J. Pineal Res.

2008; 45:422–429  2008 The Authors

Journal compilation  2008 Blackwell Munksgaard
Doi:10.1111/j.1600-079X.2008.00610.x
Journal of Pineal Research

Melatonin and the circadian entrainment of metabolic and

hormonal activities in primary isolated adipocytes

Abstract: The aim of this work was to investigate the effect of the in vitro Maria Isabel Cardoso Alonso-
circadian-like exposure to melatonin [in the presence or absence of insulin Vale, Sandra Andreotti, Paula
(Ins)] on the metabolism and clock gene expression in adipocytes. To Yuri Mukai, Cristina das Neves
simulate the cyclic characteristics of the daily melatonin profile, isolated rat Borges-Silva, Sidney Barnabé
adipocytes were exposed in a circadian-like pattern to melatonin added to the Peres, José Cipolla-Neto and
Fabio Bessa Lima
incubating medium for 12 hr (mimicking the night), followed by an equal
period without melatonin (mimicking the day) combined or not with Ins. Department of Physiology and Biophysics,
Institute of Biomedical Sciences, University of
This intermittent incubation was interrupted when four and a half 24-hr
Sao Paulo, Sao Paulo, SP, Brazil
cycles were fulfilled. At the end, either during the induced night (melatonin
present) or the induced day (melatonin absent), the rates of lipolysis and
D-[U-14C]-glucose incorporation into lipids were estimated, in addition to
the determination of lipogenic [glucose-6-phosphate dehydrogenase and fatty
acid synthase (FAS)] and lipolytic (hormone sensitive lipase) enzymes and
clock gene (Bmal-1b, Clock, Per-1 and Cry-1) mRNA expression. The leptin
release was also measured. During the induced night, the following effects Key words: clock genes, insulin, leptin,
were observed: an increase in the mRNA expression of Clock, Per-1 and lipogenesis, lipolysis, primary culture of
adipocytes
FAS; a rise in lipogenic response and leptin secretion; and a decrease in the
Address reprint requests to Fabio Bessa Lima
lipolytic activity. The intermittent exposure of adipocytes to melatonin MD, PhD, Department of Physiology and
temporally and rhythmically synchronized their metabolic and hormonal Biophysics, Institute of Biomedical Sciences,
function in a circadian fashion, mimicking what is observed in vivo in University of Sao Paulo, 1524 Prof. Lineu
Prestes Ave., 05508-900, Sao Paulo, SP,
animals during the daily light–dark cycle. Therefore, this work helps to
Brazil.
clarify the physiological relevance of the circadian pattern of melatonin E-mail: fabio@icb.usp.br
secretion and its interactions with Ins, contributing to a better understanding Received April 4, 2008;
of the adipocyte biology. accepted June 3, 2008.

The role of clock genes is to control the transcription of

Introduction
Circadian molecular clocks driven by autoregulatory tran-
scription–translation feedback loops consist of a pair of
activator proteins [circadian locomotor output cycles kaput
(CLOCK) and brain and muscle Arnt-like protein 1
(BMAL-1) in mammals] that induce the transcription of a
pair of repressor genes [Period (Per) and Cryptochrome
(Cry)], additionally regulated by modifiers [1]. These
proteins have been found in the neural circadian master
clock, the suprachiasmatic nuclei (SCN), and in several
peripheral tissues including the adipose [2]. These peri-
pheral clock genes are similar to those present in the SCN
neurons, although only the latter seem to be self-sustained.
It is still unclear how these peripheral clocks are synchro-
nized by the central SCN clock.
Among the many circadian rhythms in the body that are
driven by SCN output, melatonin, produced by the pineal
gland, functions as messager encoding the duration of
darkness. Dissemination of at least some of this circadian
information relies on the activation of melatonin receptors,
which are expressed in almost all cells of the organism. A
deficient melatonin synthesis or receptor expression should
therefore have consequences for circadian biology.
other genes, named clock-controlled genes. Recent molecu-
lar studies revealed the direct coupling of clock genes and the
regulation of metabolism [3]. Genetic mutations or deletions
have implicated the peripheral clock genes in the regulation
of some aspects of cellular function, including the control of
glucose homeostasis [4], lipid synthesis [5] and adipogenesis
[6], which are associated with obesity and type 2 diabetes
mellitus. On the other hand, melatonin also plays an
important role in regulating body mass and energy balance
[7–10]. Previous studies have reported that young people
with nocturnal lifestyle, whose melatonin secretion was
shown to be altered, increased their risk of health problems,
including eating disorders, obesity and diabetes [11].
We previously showed that the circadian pattern of
metabolic responses observed in adipose tissue of intact
animals is altered and/or impaired in pinealectomized rats,
in addition to a persistent picture of insulin (Ins) resistance
[12–15]. Moreover, we found that melatonin also influences
the adipose tissue endocrine activity by modulating the
leptin synthesis and secretion [16], which exhibits a circadian
expression. This effect was the result of melatonin binding to
its Gi-protein-coupled MT1 membrane receptor. Addition-
ally and as important as the direct effect on leptin secretion,

