Food additives such as sodium sulphite, sodium benzoate and curcumin
inhibit leptin release in lipopolysaccharide-treated murine adipocytes in vitro
Christian Ciardi1, Marcel Jenny2, Alexander Tschoner1, Florian Ueberall3, Josef Patsch1,
Michael Pedrini1, Christoph Ebenbichler1 and Dietmar Fuchs2*
1Department of Internal Medicine I, Anichstrasse 35, Innsbruck Medical University, A-6020 Innsbruck, Austria
2Division of Biological Chemistry, Biocenter, Fritz-Pregl-Straße 3, Innsbruck Medical University, A-6020 Innsbruck, Austria
3Division of Medical Biochemistry, Biocenter, Fritz-Pregl-Straße 3, Innsbruck Medical University, A-6020 Innsbruck, Austria
(Received 7 January 2011 – Revised 7 June 2011 – Accepted 7 June 2011 – First published online 1 August 2011)
Abstract
Obesity leads to the activation of pro-inflammatory pathways, resulting in a state of low-grade inflammation. Recently, several studies have
shown that the exposure to lipopolysaccharide (LPS) could initiate and maintain a chronic state of low-grade inflammation in obese
people. As the daily intake of food additives has increased substantially, the aim of the present study was to investigate a potential influence
of food additives on the release of leptin, IL-6 and nitrite in the presence of LPS in murine adipocytes. Leptin, IL-6 and nitrite concentrations
were analysed in the supernatants of murine 3T3-L1 adipocytes after co-incubation with LPS and the food preservatives, sodium
sulphite (SS), sodium benzoate (SB) and the spice and colourant, curcumin, for 24 h. In addition, the kinetics of leptin secretion was analysed.
A significant and dose-dependent decrease in leptin was observed after incubating the cells with SB and curcumin for 12 and 24 h,
whereas SS decreased leptin concentrations after 24 h of treatment. Moreover, SS increased, while curcumin decreased LPS-stimulated
secretion of IL-6, whereas SB had no such effect. None of the compounds that were investigated influenced nitrite production. The
food additives SS, SB and curcumin affect the leptin release after co-incubation with LPS from cultured adipocytes in a dose- and timedependent
manner. Decreased leptin release during the consumption of nutrition-derived food additives could decrease the amount of
circulating leptin to which the central nervous system is exposed and may therefore contribute to an obesogenic environment.
Key words: Adipocytes: Adipokines: Antioxidants: Leptin
Changes in lifestyle including overnutrition and physical inactivity
have led to a rise of excess body weight during the last
decades. Severe obesity is the sixth most important risk factor
contributing to the overall burden of diseases worldwide due
to the main adverse consequences such as CVD, metabolic
disorders and several types of cancer(1). It is well recognised
that obesity is associated with a state of chronic inflammation
due to different pathogenic mechanisms(2). Lipid accumulation
leads to adipocyte hypertrophy, cellular stress,
increased lipolysis and activation of pro-inflammatory pathways,
resulting in an increased production and secretion
of pro-inflammatory cytokines by adipocytes and macrophages
(3). Moreover, new evidence supports the idea that
some key aspects of the mammalian host–gut microbial
relationship could play a major role in obesity. LPS is continuously
produced within the gut by the death of Gram-negative
bacteria and is absorbed into the intestinal capillaries to be
transported by lipoproteins leading to a state of metabolic
endotoxaemia, which seems to be a potential pathway to
initiating and maintaining a state of low-grade inflammation
associated with obesity(4 – 6).
Among the inflammation-related cytokines, adipocytes
secrete various adipokines, which play a major role in energy
homeostasis by exerting multiple favourable effects on lipid
and carbohydrate metabolism(7). It is recognised that these beneficial
effects are removed in states of severe obesity. Leptin,
which is produced in proportion to fat stores, plays a crucial
role in the regulation of appetite, food intake and energy
homeostasis by signalling the information of available energy
within the central nervous system(8 – 10). Various studies have
investigated a potential effect of food composition on circulating
leptin levels over the last years(11 – 13). However, studies
analysing the influence of food preservatives, which are
widely used to preserve aliments, are scarce.
The addition of food preservatives such as antioxidants and
food colourants prevents the growth of bacteria, fungi and
* Corresponding author: D. Fuchs, fax þ43 512 9003 73330, email dietmar.fuchs@i-med.ac.at
Abbreviations: LPS, lipopolysaccharide; SS, sodium sulphite; SB, sodium benzoate.
