Archives of Biochemistry and Biophysics 686 (2020) 108365

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Archives of Biochemistry and Biophysics

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Pelargonidin suppresses adipogenesis in 3T3-L1 cells through inhibition of T

PPAR-γ signaling pathway
Lu Guoa, Jum Soon Kanga, Nam Jun Kangc, Byoung Il Jea, Yong Jae Leea, Young Hoon Parka,
Young Whan Choia,b,∗
a
Department of Horticultural Bioscience, Republic of Korea
b
Life and Industry Convergence Research Institute, Pusan National University, Miryang, 50463, Republic of Korea
c
Department of Horticulture, Gyeongsang National University, Jinju, 52828, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Pelargonidin is a natural compound that exists widely in fruits, and exerts antioxidant, anti-atherosclerotic, anti-
Pelargonidin inflammatory, anti-hyperglycemic, and anti-diabetic activities. However, there have not been any studies con-
3T3-L1 cells cerning its anti-obesity potential to date. Therefore, we evaluated the anti-obesity potential of pelargonidin via
Adipogenesis inhibition of adipogenesis in 3T3-L1 cells. The cellular oil droplet content was decreased to 68.14%, 56.75%, and
Triglyceride
48.39% and triglyceride accumulation decreased to 74.53%, 61.54%, and 47.86% after incubation with 5 μM,
PPAR-γ
10 μM, and 20 μM pelargonidin, respectively, when compared with DMSO group. Furthermore, pelargonidin
treatment led to decrease in glucose consumption. Western blot assay illustrated that the expression of PPAR-γ
was suppressed to 63.25%, 47.52%, and 21.23% after incubation with 5 μM, 10 μM, and 20 μM pelargonidin
when compared with DMSO group. Then, we measured the expression of some target proteins of PPAR-γ, and
found that pelargonidin decreased the expressions of HMGCR, LPL, Glut4, and A-FABP. Besides, the result of
Luciferase Reporter Assay indicated that pelargonidin inhibited PPAR-γ transcription activity. These results
indicated that pelargonidin exerts anti-adipogenic activity in 3T3-L1 cells through inhibition of PPAR-γ signaling
pathway, and pelargonidin could be used as a potential anti-obesity agent.

1. Introduction adipocyte tissue in vivo [14]. The 3T3-L1 cell line, a preadipocyte cell
line, is broadly used for studying anti-adipogenic approaches in vitro
Anthocyanins are a subgroup of water-soluble flavonoids that are [15].
responsible for colors of many flowers and fruits [1]. Previous studies Adipogenesis is regulated by many transcript factors these affect the
have revealed that anthocyanins, such as pelargonidin, delphinidin, progress of differentiation from preadipocytes to adipocytes.
malvidin, and cyanidin, exhibit a plenty of health benefits and con- Peroxisome proliferator activated receptor-γ (PPAR-γ), a member of
tribute to prevention of cancer, cardiovascular diseases, and diabetes PPAR family of nuclear hormone receptors, is considered to regulate
[2]. Pelargonidin is well known to rich in several fruits, such as adipogenesis both in vitro and in vivo. [16] Previous studies have in-
strawberry, grape, and mulberry [3–5]. It has previously been shown dicated that PPAR-γ regulates the activities of glucose transporter type
that pelargonidin exerted anti-inflammatory and antioxidant activities 4 (Glut4), lipoprotein lipase (LPL), adipocyte fatty-acid binding protein
[6,7]. Besides, it has been reported that pelargonidin presented po- (A-FABP), and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase
tential preventive action toward atherosclerosis and ameliorating ac- (HMGCR) during preadipocyte differentiation to adipocytes [17–22].
tivity against hyperglycemia and diabetes [8,9]. Insulin, rosiglitazone, and dexamethasone are effective inducible fac-
Obesity is an increasing medical problem all over the world and it tors for differentiation in 3T3-L1 cells [23].
has significantly affected the quality of life in the last few decades [10]. In this study, the mechanism of the PPAR-γ signaling pathway in
Patients with obesity tend to develop several health issues, such as adipocyte inhibited by pelargonidin brings great interest to test the
dyslipidemia, insulin resistance, hypertension, kidney disease, and fatty inhibiting effects of pelargonidin on the adipogenesis in 3T3-L1 cell.
liver [11–13]. Adipocytes are the major components of white adipocyte Furthermore, how pelargonidin inhibit adipogenesis in 3T3-L1 cell is to
tissue. Increasing preadipocytes adipogenesis causes increase in mass of be investigated by evaluating the target genes of PPAR-γ, glucose

∗
Corresponding author. Department of Horticultural Bioscience, Pusan National University, Miryang, 50463, Republic of Korea.
E-mail address: [email protected] (Y.W. Choi).

https://doi.org/10.1016/j.abb.2020.108365
Received 3 March 2020; Received in revised form 8 April 2020; Accepted 8 April 2020
Available online 18 April 2020
0003-9861/ © 2020 Elsevier Inc. All rights reserved.
L. Guo, et al. Archives of Biochemistry and Biophysics 686 (2020) 108365

consumption, and triglycerides (TG) accumulation.