422

it was observed that the chronic modulatory action of
melatonin occurred only when adipocytes were intermit-
tently but not continuously exposed to melatonin [17]. The
nature of the temporal information transmitted by the
intermittent melatonin treatment to the adipose cells is not
known but our main hypothesis is that the adipose tissue is a
target for melatonin and that the circadian oscillation of its
endocrine function is driven by the indoleamine.
Here, we report the effects of the in vitro circadian-like
exposure of adipocytes to melatonin (and its interaction with
Ins) on clock genes expression, emphasizing the different
responses that occur in the induced night and induced day
periods, indicated by the in vitro melatonin presence or
absence, respectively. Additionally, the metabolic function
of these cells was assessed by the quantification of lipolytic
and lipogenic responses, as well as the expression of
important enzymes involved in these process. Leptin secre-
tion was also characterized. The results of this work help to
clarify the physiological relevance and the repercussions of
the in vivo circadian pattern of melatonin secretion.
Materials and methods
Adipocyte preparation
Male adult Wistar rats (8-wk old) from the Animal
Resource of the Institute of Biomedical Sciences, University
of Sao Paulo, Sao Paulo, Brazil, were kept on a 12 h/12 h
light–dark cycle (lights on at 0700 hr) in a temperature
controlled room (23 ± 1C). Animals were housed in
groups of three to four in plastic cages with food (balanced
chow pellet diet; Nuvilab CR1, Nuvital SA, Colombo, PR,
Brazil) and water ad libitum. Rats were killed at 18:00 hr by
decapitation. The abdominal wall was opened and the
epididymal fat pads were excised and processed for
adipocyte isolation. All the procedures followed the insti-
tutionally approved protocol in agreement with the Ethical
Principles in Animal Research adopted by the Ethical
Committee for Animal Research (CEEA) of the Institute of
Biomedical Sciences (no. 180/01).
The adipocyte isolation was performed according to
Rodbell [18] with some slight modifications. Briefly,
epididymal fat pads were minced with fine scissors and
added to a flask (approximately 4 g per flask) containing
10 mL of sterile DulbeccoÕs modified EagleÕs medium
(DMEM) (Invitrogen, Carlsbad, CA, USA) with 20 mM
4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, 5 mM
glucose, 1% bovine serum albumin (BSA) (fraction V;
Sigma Chemical Co., St. Louis, MO, USA), antibiotics
(penicillin 100 lU/mL; streptomycin 100 lg/mL – Invitro-
gen) and 1 mg/mL collagenase type II (Sigma), pH 7.4. The
mixture was incubated for 30 min at 37C in an orbital
shaker (New Brunswick Scientific, Edison, NJ, USA) at
150 rpm. The isolated adipocytes were filtered through a
fine plastic mesh (150 lm), transferred to a conical (50 mL)
plastic tube, and washed three times in the same buffer
without collagenase (25 mL per wash). After the final wash,
the medium was thoroughly aspirated, and the cells were
harvested as described later. Adipocyte viability was tested