British Journal of Nutrition (2012), 107, 826–833 doi:10.1017/S0007114511003680
q The Authors 2011
British Journal of Nutrition
other micro-organisms. Furthermore, these additives decelerate
oxidation of fats preventing rancidity, and inhibit ageing
and discolouration of food. The daily intake of food has
increased substantially and is responsible for the dramatic
rise of the prevalence of obesity inducing metabolic disorders
such as insulin resistance, diabetes mellitus, inflammation and
blood lipid disorders(1,14). As a consequence of increased food
ingestion, the intake of antioxidant food supplements has
increased exponentially.
The aim of the present study was to analyse the effect of the
widely used food preservatives sodium sulphite (E221, SS),
sodium benzoate (E211, SB) and the spice and food colourant
curcumin (E100) on the leptin release of unstimulated and
LPS-stimulated adipocytes, in order to investigate a potential
contribution of these diet-derived agents to the development
of obesity-related metabolic perturbations.
Experimental methods
Cell culture
Murine 3T3-L1 fibroblasts cells were obtained from the
American Type Culture Collection (ATCC-CL-173; Manassas,
VA, USA) and cultured in 5% CO2 at 378C. The cells were
maintained in Dulbecco’s modified Eagle’s medium (GIBCO,
Karlsruhe, Germany) supplemented with 5mM-glucose, 10%
heat-inactivated bovine serum, 2mM-L-glutamine, 100 U/ml
penicillin and 100 mg/ml streptomycin. The medium was
changed every 2 d. At 2 d after reaching confluence, the
pre-adipocytes were treated with a medium to induce differentiation
as described previously(15). In short, 2 d post-confluence
cultured cells were supplemented with 0·5mM-1-methyl-3-
isobutylmethylxanthine þ 1·0mM-dexamethasone þ 10mg/ml
insulin þ 10% fetal bovine serum for another 2 d. Then
the cells were kept in a culture medium with 10 mg/ml
insulin þ 10% fetal bovine serum for another 2 d. After
differentiation, the culture medium was replaced every
2nd day using a culture medium with 10% fetal bovine
serum. As determined by light microscopy, over 90% of
the cells that were used for the experiments were differentiated.
All experiments were performed within 10–14 d postdifferentiation.
Experimental procedures
First, the adipocytes were incubated with increasing concentrations
of SS, SB and curcumin for 24 h to analyse a possible
dose-dependent effect on leptin secretion. Accordingly, the
cells were washed twice with PBS and incubated with 1mMand
10mM-SS, 10mM- and 20mM-SB, or 10mM- and 50 mMcurcumin.
After the incubation of cells with food additives, we stimulated
the adipocytes with 1mg/ml LPS (Sigma-Aldrich, Munich,
Germany) and incubated for 24 h before the measurement
of leptin for the dose–response experiments. To assess the
effect of LPS on the secretion of pro-inflammatory cytokines,
IL-6 concentrations were measured in the cell culture
supernatant. Furthermore, the concentration of the nitrite in
the cell culture supernatant was determined, which represents
a stable end product of nitrite oxide and thus an estimate of
NO synthase activity.
Finally, time–course experiments were conducted using
1mM-SS, 10mM-SB and 50 mM-curcumin. We incubated the
cells for 6, 12 and 24 h with and without LPS, and measured
the leptin levels in the supernatant. All the experiments
were replicated three times in triplicates.
Cell viability
To measure the possible effects of the food additives or LPS on
the viability of 3T3-L1 adipocytes during the experiments, we
used the lactate dehydrogenase release assay. Lactate dehydrogenase
was measured in the cell culture supernatant by
using an autoanalyser (ABX-Cobas Mira; Roche Diagnostic,
Mannheim, Germany) at the beginning and at the end of all
incubations according to the manufacturer’s instructions.
Lactate dehydrogenase is an ubiquitous, intracellular-located
enzyme, which is detectable in the cell culture supernatant
only after cell lysis due to cytotoxicity.
Analysis of leptin secretion
Leptin concentrations were measured in the cell culture supernatant
using an ELISA kit for mouse leptin from Research
and Diagnostic Systems (Quantikinew M Murine; R&D
Systems, Minneapolis, MN, USA; inter-assay CV% ,5; intraassay
CV% ,3·8).
