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Original Research

Melatonin reduces obesity and restores adipokine

patterns and metabolism in obese (ob/ob) mice

Gaia Favero a , Alessandra Stacchiotti a, b , Stefania Castrezzati a , Francesca Bonomini a, b ,

Massimo Albanese c , Rita Rezzani a, b,⁎, Luigi Fabrizio Rodella a, b
a
Anatomy and Physiopathology Division, Department of Clinical and Experimental Sciences, University of Brescia, 25123 Brescia, Italy
b
Interdipartimental University Center of Research “Adaption and Regeneration of Tissues and Organs-(ARTO)”
c
Department of Oral and Maxillofacial Surgery, University of Verona, Verona, Italy

ARTI CLE I NFO A BS TRACT

Article history: The increasing incidence of obesity, leading to metabolic complications, is now recognized
Received 17 March 2015 as a major public health problem. The adipocytes are not merely energy-storing cells, but
Revised 28 May 2015 they play crucial roles in the development of the so-called metabolic syndrome due to the
Accepted 3 July 2015 adipocyte-derived bioactive factors such as adipokines, cytokines, and growth factors. The
deregulated production and secretion of adipokines seen in obesity is linked to the
Keywords: pathogenesis of the metabolic disease processes. In this study, we hypothesized that
Adipokine dietary melatonin administration would support an anti-inflammatory response and play
Adiponectin an important role in energy metabolism in subcutaneous and visceral adipose tissues of
Inflammation obese mice and so may counteract some of the disruptive effects of obesity. Lean and obese
Melatonin mice (ob/ob) received melatonin or vehicle in drinking water for 8 weeks. Thereafter, they
ob/ob mice were evaluated for morphologic alteration, inflammatory cell infiltration, and the adipokine
Resistin patterns in visceral and subcutaneous white fat depots. In obese mice treated with vehicle,
Visfatin we observed a significant increase in fat depots, inflammation, and a deregulation of the
adipokine network. In particular, we measured a significant reduction of adiponectin and
an increase of tumor necrosis factor α, resistin, and visfatin in adipose tissue deposits.
These changes were partially reversed when melatonin was supplemented to obese mice.
Melatonin supplementation by regulating inflammatory infiltration ameliorates obesity-
induced adipokine alteration, whereas melatonin administration in lean mice was
unaffected. Thus, it is likely that melatonin would be provided in supplement form to
control some of the disruptive effects on the basis of obesity pathogenic process.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction changes in nutrition and lifestyle have led to a sharp increase

in the prevalence of obesity and its complications. Actually, it
Obesity is a medical condition potentially affecting all ages has reached epidemic proportions in many parts of the world
and socioeconomic groups [1]. Over the past decade, profound and this trend is expected to continue [1,2].

Abbreviations: SAT, subcutaneous adipose tissue; TNF-α, tumor necrosis α; VAT, visceral adipose tissue.
⁎ Corresponding author at: Anatomy and Physiopathology Division, Department of Clinical and Experimental Sciences, University of
Brescia, Viale Europa 11, 25123 Brescia, Italy. Tel.: +39 0303717483; fax: +39 0303717486.
E-mail address: rita.rezzani@unibs.it (R. Rezzani).

