5-Demethylnobiletin and 5-Acetoxy-6,7,8,3′,4′-pentamethoxyflavone Suppress Lipid Accumulation by Activating the LKB1-AMPK Pathway in 3T3-L1 Preadipocytes and High Fat Diet-Fed C57BL/6 Mice

Article abstract of DOI:10.1021/acs.jafc.6b00706

Polymethoxyflavones (PMFs) and hydroxylated polymethoxyflavones (HPMFs), such as nobiletin (Nob) and 5-demethylnobiletin (5-OH-Nob), are unique flavonoids that are found exclusively in citrus peels. Nobiletin has been shown to suppress adipogenesis in vit

Full text of DOI:10.1021/acs.jafc.6b00706

Article
pubs.acs.org/JAFC
5‑Demethylnobiletin and 5‑Acetoxy-6,7,8,3′,4′-pentamethoxyflavone
Suppress Lipid Accumulation by Activating the LKB1-AMPK Pathway
in 3T3-L1 Preadipocytes and High Fat Diet-Fed C57BL/6 Mice
Yen-Chen Tung,†,§ Shiming Li,# Qingrong Huang,† Wei-Lun Hung,† Chi-Tang Ho,† Guor-Jien Wei,*,⊗
and Min-Hsiung Pan*,#,§,⊥,Δ
†Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States
§Institute of Food Sciences and Technology, National Taiwan University, Taipei 106, Taiwan
#Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization and Hubei
Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang Normal University,
Huanggang, Hubei China
⊗Department of Nutrition and Health Sciences, Kainan University, Taoyuan 33857, Taiwan
⊥Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan
ΔDepartment of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan
S
* Supporting Information
ABSTRACT: Polymethoxyflavones (PMFs) and hydroxylated polymethoxyflavones (HPMFs), such as nobiletin (Nob) and
5-demethylnobiletin (5-OH-Nob), are unique flavonoids that are found exclusively in citrus peels. Nobiletin has been shown
to suppress adipogenesis in vitro, but the antiadipogenic activity of 5-OH-Nob has not been investigated. Both nobiletin and
5-OH-Nob have poor aqueous solubility and low oral bioavailability. We employed chemical modification to produce the acetyl
derivative of 5-OH-Nob, that is, 5-acetyloxy-6,7,8,3′,4′-pentamethoxyflavone (5-Ac-Nob), to improve its bioavailability and
bioactive efficiency. We found that 5-Ac-Nob reduced triacylglycerol (TG) content to a greater extent than 5-OH-Nob in 3T3-L1
preadipocytes. Orally administered 5-Ac-Nob resulted in a significant reduction in body weight, intra-abdominal fat, plasma and
liver TG levels, and plasma cholesterol level in high fat diet-induced obese male C57BL/6J mice. The 5-Ac-Nob treatment
decreased lipid accumulation by triggering the adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway to alter
transcriptional factors or lipogenesis-related enzymes in vivo and in vitro.
KEYWORDS: 5-acetyloxy-6,7,8,3′,4′-pentamethoxyflavone, high-fat diet, 3T3-L1 preadipocytes, LKB1-AMPK pathway
differentiation.4,6,7 In addition, AMPK is a major regulator of
INTRODUCTION
■
cellular energy balance that switches on the catabolic pathway
In 2015, the World Health Organization estimated that >1.9
and switches off the anabolic pathway.8 AMPK can regulate several
billion adults are overweight and >600 million people are obese.1
metabolic pathways, including glucose transport, gluconeogenesis,
An energy imbalance is the major cause of obesity. During
lipogenesis, and lipolysis.9 AMPK can control lipid metabolism
adipogenesis, excess calories are stored as triacylglycerols (TGs),
by phosphorylating acetyl-CoA carboxylases (ACC) 1 and 2 or
and the number and size of adipocytes are increased. In
regulating the long-term effects of phosphorylated SREBP-1 and
lipogenesis, the syntheses of fatty acids and TGs occurs in the
liver and adipose tissues, which eventually expands the adipose
the loss of expression of lipogenic enzymes.10
tissues.2,3 The function of brown adipose tissues is different from
Polymethoxyflavones (PMFs) and hydroxylated polymethox-
yflavones (HPMFs) are flavonoids found in citrus peels. They
that of white adipose tissues (WAT) in the body. WAT is not
only a major TG storage tissue that can be deposited under the
have several biological benefits, such as anti-inflammatory
and anticancer activities and regulation of lipid metabolism.11
skin and in the intra-abdominal area but also an endocrine organ
Nobiletin (Nob) and HPMFs can suppress lipid accumulation in
that regulates whole-body energy homeostasis. When there is a
3T3-L1 preadipocytes and high-fat diet-induced obese animal
positive energy balance, adipose tissue dysfunction may develop,
models,12,13 but the antiadipogenic effects of 5-demethylnobiletin
and ectopic fat, especially intra-abdominal fat, will be deposited
in several organs, such as the liver, pancreas, and heart, affecting
(5-OH-Nob) are still unknown. It has been reported that both
their functions.2,4 Therefore, obesity plays a key role in metabolic
PMFs and HPMFs are highly lipophilic and have poor aqueous
solubility and low oral bioavailability.14 Prodrugs are derivatives of
syndromes and is strongly associated with diabetes, cardiovas-
cular disease, fatty liver disease, and cancer.4,5 Transcription
factors play a crucial role in controlling glucose, amino acid, and
lipid metabolic homeostasis. Sterol regulatory element-binding
protein-1 (SREBP-1) is an important transcription factor
involved in cellular lipogenesis, lipid homeostasis, and adipocyte
Received: February 12, 2016
Revised: April 2, 2016
Accepted: April 4, 2016
© XXXX American Chemical Society
A
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6.96 (1H, d, J = 8.5 Hz, H-2′), 7.36 (1H, d, J = 2.1 Hz, H-5′), and 7.52
(1H, dd, J = 2.1, 8.5 Hz, H-6′) (Supplementary Figures 3−6). The
structures of 5-OH-Nob and 5-Ac-Nob are given in Figure 1.
