Journal of Functional Foods 84 (2021) 104581

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Heracleum dissectum Ledeb. ethanol extract attenuates metabolic syndrome

symptoms in high-fat diet-induced obese mice by activating adiponectin/
AMPK signaling
Yang-Ju Son a, Da Seul Jung a, b, Ji Min Shin a, c, Saruul Erdenebileg a, c, Chu Won Nho a, c, *
a
Smart Farm Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Gangwon-do 25451, Republic of Korea
b
Department of Biology, College of Natural Sciences, Gangneung-Wonju National University, Gangneung Gangwon-do 25457, Republic of Korea
c
Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: The incidence of obesity has increased worldwide, leading to metabolic disorders such as hyperlipidemia and
Adiponectin insulin resistance. Therefore, novel functional foods and therapeutic agents against obesity-related metabolic
AMPK disorders are needed. We explored the effectiveness of the edible herb Heracleum dissectum Ledeb. (HD) against
Energy expenditure
metabolic syndrome symptoms in a high-fat diet (60% of kcal) obesity mouse model. Metabolic syndrome was
Heracleum dissectum Ledeb.
Metabolic syndrome
induced after 8 weeks of high-fat diet feeding, and HD extract (50 mg/kg) was administered by oral gavage
during feeding. HD extract effectively decreased body weight gain and ameliorated the serum lipid status. HD
extract also upregulated adiponectin/AMP-activated protein kinase (AMPK) signaling, and had anti-oxidation,
anti-inflammation, and anti-insulin resistance effects. HD extract further elevated phosphorylation of AMPK
and mitochondria biogenesis in the adipose tissue, resulting increased energy expenditure via the triglyceride/
fatty acid cycle. Thus, HD extract could be considered a multi-targeting functional food candidate with novel
molecular mechanisms.

1. Introduction representing a serious public health issue, causing heavy social and
economic burdens (Jia & Lubetkin, 2005). Moreover, obesity could
Recent environmental changes in lifestyle have drastically led to an progress to several complications, including metabolic syndrome,
increased prevalence of obesity, especially in developed countries, leading to a more complex pathophysiological state. Metabolic syn­
owing to affluence, excessive food consumption, and lack of exercise drome is diagnosed when patients meet three or more criteria of the
(Jeffery & Utter, 2003). In particular, the United States has been following five criteria: abdominal obesity, hypertension, high triglyc­
struggling with an explosive epidemic of obesity for decades, eride (TG), low high-density lipoprotein-cholesterol (HDL-C), and

Abbreviations: acyl-CoA, acyl coenzyme A; ALT, alanine aminotransferase; AdipoR1, adiponectin receptor 1; AdipoR2, adiponectin receptor 2; AMPK, AMP-
activated protein kinase; Appl1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1; AST, aspartate aminotransferase; Atgl, adipose
triglyceride lipase; ATP, adenosine triphosphate; AUC, area under the curve; BAT, brown adipose tissue; BSA, bovine serum albumin; C/EBPα, CCAAT/enhancer-
binding protein α; CS, citrate synthase; CVD, cardiovascular disease; CPT-1, carnitine palmitoyltransferase-1; DIO, diet-induced obesity; DMEM, Dulbecco’s modified
Eagle medium; DsbA-L, disulfide-bond A oxidoreductase-like protein; ELISA, enzyme-linked immunosorbent assay; eWAT, epididymal white adipose tissue; FA, fatty
acid; FBS, fetal bovine serum; GLUT4, glucose transporter type 4; GSK-3β, glycogen synthase kinase 3 beta Heracleum dissectum Ledeb; HDL-C, high-density li­
poprotein cholesterol; HFD, high-fat diet; HO-1, heme oxygenase 1; Hsl, hormone-sensitive lipase; ICCT, Institute of Chemistry & Chemical Technology; IL-1β,
interleukin-1β; IPITT, intraperitoneal insulin tolerance test; LDL-C, low-density lipoprotein cholesterol; MDA, malondialdehyde; MPO, myeloperoxidase; ND, normal
diet; NHANES, National Health and Nutrition Examination Survey; NQO1, NAD(P)H quinone dehydrogenase 1; OGTT, oral glucose tolerance test; Pepck, phos­
phoenolpyruvate carboxykinase; PPARγ, peroxisome proliferator-activated receptor gamma; PRDM16, PR domain containing 16; qPCR, quantitative real-time po­
lymerase chain reaction; Serca, Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; SOD, superoxide dismutase; SREBP-1, sterol regulatory element-binding
transcription factor 1; TBST, Tris-buffered saline with Tween 20; TC, total cholesterol; TG, triglyceride; UCP1, uncoupling protein 1; UPLC-QTOF-MS/MS, ultra-
high-performance liquid chromatography and quadrupole time-of-flight mass spectrometry; WAT, white adipose tissue.
* Corresponding author at: Smart Farm Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Gangwon-do 25451, Korea.
E-mail address: [email protected] (C.W. Nho).

