/60 min. each experiment was run in triplicate, and the results rep-
resent the means of experiments from separate animals done on different days. 
the different treatment potencies were compared based on their eC50, that is, the 
concentration of agonist inducing 50% of maximal lipolysis, expressed as pd2(2log 
eC50). emax is the maximal response minus the basal lipolysis. statistical compari-
sons were done using anoVafollowed by the newman–Keuls test: Basal.
*p  0.0276 compared with the other groups emax.
#p  0.029.
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JOURNAL OF COSMETIC AND LASER THERAPy 5
phosphodiesterase enzyme activity (16). Studies of glycerol pro-
duction induced by isoprenaline in isolated pig adipocytes are 
done using theophylline at medium bath. In these conditions, 
Gardan et al. (40) show a basal of 35 nmol glycerol/105cells/2 
h and 75 nmol glycerol/105cells/2 h when adipocytes were 
stimulated by isoproterenol 10 mM plus theophylline 1 mM, 
around 100% of glycerol production at maximal concentration 
of isoprenaline. Mersmann (41), in turn, showed in adipose tis-
sue slices a stimulation induced by isoprenaline without theo-
phylline of 320%, and 190% in the presence of theophylline. In 
these assays, the presence of theophylline induced an increase 
of 198% in basal lipolysis after 120 min of incubation. In most 
cases, when isolated adipocytes and adipose tissue slices from 
the same animal were stimulated with various lipolytic agents 
(adrenergic agonists, theophylline, and adenosine deaminase), 
the qualitative response was similar. Isolated adipocytes were 
not more sensitive than tissue slices to stimulation by lipolytic 
agents (42). The maximal responses induced in our conditions 
are 1.14  0.17 mmol of glycerol/106 cells/60 min and basal of 
0.76  0.11 mmol of glycerol/106 cells/60 min, around 56% of 
stimulation. The main difference concerns on how to get xan-
thine effects. Gardan et al. (40) and Mersmann (41) directly used 
adipocytes already isolated and we used TDD of adipocytes.
Considering the different densities of ARs it is also possible 
to get different amounts of glycerol induced by isoprenaline 
while stimulation achieved by isolated adipocytes from subcu-
taneous adipose tissue from pig is around 50–100%, in adipo-
cytes isolated from epididymal adipose tissue it is possible to get 
a 200% or 300% stimulation (25, 43). These differences under-
score the importance of studying pigs as a source of fat cells in 
order to evaluate the efficiency of drug-dispensing studies by 
lipase involved in lipolysis induced both by catecholamines and 
by the atrial natriuretic factor, while adipose triacylglycerol 
lipase mediates the hydrolysis of triacylglycerides during basal 
lipolysis (10). It is known that, as a paracrine mediator, adenos-
ine has an important inhibitory effect on basal lipolysis (36). 
Being endogenously released, adenosine acts by binding to an 
A1 type membrane receptor expressed in the plasma membrane 
of mature adipocytes, and this binding triggers the inhibition 
of adenylyl cyclase, which has the ultimate effect of reducing 
lipolysis (11, 12). In addition, since caffeine inhibits the action 
of adenosine, consequently, the effect of increased basal lipoly-
sis can be inferred as coming from this caffeine effect that was 
only seen when the application was associated with US.
Besides, adenosine, acting through A1R, has been impli-
cated in many other physiological functions of adipocytes. 
It can be considered as a possible target for inclusion in the 
overall management of obesity. The A1R also has a physiologi-
cal role in protecting against obesity-related insulin resistance 
(37), and A1R agonism has been shown to lower plasma-free 
fatty acids and glycerol in obese Zucker rats (38). In addition, 
consumption of large quantities of caffeine has been reported 
to be beneficial and associated with a reduced risk of type 2 dia-
betes (39); however, the mechanisms involved remain unclear.