drug molecules. During metabolism, the active parent drugs
derived from prodrugs will be released. The formulation of a
prodrug can improve aqueous solubility, chemical stability,
presystemic metabolism, and brain penetration.15 Chemical
modification is one of the methods of prodrug formation.16
Recently, Wang et al. used chemical modification to obtain
5-acetyloxy-6,7,8,4′-tetramethoxyflavone (5-Ac-Tan), which had
improved anti-breast cancer activity compared to tangeretin.17
In our current study, based on the prodrug concept, we prepared
5-OH-Nob and 5-acetyloxy-6,7,8,3′,4′-pentamethoxyflavone
(5-Ac-Nob) from nobiletin by chemical modification, and their
antiadipogenic activities in the 3T3-L1 preadipocytes and a
high-fat diet-induced obese animal model, as well as the related
molecular mechanisms, were investigated.
Figure 1. Chemical structures of (A) 5-OH-Nob and (B) 5-Ac-Nob.
Cell Culture and Cell Differentiation. Mouse 3T3-L1 preadipo-
cytes purchased from the American Type Culture Collection (Rockville,
MD, USA) were grown in DMEM supplemented with 2 mM glutamine
(Gibco BRL), 1% penicillin/streptomycin (10000 units penicillin/mL
and 10 mg streptomycin/mL), and 10% FCS in a 10 cm dish (Nunc
Thermo, Waltham, MA, USA) at 37 °C under a humidified atmosphere
containing 5% CO2. After incubation with differentiation medium
(DM) containing insulin, IBMX, DEX, and rosiglitazone for 8 days,
3T3-L1 preadipocytes transformed to mature adipocytes. The cells were
seeded into a 96-well (2.5 × 104/mL) plate or a 10 cm dish and cultured
as described above. After 2 days, the cell medium was removed and
replaced by 10% FBS DMEM. When the cells were confluent, defined
as day 0, the cells were incubated in DM containing 5 μg/mL insulin,
0.5 mM IBMX, 1 μM DEX, and 2 μM rosiglitazone in the DMEM
supplemented with 10% FBS for 48 h. After 2 days, the medium was
removed and the cells were incubated in the DMEM containing 10%
FBS and 5 μg/mL insulin for another 2 days, defined as day 2. Starting at
day 4, the cells were incubated in DMEM containing 10% FBS, and
the medium was changed every 2 days until day 8 as the DM positive
[DM (+)] group. Following this protocol, different concentrations of
5-OH-Nob and 5-Ac-Nob were additionally added in the cell for another
2 days from day 0 to day 8 in the cell. The control group cells were not
cultured with insulin, IBMX, DEX, and rosiglitazone from the beginning
to the end of the experiment as the DM negative [DM (−)] group.
Cell Viability. The cell viability was determined by the CytoTox 96
Non-Radioactive Cytotoxicity Assay (Promega Co., San Luis Obispo,
CA, USA). The 3T3-L1 preadipocytes were treated with different
concentrations of 5-OH-Nob and 5-Ac-Nob in the DMEM with 10%
FCS for 48 h, and then the cell viability was determined by lactate
dehydrogenase (LDH) assay. LDH is a stable cytosolic enzyme. When
cell membranes are damaged, LDH will leak out into the medium.18
Briefly, 50 μL of cell supernatant was transferred to a 96-well plate and
50 μL of reconstituted substrate mix was added and incubated for 30 min
at room temperature. To each well was added 50 μL of stop solution
prior to determination of the wavelength absorbance at 490 nm.
Maximum LDH release was cell treated with lysis buffer to determine
the maximum LDH in cells. The percentage of cell death was calculated
according to the equation
MATERIALS AND METHODS
■
Chemicals and Reagents. Powder of PMFs was obtained from
BioGin Biochemicals Co., Ltd. (Chengdu, Sichuan, China). Methanol,
ethanol, water, acetonitrile, chloroform, ethyl acetate, hexanes, 2-propanol,
diethyl ether, and acetone were of HPLC grade and purchased from
Fisher Scientific (Springfield, NJ, USA). Thin layer chromatography
(TLC) plates were purchased from Analtech (Newark, DE, USA).