https://doi.org/10.1016/j.jff.2021.104581
Received 18 February 2021; Received in revised form 4 June 2021; Accepted 11 June 2021
Available online 14 June 2021
1756-4646/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y.-J. Son et al. Journal of Functional Foods 84 (2021) 104581

insulin resistance related to type 2 diabetes mellitus (Ascaso et al., Institute of Chemistry & Chemical Technology (ICCT) of Mongolia. The
2003). Metabolic syndrome not only increases the occurrence of dia­ plant samples were classified by Dr. Batsuren of the ICCT, and a voucher
betes and cardiovascular disease (CVD) but is also associated with psy­ specimen is stored at the Mongolian Academy of Sciences (2019/054).
chiatric illnesses such as depression (Reagan-Shaw, Nihal, & Ahmad, Approximately 200 g of dried powder of the entire epigeal part of the HD
2008; Simon et al., 2006). In the United States, almost one-fourth of the plant was mixed with 2 L of ethanol, and stored in the dark for 7 d at
adult population suffers from metabolic syndrome, as per National 20℃. After filtration of the liquid part, the extraction was repeated once
Health and Nutrition Examination Survey (NHANES) data (Azizi, Salehi, more for the remaining powder using 2 L of ethanol. The final HD extract
Etemadi, & Zahedi-Asl, 2003). Given the pervasiveness of obesity was obtained by completely evaporating the ethanol using a rotary
worldwide, the development of anti-obesity drugs has progressed, with evaporator (RE111; Büchi, Flawil, Switzerland).
two main types of drugs now available patients: appetite suppressants
that regulate noradrenergic or serotonergic signals, and lipase inhibitors 2.2. Animal model and treatment
that target the gastrointestinal tract (Yanovski & Yanovski, 2014).
However, the side effects of these drugs with long-term use requires A total of 44 eight-week-old male C57BL/6N mice were provided by
continued research to find safe functional food items for routine intake. Orient Bio (Sungnam, Korea). The mice were placed in isolation cage
Although the therapeutic efficacy of appetite suppressors and lipase and acclimated to the laboratory environment for 1 week under a 12-h
inhibitors have been proven in patients with obesity, energy light and 12-h dark cycle in a room maintained at 24 ± 0.5 ℃. The mice
metabolism-regulating agents have recently come into the spotlight as a were then divided into four groups based on their body weight to
novel target. For example, hydroxycitric acid derived from Garcinia standardize the initial body weight condition across experimental group.
plant is a typical energy metabolism regulator that disturbs de novo During the 8-week experimental period, the normal diet (ND) group (n
lipogenesis and enhances fatty acid oxidation, and its use resulted in = 12) was provided Teklad 10% kcal adjusted calories diet (TD.06416;
significant weight loss in clinical studies (Tomar et al., 2019). Adipo­ Envigo, Indianapolis, IN, USA). The high-fat diet (HFD) group (n = 12),
nectin, a major adipokine generated and secreted from the adipose tis­ orlistat (positive control) group (n = 12), and HD group (n = 8) were
sues, is one of the key energy homeostasis regulators in the body, and provided Teklad 60% kcal adjusted calories diet (TD.06414; Envigo).
patients with obesity have been reported to have reduced adiponectin Diet and water were provided ad libitum throughout the experiment.
levels and reduced adiponectin sensitivity (Kadowaki & Yamauchi, To determine the dosage of HD extract, we first searched the relevant
2005). In a case study of a human population, the serum adiponectin literature. The HD is mostly known for an edible plant and it has been
level and body mass index showed a prominent negative correlation, used as a vegetable in China. We found only one animal study using HD
and low adiponectin levels were associated with an increased incidence extract, which reported that the 50% lethal dose of HD was over 5,000
of type 2 diabetes mellitus (Arita et al., 1999). In addition, the plasma mg/kg (Zhang et al., 2017). In addition, they fed 125–250 mg/kg HD to
adiponectin level and TG content in the blood show a negative rela­ mice daily for a total of 3 weeks without toxic effects. Referring to this
tionship associated with an increased CVD risk (Zhang et al., 2018). A previous study, we set the dosage of HD extract for the mice to 50 mg
critical role of adiponectin is activating AMP-activated protein kinase kg− 1 d-1, which corresponds with a dose of 4.05 mg kg− 1 body weight
(AMPK) in energy homeostasis-related tissues such as the liver, muscle, per day for an adult human (Reagan-Shaw et al., 2008). In turn, the
and adipose tissue, and expedites glucose uptake and fatty acid (FA) vehicle solution (0.5% carboxymethyl cellulose) was administered to
oxidation in these tissues (Bonnard, Durand, Vidal, & Rieusset, 2008). In the ND and HFD groups, and a vehicle solution containing orlistat (30
a series of energy metabolism mediation processes, AMPK promotes mg/kg) or HD ethanol extract (50 mg/kg) was administered to the
adenosine triphosphate (ATP) production by regulating glucose, pro­ orlistat or HD group, respectively. Orlistat was used as a positive control
tein, and lipid metabolism, and enhancing biogenesis of the mitochon­ drug owing to its broad use in the human population. The administration
dria (Herzig & Shaw, 2018; Ke, Xu, Li, Luo, & Huang, 2018; Wang, Liu, of each solution was performed once a day by oral gavage throughout
Zhai, Zhang, & Tian, 2018). Moreover, AMPK activation improves anti- the 8-week experimental period.
oxidative and anti-inflammatory responses in the cellular system (Wu After the 8-week experimental period, the mice were anesthetized by
et al., 2018), and relieves multiple symptoms of metabolic syndrome, intraperitoneal injection of a mixture of ketamine (80 mg/kg) and
especially insulin resistance (Teng, Chen, Fang, Yuan, & Lin, 2017). xylazine (12 mg/kg). Blood was collected from the heart while under
Heracleum dissectum Ledeb. (HD) is a traditional vegetable of China, anesthesia, and the mice were euthanized thereafter. The animal study
belonging to the Apiaceae family, and has long been used for treating was planned and executed as per the National Institutes of Health
rheumatoid diseases and headaches in folk medicine in China and guidelines. The animal experiments were approved by the Institutional
Mongolia (Sun & Liu, 2007), and its anti-diabetic effect was revealed in a Animal Care and Use Committee of Korea Institute of Science and
recent study (Zhang et al., 2017). We recently showed that HD extract Technology (Approval no.: KIST-2019–011).
had an anti-obesity effect based on in vitro screening; thus, we hypoth­
esized that it would also be capable of mitigating metabolic syndrome 2.3. Assessment of adipocyte cell size in the epididymal white adipose
symptoms. To test this hypothesis, we established a mouse model of diet- tissue (eWAT)
induced obesity (DIO); although this model takes longer to establish
than other obesity models, it mimics the pathogenesis of obesity and The eWAT was fixed in a paraffin block and a section of the eWAT
metabolic syndrome in humans fairly well (Kuraji, Fujita, Ito, Hashi­ was stained using hematoxylin and eosin. The images of tissues were
moto, & Numabe, 2018). Using a DIO model, we aimed to verify the reconstituted and assessed with ImageJ software (National Institutes of
effectiveness of HD in treating obesity and metabolic syndrome, along Health, Bethesda, MD, USA) as per a previous study (Parlee, Lentz, Mori,
with the underlying molecular mechanisms. These findings can provide & MacDougald, 2014).
a scientific basis for developing HD as a multi-targeting functional food
to alleviate obesity and metabolic syndrome. 2.4. Colorimetric and enzyme immunosorbent assay (ELISA) kit analysis

