Ramalin inhibits differentiation of 3T3-L1 preadipocytes and suppresses adiposity and body weight in a high-fat diet-fed C57BL/6J mice

Article abstract of DOI:10.1016/j.cbi.2016.07.034

Obesity is a serious global health problem. Natural substances that could be effective remedies for treatment of obesity, and which are relatively safe, are desired. The aim of this study was to examine the anti-obesity effect and the mechanism of ramalin in 3T3-L1 preadipocytes and high fat diet (HFD)-induced obese mice. In this study, 3T3-L1 cells were treated with various concentrations of ramalin (1, 5, and 10?μg/ml). Ramalin reduced the accumulation of intracellular lipid droplets in 3T3-L1 cells. In addition, ramalin inhibited 3T3-L1 adipocyte differentiation by blocking adipogenic gene expression including CCAAT enhancer binding proteins (C/EBPs), peroxisome proliferator-activated receptors γ (PPARγ), adipocyte fatty acid-binding protein (aP2), and leptin. The suppression of adipogenesis by ramalin was mediated through the inhibition of MAPK pathways. Ramalin also reduced the secretion of TNF-α and IL-6 in 3T3-L1 adipocytes. Oral administration of ramalin (50 and 100?mg/kg) to HFD-fed mice reduced body weight gain and abdominal fat accumulation without changes in food intake. Ramalin also attenuated organ weight and basal serum level by inhibiting Iiver X receptors (LXRs), sterol regulatory element-binding protein-1c (SREBP-1c), and lipoprotein lipase (LPL) mRNA expression in HFD-fed mice. Taken together, these results indicate that ramalin inhibits adipogenesis in 3T3-L1 preadipocytes and prevents HFD-induced obesity. The present study also provides insight into the mechanisms underlying the anti-obesity activity of ramalin and suggests that ramalin has the potential to prevent obesity.

Full text of DOI:10.1016/j.cbi.2016.07.034

Chemico-Biological Interactions 257 (2016) 71e80
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Ramalin inhibits differentiation of 3T3-L1 preadipocytes and
suppresses adiposity and body weight in a high-fat diet-fed C57BL/6J
mice
Seo-Young Kim 1, Yeon Jeong Jang , Bongkyun Park , Joung-Han Yim ,
a
,
a
,
1
a
b
b
a
a, *
Hong-Kum Lee , Dong-Kwon Rhee , Suhkneung Pyo
a
School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, 16419, Republic of Korea
Polar BioCenter, Korea Polar Research Institute, KORDI, Incheon, 21990, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 May 2016
Received in revised form
Obesity is a serious global health problem. Natural substances that could be effective remedies for
treatment of obesity, and which are relatively safe, are desired. The aim of this study was to examine the
anti-obesity effect and the mechanism of ramalin in 3T3-L1 preadipocytes and high fat diet (HFD)-
induced obese mice. In this study, 3T3-L1 cells were treated with various concentrations of ramalin (1, 5,
22 July 2016
Accepted 28 July 2016
Available online 30 July 2016
and 10
addition, ramalin inhibited 3T3-L1 adipocyte differentiation by blocking adipogenic gene expression
including CCAAT enhancer binding proteins (C/EBPs), peroxisome proliferator-activated receptors
PPAR ), adipocyte fatty acid-binding protein (aP2), and leptin. The suppression of adipogenesis by
ramalin was mediated through the inhibition of MAPK pathways. Ramalin also reduced the secretion of
TNF- and IL-6 in 3T3-L1 adipocytes. Oral administration of ramalin (50 and 100 mg/kg) to HFD-fed mice
mg/ml). Ramalin reduced the accumulation of intracellular lipid droplets in 3T3-L1 cells. In
g
Keywords:
Ramalin
(
g
3T3-L1 adipocytes
High-fat diet
Adipogenesis
Obesity
a
reduced body weight gain and abdominal fat accumulation without changes in food intake. Ramalin also
attenuated organ weight and basal serum level by inhibiting Iiver X receptors (LXRs), sterol regulatory
element-binding protein-1c (SREBP-1c), and lipoprotein lipase (LPL) mRNA expression in HFD-fed mice.
Taken together, these results indicate that ramalin inhibits adipogenesis in 3T3-L1 preadipocytes and
prevents HFD-induced obesity. The present study also provides insight into the mechanisms underlying
the anti-obesity activity of ramalin and suggests that ramalin has the potential to prevent obesity.
©
2016 Elsevier Ireland Ltd. All rights reserved.
1
. Introduction
and animal studies on the development of obesity have revealed
that dietary factors can change the number of adipocytes and
adipocyte size [2]. Adipose tissue stores excess energy and is an
endocrine organ [3].
In response to an adipogenic cocktail (MDI) consisting of 3-
isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin,
postconfluent 3T3-L1 preadipocytes undergo a series of transcrip-
tional activations [4]. The process begins with CCAAT/enhancer
Obesity is a major risk factor for several metabolic disorders
including type 2 diabetes, hypertension, cardiovascular diseases
and osteoarthritis [1]. Obesity is increasing in prevalence globally
and obesity has become a significant global health threat. Cellular
binding protein beta (C/EBP
peroxisome proliferator-activated receptor gamma (PPAR
EBP , which play an important role in adipocyte phenotype [5,6].