http://dx.doi.org/10.1016/j.nutres.2015.07.001
0271-5317/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Favero G, et al, Melatonin reduces obesity and restores adipokine patterns and metabolism in obese
(ob/ob) mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.07.001
2 N UTR IT ION RE S EA RCH XX ( 2 01 5 ) X XX– X XX
Obesity is characterized by a chronic inflammatory pro-
cess, well evident in the intra-abdominal compartment
(visceral adipose tissue, or VAT). It is known that an increase
of VAT is highly correlated with diseases, whereas an
augmentation of subcutaneous adipose tissue (SAT) has
minor metabolic consequences [3]. Although the exact
mechanisms underlying the differential role of VAT vs SAT
in metabolic diseases have yet to be identified, many studies
indicate a higher inflammatory potential of VAT compared
with SAT in terms of increased production of proinflamma-
tory mediators and reduced expression of the protective
adipokine. Patsouris et al [4] documented that the accumula-
tion of proinflammatory macrophages in VAT of obese
subjects is linked to the development of insulin resistance.
Adipose tissue is a secretory and endocrine active organ
producing a variety of bioactive proteins, defined as
adipokines, that may regulate energy metabolism, food
intake, and insulin sensitivity [5]. Several studies have
shown that the production of some adipokines is altered in
obesity, in type 2 diabetes, and in the metabolic syndrome [6],
modulating adipocyte size/number and angiogenesis via
paracrine mechanisms and exerting a major role in the
regulation of fat mass [7,8]. The increase in adipose tissue
mass dysregulates both adipokine and adipocytokine secre-
tion patterns [9], which play a pivotal roles in the pathogen-
esis of cardiovascular damages through adverse effects on
hemostatic balance and vascular function [7,10].
Among the adipokines, herein we report on the perturbed
level of adiponectin, resistin, and visfatin in obese mice and the
effect of melatonin administration. Adiponectin is a protein
synthesized in the adipocyte [11,12], and it is released by both
brown and white adipose tissue [13]. Hypoadiponectinemia
causes endothelial dysfunction by increasing superoxide anion
production [14], promoting the synthesis of adhesion molecules
in endothelial cells and the proliferation of vascular smooth
muscle cells [15,16]. Resistin is a cysteine-rich polypeptide
adipokine with proinflammatory activity that is synthesized by
adipose tissue and may act both in paracrine and in endocrine
fashions [15,17]. Also, circulating monocytes and macrophages
seem to be responsible for resistin production in humans [18].
Visfatin is a novel adipokine, released from VAT and
perivascular adipose tissue, which has an insulin-mimetic
effect [17,19]. Visfatin has multiple functions in the vasculature:
it stimulates growth of vascular smooth muscle cells and
endothelial angiogenesis, and it can also directly affect vascular
contractility. Moreover, it amplifies adipocyte differentia-
tion [6,15].
It is evident that maintenance of a “normal” amount of
adipose tissue is essential because an imbalance can cause
serious health problems. Melatonin, a pineal indolamine
secreted in a circadian pattern, has well-known antioxidant
[20,21] and anti-inflammatory effects [22,23], and it is
involved in energy expenditure and body fat mass regulation
[24,25]. Interestingly, it was shown that the amplitude of the
nocturnal pineal [26] and serum [27] melatonin peaks
decreases significantly in obese animals [28].
In this study, we hypothesized that dietary melatonin
administration would support an anti-inflammatory re-
sponse in adipose tissue of obese mice that, in turn, inhibit
obesity-induced alterations. To test this hypothesis, we
Please cite this article as: Favero G, et al, Melatonin reduces obesity and restores adipokine patterns and metabolism in obese
(ob/ob) mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.07.001
investigated a number of factors such as body weight, fat
depots, white adipose tissue hyperplasia, and inflammation
as well as levels of the adipokines, mentioned above, and
inflammatory cells in a murine model of obesity. Because
prior evidence indicates that obesity changes these end
points, we have been characterized subcutaneous and VAT
alterations and investigated the beneficial effects of melato-
nin on adipose tissue responses in leptin deficient ob/ob mice.
Based on these results, we proposed melatonin supplemen-
tation in drinking water as a suitable dietary addition to
temper obesity.
2. Methods and materials
2.1. Animal model
In the current study, 20 lean (B6.-(lean)/OlaHsd) and 20 obese
mice (B6.V-Lepob/OlaHsd), defined as ob/ob mice, at 4 weeks of
age, obtained from Harlan Laboratories S.r.l. (Udine, Italy),
were housed in standard cages in a temperature-controlled
animal facility (+20°C) with a 12-hour/12-hour light-dark
cycle. All animals were fed ad libitum with normal chow
and with free access to tap water.
The ob/ob mice, genetically leptin deficient, are an impor-
tant model for studying adipose organs and are widely used
for obesity and diabetes research [29,30].
2.2. Experimental design
Mice were randomly divided into 4 groups of 10 animals each:
(a) control lean mice treated with vehicle, (b) lean mice treated
with melatonin for 8 weeks, (c) control obese mice treated with
vehicle, and (d) obese mice treated with melatonin for 8 weeks.
Treated groups, instead of tap water with vehicle, were given
melatonin dissolved in drinking water; in particular, melatonin
(kindly provided by Chronolife S.r.l., Roma, Italy) was dissolved
in a minimum volume of ethanol and diluted in drinking water
to yield a dose of 100 mg/kg body weight per day, as described in
our previous studies [31–33]. Fresh melatonin and vehicle
solutions were prepared twice a week, and the melatonin dose
was adjusted to the body weight throughout the study period.
Drinking bottles were covered with aluminium foil to protect
melatonin from light [25].