2. Materials and methods The contents of alanine aminotransferase (ALT), aspartate amino­
transferase (AST), TG, total cholesterol (TC), HDL-C, and ghrelin were
2.1. Plant materials determined using assay kits from Elabscience (Houston, TX, USA). The
low-density lipoprotein cholesterol (LDL-C), malondialdehyde (MDA),
HD was collected from the vicinity of Ulaanbaatar, Mongolia. The and myeloperoxidase (MPO) were analyzed with assay kits from Bio­
collection of plant materials was conducted with the approval of Vision (Milpitas, CA, USA). The citrate synthase (CS) activity was

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Y.-J. Son et al. Journal of Functional Foods 84 (2021) 104581

detected using a kit from Sigma-Aldrich (St. Louis, MO, USA). The obtained from Abcam (Cambridge, UK).
contents of leptin, insulin, interleukin-1β (IL-1β), and adiponectin were
determined using assay kits from RayBiotech (Peachtree Corners, GA, 2.8. Quantitative real-time polymerase chain reaction (qPCR)
USA).
RNA was extracted from the mouse eWAT tissue using an RNeasy
2.5. Cell culture and adipogenesis induction Mini kit (Qiagen, Hilden, Germany) following the manufacturer in­
structions. The cDNA was synthesized using a PrimeScript 1st Strand
Murine pre-adipocytes (3T3-L1) were obtained from American Type cDNA Synthesis kit (Takara, Kusatsu, Japan). The gene expression of
Culture Collection (Manassas, VA, USA). The cells were maintained samples was analyzed by qPCR analysis with a LightCycler 480 Real-
using Dulbecco’s modified Eagle medium (DMEM; Hyclone, Logan, UT, Time PCR System (Roche, Basel, Switzerland) using SYBR Green Mas­
USA) containing 10% bovine calf serum (Thermo Fisher Scientific, ter Mix solution (Hoffmann-La Roche Ltd., Schweiz, Switzerland). The
Waltham, MA, USA) and 1% penicillin–streptomycin (Hyclone). For the primer sequences used in this study are shown in Table S1.
induction of adipogenesis, cells were seeded in a 6-well plate (1.5 × 105
cells/well). Two days after the cells reached 100% confluence, the cul­ 2.9. Glucose and insulin tolerance test
ture medium was replaced by DMEM containing 10% fetal bovine serum
(FBS; Thermo Fisher Scientific), 1% penicillin–streptomycin solution, The oral glucose tolerance test (OGTT) and intraperitoneal insulin
0.1% 0.5 mM 3-isobutyl-1-methylxanthine, 0.05% 2.5 mM dexametha­ tolerance test (IPITT) were both performed on the 7th week of the
sone, and 0.05% 10 mg/mL insulin. After day 3, the culture medium was experiment. Before testing, the mice were fasted for 6 h and 2 g/kg
changed to DMEM containing 10% FBS, 1% penicillin–streptomycin, glucose solution was orally administered for the OGTT and 0.8 U/kg
and 0.1% 10 mg/ml insulin, which was then maintained until day 9 with insulin was injected intraperitoneally for the IPITT. Glucose levels were
refreshing the same medium at day 6. The cells were treated with HD tested in the tail vein blood using Accu-Chek meter (Roche) at 0, 30, 60,
extract or bergenin (ChemFaces, Hubei, China) throughout the 120, and 180 min after treatment.
differentiation-inducing periods; that is, the cells were treated in
differentiation-inducing media from day zero to day nine. 2.10. Ultra-high-performance liquid chromatography and quadrupole
time-of-flight mass spectrometry (UPLC-QTOF-MS/MS)
2.6. Determination of lipid accumulation in 3T3-L1 cells
Chemical analysis of the HD extract was performed using UPLC-
The accumulation of lipids in 3T3-L1 cells was examined using Oil QTOF-MS/MS. The chromatogram of the HD extract was examined by
red O staining (Ramirez-Zacarias, Castro-Munozledo, & Kuri-Harcuch, UltiMate 3000 (Thermo Fisher Scientific) with a Phenomenex Kinetex
1992). To quantify lipid accumulation, the stained Oil red O solution C18 column (2.1 × 150 mm, 1.7 μm). Solvent A was water with 0.1%
was reconstituted with isopropanol and the absorbance was measured at formic acid and solvent B was acetonitrile with 0.1% formic acid. The