The process of adipocyte differentiation is divided into the early,
intermediate, and late stages [7]. The early stage of adipogenesis is
b
) expression, followed by C/EBP
d,
Abbreviations: CCAAT/enhancer-binding protein alpha, C/EBP
enhancer-binding protein beta, C/EBP ; Peroxisome proliferator-activated receptors
gamma, PPAR ; Adipocyte fatty acid-binding protein, aP2; Liver X receptor alpha,
LXR Liver X receptor beta, LXR Lipoprotein lipase, LPL; Sterol regulatory
element-binding protein-1c, SREBP-1c; Mitogen-activated protein kinase, MAPK;
a
;
CCAAT/
g), and C/
b
a
g
a
;
b;
Interleukin-6, IL-6; Tumor necrosis factor-alpha, TNF-a; Normal fat diet, NFD; High
mainly controlled by C/EBP
increased by inducers of cell division and hormonal stimulation [8].
Increased levels of C/EBP and C/EBP trigger changes in the ex-
pressions of PPAR and C/EBP , which are involved in the
d and C/EBPb, which are rapidly
fat diet, HFD; 3-isobutyl-1-methylxanthine, IBMX.
*
Corresponding author.
d
b
E-mail address: snpyo@skku.edu (S. Pyo).
These authors contributed equally to this work.
1
g
a
http://dx.doi.org/10.1016/j.cbi.2016.07.034
009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
0

72
S.-Y. Kim et al. / Chemico-Biological Interactions 257 (2016) 71e80
regulation of the late stage of adipogenesis [9]. The activation of
PPAR and C/EBP regulates the expression of multiple adipogenic
genes necessary for fat accumulation.
Intracellular mitogen-activated protein kinase (MAPK) signaling
pathways play a critical role in the regulation of cell proliferation
and differentiation [10]. MAPK pathways are involved in the early
stage of adipocyte differentiation and regulate the expression of C/
synthesis of ramalin was already patented (Fig. 1) [17]. Briefly, 1.2-
protected L-glutamic acid and 1-benzyl 2-aminophonol were the
starting materials. Total synthesis of ramalin required four steps.
The first step was 1 carboxyl acid and 2 amino site protected
glutamic acid activation step for coupling reaction. Another
coupling agent, phenyl hydrazine, was synthesized from 1-benzyl
g
a
0
0
2-aminophonol using SnCl
2
reagent. Coupling reaction with GPH-
EBP
tion of downstream proteins related to adipocyte differentiation in
T3-L1 cells [8,10]. Therefore, it is plausible that MAPK pathways are
a and PPARg, contributing to the expression and phosphoryla-
09 and GPH-04 produced GPH-11. Final deprotection reaction
with palladium (on carbon) and H gas produced ramalin.
2
3
a potential target for the treatment of obesity. Understanding the
mechanism by which preadipocytes differentiate into adipocytes
would aid in developing therapeutic strategies to prevent obesity.
Various food materials or natural compounds isolated from
edible plants that show an anti-obesity effect have been screened for
use as functional foods or dietary supplements. Metabolites
extracted from Antarctic lichen reportedly have a variety of bio-
activities that include antibiotic, anti-mycobacterial, anti-viral, anti-
inflammatory, analgesic, anti-pyretic, anti-proliferative, and cyto-
toxic effects [11]. In addition, they have been applied in natural
cosmetics and medicines, and have demonstrated fewer side effects
compared to industrial products. However, there is still no adequate
information pertaining to the health-promoting properties of the
bioactive compounds in lichen and their pharmaceutical potential.
Ramalin, isolated from the Antarctic lichen, Ramalina terebrata
2.3. Measurement of cell viability
3T3-L1 preadipocytes were seeded at a concentration of
3
8 ꢀ 10 cells/well in 96-well tissue culture plates and treated with
the indicated concentrations of ramalin for 48 h. Cell viability was
measured by a quantitative colorimetric assay with MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which
indicates the mitochondrial activity of living cells. The extent of
reduction of MTT to formazan within cells was quantified by
measuring the optical density at 540 nm using a microplate reader
(Molecular Devices, Sunnyvale, CA).
2.4. Determination of lipid accumulation by Oil Red O staining
Oil Red O staining was performed 8 days following ramalin
exposure to stain the accumulated lipid droplets in the differenti-
ated adipocytes. The cells were washed with phosphate buffered
saline (PBS) and fixed with 10% formalin for 1 h at room temper-
ature. The cells were rinsed with 60% isopropanol and stained with
filtered Oil Red O solution for 20 min at room temperature. After
removing the staining solution, the stained cells were washed with
distilled water and dried. The stained lipid droplets were micro-
scopically examined and photographed. To quantify intracellular
lipid accumulation, the stained lipid droplets were dissolved with
100% isopropanol for 10 min. The optical density was measured at
490 nm using the aforementioned microplate reader.