Enhanced chemiluminescent-based detection system (ECL) and
Hybond-polyvinylidene difluoride (PVDF) membrane were purchased
from Millipore (Billerica, MA, USA). Acrylamide, prestained protein
markers, dimethyl sulfoxide (DMSO), glycerol, and Tris-HCl were
purchased from Merck (Whitehouse Station, NJ, USA). Bio-Rad protein
assay reagent was purchased from Bio-Rad Laboratory (Hercules, CA,
USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin−
streptomycin, fetal calf serum (FCS), and fetal bovine serum (FBS) were
purchased from Gibco BRL (Grand Island, NY, USA). Anti-β-actin
antibody, insulin, 3-isobutylmethylxanthine (IBMX), dexamethasone
(DEX), and isopropanol were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Anti-phosphorylated AMPK (pAMPKα), anti-
AMPKα, anti-LKB1, anti-phosphorylated LKB1 (pLKB1), and anti-fatty
acid synthase (FAS) antibodies were purchased from Cell Signaling
Technology (Beverly, MA, USA). Other antibodies used in this study
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Preparation of 5-OH-Nob and 5-Ac-Nob. PMF crude extracts
were purified by silica gel column chromatography with a mixture of
solvent (hexane/ethyl acetate = 4:1) eluents to obtain nobiletin as a
white powder. 5-OH-Nob was prepared by refluxing nobiletin with
concentrated HCl in 95% aqueous ethanol for 16 h, followed by con-
centration, acidification, extraction with ethyl acetate, and concentration
again prior to another silica gel column chromatography yielding 5-OH-
Nob with a minimum purity of 99%. By employing the same synthetic
methodology described in a previous paper,17 the preparation of
5-Ac-Nob started with refluxing of 5-OH-Nob with acetic anhydride in
methylene chloride with pyridine as a base overnight, then concentra-
tion and aqueous workup with ethyl acetate to yield a crude product
after concentration in vacuo. The crude mixture from the reaction was
subject to purification on a flash silica gel column with hexanes and ethyl
acetate as eluting solvents to yield 5-Ac-Nob as a pale yellow solid with
a purity score of ≥99%. NMR spectra were recorded on a Bruker
AVIII 500 MHz FT-NMR (Bruker, Rheinstetten, Germany) in CDCl3.
LC-MS was performed by a Thermo Scientific linear ion trap mass
spectrometer (LXQTM) in positive ionization mode (Thermo
Scientific, Waltham, MA, USA). The results of NMR and LC-MS are
also given in the Supporting Information. In MS positive mode, 5-OH-
Nob and 5-Ac-Nob exhibited protonated molecules of [M + H]+ at m/z
389.28 and 431.08, respectively (Supplementary Figures 1 and 2). The
proton NMR spectra of 5-OH-Nob was as follows: 1H NMR (CDCl3) δ
3.96 (3H, s, OCH3), 3.98 (3H, s, OCH3), 3.99 (6H, s, OCH3), 4.12 (3H,
s, OCH3), 6.61 (1H, s, H-3), 7.00 (1H, d, J = 8.4 Hz, H-2′), 7.42 (1H, d,
J = 2.0 Hz, H-5′), 7.59 (1H, dd, J = 2.0, 8.4 Hz, H-6′), and 12.55
100% − % cytotoxicity = exptl LDH release/max LDH release
Cell Cycle Analysis. 3T3-L1 cells were cultured in a 24-well plate
followed by the cell differentiation procedure. The cells were harvested
at 24 h after day 2 by trypsinization and fixed by the addition of 800 μL of
100% ice-cold ethanol. Cells were stored at −20 °C until analysis. Before
analysis by Beckman Coulter FC500 flow cytometry (Indianapolis, IN,
USA), the cells were washed with PBS and centrifuged at 400g for 5 min.
After centrifugation, the cells were suspended in a 500 μL propidium
iodine solution (0.2 mg/mL propidium iodide, 0.5% Triton X-100 in
PBS, and 0.5 mg/mL RNase) and incubated at 37 °C for 30 min.
Fluorescence intensity was quantified by flow cytometry.
Oil Red O Staining. Lipid accumulation of the 3T3-L1 cells at day 8
was determined by staining with Oil Red O. The cells were washed
twice with PBS and then fixed with 10% formalin at 4 °C overnight.
The Oil Red O stock solution was diluted with isopropanol (5 mg/mL)
and subsequently filtered through a 0.22 μm MCE filter. The cells were
stained with Oil Red O solution diluted with distilled water for 5 min at
1
(1H, s, OH). H NMR (CDCl3) spectra of 5-Ac-Nob: δ 2.45 (3H, s,
(CO)CH3), 3.86 (3H, s, OCH3), 3.93 (3H, s, OCH3), 3.95 (3H, s,
OCH3), 4.03 (3H, s, OCH3), 4.08 (3H, s, OCH3), 6.52 (1H, s, H-3),
B
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room temperature. After three washings with PBS, cell lipids were
extracted by isopropanol and the absorbance was quantified at 510 nm
wavelength.
Table 1. Effects of 5-OH-Nob and 5-Ac-Nob on Cell Viability
in 3T3-L1 Cells
a
group
5-OH-Nob
5-Ac-Nob
Animal Experiments. Male C57BL/6J mice were purchased from
the BioLASCO Experimental Animal Center (Taiwan Co., Ltd., Taipei,
Taiwan) at 4 weeks of age. The mice were housed in a controlled
temperature of 24 2 °C and 55% relative humidity with a 12 h light/
dark cycle, and the mice had free access to water and food. After 1 week
of acclimation, the animals were randomly divided into four groups as
follows: control diet (control, 15% of calories from fat), high-fat diet
(HFD; 45% of calories from fat (19.6 g of lard, 2 g of soybean oil, and
0.5% cholesterol per 100 g in the normal diet)), oral gavage at a dose of
25 mg/kg of 5-Ac-Nob with HFD as a LAN group, and oral gavage of
50 mg/kg 5-Ac-Nob with HFD as a HAN group for 13 weeks. Each
group contained eight mice. The experimental diets were modified from
the LabDiet 5001 standard diet (LabDiet, St. Louis, MO, USA); the
nutritional composition of the food is listed in Supplementary Table 1.