On the other hand, the agonists involved in lipolysis stimula-
tion are well known: the catecholamines, which use the stimula-
tion by the activation of adenylyl cyclase (adrenoceptors), and 
more recently the natriuretic peptides via stimulation of guany-
late cyclase (10). As a result, isolated adipocytes from the skin 
area treated with caffeine associated with US showed significant 
increase in lipolysis stimulated by isoprenaline. Studies confirm 
that caffeine and its derivatives stimulate lipolysis by inhibiting 
(A) (B)
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Figure 4. Concentration–response curves for the stimulation of glycerol released by subcutaneous adipocytes of pigs isolated from dorsal regions exposed to different 
treatments: control (▫), gel (▪), us (▴), caffeine (▾), and caffeine plus us (♦). in all cases, the results were normalized relative to the maximal response of control adipocytes 
(considered to be 100%). the points are the mean  sem of six experiments, each done in triplicate.
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6 M. S. M. PIRES-DE-CAMPOS ET AL.
US 3 MHz, with 2-min applications and intensities above 1 
W/cm2 up to 2 W/cm2 caused a significant increase in the epi-
dermis thickness, the presence of a mild inflammatory infiltra-
tion below the epidermis, and also a change in collagen fibers, 
making them thinner and numerous in epithelial tissue of 
Wistar rats. This researcher group warns on the use of US in 
high doses, especially in esthetic treatments. Furthermore, the 
intensity of US shall be chosen according to the target tissue 
absorption coefficient as well as with the attenuation coeffi-
cient, which is related to tissues through which the ultrasonic 
wave passes before reaching the target tissue, as described in 
the methodology. This study, in turn, demonstrates that US was 
not efficient in changing the lipolysis or lipolytic response in 
adipocytes; however, it was extremely effective in increasing 
skin permeation to caffeine that induced local effects. The litera-
ture reports that this increase in permeation is predominantly 
mechanical, through cavitations, radiation forces, and acoustic 
microflow (7, 8, 55).
A limitation of this study is that treatment is performed on 
consecutive days, which usually does not happen in clinical 
practice. Although this study focused on the investigation of the 
basal lipolytic response of phonophoresis with caffeine, as well 
as the one stimulated by isoprenaline, in addition to the sensi-
tivity analysis of beta adrenergic receptors, other studies should 
consider the evaluation of adenosine receptors, and adenosine 
production, including adenosine deaminase in the incubation 
buffer and analyze global responses.
This work confirms the data obtained in previous morpho-
logical research demonstrating that the lipolytic effect of topical 
application of caffeine needs association with US therapeutic. 
The results demonstrate that increased lipolysis of caffeine  US 
is due to an increase in basal and AR response by caffeine, and 
caffeine’s effect is local—avoiding unwanted effects. 
Acknowledgement
We would like to thank Espaço da Escrita, General Coordination of the 
University UNICAMP, for the language services provided.
Funding
We would like to thank the University of Campinas (UNICAMP); the 
Foundation for Research Support of the State of São Paulo—FAPESP, 
Teaching and Research Support Foundation (FAEP), and the Research 
Support Fund (FAP) of the University Methodist of Piracicaba (UNIMEP) 
for the financial support.
Declaration ofinterest
The authors report no declarations of interest. The authors alone are 
responsible for the content and writing of the paper.
References
1. Wanner M, Avram M. An evidence-based assessment of treatments 
for cellulite. J Drugs Dermatol. 2008;7:341–345.