(
[
Ramalinaceae) has anti-oxidant and anti-inflammatory activities
12,13]. There is increasing evidence that inflammation plays a
central role in the metabolic consequence of obesity [14]. Recent
reports also showed that the plasma levels of inflammatory me-
diators, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha
(
TNF-a), are increased in the insulin resistant states of obesity and
type 2 diabetes [15]. In preliminary experiments, we have shown
that the exposure of 3T3-L1 preadipocytes to ramalin induces sig-
nificant changes in the expression pattern of adipogenesis-related
genes [16]. These findings lead us to explore the effect of ramalin
on the regulation of lipid accumulation in vitro and in vivo. In the
present study, we examined the effects of ramalin on adipogenesis
in 3T3-L1 cells in vitro and whether ramalin can decrease obesity in
high fat diet (HFD)-fed C57BL/6J mice in vivo. The results demon-
strate that ramalin inhibits adipogenesis and reduces obesity in
HFD-fed mice.
2.5. Western blot analysis
After cell lysis, lysates were clarified by centrifugation at
ꢁ
13,000 rpm for 15 min at 4 C. Protein concentration was deter-
mined using the Bio-Rad DC protein assay with bovine serum al-
bumin (BSA) as the standard. The whole cell lysates were resolved
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The
fractionated proteins were electrophoretically transferred to PVDF
membranes (Amersham, Arlington Hights, IL) and probed with
antibodies to extracellular signal-regulated kinase (ERK), phospho-
ERK, p38, phospho-p38, c-Jun N-terminal kinase (JNK), phospho-
JNK, phospho-C/EBPb, PPARg, C/EBPb, cyclin A, CDK2, p27, and b-
actin. The blots were developed using an enhanced chem-
iluminescence (ECL) kit. In all immunoblotting experiments, the
2
. Materials and methods
2.1. Reagents
Unless otherwise indicated, all chemicals used in this study
were purchased from Sigma-Aldrich Co. (St Louis, MO). Orlistat was
were purchased from Cayman Chemical (Ann Arbor, MI). Bio-Rad
DC protein assay was obtained from Bio-Rad (Hercules, CA). Anti-
bodies against extracellular signal-regulated kinase (ERK),
phospho-ERK, p38, phospho-p38, c-Jun N-terminal kinase (JNK),
blots were reprobed with an anti-
protein loading.
b-actin antibody as a control for
phospho-JNK, phospho-C/EBP
Signaling Technology (Beverly, MA). Antibodies against C/EBP
cyclin A, CDK2, p27, and -actin were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence
ECL) kit was obtained from Amersham (Arlington Hights, IL). Trizol
Reagent and SuperScript II cDNA synthesis kit were purchased from
Invitrogen (Carlsbad, CA). Mouse TNF- and IL-6 Enzyme-linked
b, and PPARg were obtained from Cell
b
,
b
2.6. Cell cycle progression analysis
(
Cell cycle progression was measured by flow cytometric analysis
after propidium iodide (PI) staining. Post-confluence preadipocytes
a
were treated with 0.5 mM 3-isobutyl-1-methylxanthine, 1
dexamethasone, and 10 g/ml insulin in the presence or absence of
1, 5, and 10 g/ml ramalin and 100
mM
immunoassay (ELISA) MAX™ Standards were obtained from Bio-
legend, Inc. (San Diego, CA).
m
m
m
g/ml orlistat for 24 h. A sus-
ꢁ
pension of the cells was fixed overnight with 70% ethanol at 4 C,
2.2. Synthesis of ramalin
and then incubated with 10 mg/ml of RNase A and 50 mg/ml of
propidium iodide staining buffer for 30 min at room temperature in
the dark. Then, 10,000 cells per experiment were analyzed using a
Ramalin was synthesized as described previously and the

S.-Y. Kim et al. / Chemico-Biological Interactions 257 (2016) 71e80
73
Fig. 1. Schematic representation of the synthesis route of ramalin. (U.S. Patent 8,865,934).
FACS Canto™ II apparatus (BD Biosciences, San Jose, CA) and cell
cycle progression was measured with FCS Express 4 Flow Cytom-
etry software (De Novo Software, Los Angeles, CA).
Blood was drawn from the abdominal aorta into an ethylenedi-
amine tetra-acetic acid (EDTA)-coated tube, and the serum was
obtained by centrifuging the blood at 2000 rpm for 15 min at 4 C.
ꢁ
Livers, kidneys, testis, and abdominal fat-pads were removed,
rinsed with PBS, and weighed. The serum, livers, kidneys, testis, and
2
.7. Isolation of total RNA and quantitative real-time PCR
ꢁ
abdominal fat-pad samples were stored at ꢂ70 C until they were
Following the manufacturer's protocol, total RNA was extracted
analyzed. This study was approved by the animal care and use
committee of Sungkyunkwan University.
from liver tissue isolated from HFD-fed mice orally administered
ramalin, and from 3T3-L1 cells using Trizol Reagent. Isolated RNA
(
1
mg/m
l) was reverse transcribed using a SuperScript II kit for cDNA
2.9. Statistical analysis
synthesis. The cDNA was subjected to quantitative real-time (qRT)-
PCR using thermocyclers from Applied Biosystems (Franklin Lakes,
NJ). The sequences of the primers corresponding to the mouse
adipogenic genes analyzed in this study are shown in Table 1.