with trypan blue and cell number was determined as
previously described [19].
Melatonin and adipocyte circadian metabolism
Adipocyte incubation
Incubations were performed in culture flasks (25 cm2 of free
surface area). From the resultant adipocyte pack, an aliquot
of cells was added to the flasks containing 10 mL of
incubation buffer (similar to the digestion buffer except that
collagenase was absent and 10% fetal bovine serum was
added) in order to obtain a final cell concentration close to
4 · 105 cells per mL, followed by the addition of 1 nM
melatonin (similar to the physiological plasma content found
at its nocturnal peak in living rats [20]). Low or basal
concentrations (0.5 nM) of Ins was also added in the
beginning of the incubations in the groups denominated
ÔInsÕ in the figures, and was continuously present in the
incubations until being replaced by another medium con-
taining higher concentration of Ins (2.5 nM) for the last 6-hr
period that precedes the experiments, as described in the
following. All drugs were purchased from Sigma.
Incubations were carried out at 37C in a 5% CO2
atmosphere up to 114 hr. All the incubations started at
20:00 hr. The medium and the hormone content were changed
every 24 hr. Melatonin was added intermittently for 12 hr in a
24-hr period. Two incubation protocols were followed: (i)
12h+/12h-, cells started the incubation in the presence of
melatonin; and (ii) 12h-/12h+, in its absence for the next
12 hr. After this period, the medium containing melatonin was
washed out and replaced by the same medium without the
hormone, while in the other group, melatonin was added to
the flask. Every 12 hr, the procedure of washing out and
adding melatonin was repeated in order to fulfill a complete
24 hr melatonin+/melatonin- cycle. This procedure was
interrupted when four and a half cycles in the intermittent
presence of melatonin (alone or in combination of low
concentrations of Ins) were achieved (108 hr of incubation).
At this moment, the adipocytes were washed, the medium was
replaced and an additional 6-hr period of incubation pro-
ceeded (in the presence of melatonin for cells, which in the
preceding 12 hr were not incubated with the hormone, while
for the other group bathed with melatonin, the medium was
replaced without it). Therefore, mimicking the night with the
addition of melatonin or the day with its absence, we intended
to induce changes in the cell metabolism. The expressions
Ôinduced nightÕ and Ôinduced dayÕ established here will describe
the events occurring in the presence or absence of melatonin,
respectively. Higher concentration of Ins (2.5 nM) also
replaced the low or basal concentrations in the corresponding
groups of treatment for the last 6 hr period. In the end, cells
were prepared for metabolic studies (lipogenesis and lipolysis),
and samples were also removed for evaluation of the glucose-
6-phosphate dehydrogenase (G6PDH), fatty acid synthase
(FAS), hormone sensitive lipase (HSL), Bmal-1b, Clock, Per-1
and Cry-1 mRNA expression. Aliquots of the medium
were collected for the determination of leptin released by
