JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2005, 56, Supp 6, 91–99
www.jpp.krakow.pl
K. ZWIRSKA-KORCZALA1, J. JOCHEM1, M. ADAMCZYK-SOWA1, P. SOWA1,
R. POLANIAK2, E. BIRKNER2, M. LATOCHA3, K. PILC1, R. SUCHANEK1
INFLUENCE OF MELATONIN ON CELL PROLIFERATION,
ANTIOXIDATIVE ENZYME ACTIVITIES AND LIPID PEROXIDATION
IN 3T3-L1 PREADIPOCYTES - AN IN VITRO STUDY
Department of Physiology, Zabrze, 2Department of Biochemistry, Zabrze, 3Department of Molecular
Biology, Biochemistry and Biopharmacy, Sosnowiec, Medical University of Silesia, Katowice,
Poland, 4Department of Physiology, Jagiellonian University Medical College, Kraków, Poland
1
Melatonin, acting via MT1, MT2 and MT3 membrane receptors, influences central and
peripheral regulatory mechanisms of energy homeostasis in mammals. In peripheral
tissues, it evokes the pro-proliferative effect in a number of normal cells. Moreover,
this hormone inhibits lipolysis in subcutaneous adipocytes in vitro and reduces free
oxygen metabolites-induced damage acting directly, as a free radical scavenger, and
indirectly, by stimulation of antioxidative enzyme activities. The aim of the study was
to examine the effects of melatonin on cell proliferation, antioxidative enzyme
activities and malondialdehyde (MDA) concentration in 3T3-L1 preadipocyte cell
culture. We found that melatonin (10-3 and 10-6 M/L) stimulated cell proliferation in
dose- and time-depending manner, and this effect was inhibited by a relatively selective
MT2 receptor antagonist - luzindole (10-4 M/L). Melatonin, increased activities of
manganese containing and copper-zinc containing superoxide dismutase (MnSOD and
Cu/ZnSOD) isoenzymes, catalase, glutathione reductase and glutathione peroxidase
after 24 h of incubation. In contrast, after 48 h of incubation, activities of all studied
enzymes were lower than in the control group. There were no changes in MDA
concentrations after 24 h of incubation, whereas, in melatonin-treated media, after 48
h of the experiment, MDA level was significantly decreased. Our results demonstrate
that melatonin, acting via MT2 receptors, stimulates proliferation of 3T3-L1
preadipocytes and this action could be due to the enhancement in antioxidative enzyme
activities and attenuation of lipid peroxidation by this indole.
K e y w o r d s : 3T3-L1 preadipocytes, melatonin, luzindole, antioxidative enzyme activities,
malondialdehyde, cell proliferation
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INTRODUCTION
The adipose tissue metabolism is influenced mainly via humoral factors, such as
leptin, cholecystokinin, ghrelin, secretin and adiponectin and neural mechanisms,
for instance, the activation of the sympathetic nervous system (1-4). Melatonin (Nacetyl-5-methoxytryptamine) is a pineal hormone with structural similarities to 5hydroxytryptamine (5). It participates in the regulation of energy balance, body
weight and fat mass in mammals, however, particular mechanism of action of this
indole on cell of adipose tissue remains still not clear (6). An influence of melatonin
on body mass regulation is mediated predominantly centrally, and results in change
of metabolic rate via activation of the sympathetic nervous system and/or altered
feeding behavior (7). Moreover, melatonin is able to act on target tissues via MT1,
MT2 and MT3 membrane receptors and retinoid nuclear receptor, called RZR/ROR
receptor (8). Recent studies revealed the expression of MT1 and MT2 receptor
mRNA in human brown adipose cell line PAZ6 and in white adipose tissue cells (8).
Activation of both types of receptors results in the inhibition of adenyl cyclase and,
consequently, in a decrease in 3',5'-cAMP levels. Experimental data demonstrate a
decrease in glycogenesis, followed by an increase in plasma glucose and glucagon
concentrations and lowered insulin level after pinealectomy in rodents (9).
Moreover, melatonin decreases GLUT4 protein concentration and glucose uptake
in PAZ6 adipocytes (8).
Melatonin influences cell proliferation and differentiation, and the effect of
stimulation or suppression of cell division appears to depend on its concentration
and cell type examined (10). Anti-proliferative effects of melatonin have been
demonstrated in vivo and in vitro in a number of cancer cells (11, 12). In contrast,
it stimulates proliferation of normal cells, for example human bone cells (HOBM) (13) and splenocytes (14).