Each result is reported as mean ± S.E.M. One-way analysis of
variance (ANOVA) was used to determine significance among the
groups, after which the modified tetest and two-way ANOVA were
used for comparison between individual groups. Significant values
(p < 0.05) are represented by an asterisk.
2.8. Animals and diets
Male, 4-week-old C57BL/6J mice were purchased from SLC Inc.
3. Results
(
Hamamatsu, Shizuoka). Upon arrival, mice were fed a normal-fat
diet (NFD) for 1 week. The animals were housed in individual
cages in a temperature-controlled room with a 12 h light/dark cy-
cle. Mice were divided into five groups (n ¼ 8 in all groups) at 5
weeks of age. Mice in a group were fed the NFD and orally
3.1. Effect of ramalin on lipid accumulation and expression of
adipocyte differentiation-related genes in MDI-stimulated 3T3-L1
preadipocytes
administered 200
fed the HFD containing 60% fat (Research Diets Inc., New Bruns-
wick, NJ). One group was orally administered 200 l PBS every 3
days as a negative control. Another group of mice was orally
administered 200 l orlistat (50 mg/kg) every 3 days as a positive
control. The remaining two groups were orally administered 200
m
l PBS every 3 days. The other four groups were
We investigated the effect of ramalin on cytotoxicity in 3T3-L1
preadipocytes using the MTT assay at the indicated concentration
of ramalin for 48 h. Ramalin was cytotoxic at concentrations
m
exceeding 50
mg/ml (Fig. S1). In subsequent experiments, cells were
m
treated with 1, 5, and 10
mg/ml ramalin. Next, we examined the
ml
effect of ramalin on intracellular lipid accumulation during adipo-
cyte differentiation using Oil red O staining. As shown in Fig. 2A,
treatment of confluent 3T3-L1 with MDI resulted in a 3.3-fold
containing 50 mg/kg or 100 mg/kg ramalin every 3 days. Food
intake and body weight were monitored every 3 days for 8 weeks.
Table 1
Quantitative real-time-PCR primer sequences.
0
0
Gene
PPAR
Forward primer (5’/3 )
Reversed primer (5’/3 )
g
CGC TGA TGC ACT GCC TAT GA
AGA GGT CCA CAG AGC TGA TTC C
C/EBP
aP2
Leptin
a
GCG AGC ACG AGA CGT CTA TAG A
CCG CAG ACG ACA GGA AGG T
TCG GTA TCC GCC AAG CA
GAG TGT CGA CTT CGC AAA TGC
CAG GCT TGC AGG TGG AAT TC
ATC GGA GAA CTG CTC ATG ATGA
GGC TAT TCC GTG AAC ATC TCC TA
TGC ATC CTG CAC CAC CAA
GCC AGG AAC TCG TCG TTG AA
AGG GCC CCG CCA TCT
GGT GAA GCC CAG CAA TGA AG
CCT CTT CTT GCC GCT TCA GT
ATG GCG ATAAGC AAG GCA TACT
CGG ATC CTC TCG ATG ACG AA
ATC CAA GGG CAT CTG AGA ACT C
TCC ACG ATG CCA AAG TTG TC
LXR
LXR
LPL
a
b
SREBP-1c
GAPDH

74
S.-Y. Kim et al. / Chemico-Biological Interactions 257 (2016) 71e80
increase in lipid accumulation compared to untreated cells. In
contrast, treatment of the cells with ramalin (1e10 g/ml) for 8
days remarkably decreased intracellular lipid accumulation in a
concentration-dependent manner. Orlistat (100 g/ml), which is a
the early (0e2 days) stages were decreased by ~45.6% and 43.4%,
respectively. These results indicated that the effect of ramalin on
the inhibition of adipocyte differentiation occurs during the early
m
m
stage. C/EBP
b expression is crucial for the induction of mitotic
prescription medicine for obesity, inhibited lipid accumulation.
These results correlated exactly with the absorbance values of
eluted Oil Red O staining solution in differentiation-induced adi-
clonal expansion (MCE) [18]. In addition, the phosphorylation of C/
188
EBP
involved in adipogenesis [8]. Therefore, we examined the effects of
ramalin on C/EBP expression and the phosphorylation of C/EBP
Ramalin did not affect the protein expression of C/EBP , whereas
phosphorylation of C/EBPb at Thr was significantly attenuated by
b
at Thr
by MAPK is necessary for the induction of genes
pocytes. PPAR
tors, regulating the terminal process of adipocyte differentiation. To
investigate whether ramalin inhibits the expression of PPAR and
C/EBP in MDI-induced 3T3-L1 cells, we treated 3T3-L1 cells with 1,
, and 10 g/ml of ramalin and the positive control orlistat. Protein
and mRNA expression of PPAR and C/EBP were significantly
g
and C/EBP
a
are the two critical transcription fac-
b
b.