2. Materials and methods

2.1. Reagents

Pelargonidin chloride, rosiglitazone, NaHCO3, dexamethasone, in-

sulin, HEPEs solution, DMSO, and oil red O were bought from Sigma-
Aldrich (St. Louis, MO, USA). Antibodies and RIPA buffer were pur-
chased from Santa Cruz Biotechnology (Paso Robles, CA, USA).
Paraformaldehyde was procured from Tokyo Chemical Industry (Tokyo,
Japan). Isopropanol and triton X-100 were supplied by GENEray
Fig. 1. Pelargonidin did not show cytotoxicity when concentration is lower
(Shanghai, China). Western loading buffer, protein marker, and Bio-Rad
than 200 μM. Structure of pelargonidin (A). Cells were seeded in 96 well plate
protein assay reagent were obtained from BioRad (Hercules, CA, USA).
and grown to full confluence, then pelargonidin was added to differentiation
medium. 8 days later, an MTT assay was performed to check cell viability (B).
2.2. Cell culture Values represent mean ± SD (n = 3), * as p < 0.05 and ** as p < 0.01 vs.
DMSO group.
The 3T3-L1 cell line was bought from Korea Cell Line Bank (Seoul,
Korea) and was grown and maintained in DMEM (Gibco, Gland Island,
kit (Asan Pharmaceutical, Seoul, Korea).
NY, USA) containing 10% FBS (Welgene, Seoul, Korea), 25 mM
NaHCO3 (Sigma), and 25 mM HEPEs (Sigma) at 37 °C with 5% CO2. For
2.7. Triglycerides (TG) content determination
inducing adipogenesis, 3T3-L1 cells were seeded and grown in 48-well
plates to full confluence. Then adipogenesis of adipocytes was induced
To investigate whether pelargonidin affects TG accumulation in
with differentiation medium (DM), DMEM supplemented with 10%
vivo, differentiation method was performed in 6-well plate as described
FBS, 25 mM NaHCO3, 25 mM HEPES, 10 μM rosiglitazone (ROS),
in cell culture section. TG were extracted using 5% triton X-100 on day
10 μg/ml insulin (In), and 1 μM dexamethasone (DEX). 2 days later, the
8 and quantitative analysis was performed according to manufacturer's
medium was replaced with fresh differentiation medium, or differ-
protocol using a TG kit (Asan Pharmaceutical).
entiation medium with DMSO, or differentiation medium with pe-
largonidin (5 μM, 10 μM, and 20 μM) for an additional 2 days, and then,
the medium was replenished every 2 days. 2.8. Western blotting