The food intake was measured every day, and the body weight was
recorded every week for 13 weeks. At the end of the study, all of the
animals were fasted overnight and then sacrificed by CO2 anesthesia.
Blood samples were collected from the heart for biochemical analysis.
Liver, spleen, kidney, and intra-abdominal fat (perigonadal, retroper-
itoneal, and mesenteric fat) were collected and weighed. The tissue
samples were stored at −80 °C before analysis.
control
2.5 μM
5 μM
87.42 1.51a
87.58 13.64a
84.54 15.55a
87.45 14.11a
84.82 17.78a
87.42 1.51a
91.98 13.45a
89.80 15.25a
83.78 15.37a
87.73 11.72a
10 μM
20 μM
a
The 3T3-L1 cells were treated with 5-OH-Nob and 5-Ac-Nob for
48 h. The viability of 3T3-L1 cells was determined by the LDH assay.
Data are expressed as the mean SD (n = 3) and were statistically
analyzed using a one-way ANOVA followed by Duncan’s multiple-
range test. Different letters (a, b) represent significant differences
among treatments when p < 0.05.
Biochemical Analysis and Hepatic TG Level. Plasma samples
were separated by centrifugation at 857g for 10 min at 4 °C and stored at
−80 °C until analysis. Plasma levels of total plasma TG, cholesterol,
aspartate aminotransferase (AST), and alanine aminotransferase (ALT)
were measured by a commercial assay kit. Plasma samples were spotted
onto separate Fujifilm Dri-Chem slides (Fujifilm, Kanagawa, Japan), and
biochemical parameters were determined by a Fujifilm Dri-Chem 3500s
blood biochemistry analyzer (Fujifilm) according to the manufacturer’s
protocol. Liver TG levels were measured using a commercial kit
(Cayman Chemical Co., Ann Arbor, MI, USA). Extraction of hepatic
lipid extraction followed our previous study19 from homogenization
of liver tissue in 5% Triton X-100 solution and the heated homogenates
at 80 °C for 5−10 min to solubilize the TGs. The supernatant was
collected after centrifugation at 10000g for 10 min, and the TG levels
were determined according to the manufacturer’s protocol.
Histological Analysis of Adipose Tissue. The method of
determination of adipocyte size followed our previous study.19
Perigonadal fat was fixed in 10% formalin for 24 h and then dehydrated
with a sequence of ethanol solutions and embedded in paraffin. Tissue
sections (5−6 μm) were cut and stained with hematoxylin and eosin
(H&E). Images were taken by an Olympus microscope under 200×
magnification, and adipocyte size was determined by Olympus Cellsens
Standard.
Western Blotting. 3T3-L1 cells were harvested at day 8. Liver
tissues were collected at the end of the animal experiment. Liver tissue
and cell proteins were extracted by the addition of a lysis buffer (50 mM
Tris-HCl, pH 7.4; 1 mM NaF; 150 mM NaCl; 1 mM EGTA; 1 mM
phenylmethanesulfonyl fluoride; 1% NP-40; and 10 μg/mL leupeptin)
on ice for 1 h and then centrifuged at 10000g for 30 min at 4 °C.
Quantification of cell protein was carried out using a Bio-Rad Protein
Assay (Bio-Rad Laboratories, Munich, Germany). A total of 25 μg of
protein was mixed with 5× sample buffer and incubated at 100 °C for
10 min. The cell protein was subjected to 10% sodium dodecyl
sulfate−polyacrylamide gel electrophoresis (SDS-PAGE). After 3 h, the
SDS-PAGE was transferred on the PVDF membrane (Millipore Corp.,
Bedford, MA, USA) with transfer buffer containing 25 mM Tris-HCl
(pH 8.9), 192 mM glycine, and 20% methanol. The membranes were
put in the blocking solution containing 20 mM Tris-HCl buffer with 1%
of bovine serum albumin at room temperature for 1 h, and then primary
antibodies including AMPK, pAMPK, LKB1, pLKB1, FAS, and β-actin
(Cell Signaling Technology) were applied. The membranes were
washed three times with PBST buffer for 10 min and then incubated
with 1:5000 dilution of the horseradish peroxide (HRP)-conjugated
secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA)
and washed three times with PBST buffer. The transferred proteins were
Figure 2. Effects of different concentrations of (A) 5-OH-Nob and (B)
5-Ac-Nob on triacylglycerol content in the 3T3-L1 preadipocyte model.
DM (−), without differentiation medium; DM (+), with differentiation
medium containing insulin, IBMX, DEX, and rosiglitazone. The cells
were treated with different concentrations of 5-OH-Nob and 5-Ac-Nob
for 8 days and then dyed with Oil Red O to evaluate triacylglycerol
content in the cells. The cells were photographed under 200×
magnification. Data are expressed as the mean SD (n = 3) and were
statistically analyzed using a one-way ANOVA followed by Duncan’s
multiple-range test. Different letters (a−d) represent significant
differences among groups, p < 0.05.
visualized with an enhanced chemiluminescence detection kit (ECL)
(Millipore Corp., Bedford, MA, USA).