b
188
g
a
ramalin in a concentration-dependent manner (Fig. 3B). When
growth-arrested preadipocytes were treated with adipogenic
inducer, the number of adipocytes increased 4e5-fold during the
early stage [19]. DNA replication and the changes in chromatin
structure occur during the mitotic clonal expansion of adipogensis
[20]. We investigated whether ramalin affects the inhibition of cell
cycle progression in MCE. Flow cytometric assay revealed that
ramalin inhibited adipocyte proliferation during the early stage of
5
m
g
a
inhibited by ramalin in a concentration-dependent manner (Fig. 2B
and C). In addition, quantitative real-time PCR analysis showed that
the level of aP2 and leptin mRNA expression was significantly
reduced by ramalin during adipocyte differentiation in
a
concentration-dependent manner, compared to the MDI-induced
control (Fig. 2D). These results indicated that ramalin inhibits
adipocyte differentiation in 3T3-L1 preadipocytes through the
0 1
adipocyte differentiation; the percentage of G /G arrested cells
was increased in ramalin-treated adipocytes compared to MDI-
treated cells (Fig. 3C). However, orlistat did not affect the process
of MCE. In addition, the expression of the two cell cycle progression
proteins, Cyclin A and CDK2, were inhibited by ramalin at the
highest concentration, whereas the expression of the CDK inhibitor,
p27, was significantly recovered (Fig. 3D). The results suggested
that ramalin treatment could suppress the cells from re-entering
the cell cycle by increasing p27 expression, which inhibited MCE,
during the early stage of adipocyte differentiation.
down-regulation of PPARg, C/EBPa, aP2, and leptin.
3.2. Ramalin suppresses the early stage of adipogenesis
Adipocyte differentiation begins at the preadipocyte stage and
progresses through early, intermediate, and late stages [7]. We
investigated whether ramalin affects stage-dependent inhibition
during adipocyte differentiation. The lipid accumulation in adipo-
cytes was completely inhibited when cells were treated with
ramalin (10
m
g/ml) during the early stage (Fig. 3A). The decreased
3.3. Ramalin inhibits the phosphorylation of MAPK in MDI-induced
3T3-L1 cells
formation of lipid droplets in the early stage (0e2 days) after
ramalin treatment was similar to that seen during the entire period
of adipocyte differentiation (0e6 days). Relative lipid contents in
adipocytes during the entire differentiation period (0e6 days) or
The observations that ramalin affected the early stage of
adipocyte differentiation, MCE, and C/EBP
b phosphorylation
Fig. 2. Effect of ramalin on lipid accumulation and expression of adipocyte differentiation-related genes in MDI-induced 3T3-L1 preadipocytes. (A) Differentiated 3T3-L1 cells with
visible lipid accumulations were observed by Oil red O staining at 8 days. Lipid accumulation was quantified by a microreader at 490 nm. (B) Expression of PPAR in MDI-induced
T3-L1 cells with the indicated concentration of ramalin for 6 days. Densitometry analysis showed as relative ration of -actin considered an internal control. (C, D) mRNA
expression of PPAR , C/EBP , aP2, and leptin in MDI-induced 3T3-L1 with the indicated concentration of ramalin for 6 days. Data are means ± S.E.M of three independent ex-
periments. *, #P < 0.05, significantly different from MDI-treated cells.
g
3
b
g
a

S.-Y. Kim et al. / Chemico-Biological Interactions 257 (2016) 71e80
75
Fig. 3. Effect of ramalin on the phosphorylation of C/EBP
ramalin (10 g/ml) and MDI for 6 days was visualized by Oil red O staining. Lipid accumulation was quantified by a microreader at 490 nm. (B) Post-confluent 3T3-L1 cells were
treated with MDI for 6 h with co-treatment of ramalin at 1, 5, and 10 g/ml. Expression of C/EBP and phospho-C/EBP was analyzed by Western blot analysis and quantified by
scanning densitometry. (C) Cell cycle progression was subjected to the differentiation medium containing 1, 5, and 10 g/ml of ramalin for 24 h by flow cytometry. (D) Expression of
cyclin A, CDK2, and p27 in MDI-induced 3T3-L1 with ramalin (10 g/ml) for 24 h was subjected to Western blot assay. The -actin protein level was considered an internal control.
Data are means ± S.E.M of three independent experiments. *P < 0.05, significantly different from MDI-treated cells.
b and cell proliferation during mitotic clonal expansion. (A) Lipid accumulation in 3T3-L1 cells after co-incubation with
m
m
b
b
m
m
b
prompted us to examine whether ramalin inhibited the early stage
of adipogenesis by regulating MAPK pathways. Cells were treated
with MDI for various times. ERK, p38, and JNK were rapidly and
transiently phosphorylated within 1 or 2 h by MDI treatment
ramalin inhibits the secretion of proinflammatory cytokines using
ELISA. As shown in Fig. 5, ramalin reduced the production of TNF-
and IL-6 in a concentration-dependent manner, suggesting the
potential efficacy of ramalin on glucose homeostasis and lipid
metabolism.
a
(Fig. 4A). However, the activation of MAPK was decreased gradually
within 8 h. When treated with ramalin for 2 h after the induction
with MDI, the phosphorylation of MAPK was significantly reduced
in a concentration-dependent manner (Fig. 4B). These results
suggested that ramalin suppresses adipocyte differentiation via the
3
.5. Effect of ramalin on body weight, fat weight, serum levels, and
lipid metabolism-related genes in HFD fed C57BL/6J mice
inhibition of MCE and C/EBP
lation of MAPK pathways.