2. De Godoy JMP, De Godoy MFG. Treatment of cellulite based on 
the hypothesis of a novel physiopathology. Clin Cosmet Investig 
Dermatol. 2011;4:55–59.
the transdermal route delivery. TDD has several significant 
advantages compared with oral drug delivery, including elimi-
nation of pain and sustained drug release. However, the use of 
TDD is limited by low skin permeability due to the stratum cor-
neum, the outermost layer of the skin. Phonophoresis is a tech-
nique that temporarily increases skin permeability such that 
various medications can be delivered noninvasively. The acous-
tic phenomena occurring between the skin and the transducer 
US, such as refraction, reflection, absorption, and scattering 
produce disturbance in lipid and protein configuration of the 
cellular membranes, altering the barrier skin. Based on these 
various studies, several possible mechanisms of phonophore-
sis have been suggested. For example, cavitation is believed to 
be the predominant mechanism responsible for drug delivery 
in phonophoresis (5). Some authors suggest that the increase 
in cutaneous temperature induces acceleration in permeation 
due to the formation of intracellular spaces and pores (44–46) 
as well as by increasing local circulation (47). However, in stud-
ies with massage, even inducing hyperemia and, consequently, 
increased temperature did not alter the lipolytic response of adi-
pocytes to isoprenaline, suggesting that caffeine was not perme-
ated enough to induce its effect. These results differ from those 
of Velasco et al. (19) who reported a study with topical appli-
cation of caffeine gel that induced a reduction in diameter of 
adipocytes in Wistar female mice.
The use of US has also been reported for body contouring 
(48, 49). This effect is related to the US’s ability to induce cavi-
tation. Cavitations are easily observed in liquid medium in 
response to local variation in the acoustic pressure induced by 
the US wave that generates small bubbles. They can be stable in 
response to regular pressure changes or unstable when there is 
a violent implosion of bubbles associated with an intense peak. 
Unstable cavitations induce the production of free radicals (47). 
Rohrich et al.(48), using histological and enzyme analyses, did 
not find changes in adipocytes after using US and massage. 
These results confirm that US only produces the effect on tissues 
that absorb its waves and where the adipose tissue absorption 
coefficient is very low (3, 24).
Glick et al. (50) also found no significant changes in cAMP 
content in the skin, lungs, and peritoneal cells of mice subjected 
to US.
The spread of US in biological tissue is longitudinal, where 
the tissue particles vibrate parallel to the direction of wave 
propagation (51). The US sound field is divided into 2 parts. 
The Fresnel zone is the closest zone to the transducer and has a 
very irregular beam, with spatial and temporal peaks of energy. 
Fraunhofer zone, also called the “far field,” as the farthest from 
the transducer, has more regular behavior (52). It is known 
that physical therapy treatments, especially those that want 
to achieve fat, are performed in the next field, which makes it 
difficult to evaluate the wave behavior in the tissue. Another 
important aspect to be considered is that the depth of the tissue 
that is desired to be reached also determines the choice of the 
frequency of the US since the 3-MHz frequency is more shal-
low (approximately 2-cm depth), while 1 MHz is indicated for 
deeper tissues (4–5 cm) (53). Although the US is often used for 
therapeutic and esthetic purposes, research shows unfounded 
data on the intensity, exposure time, and calibration conditions 
for US players (54). Bem et al. (54) observed that the ongoing 
D
ow
nl
oa
de
d 
by
 [
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az
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Licenciado para - Franciane Cardoso Ramos - 03185733002 - Protegido por Eduzz.com
JOURNAL OF COSMETIC AND LASER THERAPy 7
25. Grassi-Kassisse DM, Wolf-Nunes V, Miotto AM, Farias-Silva E, 
Souza Brito ARM, Nunes DS, et al. Sensitivity to b-adrenoceptor 
agonists of adipocytes from rats treated with an aqueous extract of 
Croton cajucara Benth. J Pharm Pharmacol. 2003;55:253–257.
26. Crege DRX de O, Silveira HJV, Chaim ÉAC, Pareja JC, Géloen A, 
Grassi-Kassisse DM. Sex difference in lactate production by adipo-
cytes from lean humans. Open J Endocrine Metab Dis. 2014;4:52–58.
27. McGowan MW, Artiss JD, Strandbergh DR, Zak B. A peroxidase 
coupled method for the colorimetric determination of serum triglyc-
erides. Clin Chem. 1983;29:538–542.
28. Rice JA. Matematical statistical and data analysis. Belmont: Thomson; 
2007.