radioimmunoassay.
Reverse transcriptase-polymerase chain reaction
(RT-PCR) assay for evaluation of clock genes and
enzyme expression
All reagents were purchased from Invitrogen. Total RNA
was extracted from isolated adipocytes using guanidine
423
Alonso-Vale et al.
isothiocyanate based TRIzol reagent according to the
manufacturerÕs specifications, and quantified spectrophoto-
metrically at 260 nm with acceptable 260/280 ratios
between 1.7 to 2.0. The RNA quality was also checked by
1% agarose gel electrophoresis, stained with 1 lg/mL
ethidium bromide. Superscript II RT was used to reversely
transcribe 5 lg of total RNA isolated using an oligo-
(dT)10n primer in a total reaction volume of 50 lL. After
the RT reaction, 4 lL aliquots were used as cDNA
template for PCR amplifications with Taq DNA polymer-
ase and 2.5 mM MgCl2. The thermal cycling parameters
used were: 25 cycles, 45 s at 95C, 45 s at 60C and 30 s at
72C using a Gradient Mastercycler (Eppendorf AG,
Hamburg, Germany). The primer sequences used and the
respective fragments obtained were as follows: rat G6PDH:
sense, 5Õ-CCA TAG ACA TAC GGG ATG GG-3Õ; and
antisense, 5Õ-CAA CCC TGA GGA GTC TGA GC-3Õ, 216
base pair (bp); rat FAS: sense, 5Õ-AAG CCA GGA AGA
GTG GGA GAG C-3Õ; and antisense, 5Õ-GGT TGG CAG
CAG GAT ACA CCG-3Õ, 311 bp; rat HSL: sense, 5Õ-AGC
CTA CCC AGT TAC CAC CCT G-3Õ; and antisense,
5Õ-AAG GAG TTG AGC CAT GAG GAG G-3Õ, 240 bp;
rat BMAL-1b: sense, 5Õ-TGC CGA GGA AAT CAT GGG
AAT C-3Õ; and antisense, 5Õ-CGC ATC TGC TTC CAA
GAG GCT CG-3Õ, 337 bp; rat Clock: sense, 5Õ-TCC CGA
TTC CAT CCA GTA TGC C-3Õ; and antisense, 5Õ-CTG
TGC CTG CTG CTG TTG GTG-3Õ, 221 bp; rat Per-1:
sense, 5Õ-ACC CAA GGA CCG AGA CCA TCA C-3Õ; and
antisense, 5Õ-CCA GTT TCG ACA GTC TGT GGC-3Õ,
461 bp; rat Cry-1: sense, 5Õ-AAC GGA GGG CTC ATG
GGC TAT G-3Õ; and antisense, 5Õ-TGC TCT GTC GCT
GGA CTT TGG G-3Õ, 232 bp. As an internal control for
the integrity of the mRNA in each sample, additional
oligonucleotide primers for rat RPL-37a: sense, 5Õ-CAA
GAA GGT CGG GAT CGT-3Õ; and antisense, 5Õ-ACC
AGG CAA GTC TCA GGA GGT G-3Õ, resulting in a
product of 290 bp, were included in the PCR reactions.
Aliquots (5 lL) from the RT reactions were used in the
quantitative measurements. The reaction products were
separated by agarose gel (2.0%) electrophoresis, stained
with ethidium bromide, and analyzed by scanning densi-
tometry [Eagle Eye – Stratagene, model 401304, software
Eagle Sight 3.2 (La Jolla, CA, USA)]. Samples were
normalized to the quantity of RPL-37a signal produced
by RT-PCR and presented as arbitrary units of the various
genes mRNA relative to control. The DNA amplification
product levels were carefully measured on the linear portion
of the amplification curve.
D-[U-14C]-glucose incorporation into lipids
From a 10% adipocyte suspension in Krebs/Ringer/phos-
phate buffer pH 7.4, with 1% BSA and 2 mmol/L glucose, at
37C and saturated with a gas mixture of CO2 (5%)/O2
(95%), 450 lL aliquots were transferred to polypropylene
test tubes containing 5 lL (0.05 lCi per tube) of d-[U-14C]-