2.3. MTT assay After treatment with DMSO or different concentrations of pelargo-
nidin in differentiation medium for 8 days, cells were collected, and
3T3-L1 cells were seeded in 96 well plates with DMEM medium and then, lysed with RIPA buffer on ice for 30 min. Protein concentrations
grown to full confluence, then different concentrations (5, 10, 20, 50, were detected using a Bio-Rad protein assay reagent after lysates were
100, 200 μM) of pelargonidin were added to differentiation medium, centrifuged at 12 000 g at 4 °C for 15 min. Then, proteins, mixed with
and the cells were incubated for 8 days. MTT solution (50 μl of 2 mg/ 4 × loading buffer and 2-mercaptoethanol, were heated at 95 °C for
ml) was added to each well, and the cultures were incubated for an 7 min. 12% SDS-PAGE was used to separate 40 μg of proteins for each
additional 4 h. After removing incubation medium, formazan crystals sample along with a protein marker. Then proteins were electro-
were dissolved in 150 μl solution of DMSO (VWR, Solon, OH, USA). transferred to a PVDF membrane under 20 V for 30 min using a Semi
MTT reduction was quantified by measuring at 570 nm using an ELISA Dry Transfer system. The membranes were blocked with blocking buffer
reader (BioTek Instruments, VT, USA). at room temperature for 1 h, and then, incubated with primary anti-
bodies overnight at 4 °C. The membranes were then washed with PBST
2.4. Oil red O staining solution three times, for 5 min each time and incubated with secondary
antibodies for another 1 h at room temperature. Immuno-reactive
On day 8, cells were washed by PBS and fixed using 4% paraf- bands were detected using an ECL kit (Thermo Scientific, Rockford, IL,
ormaldehyde (Tokyo Chemical Industry) at room temperature for USA) according to the instructions after the membranes were washed
20 min, then the cells were stained by oil red O at room temperature for with PBST solution. Bands were analyzed by ImageJ software (Wayne
30 min. Subsequently, excessive dye was discarded, and cells were Rasband, NIH, USA).
washed by PBS. After which, images were captured using camera and
microscope at 100 × and 200 × magnification (Moitc, Xiamen, Fujian, 2.9. Luciferase reporter assay
China).
The Dual-Luciferase Reporter assay system (Promega, Madison, WI,
2.5. Oil red O OD value detection USA) was performed to detect whether pelargonidin is an antagonist of
PPAR-γ. The expression plasmid for the pCMXGal-hPPAR-γ-ligand
PBS was discarded after capturing images. Subsequently, 400 μl binding domain (LBD) and the Gal4 reporter vector MH100-4-TK-Luc
isopropanol was used to elute oil red O for each well, following transfer were co-transfected with 0.1 μg of pREP7 (Renilla luciferase) reporter
of 100 μl isopropanol from each well to a 96-well plate, and then, ab- for normalizing transfection efficiencies to HEK293T cells (KCLB) using
sorbance was observed at 520 nm using an ELISA reader (BioTek FuGENE-HD (Roche, Switerland) for 24 h. Then the PPAR-γ agonist,
Instruments, Winooski, VT, USA). rosiglitazone (10 μM) and pelargonidin were added to medium, and the
cells were incubated for another 24 h to determine luciferase activity.
2.6. Glucose consumption determination
2.10. Statistical analysis
Adipogenesis was induced in 48-well plates as described in cell
culture section. Glucose concentration in each well was determined All data are expressed as the means ± SD. The one-way analysis of
according to manufacturer's protocol on day 8 using a glucose quantify ANOVA was performed using SPSS 25.0 to detect differences among

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L. Guo, et al. Archives of Biochemistry and Biophysics 686 (2020) 108365

Fig. 2. Pelargonidin inhibited adipo-

genesis in 3T3-L1 cell line. 3T3-L1 cell
line was cultured in DMEM containing
10% FBS, 25 mM NaHCO3, and 25 mM
HEPES (culture medium, Control) and
adipogenesis was induced in the cells
using culture medium containing 10 μM
rosiglitazone, 10 μg/ml insulin, and
1 μM dexamethasone (differentiation
medium, DM). DMSO or pelargonidin
dissolved in DMSO was added to the
medium during the whole inducing
period. Images were captured after oil
red O staining on day 8 (A). OD value of
each sample at 520 nm was measured
using an ELISA reader (B). Images were
captured at 100 × and 200× magnifi-
cation before and after oil red O
staining (C). Values represent
mean ± SD (n = 3), ** as p < 0.01
vs. DM with DMSO group. (For inter-
pretation of the references to color in
this figure legend, the reader is referred
to the Web version of this article.)

Fig. 4. Pelargonidin inhibited triglycerides (TG) accumulation in 3T3-L1 cell

Fig. 3. Pelargonidin inhibited glucose consumption in 3T3-L1 cell line. 3T3-L1
line. 3T3-L1 cell line was cultured in DMEM containing 10% FBS, 25 mM
cell line was cultured in DMEM containing 10% FBS, 25 mM NaHCO3, and
NaHCO3, and 25 mM HEPES (culture medium, Control) and adipogenesis in-
25 mM HEPES (culture medium, Control) and adipogenesis was induced using
duced using culture medium containing 10 μM rosiglitazone, 10 μg/ml insulin,
culture medium containing 10 μM rosiglitazone, 10 μg/ml insulin, and 1 μM
and 1 μM dexamethasone (differentiation medium, DM). DMSO or pelargonidin
dexamethasone (differentiation medium, DM). DMSO or pelargonidin dissolved
dissolved in DMSO was added to medium during the whole inducing period.
in DMSO was added to medium during the whole inducing period. Medium of
Cells were collected on day 8, TG was extracted with 5% Triton X-100 and the
each well was collected to measure glucose content on day 8, and then, glucose
quantity was determined using a commercial TG kit, according to manufac-
content was using a commercial glucose determination kit. Pelargonidin sup-
turer's protocol. Pelargonidin decreased TG content in differentiated 3T3-L1 cell
pressed glucose uptake in 3T3-L1 cells. Values represent mean ± SD (n = 3),
line. Values represent mean ± SD (n = 3), * as p < 0.05 and ** as p < 0.01
** as p < 0.01 vs. with DMSO group.
vs. DMSO group.