Analysis of IL-6 secretion
IL-6 concentrations were determined in the cell culture
supernatant using an ELISA kit for mouse IL-6 (Quantikinew
Mouse IL-6; R&D Systems; inter-assay CV% ,7·6; intra-assay
CV% ,3·5).
Analysis of nitrite
Nitrite concentrations in cellular supernatants were determined
photometrically using the Griess reaction. Thereby,
nitrite in the samples was quantitatively converted to a
diazonium salt, which was then coupled with N(1-naphthyl)
ethylenediamine dihydrochloride, forming an azo dye that
was read at 540nm in a spectrophotometer.
Statistical analysis
Data are expressed as means and standard deviations unless
otherwise indicated. Normality of data was assessed using
the Shapiro–Wilk test. Differences between groups were
analysed by one-way ANOVA. Post hoc adjustment for
multiple comparisons was applied according to the method
of Bonferroni.
For the comparison of mean values within the groups
during the time–course experiment, one-way ANOVA for
repeated measures was used. In the case of significant
differences, Bonferroni’s post hoc tests were performed. The
Effect of food additives on leptin release 827
British Journal of Nutrition
Greenhouse–Geisser correction was applied when indicated
by Mauchly’s test for sphericity. P values #0·05 were considered
statistically significant.
All analyses were performed using SPSS 15.0 for Windows
(SPSS, Chicago, IL, USA).
Results
Effect of lipopolysaccharide and toxicity tests
At 10 d after the initiation of differentiation of murine 3T3-L1
fibroblasts into adipocytes, the cells secreted a significant
amount of leptin into the cell culture supernatant. In all
the experiments, we used untreated cells as a negative
control and LPS (1 mg/ml)-stimulated cells as a positive control.
The treatment of adipocytes with LPS for 24 h led to a
significant diminution of leptin concentrations by approximately
30% and a significant increase in IL-6 secretion by
nearly 800% compared with control cells in the absence of
LPS. By the measurements of lactate dehydrogenase in the
cell culture supernatants at the beginning and at the end
of all incubation procedures, we did not observe any
influence of LPS or the food additives on cell viability
(data not shown).
First, we analysed the release of leptin, IL-6 and nitrite after
the treatment of unstimulated and LPS-stimulated adipocytes
with SS, SB and curcumin at different concentrations for
24 h. The incubation of cells with food additives in the
absence of LPS did not affect leptin levels in the supernatants
of adipocyte cultures (data not shown).
The antioxidant, sodium sulphite, reduces leptin release
after co-incubation with lipopolysaccharide
Co-incubation of cells with LPS and 1mM-SS induced a stronger
decrease in leptin concentrations by 30% compared with
the LPS-stimulated control in the absence of this food additive
(P#0·001; Fig. 1(a)). Increasing the SS concentration to 10mM
did not further decrease leptin concentrations significantly.
However, a significant increase of 57% of IL-6 was found by
augmenting the concentration of SS (P,0·01; Fig. 1(b)).
With regard to nitrite formation, we found no effect after treatment
with SS (Fig. 1(c)).
Sodium benzoate decreases leptin release after
co-incubation with lipopolysaccharide
Co-treatment of LPS-stimulated adipocytes with 10mM-SB
decreased leptin levels by 49% (P,0·001; Fig. 2(a)), which
was even more pronounced by increasing the concentration
of SB to 20mM (270 %; P,0·001; Fig. 2(a)). No significant
effects could be detected on IL-6 and nitrite concentrations
after the incubation of LPS-stimulated cells with SB (Fig. 2(b)
and (c)).