b phosphorylation through the regu-
We investigated the inhibitory effects of ramalin in vivo using
HFD fed C57BL/6J mice. Body weight and food intake were moni-
tored every
3 days. HFD-induced weight was significantly
3
.4. Ramalin inhibits the production of proinflammatory cytokines
decreased with oral ramalin administration starting from 9 days
(Fig. 6A), with no association with the amount of food intake
in MDI-induced 3T3-L1 adipocytes
(Fig. 6B). Abdominal fat weight was also remarkably inhibited by
It is widely accepted that the levels of TNF-
elevated in obesity, leading to regulate glucose homeostasis and
lipid metabolism during adipogenesis [15]. We examined whether
a
and IL-6 are
orlistat and ramalin at 50 or 100 mg/kg (Fig. 6C). An increase in
abdominal fat weight was reduced by 31% in mice receiving
100 mg/kg ramalin. Changes in organ weight and serum

76
S.-Y. Kim et al. / Chemico-Biological Interactions 257 (2016) 71e80
Fig. 3. (continued).
parameters among the five groups are shown in Tables 2 and 3.
Significant differences in liver, kidney, and testis weights but not
heart weights in orlistat or ramalin oral-administered groups
compared with the HFD group (Table 2). Oral administration of
ramalin at 50 or 100 mg/kg significantly suppressed the concen-
tration of glucose compared to the HFD group. In addition, the
levels of triglyceride (TG), total cholesterol (TC), and low density
lipoprotein (LDL)-cholesterol were significantly increased and high
density lipoprotein (HDL)-cholesterol level was decreased in the
HFD group compared with the NFD group. These changes associ-
ated with HFD were suppressed by ramalin and the results were
similar to the effects of orlistat (Table 3). Taken together, these
findings suggest that ramalin inhibits liver, kidney, and testis
weight gain by suppressing the levels of glucose, TG, TC, and LDL-
cholesterol, but improves the level of HDL-cholesterol.
suppressed in the ramalin and orlistat-administered mice (Fig. 6D
and E), indicating that oral administration with ramalin was
effective in preventing HFD-induced body weight gain and
adiposity through the regulation of cholesterol, fatty acid, and
glucose homeostasis.
4. Discussion
In this study, we evaluated the in vitro and in vivo anti-obesity
properties of ramalin in 3T3-L1 adipocytes and HFD-fed mice.
The results indicate that inhibition of lipid accumulation by ramalin
could be useful in the treatment of obesity. In vitro, ramalin reduced
lipid accumulation in a concentration-dependent manner in MDI-
stimulated 3T3-L1 cells during the early stage of adipocyte differ-
entiation by regulating the phosphorylation of C/EBP
b and
We next examined the effect of ramalin on the mRNA level of
Liver X receptors (LXRs) and LXR-related gene expression in liver
tissues isolated from the HFD fed and ramalin-administered mice
groups. LXRs play a crucial role in the regulation of metabolism of
cholesterol, fatty acid, and glucose by activating the expression of
target genes including lipoprotein lipase (LPL) which are regulated
by sterol regulatory element-binding protein-1c (SREBP-1c) [21].
expression of CDK2, cyclin A, and p27. In vivo, ramalin attenuated
obesity by inhibiting body, abdominal fat, and organ weights,
obesity-associated serum markers, and lipid synthesis-related
factors in HFD-fed C57BL/6J mice.
Adipocyte differentiation is separated into early, intermediate,
and terminal stages. Each stage of adipogenesis in 3T3-L1 cell line
model is accompanied by a complex network of key transcription
The levels of LXR
a
, LXR
b
, LPL, and SREBP-1c were remarkably
factors that include PPARg and members of the C/EBP family [9].

S.-Y. Kim et al. / Chemico-Biological Interactions 257 (2016) 71e80
77
Fig. 4. Effect of ramalin on the phosphorylation of MAPKs in MDI-induced 3T3-L1 preadipocytes. (A) Post-confluent 3T3-L1 cells were treated with MDI for the indicated times
0e12 h). (B) Post-confluent 3T3-L1 cells were subjected to the differentiation medium containing 1, 5, and 10 g/ml of ramalin for 2 h. The whole cell lysates were analyzed by
(
m
western blot analysis using phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, and JNK specific antibodies. Data are mean ± S.E.M of three independent experiments. B, :, ,
P < 0.05, significantly different from untreated cells. *, #, & P < 0.05, significantly different from MDI-treated cells.
Fig. 5. Effect of ramalin on the secretion of proinflammatory cytokines in MDI-induced 3T3-L1 adipocytes. 3T3eL1 preadipocytes were cultured in differentiation medium con-
taining 1, 5, and 10
mg/ml of ramalin for 7 days. The supernatants of the cells were then measured for TNF-a (A) and IL-6 (B) by ELISA. Data are means ± S.E.M of three independent
experiments. *p < 0.05, significantly different from MDI-treated cells.