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L. Guo, et al. Archives of Biochemistry and Biophysics 686 (2020) 108365

Fig. 5. Pelargonidin inhibited expres-

sion of PPAR-γ and its target genes in
differentiated 3T3-L1 cells. 3T3-L1 cell
line was maintained in DMEM con-
taining 10% FBS, 25 mM NaHCO3, and
25 mM HEPES (culture medium,
Control) and adipogenesis was induced
using culture medium containing 10 μM
rosiglitazone, 10 μg/ml insulin, and
1 μM dexamethasone (differentiation
medium, DM). DMSO or pelargonidin
dissolved in DMSO was added to the
medium during the whole inducing
period. The total proteins were ex-
tracted with RIPA buffer, and analyzed
by western blotting. Pelargonidin in-
hibited expression of PPAR-γ and its
target genes after 8 days. Values re-
present mean ± SD (n = 3), * as
p < 0.05 and ** as p < 0.01 vs. DMSO
group.

groups. Statistical significance was regarded when p < 0.05. treatment led to inhibition of adipogenesis in a dose-dependent manner
(Fig. 2A and C). After capturing images, OD detection was performed.
The results indicated that pelargonidin treatment led to a significantly
3. Results
decrease of OD (Fig. 2B). Specifically, treatment with 5 μM, 10 μM, and
20 μM led to a decrease to 68.14 ± 1.77%, 56.75 ± 3.03% and
3.1. Cell viability of 3T3-L1 cells treated with pelargonidin
48.39 ± 1.11%, respectively, when compared to DMSO group.
To detect whether pelargonidin has cytotoxicity in 3T3-L1 cells, an
MTT assay was performed. Cells were treated with 5, 10, 20, 50, 100,
3.3. Pelargonidin inhibited glucose consumption of 3T3-L1 cells
200 μM of pelargonidin and incubated for 8 days in the differentiation
medium. Pelargonidin did not show cytotoxicity when concentration is
In order to investigate whether pelargonidin affects glucose con-
lower than 200 μM (Fig. 1B).
sumption, we measured the concentration of glucose in the medium.
The results showed that the glucose concentrations of pelargonidin-
3.2. Pelargonidin inhibited adipogenesis in 3T3-L1 cells treated groups were notably higher than DMSO group's (Fig. 3). Spe-
cifically, glucose concentrations in culture medium were 4.49 ± 0.10,
To investigate whether pelargonidin affects adipogenesis in 3T3-L1 6.20 ± 0.15, and 7.15 ± 0.10 folds of DMSO group following
cells, cells were incubated with differentiation medium plus 5 μM, treatment with 5 μM, 10 μM, and 20 μM pelargonidin, respectively.
10 μM, and 20 μM pelargonidin from day 0 to day 8. On day 8, cells These indicated that pelargonidin significantly suppressed glucose
were photographed using a camera and a microscope at 100 × and consumption of 3T3-L1 cells a dose-dependent manner.
200 × magnification after staining with oil red O. Pelargonidin

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L. Guo, et al. Archives of Biochemistry and Biophysics 686 (2020) 108365