(a) 1·5
Leptin (fold induction)
1·0
0·5
0·0
LPS
*
**
**
SS (mM)
–
–
+
–
+
1
+
10
2·0
1·5
(b)
IL-6 (fold induction)
1·0
0·5
0·0
LPS
*
** *
SS (mM)
–
–
+
–
+
1
+
10
1·5 (c)Nitrite (
fold induction)
1·0
0·5
0·0
LPS
SS (mM)
–
–
+
–
+
1
+
10
Fig. 1. Influence of sodium sulphite (SS) in lipopolysaccharide (LPS;
1 mg/ml)-treated 3T3-L1 cells on (a) leptin secretion, (b) IL-6 release and
(c) NO formation after 24 h of treatment. The control conditions in the
absence or presence of 1mg/ml LPS are shown. Fold induction was related
to the control group with LPS in the absence of SS. All experiments were
performed in triplicates. Values are means, with their standard errors
represented by vertical bars. Mean values were significantly different as
assessed by one-way ANOVA with Bonferroni’s adjustment: *P#0·01 and
** P#0·001.
828 C. Ciardi et al.
British Journal of Nutrition
The food colourant and antioxidant curcumin
decreases leptin secretion after co-incubation
with lipopolysaccharide
A significant decrease in leptin release by 18% was detected
after the incubation of the cells with 10mM-curcumin
(P,0·01; Fig. 3(a)), which was further suppressed with
50 mM by 87% (P,0·001; Fig. 3(a)). In contrast to SS and SB,
co-treatment of LPS-stimulated adipocytes with 50 mM-curcumin
significantly decreased IL-6 concentrations by 57% in
the supernatant of the cells (Fig. 3(b)). Again, no effect of
curcumin was detected on the formation of nitrite (Fig. 3(c)).
Antioxidants influence leptin secretion over time
Next, we investigated the effect of food additives on
leptin secretion into the cell culture supernatant as a function
of time (Fig. 4). In unstimulated cells, leptin concentrations
rose from 49·6 (SD 28·3) pg/ml after 6 h to 185·7
(SD 55·1) pg/ml after 12 h, and to 854·4 (SD 203) pg/ml after
24 h of treatment (all P,0·001 v. 6 h). Incubation of the
cells with LPS diminished leptin levels significantly after
12 and 24 h of treatment to 115·5 (SD 40·9) pg/ml and 529·2
(SD 138·9) pg/ml, respectively (all P,0·01 when compared
with unstimulated cells). After 6 h of treatment, no significant
changes in leptin levels could be detected on treatment
with LPS alone or in combination with the food additives.
Co-treatment of LPS-stimulated cells with SS further diminished
leptin levels slightly but significantly in relationship
with treated cells with LPS after 24 h of treatment (P,0·05;
Fig. 4(a)). In contrast, co-treatment of cells with SB or curcumin
resulted in a substantial suppression of leptin release
into the supernatant after 12 and 24 h compared with
LPS-stimulated adipocytes (Fig. 4(b) and (c)).
Discussion
In the present study, the food preservatives SS and SB as well
as the food colourant, curcumin, diminished leptin production
in the cell culture supernatants of LPS-treated murine adipocytes
in a dose- and time-dependent fashion. All tested compounds
possessed antioxidant and/or radical-scavenging
properties, which could play a role in interfering with the
signal transduction cascades that modulate leptin production.
The function of leptin was originally perceived as a signal that
prevented obesity, since leptin-deficient ob/ob mice and
leptin-resistant db/db mice develop hyperphagia and, consequently,
severe obesity(8,16). Supplementation of leptin to
genetically deficient ob/ob mice increases their metabolic
rate, body temperature and general activity, and decreases
food intake, body weight and adiposity(8,17 – 19). Basal or fasting
leptin levels are strongly correlated with adipose tissue
mass, percentage of body fat or BMI in both healthy adults
and those with type 2 diabetes mellitus(12,20,21 – 23). Despite
these strong correlations, people with similar degrees of
adiposity have circulating leptin concentrations that vary
considerably, due to the influence of several factors on
leptin metabolism such as insulin, glucocorticoids and
2·0
1·5
(a)
Leptin (fold induction)
1·0
0·5
0·0
LPS
**
**
**
*
SB (mM)
–
–
+
–
+
10
+
20
(b) 1·5
IL-6 (fold induction)
1·0
0·5
0·0
LPS
**
SB (mM)
–
–
+
–
+
10
+
20
(c) 1·5
Nitrite (fold induction)
1·0
0·5
0·0
LPS
SB (mM)
–
–
+
–
+
10
+
20
Fig. 2. Influence of sodium benzoate (SB) in lipopolysaccharide (LPS;
1 mg/ml)-treated 3T3-L1 cells on (a) leptin secretion, (b) IL-6 release and
(c) NO formation after 24 h of treatment. The control conditions in the
absence or presence of 1mg/ml LPS are shown. Fold induction was related
to the control group with LPS in the absence of SB. All experiments were
performed in triplicates. Values are means, with their standard errors represented