Blood glucose was monitored throughout the study in blood
collected via tail-snip as previously described [31]. Protocols
were approved by the Italian Ministry of Health. All necessary
care was taken to ameliorate any potential animal suffering.
At the conclusion of an 8-week treatment period, when the
mice were 13 weeks of age, all the animals were killed by
cervical dislocation and the dorsal SAT and the abdominopelvic
VAT were removed and weighed. In Fig. 1, we depict the depots
of white tissue in mice and the areas from which we removed
these deposits.
2.3. Sample processing
Fat specimens were rinsed in physiological solution, and a
portion was fixed in buffered formalin, dehydrated, in graded
 N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX– X XX
Fig. 1 – White adipose depots in mice. Representation of major
white adipose depots (subcutaneous and visceral fat) of mice.
Purple identifies the visceral and subcutaneous fat depots
analyzed in the present study. Modified from Cinti [41].
ethanol, and then embedded in paraffin wax. Serial paraffin
sections (7 μm thick) of each sample were cut with a
microtome and then used for morphometrical evaluations
and immunofluorescences analyses [34]. The other parts of
adipose tissues were fixed in 2.5% glutaraldehyde in
cacodilate buffer 0.1 M (pH 7.4) for 3 hours at +4°C and
postfixed in 2% osmium tetroxide in cacodilate buffer for 1
hour at +4°C for the ultrastructural investigation.
2.4. Transmission electron microscopy
The adipose tissues fixed in glutaraldehyde were treated for
ultrastructural analysis according to Rezzani et al [34]. Briefly,
adipose tissues were dehydrated in increasing ethanol
concentrations and propylene oxide, followed by Araldite-
Epon resin embedding. Semithin sections (1 μm thick) were
obtained using an UltraCut E ultramicrotome, then stained by
toluidine blue and observed with a light microscope (Olym-
pus, Hamburg, Germany). Subsequently, from representative
blocks, 70- to 80-nm-thick ultrathin sections were obtained
using a diamond knife, collected on formvar coated grids,
double stained by uranyl acetate and lead citrate, and
observed with a transmission electron microscopy (Tecnai
G2 Spirit; FEI Company, Eindhoven, the Netherlands) at 80 kV.
2.5. Morphometrical analysis
Alternate paraffin sections were deparaffinized, rehydrated,
and stained with hematoxylin-eosin and then were observed
with an optical light microscope (Olympus) at a final
magnification of ×200. Digital images of SAT and VAT were
captured and the area of 100 random adipocytes was
measured for each animal, as previously described by Nicolai
et al [35]. Individual adipocyte area (μm2) was determined
using an image analyzer (Image Pro Plus 9.1 Version, Milan,
Italy) by 2 observers blinded to the treatment, whose
evaluation was assumed to be correct if the values were not
Please cite this article as: Favero G, et al, Melatonin reduces obesity and restores adipokine patterns and metabolism in obese
(ob/ob) mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.07.001
3
significantly different. In case of dispute concerning interpre-
tation, the case was reconsidered to reach an agreement.
2.6. Immunofluorescence localization of adipokines
Sections were dewaxed in xylene and hydrated using graded
ethanols, and the blocking step was performed before the
application of the primary antibodies with 1% bovine serum
albumin in phosphate-buffered saline for 1 hour in a humid
chamber. Subsequently, the sections were incubated overnight at
4°C with the following primary antibodies: rabbit polyclonal
adiponectin (diluted 1:700; AbCam, Cambridge, UK), goat poly-
clonal adiponectin receptor 1 (diluted 1: 100; Santa Cruz Biotech-
nology, Santa Cruz, CA, USA), goat polyclonal adiponectin
receptor 2 (diluted 1:100; Santa Cruz Biotechnology), goat poly-
clonal resistin (diluted 1:200; Santa Cruz Biotechnology), rabbit
monoclonal visfatin (diluted 1:250; Abcam), and goat polyclonal
tumor necrosis factor α (TNF-α; diluted 1:50; Santa Cruz Biotech-
nology). Samples were washed in phosphate-buffered saline and
labeled using specific Alexa Fluor (Invitrogen detection technolo-
gies, Leiden, The Netherlands) 488 or 543 conjugated secondary
antibodies (diluted 1:200). Finally, the samples were counter-
stained with 4′,6-diamidino-2-phenylindole, mounted and ob-
served with a confocal microscope (LSM 510 Zeiss, Munich,
Germany), as reported previously by Rodella et al [36]. Sections
without primary antibody and in the presence of isotype-matched
IgGs served as negative immunofluorescent control.
Fifteen fields (area of which was 0.04 mm2), randomly
selected from each section, were analyzed and the
immunopositivity for each primary antibody was calculated
using a software for image acquisition (Image Pro Plus 9.1
Version). The evaluation of positive immunostaining was
made by 2 blinded investigators, whose evaluation was
assumed to be correct if the values were not significantly
different. In case of dispute concerning interpretation, the
case was reconsidered to reach an agreement.
2.7. Statistical analyses
The sample size of this research plan was 5 animals per cell, 4
cells with a factorial design with 2 factors: animal model-lean
or obese (factor A) and treatment-melatonin or vehicle (factor
B) at 2 × 2 levels. This design achieves 99.64% power to
demonstrate an effect size of 1 with a combination between A
and B factors (fixed-effects analysis of variance power
analysis). At this aim, we decided to perform in duplicate
the experiment.
The data obtained in the morphometrical and immuno-
fluorescence analyses were presented as means ± SD.
Statistical analyses were performed using a 1-way analysis
of variance test corrected by Bonferroni. Differences among
groups were considered statistically significant at P < .05.
3. Results
All animals of each experimental group survived and mela-
tonin supplementation in drinking water was well tolerated.
After 8 weeks of melatonin treatment, ob/ob mice resulted in a
4 N UTR IT ION RE S EA RCH XX ( 2 01 5 ) X XX– X XX