[26,27]. Pelargonidin exhibited anti-adipogenic effects in the present

study (Fig. 2).
It is already reported that glucose consumption raises when 3T3-L1
cells treated with rosiglitazone [28]. High glucose availability promotes
pre-adipocytes differentiation to adipocytes [29]. Pelargonidin notably
inhibited glucose consumption in dose-dependent manner in the pre-
sence of rosiglitazone (Fig. 3). Besides, TG accumulation was notably
suppressed by pelargonidin (Fig. 4). This might explain why pelargo-
nidin suppressed 3T3-L1 preadipocyte differentiation to adipocytes.
PPAR-γ has been used as an anti-adipogenic drug target for drug
screening [30,31]. It is shown that PPAR-γ regulates fatty acid storage
and glucose consumption [32]. PPAR-γ antagonists may inhibit lipo-
genesis and adipocyte differentiation, reduce fat weight in the obese
mouse. Thus, expression of PPAR-γ was measured using western blot
Fig. 6. Pelargonidin inhibited the transcription activity of PPAR-γ. HEK293T assay in the present work. Pelargonidin significantly inhibited the ex-
cells were co-transfected with GAL4-PPAR-γ ligand binding domain (LBD) fu- pression of PPAR-γ when compared with DMSO group (Fig. 5). Besides,
sion plasmid and a plasmid of the GAL4UAS-luciferase reporter for 24 h. Then,
the result of Luciferase reporter assay illustrated that pelargonidin in-
pelargonidin was added to the medium with or without rosiglitazone in the
hibited the transcription activity of PPAR-γ induced by rosiglitazone
medium and remain for another 24 h. The Dual-Luciferase Reporter Assay
system were carried out. Pelargonidin inhibited the transcription activity of
(Fig. 6). It has been revealed that higher expression of PPAR-γ in adi-
PPAR-γ. Values represent mean ± SD (n = 3), * as p < 0.05 and ** as pose tissue of obese human than lean human [33]. Deletion of PPAR-γ
p < 0.01 vs. DMSO group. reduced body weight gain, serum leptin and serum adiponectin in high-
fat diet induced mice [34]. Therefore, treatment with pelargonidin can
be anticipate to prevent and ameliorate obesity, although further stu-
3.4. Pelargonidin inhibited TG accumulation 3T3-L1 cells
dies are need to verify efficacy in vivo and its safety.
Furthermore, expression of several PPAR-γ target genes which relate
Oil Red O staining assay illustrated that pelargonidin inhibited in-
to glucose consumption and lipids accumulation was measured using
tracellular lipids accumulation (Fig. 2). TG accumulation is account for
western blot assay. A-FABP is an important protein in regulation of fatty
lipids accumulation in adipocytes. In this study, TG levels of pelargo-
acid storage; therefore, blocking expression of A-FABP possibly im-
nidin-treated groups were significantly suppressed in a dose-dependent
proved obesity [35]. LPL plays an considerable role in lipids uptake
manner (Fig. 4). TG contents were decreased to 74.53 ± 3.89%,
[36]. A previous study has revealed that inhibition of expression of
61.54 ± 1.36%, and 47.86 ± 1.07% of the DMSO group's after in-
HMGCR leads to decrease in lipid droplets and intracellular TG content
cubation with 5 μM, 10 μM, and 20 μM pelargonidin, respectively.
in vitro. [37] Fig. 5 showed that expression of A-FABP, LPL and HMGCR
was inhibited after cells incubation with pelargonidin. This contributed
3.5. Pelargonidin inhibited expression of PPAR-γ pathway in differentiated to reduction of TG accumulation and lipid droplets in 3T3-L1 cells. To
3T3-L1 cells reveal the mechanism of the inhibiting glucose consumption by pe-
largonidin, the expression of Glut4 was measured. A dose-dependent
To reveal the anti-adipogenic mechanism of pelargonidin, total decrease of expression of Glut4 by pelargonidin treatment was observed
proteins were extracted for western blot assay. The results demon- (Fig. 5). Glut4 plays a considerable role in glucose transportation in
strated that the differentiation medium and DMSO groups exhibited 3T3-L1 cells when differentiate to adipocytes [38]. Inhibition of ex-
higher expression of PPAR-γ than the control group. While, expression pression of Glut4 by pelargonidin led to suppress in glucose consump-
of PPAR-γ was dose-dependently inhibited after incubation with pe- tion in 3T3-L1 cells, which contributes to suppression of adipogenesis in
largonidin for day 8, and the ratios of PPAR-γ/α-tubulin after incuba- 3T3-L1 cells.
tion with 5 μM, 10 μM, and 20 μM pelargonidin on day 8 were
63.25 ± 5.68%, 47.52 ± 2.14%, and 21.23 ± 3.41% of DMSO
group, respectively (Fig. 5). Besides, expressions of HMGCR, LPL, Glut4, 5. Conclusion
and A-FABP were detected. Fig. 5 shows that pelargonidin treatment
inhibited expressions of HMGCR, LPL, Glut4, and A-FABP in dose-de- In summary, pelargonidin significantly inhibited adipogenesis,
pendent manner. glucose consumption, and TG accumulation during the differentiation
process of 3T3-L1 cells. Such inhibiting effects are accompanied by
3.6. Pelargonidin inhibited the transcription activity of PPAR-γ suppresses expression of PPAR-γ, which consequently modulate its
down-stream genes including A-FABP, HMGCR, LPL, and Glut4. Thus,
Based on the inhibition of 3T3-L1 cells differentiation and the the results provide a cellular and molecular basis for inhibiting me-
suppression of the expression of PPAR-γ and its target genes, we hy- chanism of pelargonidin against adipogenesis in vitro.
pothesize that pelargonidin might target PPAR-γ directly. To test this
hypothesis, A Dual-Luciferase Reporter assay system was performed.
Rosiglitazone could activate PPAR-γ transcription, which was sig- Declaration of competing interest
nificantly inhibited by pelargonidin (Fig. 6).
The authors have no conflicts of interest to declare.
4. Discussion