by vertical bars. Mean values were significantly different as
assessed by one-way ANOVA with Bonferroni’s adjustment: *P#0·01 and
** P#0·001.
Effect of food additives on leptin release 829
British Journal of Nutrition
25
20
15
(a)
Leptin (fold induction)
10
5
0
6 12 24
**
**
†
25
20
15
(b)
Leptin (fold induction)
10
5
0
6 12 24
**
††
**
††
**
††
**
††
25
20
15
(c)
Leptin (fold induction)
10
5
0
6 12
Time (h)
24
Fig. 4. Time course of leptin production in unstimulated and lipopolysaccharide
(LPS; 1mg/ml)-stimulated 3T3-L1 cells, and in cultures co-incubatedwith
LPS (1 mg/ml) and (a) 1mM-sodium sulphite (–O–), control
group 2 LPS (–X–), control group þ LPS (–B–); (b) 10mM-sodium
benzoate (–O–), control group 2 LPS (–X–), control group þ LPS (–B–)
or (c) 50mM-curcumin (–O–), control group (–X–), control group þ LPS
(–B–). Experiments (n 3) were performed in triplicates. Values are
means, with their standard errors represented by vertical bars. ** Mean
values were significantly different from those of leptin levels at 6 h to levels
at 12 and 24 h in the presence of LPS and food additives within the
condition as determined by one-way repeated-measures ANOVA, Bonferroni’s
method applied (P,0·001). Mean values were significantly different
of leptin concentration after incubation with LPS in the presence of food
preservatives when compared with the control in the presence of LPS
alone as determined by one-way ANOVA with Bonferroni’s adjustment:
† P,0·05 and ††P,0·01.
2·0
1·5
(a)
Leptin (fold induction)
1·0
0·5
0·0
LPS
**
**
*
**
Curcumin (μM)
–
–
+
–
+
10
+
50
(b) 1·5
IL-6 (fold induction)
1·0
0·5
0·0
LPS
**
**
**
Curcumin (μM)
–
–
+
–
+
10
+
50
(c) 1·5
Nitrite (fold induction)
1·0
0·5
0·0
LPS
Curcumin (μM)
–
–
+
–
+
10
+
50
Fig. 3. Influence of curcumin in lipopolysaccharide (LPS; 1mg/ml)-treated
3T3-L1 cells on (a) leptin secretion, (b) IL-6 release and (c) NO formation
after 24 h of treatment. The control conditions in the absence or presence
of 1mg/ml LPS are shown. Fold induction was related to the control group
with LPS in the absence of curcumin. All experiments were performed in
triplicates. Values are means, with their standard errors represented by
vertical bars. Mean values were significantly different as assessed by oneway
ANOVA with Bonferroni’s adjustment: *P#0·01 and ** P#0·001.
830 C. Ciardi et al.
British Journal of Nutrition
catecholamines, suggesting that leptin metabolism is regulated
by a complex network(24).
Based on the current data, different mechanisms induce and
maintain a chronic state of inflammation in obese people(2).
Among others, it could be shown that in obese states the
amount of Gram-negative bacteria differs compared with
that in lean individuals. Moreover, due to the increased
decay of Gram-negative bacteria, the release of LPS and its
absorption in the gut capillaries may induce metabolic endotoxaemia
and support chronic inflammation(4 – 6).
In our model, we found no direct effect on leptin secretion
after incubation with antioxidants in the absence of LPS, at
low levels of IL-6. However, at high levels of IL-6 due to
LPS co-incubation, leptin secretion was significantly less
than in the absence of antioxidants. These results suggest
that a state of inflammation may be a prerequisite for the
observed effect on leptin secretion. In the past 30 years, SS
has become one of the leading food preservatives in the
food sector throughout the world. The measured concentrations
of SS in food vary between 0·8mM, i.e. in dried
potatoes, and 1·6mM, i.e. in wine and dried fruits(25).
In 1983, the Joint Expert Committee on Food Additives of
the FAO of the WHO established an acceptable daily intake
level of 0·7 mg/kg body weight. A constant fraction of
sulphite agents that enter the body via ingestion is metabolised
in the liver. However, a finite amount will pass through
the organ and enter the systemic circulation. Approximately,
10% of the ingested dose is excreted unchanged in the
urine(26,27).