510 nm. The TG content was examined from the cell supernatants using gradient of solvents A:B was as follows: 0–10 min, 99:1; 15 min, 90:10;
a colorimetric assay kit (Biovision). Cell viability was determined using 30 min, 70:30; 40 min, 30:70; 41–44 min, 0:100; 45–50 min, 99:1. The
EZ-Cytox reagent (DoGEN, Seoul, Korea) following the manufacturer’s flow rate was 0.2 mL/min and detection was conducted at 254 nm. The
protocol. mass signal was detected using a Triple TOF 5600 system (AB SCIEX,
Foster City, CA, USA). Both positive and negative modes were analyzed
2.7. Western blot analysis and an electrospray ionization source was used. The mass scan range
was set to 50–2,000 m/z, and was operated in full scanning mode. Ni­
The mouse eWAT and liver tissues or 3T3-L1 cells were lysed in a trogen gas was used as the collision gas and the desolvation temperature
protein inhibitors cocktail (Sigma-Aldrich) containing RIPA buffer, and was set to 500 ℃. The ion-spray voltage was 5.5 kV and 4.5 kV for the
the protein contents were quantified by the Bradford assay. Lysates were positive and negative mode, respectively. The collision-induced disso­
mixed with loading dye and electrophoresis was conducted using a so­ ciation energy was set as 35 ± 15 eV for positive mode and at –35 ± 15
dium dodecyl sulfate–polyacrylamide gel electrophoresis system. After eV for negative mode.
transferring the proteins to the polyvinylidene fluoride membrane, the
membranes were soaked in blocking solution, 3% bovine albumin serum 2.11. Statistical analysis
(BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST), for 1 h. After
washing the membranes with TBST solution for 5 min three times, they All data are expressed as mean ± standard error of the mean. Sta­
were incubated with primary antibodies in 1% BSA-TBST solution at 4℃ tistical comparison across experimental groups was conducted using
overnight. The membranes were rinsed with TBST solution three times, one-way analysis of variance and Duncan’s multiple range test. Statis­
soaked in 1% BSA-TBST solution containing anti-mouse or anti-rabbit tical analysis was conducted using SPSS Statistics v25 (SPSS Inc., Chi­
secondary antibodies for 1 h at 20℃, and then washed with TBST so­ cago, IL, USA).
lution three times again. The chemiluminescence signals were detected
using LAS-3000 Bio Imaging System (Fuji Film Co., Tokyo, Japan) by 3. Results
developing the membranes using SuperSignalTM West Femto Maximum
Sensitivity Substrate (Thermo Fisher Scientific). The sterol regulatory 3.1. HD intake decreased weights of the body and WATs in the DIO mouse
element-binding transcription factor 1 (SREBP-1), carnitine model
palmitoyltransferase-1 (CPT1), peroxisome proliferator-activated re­
ceptor gamma (PPARγ), heme oxygenase 1 (HO-1), NAD(P)H quinone After eight weeks of inducing obesity in mice, the difference in final
dehydrogenase 1 (NQO1), superoxide dismutase 1 and 2 (SOD1 and body weight between the ND and HFD groups was 8.5 g (Table 1). Both
SOD2), glycogen synthase kinase 3 beta (GSK-3β), p-GSK-3β (Ser9), and the HD extract and the positive control orlistat caused significant loss of
glucose transporter type 4 (GLUT4) primary antibodies were obtained body weight compared to the HFD (p < 0.05), and the weights of three
from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies kinds of WATs were significantly reduced in both the orlistat and HD
against CCAAT/enhancer-binding protein α (C/EBPα), AMPK, p-AMPK, groups (p < 0.05). Daily calorie intake was the highest in the orlistat
cytochrome C, Akt, and p-Akt (Ser473) were obtained from Cell group, and there was no significant difference between that in HFD and
Signaling Technology (Danvers, MA, USA). The antibodies for uncou­ HD groups. The serum levels of leptin and ghrelin were significantly
pling protein 1 (UCP1) and PR domain containing 16 (PRDM16) were decreased in the HD group compared with those of the HFD group (p <