Obesity influences people's daily life and life quality around the Acknowledgements
world [24]. The 3T3-L1 cells is universally used for screening novel
anti-obesity drugs in vitro [25]. In order to induce adipogenesis in the This study was financially supported by the Korea Institute of
3T3-L1 cells, insulin, dexamethasone, and rosiglitazone, a PPAR-γ Planning and Evaluation for Technology in Food, Agriculture, Forestry
agonist, were added to medium in the present work. These three ef- and Fisheries (IPET), South Korea (315004-05-1-HD030).
fectors have been shown to induce adipogenesis in the 3T3-L1 cells

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L. Guo, et al. Archives of Biochemistry and Biophysics 686 (2020) 108365

References [21] K.T. Iida, Y. Kawakami, H. Suzuki, H. Sone, H. Shimano, H. Toyoshima, Y. Okuda,
N. Yamada, PPAR gamma ligands, troglitazone and pioglitazone, up-regulate ex-
pression of HMG-CoA synthase and HMG-CoA reductase gene in THP-1 macro-
[1] S. Asen, R.N. Stewart, K.H. Norris, Co-pigmentation of anthocyanins in plant tissues phages, FEBS Lett. 520 (2002) 177–181, https://doi.org/10.1016/s0014-5793(02)
and its effect on color, Phytochemistry 11 (1972) 1139–1144, https://doi.org/10. 02811-9.
1016/S0031-9422(00)88467-8. [22] D. Vishwanath, H. Srinivasan, M.S. Patil, S. Seetarama, S.K. Agrawal, M.N. Dixit,
[2] J. He, M.M. Giusti, Anthocyanins: natural colorants with health-Promoting prop- K. Dhar, Novel method to differentiate 3T3 L1 cells in vitro to produce highly
erties, Annu. Rev. Food Sci. Technol. 1 (2010) 163–187, https://doi.org/10.1146/ sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose
annurev.food.080708.100754. analog, J. Cell Commun. Signal. 7 (2013) 129–140, https://doi.org/10.1007/
[3] M. Buchweitz, M. Speth, D.R. Kammerer, R. Carle, Impact of pectin type on the s12079-012-0188-9.
storage stability of black currant (Ribes nigrum L.) anthocyanins in pectic model [23] K. Zebisch, V. Voigt, M. Wabitsch, M. Brandsch, Protocol for effective differentia-
solutions, Food Chem. 139 (2013) 1168–1178, https://doi.org/10.1016/j. tion of 3T3-L1 cells to adipocytes, Anal. Biochem. 425 (2012) 88–90, https://doi.
foodchem.2013.02.005. org/10.1016/j.ab.2012.03.005.
[4] Q. Lou, L. Wang, H. Liu, Y. Liu, Anthocyanin profiles in flowers of grape hyacinth, [24] C.M. Apovian, Obesity: definition, comorbidities, causes, and burden, Am. J.
Molecules 22 (2017), https://doi.org/10.3390/molecules22050688. Manag. Care 22 (2016) s176–s185.
[5] A.M. Pawlowska, W. Oleszek, A. Braca, Quali-quantitative analyses of flavonoids of [25] F.J. Ruiz-Ojeda, A.I. Ruperez, C. Gomez-Llorente, A. Gil, C.M. Aguilera, Cell models
Morus nigra L. and Morus alba L. (Moraceae) fruits, J. Agric. Food Chem. 56 (2008) and their application for studying adipogenic differentiation in relation to obesity: a
3377–3380, https://doi.org/10.1021/jf703709r. review, Int. J. Mol. Sci. 17 (2016), https://doi.org/10.3390/ijms17071040.
[6] L.J. Duarte, V.C. Chaves, M.V.P.S. dos Nascimento, E. Calvete, M. Li, E. Ciraolo, [26] Y. Zhang, L. Yu, W. Cai, S. Fan, L. Feng, G. Ji, C. Huang, Protopanaxatriol, a novel
A. Ghigo, E. Hirsch, C.M.O. Simões, F.H. Reginatto, E.M. Dalmarco, Molecular PPAR γ antagonist from Panax ginseng, alleviates steatosis in mice, Sci. Rep. 4
mechanism of action of pelargonidin-3-O-glucoside, the main anthocyanin re- (2014) 1–12, https://doi.org/10.1038/srep07375.