Melatonin is capable of reducing of free radical damage acting directly as a
free radical scavenger, and indirectly, by stimulation of antioxidant enzyme
activities (15). It effectively protects against lipid peroxidation and decreases the
synthesis of malondialdehyde (MDA) which is an end-product of lipid
peroxidation (16, 17). Interestingly, many studies demonstrate that melatonin
influences antioxidant enzyme activities under conditions of elevated oxidative
stress, for instance after administration free radicals generating agents, such as
lipopolysaccharide (LPS), 6-hydroksydopamine or porphyrins (17).
Luzindole (N-acetyl-2-benzyltryptamine) is a relatively selective MT2
receptor antagonist, with a 25-fold higher affinity for the MT2 than for the MT1
receptor subtype. Recent studies revealed that in both in vitro and in vivo
conditions, it attenuates the ability of melatonin to enhance splenic lymphocyte
proliferation (14, 18), however, there is no information about melatonin effects on
other cell types. Thus, the aim of our present study was to examine the influence
of melatonin and luzindole on proliferation of preadipocyte 3T3-L1 cells.
Moreover, we studied the effect of melatonin on antioxidative enzyme activities
and MDA concentration in 3T3-L1 cells media.
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MATERIAL AND METHODS
Cell culture
3T3-L1 cells are preadipocytes obtained by Green and Kehinde from murine 3T3 fibroblasts by
cloning clusters of cells filled with fat droplets (19). 3T3-L1 cells were purchased from ATCC
(American Type Culture Collection, Rockville, MD, USA). Preadipocytes were plated at a density
of 1 x 106 cells per 25 cm2 flask and cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin under
an atmosphere of 95% air and 5% CO2 at 37°C.
Experimental protocol
3T3-L1 cells, with or without melatonin, were cultured for 24 or 48 h; and incubation media
were not changed during this time. After incubation periods, media were removed, centrifuged and
freezed until enzymatic measurements. Cells were trypsinized and number of cells was estimated
by direct counting, using a net micrometer with 10x objective and 10x ocular, on three square (1
mm2). Results were calculated as cell number (cells/ml) as average of count per square x 10000.
Viability of cells was estimated by trypan blue staining. The cell monolayer was discarded,
harvested and treated with trypan blue.
Enzymatic assays
Manganese-containing (MnSOD) and copper-zinc-containing superoxide (Cu/ZnSOD)
dismutase isoenzyme (EC 1.15.1.1) activities were estimated according to Oyanagui and expressed
in nitrite units/ml (NU/ml) (20, 21). Glutathione peroxidase (GSH-Px) (EC 1.11.1.19) activity was
measured according to Paglia and Valentine using enzymatic conjunction with glutathione reductase
(22). Catalase (CAT) (EC 1.11.1.6) activity was measured according to the kinetic method of Aebi
and expressed in IU/ml of medium (23). Glutathione reductase (GSSH-Rd) (EC 1.6.4.2) assay was
based on the oxidation of NADPH to NADP+ (24).
MDA concentrations were determined according to the colorimetric method by Ohkawa et al.
using the reaction with thiobarbituric acid (25).
Drugs
The following agents were used: melatonin, luzindole - Sigma Chemical CO. (St. Louis, MO,
USA), trypsin, trypan blue, Penicillin-Streptomycin Mixture, Dulbecco's Modified Eagle's Medium
and Fetal Bovine Serum (Bio Whittaker, Verviers, Belgium). Melatonin was dissolved in a minimum
amount of ethanol (95%) and diluted in the medium to obtain a stock solution of 10-3 M/L. Additional
dilutions were done in the medium to achieve a final solution of 10-6 M/L. Luzindole was also
dissolved in 0.2 ml of ethanol (95%), and then diluted with medium to reach the final concentration
of 10-4 M/L. Luzindole was added 30 min before subsequent exposure of the cells to melatonin.
Statistics
All values are given as means ± SEM. Comparison of differences between the mean values
were made using ANOVA and Student's t-test. Differences with P < 0.05 were considered as
statistically significant.
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RESULTS
Influence of melatonin and luzindole on 3T3-L1 cells proliferation
Melatonin at concentrations of 10-3 M/L and 10-6 M/L stimulated proliferation
of 3T3-L1 cells in dose- and time-depending manner from 6 to 50% compared to
the control (Fig. 1). There were no differences between the control and
melatonin-treated groups in regards to viability of the cells.
Luzindole (10-4 M/L) given alone slightly, but not significantly, increased
3T3-L1 cells number in comparison to the control (Fig. 1). In contrast, pretreatment with this antagonist in the melatonin-treated groups resulted in an
inhibition of the pro-proliferative effect of this hormone at 24 and 48 h of
incubation (Fig. 1).