78
S.-Y. Kim et al. / Chemico-Biological Interactions 257 (2016) 71e80
Fig. 6. Effect of ramalin on increased body weight, fat weight, and lipid metabolism-related genes in HFD fed C57BL/6J mice. (A) The body weight of the mice with oral admin-
istration of ramalin and orlistat was measured every 3 days. (B) Food intake measurements. No significant difference in amount of food intake was noted between the four groups.
(
C) Abdominal fat pads which were isolated at the conclusion of the study were weighed. Values are given as means ± S.E.M. n ¼ 8 for all groups. (D, E) mRNA expression of LXR
a,
LXR , LPL, and SREBP-1c in HFD-fed mice with oral administration of ramalin and orlistat. Data are the means ± S.E.M from three independent experiments. *, #P < 0.05,
b
significantly different from HFD-fed mice group. N.S, no statistical significance.
Table 2
Selected organ weights in C57BL/6J mice fed a HFD and oral-administered with ramalin or orlistat for 8 weeks.
Weight (g)
Dietary groups
NFD þ PBS
HFD þ PBS
HFD þ ramalin 50 mg/kg
HFD þ ramalin 100 mg/kg
HFD þ orlistat 50 mg/kg
Liver
1.07 ± 0.13
0.28 ± 0.44
0.12 ± 0.02
0.17 ± 0.04
2.12 ± 0.21
0.75 ± 0.04
0.16 ± 0.02
0.22 ± 0.01
1.22 ± 0.1*
0.34 ± 0.01*
0.15 ± 0.01
0.18 ± 0.03*
1.15 ± 0.03*
0.32 ± 0.01*
0.15 ± 0.01
0.17 ± 0.01*
1.11 ± 0.03*
0.45 ± 0.02*
0.16 ± 0.02
0.17 ± 0.01*
Kidney
Heart
Testis
All values are given as means ± S.E.M. n ¼ 8 for all groups. One-way analysis of variance (ANOVA) was used to analyze among the groups. *P < 0.05, significantly different from
HFD-fed mice group.
Table 3
Plasma glucose, TG, total cholesterol, HDL and LDL-cholesterol levels in C57BL/6J mice fed a HFD and oral-administrated with ramalin or orlistat for 8 weeks.
Measurements (mg/dL)
Dietary groups
NFD þ PBS
HFD þ PBS
HFD þ ramalin 50 mg/kg
HFD þ ramalin 100 mg/kg
HFD þ orlistat50 mg/kg
Glucose
Triglyceride
Total cholesterol
HDL-cholesterol
LDL-cholesterol
219.4 ± 16.4
84.2 ± 5.7
75.2 ± 8.9
17.2 ± 4.1
14.6 ± 2.3
391.0 ± 10.2
289.0 ± 7.6
191.0 ± 9.7
122.8 ± 7.1
42.6 ± 5.5
295.0 ± 4.3*
165.3 ± 11.9*
144.0 ± 8.1*
132.5 ± 1.2*
30.8 ± 4.4*
232.9 ± 10.3*
136.0 ± 4.7*
125.3 ± 3.4*
147.5 ± 2.9*
27.4 ± 1.3*
240.8 ± 5.6*
139.3 ± 5.2*
123.9 ± 4.8*
128.6 ± 7.3*
32.7 ± 6.9*
All values are given as means ± S.E.M. n ¼ 8 for all groups. One-way analysis of variance (ANOVA) was used to analyze among the groups. *P < 0.05, significantly different from
HFD-fed mice group.
Particularly, the role of C/EBP
adipocyte differentiation. C/EBP
MAPK and GSK-3 binds to the C/EBP regulatory element. After
that, C/EBP and C/EBP , the major transcription factors, activate
the expression of C/EBP and PPAR induced during intermediate
stage [18]. In our study, ramalin displayed an inhibitory effect on
the phosphorylation of C/EBP , whereas C/EBP expression in 3T3-
b
is important during the early stage of
by inhibiting the phosphorylation of C/EBP
of adipocyte differentiation.
b during the early stage
b
is sequentially phosphorylated by
b
During the early stage of adipogenesis, the cell population is
increased by two progressions of the cell cycle in the MCE process
[20]. Cell cycle progression during MCE in adipocyte differentiation
is affected by cyclin D-CDK4/6 complexes, which are essential for
progression through the early G1 phase, as well as by cyclin A- or
cyclin E-CDK2 complexes required for late G1 phase and S phase
entry. p21 and p27, which bind to cyclins/CDKs, can inhibit the
b
d
a
g
b
b
L1 cells treated with ramalin was not different from the control
cells. These results suggest that ramalin inhibits lipid accumulation

S.-Y. Kim et al. / Chemico-Biological Interactions 257 (2016) 71e80
79
activation of CDKs [19,22]. In addition, the ERK/MAPK pathway
plays a pivotal role in cell cycle progression and ERK acts as a
mitogenic signaling molecule in 3T3-L1 preadipocytes [10]. The
present results demonstrated that ramalin inhibited cell prolifera-
tion and the expression of cell cycle progression proteins, cyclin A
and CDK2, by up-regulating p27 during MCE. Moreover, ramalin
suppressed the phospholyation of ERK1/2, p38, and JNK. Based on
these findings, it is plausible that the inhibitory effect of ramalin on
the MAPK pathway could contribute to the regulation of cell pro-
liferation during MCE. Taken together, our data suggest that the
MCE is a target of ramalin for preventing adipogenesis in 3T3-L1
cells during the early stage.
levels [34]. In addition, previous results suggest that a reduction in
body weight could lead to a decrease in the levels of leptin [35]. The
data in the present study show that ramalin significantly reduced
the secretion of TNF-a and the mRNA level of leptin in a
concentration-dependent manner during adipocyte differentiation.