sponsible for the anti-inflammatory effect of strawberry fruits, Food Chem. 247 [27] L. Feng, H. Luo, Z. Xu, Z. Yang, G. Du, Y. Zhang, L. Yu, K. Hu, W. Zhu, Q. Tong,
(2018) 56–65, https://doi.org/10.1016/j.foodchem.2017.12.015. K. Chen, F. Guo, C. Huang, Y. Li, Bavachinin, as a novel natural pan-PPAR agonist,
[7] L.S. Wang, X.D. Sun, Y. Cao, L. Wang, F.J. Li, Y.F. Wang, Antioxidant and pro- exhibits unique synergistic effects with synthetic PPAR-gamma and PPAR-alpha
oxidant properties of acylated pelargonidin derivatives extracted from red radish agonists on carbohydrate and lipid metabolism in db/db and diet-induced obese
(Raphanus sativus var. Niger, Brassicaceae), Food Chem. Toxicol. 48 (2010) mice, Diabetologia 59 (2016) 1276–1286, https://doi.org/10.1007/s00125-016-
2712–2718, https://doi.org/10.1016/j.fct.2010.06.045. 3912-9.
[8] J.E. Son, H. Jeong, H. Kim, Y.A. Kim, E. Lee, H.J. Lee, K.W. Lee, Pelargonidin at- [28] R. Lennon, G.I. Welsh, A. Singh, S.C. Satchell, R.J. Coward, J.M. Tavaré,
tenuates PDGF-BB-induced aortic smooth muscle cell proliferation and migration by P.W. Mathieson, M.A. Saleem, Rosiglitazone enhances glucose uptake in glomerular
direct inhibition of focal adhesion kinase, Biochem. Pharmacol. 89 (2014) 236–245, podocytes using the glucose transporter GLUT1, Diabetologia (2009), https://doi.
https://doi.org/10.1016/j.bcp.2014.02.015. org/10.1007/s00125-009-1423-7.
[9] M. Roy, S. Sen, A.S. Chakraborti, Action of pelargonidin on hyperglycemia and [29] R.M. Jackson, B.A. Griesel, J.M. Gurley, L.I. Szweda, A.L. Olson, Glucose avail-
oxidative damage in diabetic rats: implication for glycation-induced hemoglobin ability controls adipogenesis in mouse 3T3-L1 adipocytes via up-regulation of ni-
modification, Life Sci. 82 (2008) 1102–1110, https://doi.org/10.1016/j.lfs.2008. cotinamide metabolism, J. Biol. Chem. 292 (2017) 18556–18564, https://doi.org/
03.011. 10.1074/jbc.M117.791970.
[10] F. Corica, G. Bianchi, A. Corsonello, N. Mazzella, F. Lattanzio, G. Marchesini, [30] H.-S. Park, U.-I. Ju, J.-W. Park, J.Y. Song, D.H. Shin, K.-H. Lee, L.S. Jeong, J. Yu,
Obesity in the context of aging: quality of life considerations, Pharmacoeconomics H.W. Lee, J.Y. Cho, S.Y. Kim, S.W. Kim, J.B. Kim, K.S. Park, Y.-S. Chun,
33 (2015) 655–672, https://doi.org/10.1007/s40273-014-0237-8. PPARgamma neddylation essential for adipogenesis is a potential target for treating
[11] R. Divella, A. Mazzocca, A. Daniele, C. Sabba, A. Paradiso, Obesity, nonalcoholic obesity, Cell Death Differ. 23 (2016) 1296–1311, https://doi.org/10.1038/cdd.
fatty liver disease and adipocytokines network in promotion of cancer, Int. J. Biol. 2016.6.
Sci. 15 (2019) 610–616, https://doi.org/10.7150/ijbs.29599. [31] A. Janesick, B. Blumberg, Minireview: PPARgamma as the target of obesogens, J.
[12] K. Rahmouni, M.L.G. Correia, W.G. Haynes, A.L. Mark, Obesity-associated Steroid Biochem. Mol. Biol. 127 (2011) 4–8, https://doi.org/10.1016/j.jsbmb.
Hypertension: New Insights into Mechanisms vol. 45, Hypertens., Dallas, Tex, 1979, 2011.01.005.
pp. 9–14, https://doi.org/10.1161/01.HYP.0000151325.83008.b4 2005. [32] U. Kintscher, R.E. Law, PPARγ-mediated insulin sensitization: the importance of fat
[13] J.I. Lakkis, M.R. Weir, Obesity and kidney disease, Prog. Cardiovasc. Dis. 61 (2018) versus muscle, Am. J. Physiol. Endocrinol. Metab. 288 (2005) 287–291, https://doi.
157167, , https://doi.org/10.1016/j.pcad.2018.07.005. org/10.1152/ajpendo.00440.2004.