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Y.-J. Son et al. Journal of Functional Foods 84 (2021) 104581

Table 1 and adaptor protein phosphotyrosine interacting with PH domain and

Whole-body and fat-tissue weights, daily diet intake, and hormone levels of HD- leucine zipper 1 (Appl1) in the HD group were significantly higher than
administered diet-induced obesity (DIO) mice. those of the HFD group (p < 0.05). To verify the stimulation of HD
ND HFD ORL HD extract on adiponectin/AMPK signaling and energy expenditure, the
Body weight
mitochondria content was first examined by a CS activity assay (Fig. 2C).
Initial body weight (g) 22.23 ± 22.38 ± 22.27 ± 22.36 ± The HD group showed significantly increased CS activity in the eWAT
0.28 0.37 0.23 0.37 compared with that of the other groups (p < 0.05). At the molecular
Final body weight (g) 26.95 ± 35.47 ± 32.34 ± 33.75 ± level, the phosphorylation of AMPK was significantly increased by HD
0.44c 0.73a 0.56b 1.00b
extract treatment (p < 0.05), and protein levels of PRDM 16 and cyto­
Weight gain (g) 4.72 ± 13.08 ± 10.07 ± 11.39 ±
0.35c 0.77a 0.44b 0.79b chrome C were also significantly increased (Fig. 2D). However, the level
White adipose tissue weight of UCP1, a major thermogenesis channel, was not elevated by HD
Epididymal (g) 0.75 ± 2.20 ± 1.56 ± 1.43 ± treatment. Therefore, we predicted that the HD extract upregulates
0.04c 0.12a 0.13b 0.18b thermogenesis mechanisms via AMPK stimulation rather than via UCP1-
Mesenteric (g) 0.34 ± 0.74 ± 0.51 ± 0.47 ±
0.02c 0.05a 0.04b 0.04b
dependent signaling. The stimulation of ATP synthesis in the eWAT and
Perirenal (g) 0.33 ± 0.94 ± 0.74 ± 0.63 ± UCP1-independent thermogenesis mechanisms generally require
0.02c 0.04a 0.05b 0.08b enhanced mitochondrial functions; therefore, we further evaluated the
Daily diet intake mRNA expression of genes related to mitochondrial biogenesis. The HD
Daily diet intake (g 0.10 ± 0.08 ± 0.09 ± 0.08 ±
group showed significantly elevated mRNA expression levels of mito­
body weight− 1) 0.00a 0.00b 0.00a 0.00b
Daily calorie intake 0.35 ± 0.41 ± 0.46 ± 0.41 ± chondrial biogenesis-related markers (Fig. 2E; p < 0.05), similar to the
(Kcal body weight− 1) 0.01c 0.02b 0.02a 0.02b result of CS activity. Lastly, we also determined the effects of HD extract
Hormone level in serum on lipid catabolism, FA synthesis, and glyceroneogenesis-related genes.
Leptin (pg/mL) 28.19 ± 142.50 ± 133.96 ± 96.07 ± As shown in Fig. 2F, genes related to the TG/FA cycle, such as adipose
5.42c 13.65a 13.24a 38.41b
triglyceride lipase (Atgl), hormone-sensitive lipase (Hsl), and phospho­
Ghrelin (pg/mL) 348.19 ± 442.47 ± 317.39 ± 293.68 ±
24.16b 49.47a 21.15b 3.67b enolpyruvate carboxykinase (Pepck), were significantly increased in the
HD group compared with those in the HFD group (p < 0.05). The cir­
Different lowercase superscripts within rows indicate significant differences
culation of this cycle requires futile energy input; therefore, the acti­
between groups at p < 0.05.
vation of this cycle may have caused the usage of synthesized ATPs.