glucose, in the presence or absence of Ins (10 nmol/L). These
samples were then incubated for 1 hr at 37C in a water bath
(final volume = 500 lL). After incubation, the mixture was
acidified with 0.2 mL H2SO4 (8 N) and incubated for
additional 30 min. At the end of incubation, the reaction
424
mixture was treated with 2.5 mL of DoleÕs reagent (isopro-
panol: n-heptane: H2SO4, 4:1:0.25 v/v/v) for lipid extraction
[18]. The results were expressed as nanomoles of glucose
incorporated into lipids per 106 cells per hr.
Measurement of lipolysis
From a 20% adipocyte suspension in Krebs/Ringer/Phos-
phate buffer pH 7.4, with 1% BSA, 170 lL aliquots were
transferred to microfuge tubes (1.5 mL) containing 20 lL
of adenosine deaminase (0.2 U/mL) and were incubated at
37C for 5 min to allow the degradation of endogenous
released adenosine, which is a potent inhibitor of lipolysis
[21]. After this period, these samples were incubated for
1 hr at 37C in the presence or absence of 10 lL of
isoproterenol (10)5 mol/L), in a final volume of 200 lL. At
the end of incubation, the reaction was blocked by moving
the tubes to a cold water bath followed by centrifugation at
3500 g for 5 min at 4C. The infranatant was carefully
transferred to microtubes containing 150 lL of silicone oil
and re-centrifuged at 3500 g for 2 min. The glycerol content
of the incubation medium was measured using an enzy-
matic-colorimetric assay (Free glycerol determination kit;
Sigma). It was used as an index of lipolysis and was
corrected to be expressed as nanomoles per 106 cells per hr.
Leptin secretion
The leptin levels were measured in the culture medium by
radioimmunoassay (RIA) using a commercially available
Rat leptin kit (Linco Research Inc., St. Charles, MO,
USA). The test sensitivity is 0.5 ng/mL and the intra-assay
coefficient of variation was less than 5%. The secretion rate
was expressed as picograms of leptin released per 106 cells
per 6 hr.
Statistical analysis
Statistical analysis was performed by two-way ANOVA
followed by Bonferroni post hoc tests using GraphPad
Prism version 4.03 for Windows (GraphPad Software, Inc.,
San Diego, CA, USA). The level of significance was set at
P < 0.05. Data were presented as mean ± SEM.
Results
Figure 1 illustrates the clock gene mRNA expression
during the induced night and the induced day in adipocytes
cultured for 114 hr in the intermittent presence or absence
of melatonin (alone or in combination with Ins). The results
show an increase (approximately 55%, n = 14, P < 0.05)
on the Clock mRNA expression during the induced night
(Fig. 1A). In the presence of Ins, this effect was not
observed. A similar pattern of response (however not
statistical significantly, P = 0.06) was observed to Bmal-1b
clock gene expression (Fig. 1B). On the other hand, the
clock gene Per1 mRNA expression was higher (60%,
n = 14, P < 0.05) in the presence of Ins during
the induced night (Fig. 1C). Finally, the synchronization
of the adipocytes culture by melatonin did not modulate the
expression of the clock gene Cry1 (Fig. 1D).
 Melatonin and adipocyte circadian metabolism