Statistical Analysis. Data are presented as the mean SD and
analyzed by one-way analysis of variance (ANOVA) and Duncan’s
multiple-comparison tests (SAS Institute Inc., Cary, NC, USA). p < 0.05
represents a significant difference among treatment groups.
C
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Figure 3. Effects of different concentrations of (A) 5-OH-Nob and (B) 5-Ac-Nob on the cell cycle in 3T3-L1 preadipocytes. DM (−), without the
differentiation medium; DM (+), with differentiation medium containing insulin, IBMX, DEX, and rosiglitazone.
When the cells were treated with 2.5, 5, 10, and 20 μM 5-OH-Nob
and 5-Ac-Nob during the differentiation process, we found
that both 5-OH-Nob and 5-Ac-Nob significantly decreased TG
content in the mature cells in dose-dependent manners. After
treatment with 20 μM 5-OH-Nob (Figure 2A) and 5-Ac-Nob
(Figure 2B), the TG levels decreased by 35 and 70%, respectively,
suggesting that 5-Ac-Nob is twice as potent as 5-OH-Nob in
decreasing lipid accumulation in 3T3-L1 adipocytes.
Effect of 5-OH-Nob and 5-Ac-Nob on the Cell Cycle of
3T3-L1 Preadipocytes. To evaluate the antiadipogenic effects
of 5-OH-Nob and 5-Ac-Nob, we further determined whether
5-OH-Nob and 5-Ac-Nob could affect the growth of 3T3-L1
preadipocytes by analyzing the cell cycle. The results showed that
RESULTS
■
Effects of 5-OH-Nob and 5-Ac-Nob on Cell Viability and
Lipid Accumulation in 3T3-L1 Preadipocytes. We used a
CytoTox 96 Non-Radioactive Cytotoxicity Assay kit to
determine the LDH levels, which are a measure of cell viability.
The 3T3-L1 preadipocyte cells were treated with 2.5, 5, 10, and
20 μM 5-OH-Nob and 5-Ac-Nob for 48 h. Our results showed
that treatment with 5-OH-Nob and 5-Ac-Nob at various
concentrations did not significantly alter cell viabilities compared
to the control group, indicating that both 5-OH-Nob and
5-Ac-Nob did not cause cytotoxicity in 3T3-L1 preadipocytes
(Table 1). We further investigated their effects during adipo-
genesis and determined the TG content in 3T3-L1 preadipocytes.
D
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Figure 4. Effects of 5-Ac-Nob on LKB1, AMPK, SREBP-1, and FAS protein expression in 3T3-L1 adipocytes. Protein expression levels were determined
relative to actin in three independent experiment. Data are expressed as the mean SD (n = 3) and were statistically analyzed using a one-way ANOVA
and Duncan’s test. Different letters (a−c) represent significant differences among groups when p < 0.05.
67−76% of the cells (10000 cells) were in G0/G1 phase and
7−9% in S phase in the DM (−) group, whereas 45−52% of the
cells were in G0/G1 phase and 16−30% of the cells were in
S phase in the DM (+) group. The cells in the DM (+) group
showed cell cycle progression. After treatment with 2.5, 5, 10, and
20 μM 5-OH-Nob and 5-Ac-Nob for 24 h, the results of cell cycle
analysis were similar to the DM (+) group, indicating that both
5-OH-Nob and 5-Ac-Nob did not induce 3T3-L1 cell apoptosis
or cell cycle arrest. The 5-OH-Nob and 5-Ac-Nob treatments did
not affect cell growth in the early stage of cell differentiation
(Figure 3).
Effect of 5-Ac-Nob on the LKB1-AMPK Signaling
Pathway in 3T3-L1 Preadipocytes. The 5-Ac-Nob treatment
did not affect the 3T3-L1 preadipocyte growth; therefore, its
reduction of TG levels may occur in the late stage of 3T3-L1
preadipocyte differentiation. Because 5-Ac-Nob showed a stronger
TG decrease than 5-OH-Nob, we further examined the effects
of 5-Ac-Nob on the lipid synthesis-related protein expression in
the late stage of cell differentiation. We found that the expression
of SREBP-1, phosphorylated ACC (pACC), and FAS protein
in the DM (+) group was higher than in the DM (−) group.
Treatment with 5-Ac-Nob effectivelydecreased SREBP-1 and FAS
protein levels in the late stage of cell differentiation (Figure 4)
but did not increase pACC protein expression (Supplementary
Figure 7). We also found that cells in the DM (+) group had lower
levels of LKB1, pLKB1, and pAMPKα than the DM (−) group.
Additionally, 5-Ac-Nob increased LKB1, phosphorylated LKB1
(pLKB1), and phosphorylated AMPKα (pAMPKα) protein
expression compared to the DM (+) group. Taken together,
our results indicate that 5-Ac-Nob could increase pLKB1 and
pAMPKα protein levels and subsequently affect lipid synthesis-
related protein expression in the late stage of cell differentiation
(Figure 4).