0.05), although the ratio of leptin to ghrelin remained unchanged. The

3.4. HD extract upregulated anti-oxidation-related proteins and alleviated
weights of the major organs (liver, kidney, spleen, and heart) and levels
inflammation in DIO mice
of serum ALT and AST were determined to check the potential toxicity;
there was no evident toxic effect observed in all mice groups (Table S2).
Excessive accumulation of lipids in animal tissues causes oxidative
stress and inflammatory responses. Administration of HD extract resul­
3.2. Adipocyte size in the eWAT and serum lipid parameters were ted in a decreased MDA content (a lipid peroxidation marker), and
ameliorated by HD intake in DIO mice enhanced expression of anti-oxidative-related proteins (Fig. 3A, B). In
addition, the HD group showed significantly reduced serum IL-1β and
The morphology and adipocyte size of the eWAT were verified using MPO levels in eWAT, and reduced mRNA expression levels of inflam­
images of tissue sections (Fig. 1A–C). Administration of orlistat and HD matory response genes in the eWAT (p < 0.05) (Fig. 3C–E). The total
reduced the proportion of larger adipocytes, and the average size of macrophage and M1 macrophage markers (F4/80 and CD11c) were also
adipocyte was significantly decreased in these groups compared with significantly mitigated in the HD group (p < 0.05).
that of the HFD group (p < 0.05). There was no change regarding adi­
pogenesis (PPARγ and C/EBPα), lipid metabolism (SREBP-1), and lipid 3.5. HD treatment improved insulin sensitivity in the eWAT and liver of
oxidation (CPT-1)-related protein expression in the orlistat and HD DIO mice
groups (Fig. 1D). Administration of HD extract significantly reduced TG,
TC, and LDL-C levels in the serum (p < 0.05), but there was no decrease In the OGTT, the fasting glucose level was higher in the HFD group
of serum HDL-C levels in the HD group compared with that in the HFD than in the HD group, and the HD group showed faster recovery of the
group (Fig. 1E–H). Therefore, HD extract notably improved hyperlip­ serum glucose level after oral glucose intake; the area under the curve
idemia in the DIO mouse model. (AUC) of the HD group was significantly decreased compared with that
of the HFD group (p < 0.05) (Fig. 4A, B). Similarly, HD administration
3.3. HD extract elevated adiponectin/AMPK signaling and stimulated enhanced insulin sensitivity in the IPITT, with a significant decrease in
biogenesis of the mitochondria in the eWAT the AUC compared with that of the HFD group (p < 0.05; Fig. 4C, D).
Although there was no significant difference in the fasting serum insulin
Although there was loss of weight in the HD group, there was no level, the HOMA-IR, an important marker of insulin resistance, was
significant difference in calorie intake between the HFD and HD groups significantly reduced in the HD group (p < 0.05) (Fig. 4E, F). At the
(Table 1). These results suggest that HD extract could stimulate energy molecular level, phosphorylation of Akt was upregulated in the eWAT by
expenditure mechanisms. To test this possibility, we determined the HD treatment (Fig. 4G). We also verified the glycometabolism of the
serum level of adiponectin, an adipokine that is an upstream hormone liver, and found that the HD extract upregulated the phosphorylation of
for AMPK activation (Fig. 2A). The HD extract highly increased the Akt and GSK-3β along with GLUT4 protein expression in the liver
serum adiponectin level, and also upregulated the mRNA expression of (Fig. 4H). Hence, HD extract alleviated insulin resistance in DIO mice,
adiponectin-related genes (Fig. 2B). The mRNA levels of adiponectin, the mediating related signaling in the eWAT and liver.
adiponectin synthesis-related gene, and disulfide-bond A
oxidoreductase-like protein (DsbA-L), related to the maturation and 3.6. HD extract and its major compound, bergenin, alleviated lipid
secretion of adiponectin, were significantly elevated in the HD group accumulation in vitro
compared with those of the HFD group (p < 0.05). In addition, the
mRNA expression levels of adiponectin receptor-related genes such as Using UPLC-MS/MS, we identified five major compounds in the HD
adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2), ethanol extract (Figure S1). The largest peak in the HPLC chromatogram