significative blood glucose reduction with respect to ob/ob
mice treated with vehicle; however, these mice had higher
glucose level than did lean mice treated with melatonin or
vehicle (Fig. 2A). As described in our previous studies [31–33],
ob/ob mice exhibited a body weight increase of 42.13% with
respect to control mice treated with melatonin or vehicle
(Fig. 2B). Moreover, we noted that ob/ob mice treated with
vehicle had VAT and SAT masses 91.04% and 88.16%,
respectively, greater than those of lean mice treated with
melatonin or vehicle. Melatonin orally supplementation
decreased the body weight gain of ob/ob mice treated with
vehicle by 4.43%, and the examination of mice at the gross
anatomical level indicated that ob/ob mice treated with
melatonin clearly exhibited reduced white adipose depots,
which was confirmed also by the measurement of VAT and
SAT depot weights that were lower by 53.03% and 41.11%,
respectively (Fig. 2C-H).
The histologic examination of VAT and SAT revealed that
lean mice, treated with melatonin or vehicle, contained
mature adipocytes, which were uniformly endowed with a
single fat droplet and the nucleus in the periphery (Fig. 3A, E).
In contrast, VAT and SAT adipocytes in ob/ob mice treated
with vehicle exhibited a significantly increased size compared
with the adipocyte cell area of lean mice treated with vehicle
(Fig. 3B, F). The, ob/ob mice treated with melatonin showed a
significant reduction in adipocyte cell size, an important
indicator of healthy adipose tissue (Fig. 3C, G). These results
were confirmed also by the morphometrical analyses sum-
marized in Fig. 3D and H. Interestingly, VAT depots of ob/ob
mice treated with vehicle had a significatively elevated
inflammatory infiltrate appeared as crown-like structures
with respect to SAT depots (these VAT infiltrates are
identified in the box of Fig. 3B).
The semithin section analyses confirmed the previous
morphologic evaluation of ob/ob VAT; these large cells
contained a single lipid droplet and a thin nucleus. The cells
were sometimes surrounded by an inflammatory infiltrate
(Fig. 3I). At the ultrastructural level, macrophages (Fig. 3L) and