[14] B.M. Spiegelman, J.S. Flier, Obesity and the regulation of energy balance, Cell 104 [33] A.J. Vidal-Puig, R.V. Considine, M. Jimenez-Liñan, A. Werman, W.J. Pories,
(2001) 531–543, https://doi.org/10.1016/S0092-8674(01)00240-9. J.F. Caro, J.S. Flier, Peroxisome proliferator-activated receptor gene expression in
[15] S.P. Poulos, M.V. Dodson, G.J. Hausman, Cell line models for differentiation: pre- human tissues: effects of obesity, weight loss, and regulation by insulin and glu-
adipocytes and adipocytes, Exp. Biol. Med. 235 (2010) 1185–1193, https://doi.org/ cocorticoids, J. Clin. Invest. (1997), https://doi.org/10.1172/JCI119424.
10.1258/ebm.2010.010063. [34] J.R. Jones, C. Barrick, K.A. Kim, J. Lindner, B. Blondeau, Y. Fujimoto, M. Shiota,
[16] R.M. Evans, G.D. Barish, Y.X. Wang, PPARs and the complex journey to obesity, R.A. Kesterson, B.B. Kahn, M.A. Magnuson, Deletion of PPARγ in adipose tissues of
Nat. Med. 10 (2004) 355–361, https://doi.org/10.1038/nm1025. mice protects against high fat diet-induced obesity and insulin resistance, Proc.
[17] S. Fan, L. Guo, Y. Zhang, Q. Sun, B. Yang, C. Huang, Okra polysaccharide improves Natl. Acad. Sci. U.S.A. (2005), https://doi.org/10.1073/pnas.0306743102.
metabolic disorders in high-fat diet-induced obese C57BL/6 mice, Mol. Nutr. Food [35] L. Makowski, G.S. Hotamisligil, Fatty acid binding proteins—the evolutionary
Res. 57 (2013) 2075–2078, https://doi.org/10.1002/mnfr.201300054. crossroads of inflammatory and metabolic responses, J. Nutr. 134 (2004)
[18] W. Liao, M.T.A. Nguyen, T. Yoshizaki, S. Favelyukis, D. Patsouris, T. Imamura, 2464S–2468S, https://doi.org/10.1093/jn/134.9.2464s.
I.M. Verma, J.M. Olefsky, Suppression of PPAR-γ attenuates insulin-stimulated [36] G. Ranganathan, R. Unal, I. Pokrovskaya, A. Yao-Borengasser, B. Phanavanh,
glucose uptake by affecting both GLUT1 and GLUT4 in 3T3-L1 adipocytes, Am. J. B. Lecka-Czernik, N. Rasouli, P.A. Kern, The lipogenic enzymes DGAT1, FAS, and
Physiol. Endocrinol. Metab. 293 (2007), https://doi.org/10.1152/ajpendo.00695. LPL in adipose tissue: effects of obesity, insulin resistance, and TZD treatment, J.
2006. Lipid Res. 47 (2006) 2444–2450, https://doi.org/10.1194/jlr.M600248-JLR200.
[19] F.G. Gbaguidi, G. Chinetti, D. Milosavljevic, E. Teissier, J. Chapman, G. Olivecrona, [37] Y.S. Yeh, H.F. Jheng, M. Iwase, M. Kim, S. Mohri, J. Kwon, S. Kawarasaki, Y. Li,
J.C. Fruchart, S. Griglio, J. Fruchart-Najib, B. Staels, Peroxisome proliferator-acti- H. Takahashi, T. Ara, W. Nomura, T. Kawada, T. Goto, The mevalonate pathway is
vated receptor (PPAR) agonists decrease lipoprotein lipase secretion and glycated indispensable for adipocyte survival, IScience 9 (2018) 175–191, https://doi.org/
LDL uptake by human macrophages, FEBS Lett. 512 (2002) 85–90, https://doi.org/ 10.1016/j.isci.2018.10.019.
10.1016/S0014-5793(02)02223-8. [38] E.D. Abel, O. Peroni, J.K. Kim, Y.B. Kim, O. Boss, E. Hadro, T. Minnemann,
[20] S.L. Pearson, M.A. Cawthorne, J.C. Clapham, S.J. Dunmore, S.D. Holmes, G.I. Shulman, B.B. Kahn, Adipose-selective targeting of the GLUT4 gene impairs
G.B. Moore, S.A. Smith, M. Tadayyon, The thiazolidinedione insulin sensitiser, BRL insulin action in muscle and liver, Nature 409 (2001) 729–733, https://doi.org/10.
49653, increases the expression of PPAR-gamma and aP2 in adipose tissue of high- 1038/35055575.
fat-fed rats, Biochem. Biophys. Res. Commun. 229 (1996) 752–757, https://doi.
org/10.1006/bbrc.1996.1876.