29. Mills SE, Mersmann HJ. Beta-adrenergic agonists, their receptors, 
and growth: Special reference to the peculiarities in pigs. In: Smith SB, 
Smith DR, editors. Biology of fat in meat animals: Current advances. 
Champaign, IL: American Society of Animal Science; 1995. pp. 1–34.
30. Liang W, Mills SE. Distribution of beta-adrenergic receptor subtypes 
in pig tissues. FASEB J. 1999;13:A258.
31. Huong SR, Bun H, Fourneron JD, Reynier JP, Andrieu V. Use of 
various models for in vitro percutaneous absorption studies of 
ultraviolet filters. Skin Res Technol. 2009;15:253–261.
32. Di Giancamillo A, Rossi R, Vitari F, Pastorelli F, Corino C, 
Domeneghini C. Dietaryconjugatedlinoleicacidsdecreaseleptin in 
porcine adipose tissue. J Nutr. 2009;139:1867–1872.
33. Guimarães GN, Chorilli M, Leonardi GR, Prestes OS, Garcia G, 
Pires-de-Campos MSM, Polacow MLO. Effects of formulations 
containing dimethylaminoethanol (DMAE) acetoamidobenzoate and 
pidolate on the skin. Lat Am J. 2011;30:614–616.
34. Feve B, Emorine LJ, Lasnier F, Blin N, Baude B, Nahmias C, 
Strosberg AD, Pairault J. 1991. Atypical b-adrenergic receptor in 
3T3–F442A adipocytes. J Biol Chem. 1991;266:20329–20336.
35. Langin D, Portillo MP, Saulnier-Blache J-S, Lafontan M. Coexistence 
of three b-adrenoceptor subtypes in white fat cells of various mam-
malian species. Eur J Pharmacol. 1991:199;291–301.
36. Castan I, Valet P, Quideau N, Voisin T, Ambid L, Laburthe M, 
et al. Antilipolytic effects of alpha2-adrenergic agonists, neuropep-
tide Y, adenosine, and PGE1 in mammal adipocytes. Am J Physiol. 
1994;266:R1141–R1147.
37. Dong Q, Ginsberg HN, Erlanger BF. Overexpression of the A1 ade-
nosine receptor in adipose tissue protects mice from obesity related 
insulin resistance. Diabetes Obes Metab. 2001;3:360–366.
38. Schoelch C, Kuhlmann J, Gossel M, Mueller G, Neumann-Haefelin C, 
Belz U et al. Characterization of adenosine-A1 receptor-mediated 
antilipolysis in rats by tissue microdialysis, 1 H spectroscopy and 
glucose clamp studies. Diabetes. 2004;53:1920–1926.
39. van Dam RM, Hu FB. Coffee consumption and risk of type 2 diabetes: 
A systematic review. J Am Med Assoc. 2005;294:97–104.
40. Delphine G, Gondret F, Louveau, I. Lipid metabolism and secretory 
function of porcine intramuscular adipocytes compared with subcu-
taneous and perirenal adipocytes. Am J Physiol Endocrinol Metab. 
2006;291:E372–E380.
41. Mersmann HJ. Adenosine-theophylline interactions on the lipolytic 
response to beta-adrenergic agonists in porcine adipose tissue. Camp 
Biochem Physiol. 1992; 103C(3):541–547.
42. Mersmann HJ, Shparber A. Is the lipolytic response in porcine 
adipose tissue slices equivalent to the lipolytic response in isolated 
adipocytes? Int J Biochem. 1993;25(11):1673–1679.
43. Farias-Silva E, Grassi-Kassisse DM, Wolf-Nunes V, Spadari-Bratfisch, 
RC. Stress-induced alteration in the lipolytic response to b-adrenoceptor 
agonists in rat white adipocytes. J Lip Res. 1999;40(9):1719–1727.