(A) * (B)

Bmal-1b /RPL mRNA

2

Clock /RPL mRNA

1.8 2
1.8
1.6
1.6

(AU)
1.4

(AU)
1.4
1.2 1.2
1 1
0.8 0.8
0.6 0.6
Ins (2.5 nM) – + – + Ins (2.5 nM) – + – +
Mel (1 nM) + + – – Mel (1 nM) + + – –
(C) (D)
*

Per-1 /RPL mRNA

Cry-1 /RPLmRNA
2 2
1.8 1.8
1.6 1.6

(AU)

(AU)
1.4 1.4
1.2 1.2
Fig. 1. Effect of melatonin (associated or 1 1
not to insulin) on clock genes mRNA 0.8 0.8
expression in primary culture of adi- 0.6 0.6
pocytes. Values are mean ± S.E.M. of 14 Ins (2.5 nM) – + – + Ins (2.5 nM) – + – +
independent experiments and are Mel (1 nM) + + – – Mel (1 nM) + + – –
expressed as arbitrary units (AU).
*P < 0.05 induced night versus induced Induced Induced Induced Induced
day. night day night day

Figure 2 presents the rates of glucose incorporation adipocytes to melatonin, an inhibition (approximately
into lipids. Evaluating the difference between the maximal 50%, n = 8, P < 0.05) was observed on the basal and
(or Ins stimulated) and the basal rates (DMax-Bs), we isoproterenol-stimulated lipolysis during the induced night.
found out a significant (n = 8, P < 0.05) increase in the Again, Ins and melatonin interacted to induce this effect.
Ins responsiveness during the induced night compared With regard to the gene expression of the HSL lipolytic
with the induced day when Ins was present in the enzyme, we verified that the circadian exposure of adipo-
incubations. cytes to melatonin (associated or not to Ins) did not modify
Data about the G6PDH and FAS enzyme mRNA the HSL mRNA content (Fig. 5).
expression are illustrated in Fig. 3. A tendency to During the induced night, melatonin also interacted with
increase was observed in the G6PDH enzyme mRNA Ins to promote an enhancement in leptin release (130%,
expression, which however, was not statistically signifi- n = 8, P < 0.05) (Fig. 6).
cant (Fig. 3A). A significant rise of approximately 70%
(n = 10, P < 0.05) in the expression of FAS only in
adipocytes treated with Ins during the induced night is
Discussion
shown (Fig. 3B), where the effects of Ins and melatonin This work was designed to simulate the circadian exposure
were additive. to melatonin in an in vitro model of adipocyte primary
The spontaneous (basal) and isoproterenol stimulated culture. This goal was achieved by incubating cells with
lipolytic responses were also evaluated. In accordance melatonin in an intermittent, rhythmical and circadian-like
with the data (Fig. 4), after intermittent exposure of the presentation of the hormone.
In mammals, the internal daily cycles are driven by a
*
central circadian clock (localized in the SCN) whose
D-[U14C]-glucose lipid incorporation

50.0 activity is based on the cell-autonomous rhythmic expres-

(nanomoles/106 cells/hr)

40.0 sion of clock genes. It is not completely clear however

how some peripheral cells are able to follow the rhythmic
(ΔMax-Bs)

30.0

20.0
signals spread out from this central oscillator. In a recent
10.0
work [22], it was shown that the cycling expression of the
clock gene Period1 (Per1) in rodent pituitary pars tuber-
0.0
Ins (2.5 nM) – + – + alis (PT) cells depended on the nocturnal activation of
Mel (1 nM) + + – – melatonin MT1 receptors. Removal of the pineal gland,
Induced night Induced day which is the endogenous source of circulating melatonin,
completely abolished Per1 signal in hamster PT cells [23,
Fig. 2. Effect of melatonin (associated or not to insulin) on 24]. On the other hand, Per1 mRNA signal was dimin-
incorporation of D-[U-14C]-glucose into lipids in primary culture of ished by the prolongation of melatonin presence, mimick-
adipocytes. Bs, basal; Mx, maximally stimulated by insulin. Values
are mean ± S.E.M. of eight independent experiments performed in
ing long nights as seen in winter days [23, 24]. Moreover,
duplicate and are expressed as nanomoles of glucose incorporated it was also reported [25] that the rhythmic expression of
into lipids per 106 cells per hr. *P < 0.05 induced night versus another clock gene, Cry1, is directly induced by melatonin
induced day. in the PT.

425
Alonso-Vale et al.

(A) liver) and Ins resistance (in fat) [4]. As reported in a recent
290 bp - RPL review [1], this experimental Genetics (Chronogenetics)
begins to be applied to dissect ways in which specific genes
encoding clock proteins affect energy balance and meta-
216 bp - G6PDH
bolic homeostasis.
1.8 Here, we measured the glucose incorporation into lipids
in response to Ins after a circadian exposure of the
G6PDH /RPL mRNA

1.6 adipocytes to melatonin. Glucose is an important lipogenic

1.4 substrate as it allows the synthesis of pyruvate and glycerol-
(AU)