Fig. 2 – Blood glucose, body weight, and adipose tissue depot weight. Blood glucose concentrations (A) and body weights (B) in
different experimental groups (lean mice treated with vehicle or melatonin and ob/ob mice treated with vehicle or melatonin),
visceral (C, D) and subcutaneous (E, F) adipose tissue of ob/ob mice treated with vehicle, and the weights of visceral (G) and
subcutaneous (H) fat of each experimental group (lean mice treated with vehicle or melatonin and ob/ob mice treated with
vehicle or melatonin). Scale bar = 1.4 cm. The “*” represents significant difference from the lean group treated with vehicle, “#”
represents significant difference from the lean group treated with melatonin, and “+” represents significant difference from
the ob/ob group treated with vehicle. P < .05. Values are means ± SD; n = 10 mice/group.

Please cite this article as: Favero G, et al, Melatonin reduces obesity and restores adipokine patterns and metabolism in obese
(ob/ob) mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.07.001
 N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX– X XX 5

Fig. 3 – Morphologic and transmission electron microscopy evaluations of white adipose tissue depots. Photomicrographs of VAT
(A-C) and SAT (E-G) of lean mice treated with vehicle (A, E), obese mice treated with vehicle (B, F), and obese mice treated with
melatonin (C, G). Hematoxylin-eosin staining. Scale bar: 20 μm. In the photomicrographs, the box identifies the area of
inflammatory infiltration, the “*” shows small adipocytes, and the “§” shows big adipocytes. The graphs represent, respectively,
the visceral (D) and subcutaneous (H) adipocyte sizes, expressed in μm2. The “*” represents a significant difference from the lean
group treated with vehicle, “#” represents significant difference from the lean group treated with melatonin, and “+” represents
significant difference from the ob/ob group treated with vehicle. P < .05 Values are means ± SD, n = 10 mice/group. Semithin (I) and
transmission electron microscopy photomicrographs (L-N) of VAT of ob/ob mice treated with vehicle that depict a single
macrophage near hypertrophic adipocytes (I, L) and a degranulated mast cell in the adipocyte interstitium (M, N). Transmission
electron microscopy photomicrographs of ob/ob mice treated with vehicle visceral fat (O, P) show enlarged lipid droplets with
interstitial cells and connective fibrotic deposits. a, adipocyte; m, macrophage; M, mast cell. The arrow identifies collagen in the
space between 2 adipocytes. Scale bars: 20 μm (A), 2.5 μm (B, E, and F), 10 μm (C), and 5 μm (D).

mast cells often appeared in a degranulated state (Fig. 3M, N).
After melatonin supplementation, the VAT of obese mice
treated with vehicle showed regular smaller adipocytes and,
at ultrastructural level, regular vessels associated with fibro-
blastic cells devoid of inflammatory elements (data not shown).
Interesting histopathologic evidence, best characterized by
transmission electron microscopy, showed that VAT was
associated with fibrosis in obese mice treated with vehicle.
Many fibrotic cells were located in abundant connective and
fibrotic matrix in the interstitial space (Fig. 3O, P).
Immunofluorescence analyses of TNF-α (identified in
red—Fig. 4A-D), important inflammation-related adipokine,
confirmed the previous morphologic and ultrastructural
evaluations showing at VAT level of lean mice, treated with
melatonin or vehicle (Fig. 4A, B), a negative expression with
respect to a moderate expression of ob/ob mice treated with
vehicle (Fig. 4C). Interestingly, melatonin supplementation in
ob/ob mice reduced significantly the VAT inflammatory
marker expression (Fig. 4D). Furthermore, immunofluores-
cence analyses of resistin (identified in green—Fig. 4E-H) and
visfatin (identified in green—Fig. 4I-N) showed that lean mice,
treated with melatonin or vehicle, had no or very weak
expression of both adipokines (Fig. 4E, F, I, L). By comparison,
ob/ob mice treated with vehicle showed a moderate/strong
expression of both adipokines at the level of the membrane of
visceral white adipocytes (Fig. 4G, M). Melatonin treatment for