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Y.-J. Son et al. Journal of Functional Foods 84 (2021) 104581

Fig. 1. HD treatment decreased adipocyte size of epididymal white adipose tissue (eWAT) and alleviated hyperlipidemia in DIO mice. (A) eWAT tissue section was
visualized with H&E staining (×100 magnification). (B) Distribution of adipocyte sizes and (C) average size of adipocytes of eWAT were calculated by ImageJ
software. (D) Expression of proteins related to adipogenesis, lipid synthesis, and lipid oxidation in eWAT. (E) Serum triglyceride levels. (F) Serum total cholesterol
levels. (G) Serum high-density lipoprotein cholesterol (HDL-C) levels. (H) Serum low-density lipoprotein cholesterol (LDL-C) levels. Statistical differences within
groups were analyzed by one-way analysis of variance (ANOVA) and Duncan’s multiple range test. Different lowercase superscripts indicate significant differences
between groups at p < 0.05.

was revealed to bergenin, and its effectiveness on obesity and type 2 opposite to our animal study data. Therefore, we conjecture that the
diabetes were reported in previous studies (Ambika & Saravanan, 2016; therapeutic efficacies of HD extract stemmed from an integrated effect of
Shikov et al., 2012). Since the most abundant chemical compound in the multiple constituents in HD extract, and not solely the contribution of
HD extract was bergenin, we additionally verified whether bergenin the major component, bergenin. However, further study is needed to
could alleviate lipid accumulation in the 3T3-L1 cell line. First, we verify it.
checked the cell toxicity of the HD extract and bergenin in 3T3-L1 cells,
and found no significant differences in cell viability compared with 4. Discussion
negative control cells up to 100 μg/mL or 100 μM for HD extract and
bergenin treatment, respectively (Figure S2). The HD extract decreased Metabolic syndrome represents a range of several disorders, and is
lipid accumulation and TG synthesis of 3T3-L1 cells (Fig. 5A, B), and mostly caused by obesity. Although the pathogenesis of metabolic syn­
similar effects were found with bergenin treatment (Fig. 5C, D). We also drome from obesity is not yet completely elucidated, some obesity-
found the bergenin successfully regulates adipogenesis, lipid meta­ related complications such as increased serum lipid levels and inflam­
bolism, and lipid oxidation related proteins (Fig. 5E), and the result was matory cytokines are explicit risk factors of metabolic syndrome

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Fig. 2. HD intake stimulated adiponectin/AMP-activated protein kinase (AMPK) signaling and it evoked uncoupling protein 1 (UCP1)-independent energy con­
sumption in eWAT of DIO mice. (A) Adiponectin content in serum. (B) The mRNA level of genes related with adiponectin synthesis, secretion, and its receptors in
eWAT. (C) Citrate synthase activity of eWAT. (D) Protein expression of AMPK and p-AMPK, and other energy expenditure related markers in eWAT. (E) The relative
mRNA expression of mitochondrial biogenesis and lipid oxidation related genes in eWAT. (F) The relative mRNA expression of tryglyceride (TG)/fatty acid (FA) cycle
related genes in eWAT. Statistical differences within groups were examined by ANOVA and Duncan’s multiple range test. Different lowercase superscripts indicate
significant differences between groups at p < 0.05.