Fig. 1. Influence of melatonin (10-3, 10-6 M/L) and luzindole (10-4 M/L) on cell count of 3T3-L1
preadipocytes; data are given as mean ± SEM; * P<0.05 vs. the coresponding value in the control
group; in luzindole pretreated groups # P<0.05 vs. melatonin treated groups.
Influence of melatonin on antioxidative enzyme activities in 3T3-L1 cells media
At 24 h of incubation, there were significantly higher media activities of
MnSOD, Cu/ZnSOD and CAT detected after treatment with lower melatonin dose,
while in media with higher melatonin concentration, the activities of all studied
enzymes were significantly increased in comparison to the control (Table I).
In contrast, after 48 h of incubation with melatonin (10-3 and 10-6 M/L),
activities of all studied enzymes, except for GSSG-Rd activity in melatonin
10-3 M/L concentration medium, were significantly lower than in the
corresponding control (Table I).
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Table I.Activity of manganese-containing (MnSOD) and copper-zinc-containing superoxide dismutase
(Cu/ZnSOD), glutathione peroxidase (GSH-Px), catalase (CAT), glutathione reductase (GSSG-Rd) and
levels of malondialdehyde (MDA) after 24 and 48h of incubation time with melatonin at concentrations
10-3 M/L (group 1a and 2a, respectively) and 10-6 M/L (group 1b and 2b, respectively) in 3T3-L1
preadipocyte cells culture medium; data are given as mean ± SEM from 6 experiments.
MnSOD
Cu/ZnSOD
GSH-Px
CAT
GSSG-Rd
(NU/ml medium)
(NU/ml medium)
(µmol NADPH2/
ml medium)
(IU/ml medium)
(IU/ml medium)
24h control
Group 1a
Group 1b
2.48±0.13
3.26±0.21*
4.57±0.23*
3.15±0.12
3.66±0.09*
3.85±0.2*
320.5±23.1
339.1±18.9
380.8±21.7*
86.17±6.58
156.78±11.7*
258.2±18.3*
2.37±0.12
2.37±0.09
3.82±0.14*
48h control
Group 2a
Group 2b
1.14±0.06*
0.48±0.02H
0.89±0.02H
3.31±0.08*
2.84±0.13H
2.26±0.04H
138.2±8.7*
45.5±3.9H
36.1±1.7H
157.9±9.98*
103.23±5.69H
95.7±4.38H
1.1±0.07*
1.35±0.22
1.41±0.07H
*P<0.05 vs. 24h control; HP<0.05 vs. 48h control
Influence of melatonin on MDA concentration in 3T3-L1 cells media
There were no differences in MDA concentrations in the studied media after
24 h of incubation, whereas after 48 h of incubation, MDA levels were
significantly lower in melatonin-treated cells as compared to the control (Fig. 2).
Fig. 2. MDA concentrations in media of 3T3-L1 preadipocytes after 24 and 48h of incubation time
with melatonin (10-3, 10-6 M/L); data are given as mean ± SEM; * P<0.05 vs. the control group.
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Moreover, the MDA concentrations in the media of control cells after 48 h of
incubation were significantly diminished as compared to those determined at 24
h of incubation (Fig. 2).
DISCUSSION
Our results demonstrate for the first time that melatonin acting via MT2
receptors stimulates the proliferation of 3T3-L1 preadipocytes. Moreover, we
show increased activities of antioxidative enzymes and decreased lipid
peroxidation in melatonin-treated 3T3-L1 cultures.
The cell line employed in our study offers an excellent model to study the
differentiation processes. During this process cells undergo a change from the
elongated fibroblastic shape to an oval form and accumulate small drops of lipids
within the cytoplasm (19). These lipid drops fuse into one large drop giving the
cell the aspect of a mature adipocyte of white adipose tissue (19).
Our study demonstrates that melatonin, applied at concentrations of 10-3 and
-6
10 M/L stimulates 3T3-L1 cells proliferation in dose- and time-depending
manner. Thus, the results confirm earlier findings that melatonin is able to
increase the proliferation in normal cells (10). In contrast, melatonin reveals an
antiproliferative effect in a number of cancer cells, including those of rat
pheochromocytoma PC12 cells (12).
We have shown that MT2 receptor antagonist luzindole (10-4 M/L), added 30
min before exposure of 3T3-L1cells to melatonin, abolished the pro-proliferative
effect of melatonin, which suggests the melatonin acting via receptor-dependent
mechanism of action. Similarly, melatonin enhanced the proliferative ability of
splenocytes in vitro, and this effect was significantly attenuated by luzindole (14,
18). Indeed, our results indicate that also in 3T3-L1 cells melatonin-induced
influence on proliferation rate is mediated via MT2 receptors. The particular
mechanism involved is not known, especially after results of the study by Sainz
et al., who demonstrated that melatonin caused decrease in expression of mRNA
for histone H4 in the rat thymus, and in this way, a marked fall in thymocytes
proliferation (26).