Please cite this article as: Favero G, et al, Melatonin reduces obesity and restores adipokine patterns and metabolism in obese
(ob/ob) mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.07.001
6 N UTR IT ION RE S EA RCH XX ( 2 01 5 ) X XX– X XX

Fig. 4 – Tumor necrosis factor α, visfatin, and resistin immunofluorescence analyses. VAT expression of TNF-α (A-D), resistin
(E-H), and visfatin (I-N), respectively, in lean mice treated with vehicle (A, E, I), lean mice treated with melatonin (B, F, L), ob/ob
mice treated with vehicle (C, G, M), and ob/ob mice treated with melatonin (D, H, N). Scale bars: 20 μm. In the
photomicrographs, the “*” shows small adipocyte, the “§” shows big adipocytes. The graphs summarize the
histomorphometrical analyses, at the visceral and subcutaneous fat levels, respectively, of TNF-α (O), resistin (P), and visfatin
(Q). The “*” represents significant difference from the lean group treated with vehicle, “#” represents significant difference
from the lean group treated with melatonin, and “+” represents significant difference from the ob/ob group treated with
vehicle. P < .05. Values are means ± SD; n = 10 mice/group.

ob/ob mice reduced significantly the expressions of resistin
and visfatin (Fig. 4H, N). The quantitative data related to VAT
and SAT immunostainings are summarized in Fig. 4O-Q.
An opposite pattern with respect to the expression of TNF-
α, visfatin, and resistin was shown in the immunofluores-
cence analyses of adiponectin and its relative receptors. Lean
mice, treated with melatonin or vehicle, expressed moderate
adiponectin levels (identified in green) at the level of the
membrane of visceral white adipocytes (Fig. 5A, B).
Adiponectin expression of white adipocytes of ob/ob mice
treated with vehicle was significantly decreased, often being
almost absent with respect to control mice treated with
melatonin or vehicle (Fig. 5C). Interestingly, this effect was
reversed, in part, by oral supplementation of melatonin; these
cells showed a weak adiponectin expression (Fig. 5D). The
pattern of expression of adiponectin receptor 1 (identified in
green—Fig. 5E-H) and receptor 2 (identified in red—Fig. 5I-N)
matched the level of adiponectin; in particular, ob/ob mice
treated with vehicle showed a very weak expression
compared with lean mice treated with melatonin or vehicle,
and melatonin treatment for ob/ob mice for 8 weeks induced
the up-regulation of both adiponectin receptors.
These data are displayed for quantitative analyses and
summarized in Fig. 5O-Q, which also shows the SAT
immunopositivity levels.
All these obesity-induced alterations and the melatonin
protective effects at adipose tissue level are schematically
summarized respectively in Fig. 6A and B.
4. Discussion
Dietary interventions that reduce obesity-related alterations
garner significant research interest. Melatonin supplementa-
tion is drawing increased interest in the medical and
nutritional research fields [37–39]; actually, much of the
research focus is on metabolic syndrome obtained mainly in
animal models and suggests that melatonin might increase

Please cite this article as: Favero G, et al, Melatonin reduces obesity and restores adipokine patterns and metabolism in obese
(ob/ob) mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.07.001
 N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX– X XX 7