1.2 3-phosphate that will be transformed by the action of

several enzymes, including the acetyl-CoA carboxylase and
1
FAS, in FA that will be stored as triacylglycerol into lipid
0.8 droplets within the adipocytes. The required NADPH for
0.6 the FA synthesis is produced by both G6PDH (the first
Ins (2.5 nM) – + – + enzyme of the pentose–phosphate pathway) and the malic
Mel (1 nM) + + – – enzyme [30]. These anabolic pathways are mainly under the
Ins control, a lipogenic hormone that among other actions
(B)
elevates the mRNA expression and activity of lipogenic
enzymes [31].
290 bp - RPL The lipogenic response of cells incubated with Ins and
melatonin (induced night) revealed a rise in the glucose
311 bp - FAS incorporation into lipids which was not observed during the
induced day. We have already demonstrated an interaction
* between melatonin and Ins, i.e. a modulation of the Ins
action by melatonin on metabolic responses of adipocytes
3.3
in pinealectomized rats [12–15].
2.9
FAS /RPL mRNA

Moreover, we verified a significant increase in the

2.5 lipogenic FAS mRNA expression and a tendency to a rise
2.1
(AU)

in the G6PDH expression in the Ins-treated adipocytes

1.7
1.3
0.9
0.5
Ins (2.5 nM) – + – +
Mel (1 nM) + + – –
Induced night Induced day
Fig. 3. Effect of melatonin (associated or not to insulin) on glu-
cose-6-phosphate dehydrogenase and fatty acid synthase mRNA
expression in primary culture of adipocytes. Values are mean ±
S.E.M. of 10 independent experiments and are expressed as arbi-
trary units (AU). *P < 0.05 induced night versus induced day.
In the present work, it was shown that the rhythmic
exposure of cultured adipocytes to melatonin temporally
influenced the rhythmic expression of the clock genes
Clock, Bmal1 and Per 1 and the peaks occurred during the
in vitro induced night.
The link between circadian rhythms and metabolic
activity has long been studied in vivo [26–29]. In a recent
work, it was shown that two essential components of the
circadian clock (Clock/Bmal) were involved in the diurnal
variation of glucose and triacylglycerols [4]. Furthermore,
Bmal1 was shown to regulate adipogenesis and lipid
synthesis [6]. Clock mutant mice developed abnormalities
in the timing of food intake, a decrease in energy expen-
diture and were hyperphagic, causing a significant incre-
ment in adiposity [5]. Moreover, these animals developed
abnormalities in carbohydrate metabolism, which might
have involved a mixed picture of Ins hypersensitivity (in
during the induced night.
This circadian-like distribution of the adipose tissue
metabolic function is expected in the intact animal (rats)
synchronized to the light/dark cycle; the night is the time of
feeding and, therefore, the moment of a greater input of
nutrients which demands a more adapted and appropriated
metabolic setting for energy storage.
Lipolysis, the lipid mobilization from adipocytes, con-
sists of the triacylglycerol hydrolysis and the free FA and
glycerol release. This process is triggered by lipolytic
hormones, mainly catecholamines, acting through beta-
adrenoreceptor activation and represents an important
mechanism of white adipose tissue mass regulation [32].
In this regard, our findings showed that the spontaneous
lipolytic activity during the induced day was more intense
than during the induced night. There was a synergism
between melatonin and Ins to provoke an inhibition on
lipolysis activity during the induced night. By analogy,
the upregulation of the lipolytic activity during the
induced day and its strong attenuation during the night
is also expected in rats synchronized to the natural
circadian cycle, as at night time, the storage of energy
becomes more efficient if, at the same time, lipid
mobilization is attenuated.
Concerning the lipolytic enzyme HSL mRNA expression,
we verified that it did not change in response to the
adipocyte exposure to melatonin. Nevertheless, the lipolytic
response was highly modulated by the presence and the
absence of melatonin.
It was previously shown that the modulation of
lipolytic activity is rarely followed by changes in the
HSL enzyme expression, but in consequence to changes

426
 Melatonin and adipocyte circadian metabolism

basal basal
isoproterenol isoproterenol *

4000.00

(nanomoles/106 cells/hr)
3500.00

Glycerol released
3000.00

2500.00

2000.00

Fig. 4. Effect of melatonin (associated or 1500.00

not to insulin) on lipolysis in primary
culture of adipocytes. Values are 1000.00
mean ± S.E.M. of eight independent
Ins (2.5 nM) – – + + – – + +
experiments performed in duplicate and
are expressed as nanomoles of glycerol Mel (1 nM) + + + + – – – –
released per 106 cells per hr. *P < 0.05
induced night versus induced day. Induced night Induced day