Results of the present study show that melatonin is able to influence oxidativeantioxidative balance in preadipocytes. Antioxidant properties of melatonin are
clearly recognized and they have been studied extensively during recent years (5,
15). Mechanisms of the antioxidant action of this indole include scavenge of
radicals and reactive species, induction of antioxidative enzyme activities and
inhibition of nitric oxide (NO) synthase activity (17, 27). On the other hand, free
radicals are able to alter antioxidant enzyme activities. It has been proposed that
moderate levels of toxic oxygen metabolites could induce an increase in
antioxidant enzyme activities, while very high levels of these reactants attenuate
these enzyme activities as a result of damage of the molecular machinery that is
required to induce these enzymes (8). Reactive oxygen species (ROS) are
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generated in vivo in extremely high amounts during the partial hypoxia and
subsequent reoxygenation, for example in haemorrhagic shock (28, 29), or upon
exposure to toxic environmental agents, such as those coming from ionizing
radiation (30, 31). The experimental evidence confirmed the role of melatonin as
a direct ROS scavenger and as an indirect antioxidant, since it stimulates
antioxidant enzyme activities (17, 32). Indeed, in the present study, we
demonstrated that melatonin affects antioxidative enzyme activities in media after
24 h incubation period of 3T3-L1 cell cultures. A number of studies reported that
luzindole abolishes melatonin effects in different species for instance, it produces
an augmentation of pancreatic MDA and 4-hydroxynonenal content, and evokes
marked increases of plasma levels of lipase, amylase and TNF-α in rats with
caerulein-induced pancreatitis (33).
Mechanisms of melatonin-induced effects are probably multidirectional. In
physiological concentrations (10-9 M/L), it increases mRNA levels of both SOD and
GSH-Px isoenzymes in time-dependent manner in rat pheochromocytoma PC12
cells and SK-N-SH neuroblastoma cells (34). Our study is in keeping with this
finding by demonstration that melatonin is able to increase activities of MnSOD,
Cu/ZnSOD and GSH-Px after 24 h of incubation. Similarly, exogenous melatonin
was found to increase the GSH-Px activity in the rat brain, liver and kidney (27).
Under normal conditions, melatonin has no effect on GSSG-Rd activity.
However, it protects the cells against a decrease in GSSG-Rd activity produced
by okadaic acid (35). Interestingly, in the present study, we have shown that
melatonin given at concentration of 10-6 M/L stimulates GSSG-Rd medium
activity observed after 24 h of incubation of preadipocytes.
Our results show that melatonin in both concentrations increases CAT activity
in 3T3-L1 cell medium after 24 h of incubation. Earlier papers suggested that
melatonin produced an enhancement of CAT activity in diabetic skin fibroblasts
(36). Moreover, in vivo studies have demonstrated that melatonin caused an
increase in CAT activity in rats treated with doxorubicin (37). In addition, it
prevents CAT inactivation by neutralization of alkyl-peroxyl radicals (38).
Interestingly, our results demonstrated the fall in activities of all studied
enzymes after 48 h of incubation with melatonin, which is difficult to explain. We
can only suggest that this effect can be associated with a long-lasting exposure to
melatonin and its direct action as a ROS scavenger. Lowered activities of
antioxidative enzymes could be related to a decrease in concentration of free
radicals generation induced by melatonin.
Free radicals-induced cell damage may be quantitatively determined be
measurement of MDA levels, which is an indicator of lipid peroxidation. Present
data show that there are no differences in MDA media levels after 24 h of incubation,
whereas melatonin had pronounced inhibitory effect on MDA media concentrations
after 48 h of incubation. The inhibitory effect of melatonin in MDA levels could by
attributed to the increased activities of antioxidative enzymes, which was much more
pronounced when 3T3-L1cells were incubated for 48h with this indole.
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In conclusion, our results suggest that melatonin stimulates proliferation of
3T3-L1 preadipocytes and that his effect is mediated via MT2 receptors. The
enhanced antioxidative enzyme activities and attenuation of the process of lipid
peroxidation can contribute to the melatonin-induced increase in proliferation rate
observed in 3T3-L1 preadipocytes in vitro.
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Author's address: Krystyna Zwirska-Korczala M.D.,Ph.D., Department of Physiology, Medical
University of Silesia, Jordana 19, 41-808 Zabrze, Poland