Fig. 5 – Immunofluorescence analyses of adiponectin and adiponectin receptors. VAT expression of adiponectin (A-D) and
adiponectin receptor 1 (E-H) and adiponectin receptor 2 (I-N) in lean mice treated with vehicle (A, E, I), lean mice treated with
melatonin (B, F, L), ob/ob mice treated with vehicle (C, G, M), and ob/ob mice treated with melatonin (D, H, N). Scale bars: 20 μm.
In the photomicrographs, the “*” shows small adipocyte, whereas the “§” shows big adipocytes. The graphs summarize the
histomorphometrical analyses, at visceral and subcutaneous fat levels, of adiponectin (O), adiponectin receptor 1 (P), and
adiponectin receptor 2 (Q). The “*” represents significant difference from the lean group treated with vehicle, “#” represents
significant difference from the lean group treated with melatonin, and “+” represents significant difference from the ob/ob
group treated with vehicle. P < .05. Values are means ± SD; n = 10 mice/group.

energy expenditure. In the present study, we focused on the
anti-inflammatory properties of melatonin and sought to
determine whether a melatonin-supplemented diet for 8
weeks was beneficial for attenuating inflammatory infiltra-
tion and restoring adipokine pattern in obese mice. In
particular, we show that in ob/ob mice, both VAT and SAT
increase in volume (adipocyte hyperplasia and hypertrophy)
and VAT, more than SAT, is strictly associated with local
infiltration of inflammatory cells and alterated adipokine
expression pattern, whereas interestingly as supposed by our
research hypothesis, melatonin administration to ob/ob
mice ameliorated adipose tissue morphologic alterations,
reduced adipocyte inflammation, and restored the correct
adipokines profile.
With regard to the VAT and SAT increases, our data are in
agreement with those obtained from others showing that
under conditions of positive energy balance, visceral adipo-
cytes are smaller than those in the subcutaneous deposits
[40,41]. Moreover, Fain [42] documented that human visceral
omental fat cells are smaller than subcutaneous adipocytes,
but macrophage accumulation is greatest in visceral omental
depot, suggesting that something in VAT is essential for
maintaining correct cell homeostasis [43]. We extended these
findings by showing a higher inflammatory cell infiltration
and expression of proinflammatory adipokines in VAT. Under
normal conditions, the VAT adipocytes are involved in lipid
synthesis, storage, and secretion of anti-inflammatory mole-
cules, but they can also be induced to secrete a number of
inflammatory factors [5]. Indeed, in an early study of ob/ob
mice, we observed an increased expression of both TNF-α, as
in this study, and CD68, marker of macrophages [31].
Furthermore, He et al [44] considered TNF-α as an inflammation-
related adipokine, which can modulate insulin signaling and
induce insulin resistance in adipocytes [45], and so it is
involved in the development of metabolic syndrome [46].
Also, the ultrastructural investigation confirmed that VAT