Taken together, these findings suggest that ramalin reduces body
weight and fat mass by attenuating the adipose tissue inflamma-
tion leading to potential novel therapy for reducing the risk of
obesity and hyperlipidemia.
In the present study, oral administration with ramalin signifi-
cantly inhibited the HFD-induced LXRa, LXRb, and LPL as well as
SREBP-1c mRNA level in liver tissue, indicating that ramalin is able
to regulate cholesterol, fatty acid, and glucose homeostasis. In
addition, ramalin (100 mg/kg) had significant anti-obesity effects,
compared to orlistat suggesting that ramalin treatment could have
more favorable effect than orlistat in the treatment of obesity.
In summary, our results suggest that ramalin acts as a negative
regulator of adipogenesis in 3T3-L1 cells through the inhibition of
Up-regulated PPARg and C/EBPa alone or in cooperation with
each other induce the transcription of many adipogenesis-related
genes involved in creating and maintaining the adipocyte pheno-
type in the late stage [23]. PPARg and C/EBPa, which combine in a
transcriptional complex, regulate the transcription of target genes
including aP2, LPL, leptin, and fatty acid synthase (FAS) [24]. Leptin
is an adipocyte-secreted cytokine that plays a critical role in energy
homeostasis as well as controls the fat mass through reducing food
intake and increasing energy expenditure [25]. Adipocyte fatty acid
binding protein, ap2, functions as a key mediator of intracellular
transport and metabolism of fatty acids [26]. In the present study,
ramalin remarkably inhibited adipocyte differentiation through the
C/EBPb phosphorylation and C/EBPa and PPARg expression. This
inhibitory effect could be mediated by the suppression of MAPK
signaling pathways during the early stages of adipogenesis. In vivo,
ramalin remarkably reduced body and abdominal fat weight in
HFD-fed mice through the attenuation of glucose, total-cholesterol
and LDL-cholesterol.
down-regulation of PPAR
differentiation-induced 3T3-L1 preadipocytes.
Recent studies have shown that obesity induces chronic and
low-grade inflammation in adipose tissue. As an active endocrine
organ, adipose tissue synthesizes and secretes various adipokines
g
, C/EBP
a
, aP2, and leptin mRNA levels in
The present data provide insight into the mechanisms of the
inhibitory effects of ramalin and suggest that the inhibitory activity
of ramalin indicates a potential pharmacological intervention spe-
cifically directed toward metabolic disorders including obesity.
into circulation, including TNF-
a, IL-6, angiogenic proteins, and
Conflict of interest
metabolic regulators in obesity [27]. The levels of IL-6 and TNF-
a
secreted by adipose tissues are associated with insulin resistance
which causes the derangements in whole body glucose and lipid
metabolism [15,28]. Thus, targeting proinflammatory cytokines has
been suggested as a promising treatment for insulin resistance and
obestiy. Our data showed that ramalin treatment resulted in
decreased TNF-a and IL-6 secretion, suggesting the inhibitory ef-
fects of ramalin on obesity-associated inflammation and insulin
resistance.
The authors have no conflict of interest to declare.
Acknowledgements
This research was supported by a grant from the Ministry of
Oceans and Fisheries' R&D project (PM13030) and the Korea Polar
Research Institute project (PE13040).
Development of obesity is related with the increased serum
levels of total TG, TC, and LDL-cholesterol [29]. The level of TC is the
main determinant of vascular disease risk [30]. Decreasing the level
of LDL-cholesterol reduces the risk of symptomatic vascular disease
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.cbi.2016.07.034.
[31]. Moreover, abdominal obesity has been reported to be associ-
ated with dyslipidemia, characterized by increasing TG and
decreasing HDL-cholesterol levels [32]. Additionally, a previous
study reported that decreased serum TG level is critical in weight
loss [33]. Based on these findings, a possible mechanism for the
anti-obesity effects of ramalin could be the inhibition of patho-
physiological factors related to the development of obesity. In this
study, ramalin significantly reduced the level of total and LDL-
cholesterol as well as TG in HFD-fed C57BL/6J mice. Moreover,
oral administration of ramalin resulted in a significant decrease in
body and abdominal fat weights, which is not associated with the
amount of food intake. Based on these findings, it is plausible that
the significant reduction in body weight and body fat by ramalin is
not due to a decrease of appetite.
Many lines of evidence indicate that obesity induced by high fat
food can increase the secretion of several biologically active adi-
pokines including leptin, TNF-a, and IL-6 [15]. These cytokines play
an important role in obesity-related inflammation and metabolic
diseases. The levels of cytokines are markedly elevated in obese
individuals and a reduction of adipose mass leads to a decrease in
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.cbi.2016.07.034.
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