Effect of 5-Ac-Nob on Body Weight and Intra-
abdominal Fat Weight in the High Fat Diet-Induced
Obese Animals. As described above, we found that 5-Ac-Nob
potently inhibited lipid accumulation in 3T3-L1 adipocytes
better than 5-OH-Nob, and we further investigated its effects on
lipid accumulation in vivo. The mice were fed a diet with 45% of
calories from fat for 13 weeks and administered 25 and 50 mg/kg
5-Ac-Nob by oral gavage. After 13 weeks, the body weight of the
HFD group was 29.6 2.5 g, significantly higher than that of the
E
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a
Table 2. Effects of 5-Ac-Nob on Body Weight, Food Intake, and Organ Weight
b
c
d
control
HFD
HFD+LAN
18.2 0.7a
HFD+HAN
initial wt (g)
18.4 0.8a
25.5 1.1c
7.1 1.1c
18.9 0.8a
29.6 2.5a
10.6 1.9a
3.4 0.5bc
0.23 0.06a
1.25 0.05ab
0.42 0.01a
0.06 0.01a
18.6 0.6a
27.5 0.8b
8.9 1.1b
final wt (g)
27.0 1.0bc
8.8 0.8b
wt gain (g)
food intake (g/mouse/day)
4.4 0.9a
3.0 0.3c
3.5 0.4b
e
food efficiency ratio
0.13 0.07b
1.25 0.05ab
0.42 0.01a
0.07 0.01a
0.22 0.06a
1.26 0.06a
0.41 0.02a
0.06 0.01a
0.21 0.05a
1.20 0.04b
0.42 0.02a
0.06 0.01a
liver wt (g)
kidney wt (g)
spleen wt (g)
a
Data are expressed as the mean SD (n = 8) and were statistically analyzed using one-way ANOVA followed by Duncan’s multiple-range test.
b
c
d
Different letters (a−c) represent significant differences among treatments when p < 0.05. HFD, high-fat diet. LAN, 25 mg/kg of 5-Ac-Nob. HAN,
50 mg/kg of 5-Ac-Nob. Food efficiency ratio gain of body weight (g)/food intake (g).
e
Figure 5. Effects of 5-Ac-Nob on intra-abdominal fat weight, (A) perigonadal fat; (B) histological analysis of perigonadal fat by H&E staining
(200× magnification) and adipocyte size (μm2); (C) retroperitoneal fat and (D) mesenteric fat in the HFD-induced obesity animal model. Data are
expressed as the mean SD (n = 3) and were statistically analyzed using one-way ANOVA and Duncan’s test. Different letters (a−d) represent
significant differences among groups when p < 0.05. HFD, high-fat diet; LAN, 25 mg/kg of 5-Ac-Nob; HAN, 50 mg/kg of 5-Ac-Nob.
control group (25.5 1.1 g). The body weights of the LAN and
HAN groups were 27 1.0 and 27.5 0.8 g, respectively, lower
than that of the HFD group (Table 2). During the experiment,
food intake was recorded daily, and the results showed that the
control group had a higher food intake than the HFD, LAN, and
HAN groups, whereas there were no significant differences in
food intake among the HFD, LAN, and HAN groups. HFD had
higher weight gain than the LAN and HAN groups at the same
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a
Table 3. Effects of 5-Ac-Nob on Serum Biochemical Parameters
b
c
d
control
HFD
HFD+LAN
HFD+HAN
82.1 7.1b
TG (mg/dL)
74.6 5.3b
63.9 4.8c
116.6 29.3a
41.4 3.0a
97.1 13.8a
134.4 8.1a
110.6 17.4a
27.3 2.2b
94.6 13.2a
132.8 7.3a
124.8 38.3a
38.8 18.3a
T-cholesterol (mg/dL)
AST (U/L)
117.4 11.0b
106.3 26.3a
27.6 1.9b
ALT (U/L)
a
Data are expressed as the mean SD (n = 8) and were statistically analyzed using one-way ANOVA and Duncan’s test. Different letters (a−c)
b
c
d
represent significant differences among treatments, p < 0.05. HFD, high-fat diet. LAN, 25 mg/kg 5-Ac-Nob; HAN, 50 mg/kg 5-Ac-Nob.
food efficiency ratio (Table 2). At the end of the experiment,
liver, kidney, spleen, and intra-abdominal fat, including
perigonadal fat, retroperitoneal fat, and mesenteric fat, were
collected and weighed. The organ weights did not show significant
differences among the groups (Table 2). The weight of the intra-
abdominal fat and the adipocyte size of the HFD group were
significantly higher than those of the control, LAN, and HAN
groups (Figure 5).
Effects of 5-Ac-Nob on Serum Biochemistry and
Hepatic Triacylglycerol Content in the High Fat Diet-
Induced Obese Animal Model. As shown in Table 3, the
serum AST levels did not show significant differences among the
four groups tested. The HFD and HAN groups had lower ALT
levels than the control and LAN groups. The total serum
cholesterol level of the HAN group was 117.4 11.0 mg/dL, and
that of the control group was 63.9 4.8 mg/dL, significantly
lower than that of the HFD group (134.4 8.1 mg/dL). The
serum TG level of the HFD group was 97.1 13.8 mg/dL,
significantly higher than that of the control group (74.6
5.3 mg/dL) or the HAN group (82.1 7.1 mg/dL). The total
serum cholesterol levels had a trend similar to that of the serum
TG levels (Table 3). As shown in Figure 6, the TG levels in
the HFD group had lower pAMPKα protein levels than the
control group. The HAN group showed increased pLKB1 and
pAMPKα protein levels compared to the HFD group; especially
the HAN group had significantly increased pAMPKα protein
levels relative to the HFD group. We also found that the HFD
group had lower pACC protein levels than the control group.