- RPL
dent PKA signaling [34, 35]. As the main path to
290 bp
melatonin signal transduction is through its association
240 bp - HSL with Gi protein-coupled receptors [36, 37], it is possible
that the presence of melatonin, by lowering the intracy-
2
HSL /RPL mRNA

toplasmatic cAMP content, inhibits directly the HSL

1.5 activity, and thus, the lipolytic activity in cells without
the involvement of clock genes. This hypothesis however
(AU)

1
needs future investigation.
0.5 Lastly, but not less important, is the participation of
0
adipose triglyceride lipase (ATGL), which has been
Ins (2.5 nM) – + – +
described as a protein involved in FA mobilization in
rodents [38]. In the present work, we did not address its
Mel (1 nM) + + – –
contribution to the phenomenon. However, we cannot
Induced night Induced day
exclude that melatonin could exert a modulation in the
process.
Fig. 5. Effect of melatonin (associated or not to insulin) on hor-
mone sensitive lipase mRNA expression in primary culture of We previously showed that pinealectomy induced a daily
adipocytes. Values are mean ± S.E.M. of 14 independent experi- modification on the adipocyte metabolic pattern resulting
ments and are expressed as arbitrary units (AU). in an inappropriate balance between fuel storage and
mobilization in accordance with the daily rest–activity
cyclic demands [12]. Moreover, melatonin absence impaired
* the promptness of metabolic adaptation, which is extremely
2500
necessary for the animal to face challenging conditions
(such as exercise [14] and starvation [13]). In addition, the
pg/106 cells/6 hr

2000
daily secretion of Ins stimulated by glucose intake [15] and
Leptin

1500 the Ins secretion by isolated pancreatic islets [39] are

1000 impaired in pinealectomized rats. Taking into account these
500
data and the demonstration that melatonin is able to
temporally allocate certain adipose tissue metabolic func-
0
tions to restricted periods of the day in vitro, it can be
Ins (2.5 nM) – + – +
hypothesized that the pineal gland, through its daily
Mel (1 nM) + + – – rhythmic production of melatonin, might act as an internal
Induced night Induced day
synchronizer of the energy metabolism, acting as a link
between the external environment and the animal internal
Fig. 6. Effect of melatonin (associated or not to insulin) on leptin
demands.
release in primary culture of adipocytes. Values are mean ±
S.E.M. of eight independent experiments and are expressed as We have also demonstrated that the interaction between
picograms of leptin released per 106 cells per 6 hr. *P < 0.05 melatonin and Ins not only comprises the metabolic effects,
induced night versus induced day. but it can also embrace other adipocyte functions, like its
ability to acutely produce leptin [16, 17]. Here, we
in its activity, which depends on the degree of enzyme confirmed the synergism between melatonin and Ins to
phosphorylation [33]. The HSL activity and its state of promote leptin secretion. In addition, the presence of
phosphorylation is positively modulated by cAMP-depen- melatonin was able to preserve the circadian rhythm of

427
Alonso-Vale et al.
leptin release, i.e. the highest levels during the induced night
as observed in vivo [40, 41].
In conclusion, these data suggest that melatonin modu-
lates the rhythmic expression of clock genes and regulates
some aspects of the adipocyte biology. Whether there is a
correlation between the two effects induced by the in vitro
melatonin synchronization remains to be investigated.
Moreover, understanding the mechanisms underlying the
interaction between the circadian role of melatonin secre-
tion and the modulation of adipose tissue metabolism could
contribute towards establishing and improving therapies to
control disturbances of adipose tissue biology, such as those
seen in aging, obesity and diabetes mellitus.
Acknowledgement
This work was supported by Sao Paulo State Research
Foundation (FAPESP), grant No 04/06767-2 and by
fellowship no. 04/14696-8 from FAPESP.
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