44. Rao R, Nanda S. Sonophoresis:Recent advancements and future 
trends. J Pharm Pharmacol. 2009;61:689–705.
45. Zhao YZ, Du LN, Lu CT, Jin YG, Ge SP. Potential and problems in 
ultrasound-responsive drug delivery systems. Int J Nanomedicine. 
2013;8:1621–1633.
46. Azagury A, Khoury L, Enden G, Kost J. Ultrasound mediated trans-
dermal drug delivery. Adv Drug Deliv Rev. 2014;72:127–143.
47. Polat BE, Hart D, Langer R, Blankschtein D. Ultrasound-mediated 
transdermal drug delivery: mechanisms, scope, and emerging trends. 
J Control Rel. 2011;152:330–348.
3. Akomeah FK. Topical dermatological drug delivery: Quo vadis? 
Current Drug Deliv. 2010;7(4):283–296.
4. Fellinger K, Schmid J. Klinik und merapiedes Chmnischen 
Gelenkhuematismum. Vienna, Austria: Maudrich (Austrian);1954: 
549–552.
5. Park D, Park H, Seo J, Lee S. Sonophoresis in transdermal drug deliv-
erys. Ultrasonics 2014;54:56–65.
6. Maia Filho ALM, Villaverde AB, Munin E, Aimbire F, Albertini R. 
Comparative study of the topical application of aloe vera gel, 
therapeutic ultrasound and phonophoresis on the tissue repair in 
collagenase-induced rat tendinitis. Ultrasound Med Biol. 2010;36: 
1682–1690.
7. Pires-De-Campos MSM, Polacow MLO, Granzotto TM, 
Spadari-Bratfisch RC, Leonardi GR, Grassi-Kassisse DM. Influence of 
the ultrasound in cutaneous permeation of the caffeine: In vitro study. 
Pharmacol Online. 2007;1:477–486. Retrieve from: http://pharmacol-
ogyonline.silae.it/files/archives/2007/vol1/49_Campos.pdf
8. Pires-De-Campos MSM, Leonardi GR, Chorilli M, Spadari-Bratfisch 
RC, Polacow MLO, Grassi-Kassisse DM. The effect of topical caffeine 
on the morphology of swine hypodermis as measured by ultrasound. 
J Cosm Derm 2008;7:232–237.
9. Boucaud A, Machet L, Arbeille B, Machet M, Sournac M, Mavon A, 
Patat F, Vaillant L. In vitro study of low-frequency ultrasound-
enhanced transdermal transport of fentanyl and caffeine across 
human and hairless rat skin, Int. J. Pharm. 2001;228:69–77.
10. Langin D. Adipose tissue lipolysis as a metabolic pathway to define 
pharmacological strategies against obesity and the metabolic syn-
drome. Pharmacol Res. 2006;53:482–491.
11. Dhalla AK, Chisholm JW, Reaven GM, Belardinelli L. A1 adenos-
ine receptor: role in diabetes and obesity. Handb Exp Pharmacol. 
2009;193:271–295.
12. Gharibi B, Abraham AA, Ham J, Evans BAJ. Contrasting effects 
of A1 and A2b adenosine receptors on adipogenesis. Int J Obes. 
2012;36:397–406.
13. Dias M, Farinha A, Faustino E, Hadgraft J, Pais J, Toscano C. Topical 
delivery of caffeine from some commercial formulations. Int J Pharm. 
1999;182:41–47.
14. Sarheed O, Rasool BKA. Development of an optimised application 
protocol for sonophoretic transdermal delivery of a model hydro-
philic drug. Open Biomed Eng J. 2011;5:14–24.
15. Fredholm BB, Battig K, Holmen J, Nehlig A, Zvartau EE. Actions of 
caffeine in the brain with special reference to factors that contribute to 
its widespread use. Pharmacol Rev. 1999;51:83–133.
16. Herman A, Herman AP. Caffeine’s mechanisms of action and its 
cosmetic use. Skin Pharmacol Physiol. 2013;26:8–14.