The LAN and HAN groups had elevated pACC protein levels
compared to the HFD group. The HFD group had lower
SREBP-1 and FAS protein levels than the control and LAN
groups. The LAN and HAN groups did not show decreased
SREBP-1 and FAS protein levels (Supplementary Figure 8).
These results indicate that the HAN group had decreased lipid
accumulation in the liver due to increased pLKB1 and pAMPKα
protein levels and increased pACC lipid synthesis-related protein
expression.
DISCUSSION
■
Several recent studies have reported that suppressing adipo-
genesis in the adipocytes can help control body weight. Some
plant extracts, such as raspberry ketones and PMFs and HPMFs
in citrus peel extracts, were found to regulate lipid metabolism
in 3T3-L1 adipocytes and high fat diet-induced obesity animal
models.20−22 However, PMFs and HPMFs have poor aqueous
solubility, which leads to low oral bioavailability.11,14 In this
study, we used the prodrug concept and successfully prepared
an acetylated derivative of 5-OH-Nob that can release the active
parent molecules by metabolic transformation in vivo to enhance
its oral bioavailability.15 Our current data demonstrated that
a 5-OH-Nob derivative, 5-Ac-Nob, decreased TG content to a
greater extent in the 3T3-L1 adipocytes than its parent
compound. In addition, we also found that 5-Ac-Nob effectively
decreased lipid accumulation in the high fat diet-induced obese
animal model. A previous study showed that nobiletin decreased
TG accumulation by 80% at a concentration of 100 μM in the
3T3-L1 cells.23 In this study, we found that both 5-OH-Nob and
5-Ac-Nob effectively decreased TG content at concentrations
of 5, 10, and 20 μM without any cytotoxicity (Figure 2).
Adipogenesis is a complicated process that includes early phase
growth arrest, mitotic clonal expansion, and late-phase lipo-
genesis-related enzyme expression from preadipocytes to mature
adipocytes.24 The antiadipogenic activity of HPMFs resulted
from blocking the early phase cell cycle procession and causing
cell cycle arrest in G0/G1 phase.21 In this study, when the cells
were treated with 5-OH-Nob and 5-Ac-Nob, they still underwent
cell cycle progression to S phase, and compared to the DM (−)
group, the cells stayed in G0/G1 phase (Figures 4 and 5). These
results suggest that the antiadipogenic effects of 5-OH-Nob
and 5-Ac-Nob may affect the late stage of adipogenesis. (Table 1;
Figures 4 and 5). Our results showed that 5-Ac-Nob inhibited
TG content to a greater extent than 5-OH-Nob in 3T3-L1
adipocytes. We further investigated the effects of 5-Ac-Nob
in an HFD-induced obesity animal model. Here, we found that
50 mg/kg (HAN group) 5-Ac-Nob significantly reduced the
Figure 6. Effects of 5-Ac-Nob on TG levels in the mouse liver. Data are
expressed as the mean SD (n = 8) and were statistically analyzed using
one-way ANOVA and Duncan’s test. Different letters (a−c) represent
significant differences among treatments when p < 0.05. HFD, high-fat
diet; LAN, 25 mg/kg 5-Ac-Nob; HAN, 50 mg/kg 5-Ac-Nob.
the liver were 23.5 8.7, 74.8 21.4, 68.5 17.5, and 49.6
11.9 mg/dL in the control, HFD, LAN, and HAN groups,
respectively. Therefore, the high dose of 5-Ac-Nob substantially
decreased the TG levels in both the serum and liver.
Effect of 5-Ac-Nob on the LKB1-AMPK Signaling
Pathway in the Liver. As shown in Figure 7, we found that
G
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Figure 7. Effects of 5-Ac-Nob on the phosphorylation of LKB1, AMPKα, and ACC and the protein levels of LKB1, AMPKα, and ACC. Lipid
accumulation was inhibited via activation of the LKB1-AMPK pathway and pACC protein levels in an HFD-induced obesity animal model. Protein
expression was determined relative to actin in three independent experiments. Data are expressed as the mean SD and were statistically analyzed using
one-way ANOVA and Duncan’s test. Different letters (a, b) represent significant differences among groups when p < 0.05. HFD, high-fat diet; LAN,
25 mg/kg 5-Ac-Nob; HAN, 50 mg/kg 5-Ac-Nob.
body weight, intra-abdominal adipose tissue (retroperitoneal,
perirenal, mesenteric, and perigonadal are intra-abdominal fats
in mice) weight, plasma TGs, and total cholesterol compared to
the HFD group (Figure 5; Tables 2 and 3). The liver is the major
organ that regulates glucose and lipid metabolism, controlling
energy balance and body weight.25 Excess fat around the liver
(intra-abdominal adipose) can be more easily transported into
the liver by portal circulation as free fatty acids, which would
induce TG synthesis in the liver.26 In our current study, we found
that 50 mg/kg 5-Ac-Nob significantly decreased liver TG levels
(Figure 6).