Another widely used preservative is SB, which is known for
its hydroxyl-radical scavenging, bacteriostatic and fungistatic
properties under acidic conditions(28). The Joint Expert
Committee on Food Additives of the FAO/WHO established
an acceptable daily intake level of 5 mg/kg body weight.
However, recent studies have suggested that SB is linked
to allergic reactions and, moreover, to hyperactivity in
children(29 – 31). The possible effects of SS and SB on specific
metabolic pathways are scarce, albeit SB is a well-documented
hydroxyl-radical scavenger and was recently found to suppress
Th1-type immune responses in vitro (32,33).
Another compound with a strong antioxidant capacity is
curcumin, the major component of turmeric (Curcuma
longa). In vitro and animal studies have shown that curcumin
exerts various beneficial properties such as anti-inflammatory,
anti-neoplastic, anti-cancer and anti-ischaemic effects(34 – 36).
Recently published studies have elucidated the effect of curcumin
on metabolisms of obesity and insulin resistance
in various animal models. Taken together, these studies
demonstrate decreased leptin levels after curcumin consumption
in animal models(37 – 39). In our study, we were able to
confirm and to extend these results using a cell culture
system of differentiated adipocytes, where we could show a
significant decrease in leptin levels already after 12 h of
exposure to curcumin. After an incubation time of 24 h, curcumin
also decreased LPS-induced IL-6 levels in the supernatant,
which is consistent with the reported anti-inflammatory properties
of curcumin(40). However, another possible explanation
for the strong effects of curcumin on leptin release could
be the induction of apoptosis, as described by various
studies(41,42). For SS and SB, mechanisms of apoptosis do
not explain the obtained diminution of leptin levels, as IL-6
and nitrite were not affected during the whole experiments.
As we did not observe any effect of the antioxidants on
leptin secretion in the absence of LPS, we assumed that
the activation of the inducible NO synthase could provide a
possible explanation for the affected leptin secretion in
LPS-stimulated cells only. Although LPS is well known to activate
inducible NO synthase in macrophages and dendritic
cells of the skin, thereby elevating concentrations of nitrite,
which in turn may affect leptin secretion(43 – 45), in our
model, we did not observe any effect on nitrite release
after treatment with LPS alone or in combination with the
food additives. These results are in line with previously
published studies, which show that only a combination of
LPS, TNF-a and interferon-g affects inducible NO synthase
activity in murine 3T3-L1 cells, suggesting that the induction
of this enzyme is mediated by a complex network of interactions
involving inflammatory cytokines and LPS at the
level of the adipocytes(46,47).
Taken together, our data suggest a possible effect of
the food additives SS, SB and curcumin on the release of
leptin from adipocytes in a state of chronic inflammation,
which is associated with obesity. Decreased leptin release
during the consumption of nutrition-derived food additives
would decrease the overall amount of circulating leptin to
which the central nervous system is exposed and could
thereby influence food intake and contribute to an obesogenic
effect(9).
In the murine 3T3-L1 cell culture system, we could analyse
the sole effect of the investigated compounds avoiding interfering
factors, which affect leptin metabolism, i.e. circulating
insulin, glucocorticoids or catecholamines. From the data
obtained in the present in vitro study, however, it is unclear
how food additives interfere in a complex system such as
the human organism with regard to leptin metabolism.
Therefore, it is unclear to what extent any conclusion from
the present in vitro study can be extrapolated to the in vivo
situation, and clearly more studies are needed to investigate
the potential contribution of diet-derived agents in a complex
organism and a possible influence on the development
of obesity.
Acknowledgements
We highly appreciate the expert technical assistance of Karin
Salzmann, Simone Wu¨hl and Astrid Haara. The authors’
responsibilities were as follows: C. C. and D. F. conceived
and designed the study; C. C. and A. T. were responsible for
the data analysis and interpretation; C. C. was responsible
for writing the manuscript; M. J., A. T., F. U., M. P., J. P. and
C. E. were responsible for the critical revision and its important
intellectual content; D. F. was responsible for the study
supervision. The authors have nothing to disclose. The present
study received no specific grant from any funding
agency in the public, commercial or not-for-profit sectors.
Effect of food additives on leptin release 831
British Journal of Nutrition
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