17. Siddons S. Cellulite causes and treatments. Discov Fit Health. 
2013;22:1 http://health.howstuffworks.com/skin-care/problems/
beauty/cellulite2015.htm
18. Lupi O, Semenovitch IJ, Treu C, Bottino D, Bouskela E. Evaluation 
of the effects of caffeine in the microcirculation and edema on thighs 
and buttocks using the orthogonal polarization spectral imaging and 
clinical parameters. J Cosmet Dermatol. 2007;6(2):102–107.
19. Velasco MV, Tano CT, Machado-Santelli GM, Consiglieri VO, 
Kaneko TM, Baby AR. Effects of caffeine and siloxanetriol alginate 
caffeine, as anticellulite agents, on fatty tissue: histological evaluation. 
J Cosmet Dermatol. 2008;7(1):23–29.
20. Bergen WG, MersmannHJ. Comparative aspects of lipid metabolism: 
Impact on contemporary research and use of animal models. J Nutr. 
2005;135:2499–2502.
21. McNeel RL, Mersmann HJ. Distribution and quantification of beta1-, 
beta2- and beta3- adrenergic receptor subtype transcripts in porcine 
tissues. J Anim Sci. 1999;77:611–621.
22. Ding ST, McNeel RL, O’Brian Smith E, Mersmann HJ. Modulation of 
porcine adipocyte b-adrenergic receptors by a b-adrenergic agonist. 
J Anim Sci. 2000;78:919–929.
23. Olfert ED, Cross BM, McWilliam AA. Guide to the care and use of 
experimental animals. Ottawa, Ontario: Canadian Council on Animal 
Care; 1993. p. 213.
24. Watson R. Ultrasound in contemporary physiotherapy pratice. 
Ultrasonics. 2008;48:321–329.
D
ow
nl
oa
de
d 
by
 [
G
az
i U
ni
ve
rs
ity
] 
at
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9:
19
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ar
y 
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Licenciado para - Franciane Cardoso Ramos - 03185733002 - Protegido por Eduzz.com
8 M. S. M. PIRES-DE-CAMPOS ET AL.
52. Hedrick WR, Hykes DL, Starchman DL. Ultrasound physics and 
instrumentation. Saint Louis: Mosby; 2005.
53. Altomare M, Nascimento AP, Romana-Souza B, Amadeu TP, 
Monte-Alto-Costa A. Ultrasound accelerates healing of normal wounds 
but not of ischemic ones. Wound Repair Regen. 2009;17(6):825–831.
54. Bem DM, Maciel CD, Zuanon JÁ, Neto CB, Parizotto NA. Histological 
analysis of healthy epithelium of Wistar rats in vivo irradiated with 
different intensities of therapeutic ultrasound. Rev Bras Fisioter. 
2010;14(2):114–120.
55. Goraj-Szczypiorowska B, Zajac L, Skalska-Izdebska R. Evaluation of 
factors influencing the quality and efficacy of ultrasound and phono-
phoresis treatment. Ortop Traumatol Rehabil. 2007;9:449–584.
48. Rohrich RJ, Morales DE, Krueger JE, Ansari M, Ochoa O Jr 
et al. Comparative lipoplasty analysis of in vivo-treated adipose 
tissue. Plast Reconstr Surg. 2000;105:2152–2158.
49. Garcia O Jr, Schafer M. The effects of nonfocused external ultrasound 
on tisseu temperature and adipocyte morphology. Aesthet Surg J. 
2013;33:117–127.
50. Glick D, Adamovics A, Edmonds PD, Taenzer JC. Search for 
biochemical effects in cells and tissues of ultrasonic irradiation of 
mice and of the in vitro irradiation of mouse peritoneal and human 
amniotic cells. Ultrasound Med Biol. 1979;5:23–33.
51. Leighton TG. What is ultrasound? Prog Biophys Mol Biol. 2007; 
93(1–3):3–83.
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