We demonstrated that 5-Ac-Nob could decrease TGs in both
the 3T3-L1 cells and high fat diet-induced obese mice. SREBPs
are transcription factors that are involved in cellular lipogenesis
and lipid homeostasis by regulating cholesterol, fatty acids,
TGs, and phospholipid synthesis-related enzyme expression.3,7
Generally, the activation of SREBPs involves a two-step proteo-
lytic cascade. In the first step, SREBP is cleaved from the SREBP/
SREBP cleavage-activating protein (SCAP) complex (125 kDa)
at the endoplasmic reticulum membrane, and in the second step,
cleavage releases the mature SREBPs (68 kDa) to the nucleus to
activate transcriptional activity.27 Here, we found that 5-Ac-Nob
decreased the nuclear form of the SREBP-1 protein (68 kDa) in
3T3-L1 preadipocytes, but SREBP-1 protein expression was not
decreased in the liver. Lipogenesis is a complicated process, and
several crucial enzymes, such as ACC and FAS, are involved.28
We found that 5-Ac-Nob decreased FAS protein levels, whereas
pACC protein levels in 3T3-L1 cells were not increased.
Interestingly, we have observed that 5-Ac-Nob increased the
expression of the pACC protein, a crucial enzyme involved in
the initial step of lipogenesis28 in the liver. However, 5-Ac-Nob
did not decrease the SREBP-1 and FAS protein expressions com-
pared to the HFD group in the liver. AMPK plays a key role in
energy homeostasis, and it can inhibit fatty acid synthesis by
directly phosphorylating ACC or FAS or inhibiting the pro-
teolytic cleavage of SREBP-1.29,30 LKB1 can up-regulate AMPK
by phosphorylating a threonine residue at position 172 in an
α catalytic subunit.31 Here, we found that 5-Ac-Nob increased
pAMPKα protein expression in 3T3-L1 cells and in the liver.
In conclusion, we found that both 5-OH-Nob and 5-Ac-Nob
decreased TG levels in 3T3-L1 cells effectively, but 5-Ac-Nob
was more potent. Hence, we used 5-Ac-Nob to further evaluate
the antiobesity effects in an obese animal model induced by a
high-fat diet. We observed that 5-Ac-Nob not only suppressed
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lipid accumulation to a greater extent than 5-OH-Nob in the
3T3-L1 cell model but also decreased body weight, alleviated
hyperlipidemia, and decreased liver TG content in the high fat
diet-induced obese mice. The 5-Ac-Nob treatment suppressed
lipid accumulation by activating the LKB1-AMPK pathway and
subsequently affected lipogenesis-related protein expression in
the cells and livers and the related transcriptional factor SREBP-1
in cells (Figure 8). Thus, on the basis of the in vivo results
relative to actin in three independent experiment. Data are
expressed as the mean SD (n = 3) and were statistically
analyzed using a one-way ANOVA and Duncan’s test.
Different letters (a, b) represent significant differences
among groups when p < 0.05. Figure S8: Relative protein
expression of SREBP1 and FAS in HFD-induced animal
model. Protein expression levels were determined relative
to actin in three independent experiment. Data are
expressed as the mean SD (n = 3) and were statistically
analyzed using a one-way ANOVA and Duncan’s test.
Different letters (a, b) represent significant differences
among groups when p < 0.05. Table S1: Composition of
diets. The control diets were LabDiet 5001 standard diet
bought from LabDiet (St. Louis, MO, USA). The HFD
diet was modified from LabDiet 5001 standard diet,
containing an extra 19.6 g of lard, 2 g of soybean, and 0.5 g
of cholesterol in 100 g of LabDiet 5001 standard diet
(PDF)
AUTHOR INFORMATION
Corresponding Authors
*(M.-H.P.) Phone: +886 2 33664133. Fax: +886-2-3366-1771.
E-mail: mhpan@ntu.edu.tw.
■
*(G.-J.W.) Phone: +886-3-341-2500, ext. 2512. Fax: +886-3-
270-5904. E-mail: gwei@mail.knu.edu.tw.
Funding
This study was supported by the National Taiwan University
NTU-104R7777; the Ministry of Science and Technology
101-2628-B-022-001-MY4, 102-2628-B-002-053-MY3; and a
collaboration grant of Hubei province, China (2014BHE036).
Figure 8. Possible mechanisms of 5-Ac-Nob for suppressing lipid
accumulation in the 3T3-L1 preadipocytes and mouse liver tissue.
Notes
The authors declare no competing financial interest.
obtained from our current study, we suggest that 5-Ac-Nob may
become a potential candidate for controlling obesity or delaying
the onset of fatty liver disease. Additionally, this research provides
more evidence that acetylation of hydroxylated nobiletin is an
effective method to develop a prodrug with a greater efficacy
in suppressing lipid accumulation than 5-OH-Nob, its naturally
occurring parent compound from citrus peels. Future studies
should clarify the metabolism of 5-Ac-Nob and its relationship
with 5-OH-Nob in vivo.
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Figure S1: LC-MS of 5-OH-Nob. LC-MS was performed
by a Thermo Scientific linear ion trap mass spectrometer
(LXQTM) in positive ionization mode (Thermo
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Figure S5: H NMR of 5-Ac-Nob. NMR spectra were
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