Please cite this article as: Favero G, et al, Melatonin reduces obesity and restores adipokine patterns and metabolism in obese
(ob/ob) mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.07.001
8 N UTR IT ION RE S EA RCH XX ( 2 01 5 ) X XX– X XX
Fig. 6 – Melatonin and obesity dysfunctions. Schematic representations of obesity-induced alterations (A) and of protective
effects of melatonin (B) at white adipocyte level.
expansion in ob/ob mice is associated with a local infiltration
of inflammatory cells, mainly macrophages and mast cells,
as previously reported also by Giordano et al [47]. Further-
more, Cinti et al [48] suggested that macrophages are
localized selectively at sites of necrotic-like cell death
where they appear as crown-like structures. Whether en-
hanced lysis/death of hypertrophic adipocytes is the primary
trigger that accounts for inflammation is unknown. Also,
what signal results in greater macrophage accumulation in
adipose tissue remains to be investigated; however, VAT
hypertrophy per se promotes cell death resulting in macro-
phage accumulation and aggregation around dead cells.
Moreover, it is known that there is a strict correlation
between adipocyte size and local inflammation [42] that, in
turn, promotes and increase obesity damages.
Concerning the alteration of VAT adipokines expression,
we observed that VAT expansion in ob/ob mice treated with
vehicle requires extensive tissue remodeling which, in turn,
probably contributes to the physiological balance of adipokine
expression. Adipokines, which are produced by adipocytes or
adipose tissue macrophages, induce a low-grade chronic
inflammatory state that plays a central role in obesity-related
cardiovascular complications and insulin resistance [6,15].
Therefore, fat tissue affects not only overall metabolism but
also the functionality of many organs and tissues. In vivo and
in vitro studies suggest that VAT, rather than the SAT, is the
main source of adiponectin [49]. Here, we observed that
adiponectin and its receptors 1 and 2 are expressed abun-
dantly by adipocytes of lean mice treated with melatonin or
vehicle. In ob/ob mice treated with vehicle, adiponectin and
adiponectin receptor expressions were dramatically reduced
and disappear almost completely. Typically, adiponectin is
negatively related to the increase of fat mass likely owing to
the abnormal hormonal milieu mainly caused by the inhib-
itory effects exerted by the increased local TNF-α levels (as
Please cite this article as: Favero G, et al, Melatonin reduces obesity and restores adipokine patterns and metabolism in obese
(ob/ob) mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.07.001
also observed in this study), oxidative stress, and the
proinflammatory state which prevail in central obesity [10].
With regard to other adipokines, resistin expression is
stimulated by the increased VAT expression of TNF-α and
proinflammatory factors [50], and visfatin is abundantly
expressed by VAT and macrophages [51], both of which are
increased in obesity [15]. Herein, we observed that ob/ob mice
treated with vehicle showed a higher expression of TNF-α,
resistin, and visfatin in VAT compared with lean mice, treated
with melatonin or vehicle.
A final observation in this study is that melatonin
administration for 8 weeks reduced body weight, adipose
tissue depots, adipocyte hyperplasia and hypertrophy, blood
glucose, proinflammatory factors, and restored adipokine
physiological profile expression. Our data are in agreement
with previous findings in which melatonin treatment was
reported to counteract obesity alterations and to optimize
metabolism [52–54]. The effects of melatonin in obesity have
been intensively studied in animal models of diet-induced
obesity [51,55–58]. In the current investigation, we evaluated
the effect of orally administered melatonin in an animal
model of obesity, which have a missense mutation of the
leptin gene.
The current study has demonstrated that dietary melato-
nin supplementation is associated with partial amelioration
of the pathogenesis of obesity, already in the early phases.
The biological action of melatonin in this study may have
involved (i) the activation of central receptors [59–61],
resulting in changes in metabolic rate via sympathetic
nervous activity and subsequent effects on lipolysis and
adipose tissue plasticity, and/or (ii) a direct effect of melato-
nin on adipose tissue [28,62–65]. The activation of adipose
tissue receptors may influence energy storage by modulating
adipocyte metabolism or proliferation. The findings indicate
that the adipose tissue is a peripheral target of melatonin for
 N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX– X XX
the regulation of its overall metabolism [47]. Consistent with
this, Brydon and colleagues [64] reported that MT2 receptors
are expressed in human adipose tissue and participate in
adipocyte homeostasis.
A limitation of this study may be that no significative
difference in ob/ob body weight was seen after 8 weeks of
melatonin treatment; a longer duration of treatment may be
needed to elicit such improvement. Moreover, probably
melatonin receptor expression at VAT and SAT level would
be interesting to evaluate together with adipokines expres-
sion for better understanding the melatonin mechanism of
action. These limitations will hopefully be addressed in
future studies.
Our data suggested that melatonin improves the low grade
of adipose tissue inflammation that is associated with obesity
and, together with the evidences of melatonin safety and
efficacy, make this indoleamine a potential dietary supple-
mentation to help people in the prevention of obesity,
diabetes, and associated complications. More research, in
particular clinical trials, is needed to further strengthen the
link between melatonin and obesity and the effective dose
required to modulate these metabolic responses in human.
Acknowledgment
The authors sincerely thank Russel J. Reiter for his kind
American English and editing revision. The authors also
thank Antonio Lavazza for his support in electron microscopy
analyses and Lorena Giugno for her technical support. They
also sincerely thank Chronolife S.r.l. (Roma, Italy) for courte-
ously providing melatonin. This study was supported by a
grant (ex-60%) from the University of Brescia, Italy.
The authors declare that there are no conflicts of interest.
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