(Grundy, 2004). This indicates that the first goal for treating metabolic expenditure in animal models noted the expression of UCP1-dependent
syndrome is to lose weight. Here, we showed that HD extract effectively thermogenesis in the WAT (Shabalina et al., 2013). There is a new type
reduced body weight in DIO mice and also decreased the weights of of adipocyte tissue proposed, named “beige” or “brown-like”, whose
WATs from different locations. HD extract also mitigated the elevated function resembles that of the BAT but is converted from the WAT
TG and TC content in the serum, and decreased the serum LDL-C level (Desjardins & Steinberg, 2018). PRDM16 is a key mediator of brown fat
(compared with those of the HFD group) but without a decrease in the differentiation that is selectively expressed in the BAT (Seale et al.,
HDL-C level. A high TG content and low HDL-C level are the main 2007); therefore, PRDM16 is commonly used as a marker of switching
criteria for a diagnosis of metabolic syndrome, and are closely related to energy expenditure mechanisms in the WAT to the brown-like adipose
CVD risk (de Freitas et al., 2011). The ratios of TC/HDL-C and LDL-C/ tissue (Hilton, Karpe, & Pinnick, 2015). In this study, the HD extract
HDL-C have been used as CVD risk factors (Millán et al., 2009), and strongly stimulated the expression of PRDM16 in the eWAT, and cyto­
HD administration ameliorated both of these factors. Interestingly, both chrome C protein expression and AMPK phosphorylation were also
obesity and serum lipid status were improved in the HD group, whereas significantly elevated. The increase of cytochrome C represents excita­
adipogenesis and lipid synthesis-related protein expression remained tion of the electron transport system, further supporting that HD extract
unchanged. For this reason, we suspect that the loss of weight in the HD stimulates energy consumption in the WAT. However, the expression
group was mainly due to energy expenditure mechanisms rather than by level of UCP1 remained unchanged despite the increase of PRDM16 in
disturbing adipogenesis and lipid synthesis in the WAT. The increased the HD group, which generally accompanies activation of thermogenesis
burn in calories in HD group is in line with the diet intake results since reactions via the UCP1 channel. Hence, we concluded that HD extract
there was no difference in total energy intake between the HFD and HD likely stimulates the electron transport system, with the translocated H+
groups. ions mainly passing through ATP synthase rather than the UCP1 channel
There are two major types of adipose tissues, namely the WAT and (Letts & Sazanov, 2017). This pathway is known as UCP1-independent
the brown adipose tissue (BAT), with opposite roles of fat storage and energy expenditure and its mechanism is unusual and unclear; howev­
energy expenditure with thermogenesis, respectively (Marlatt & Rav­ er, a previous study indicated that Ucp1-/- mice can also exploit ther­
ussin, 2017). However, some previous studies that investigated energy mogenic responses in a cold-acclimated mice model (Ukropec,

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Fig. 3. HD extract upregulated anti-oxidation related proteins and mitigated inflammatory responses in DIO mice. (A) Expression of anti-oxidative proteins in eWAT
(B) The malondialdehyde content of eWAT. (C) Serum IL-1β content. (D) Myeloperoxidase amounts in eWAT. (E) The relative mRNA expression of cytokine, che­
mokine, adhesive molecules, and macrophage markers in eWAT. Statistical differences within groups were determined by ANOVA and Duncan’s multiple range test.
Different lowercase superscripts indicate significant differences between groups at p < 0.05.

Anunciado, Ravussin, Hulver, & Kozak, 2006). Another study suggested Ca2+-ATPase (Serca) 1 or 2 is a central mediator of this signaling, and
that UCP1-independent energy expenditure may involve ATP-related stimulates respiratory reactions in the mitochondria, accompanied by
mechanisms through chronic AMPK activation, which increases mito­ enhanced tricarboxylic acid metabolism and glycolysis (Pollard et al.,
chondrial biogenesis and the oxidative capacity of the WAT (Ceddia, 2019). However, we identified another possible energy consumption
2013). In line with this proposed mechanism, HD extract treatment mechanism in this study related to AMPK signaling given the strong
upregulated the mRNA expression of Pgc-1α/Nrf1/Tfam, which is a key AMPK activation in the HD group.
signaling pathway of mitochondrial biogenesis (Chen, Tao, Li, & Yao, The futile TG/FA cycle is a UCP1-independnt energy expenditure
2018; Kang, Chu, & Kaufman, 2018), and the increased CS activity mechanism, which mainly involves AMPK as a key modulator (Flachs,
further indicated an increase in mitochondria abundance in the eWAT. Rossmeisl, Kuda, & Kopecky, 2013; Mottillo et al., 2014). Previous
The swelling of mitochondria enables the WAT to generate energy by studies using omega-3 FAs or palmitoleic acid also proposed the model
increasing the oxidative capacity; however, it is still unclear how ATP that upregulation of the TG/FA cycle is associated with abundant ATPs
could be consumed in the WAT in this study. induced by elevated AMPK phosphorylation in the WAT (Cruz et al.,
Recent studies have proposed some of possible UCP1-independent 2018; Flachs et al., 2013). There are two opposite metabolic functions
thermogenesis mechanisms related to the consumption of energy for FA homeostasis in the WAT: lipolysis and re-esterification. Their co-
through specific futile cycles (Chang, Song, Choi, Yun, & Park, 2019; activation initiates the TG/FA cycle, and their net rates control the
Ikeda & Yamada, 2020). For example, Ca2+ cycling-mediated thermo­ amount of FAs released from the WAT (Cadoudal et al., 2005).
genesis via Ca2+-ATPase transport in the WAT is a well-established Approximately 20–30% of FAs are recycled by co-activation of these
mechanism (Ikeda et al., 2017). Sarcoplasmic/endoplasmic reticulum antagonistic reactions in the WAT in a normal state, but the increment of

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Y.-J. Son et al. Journal of Functional Foods 84 (2021) 104581

Fig. 4. HD treatment improved insulin sensitivity and ameliorated glycometabolism of eWAT and liver in DIO mice. (A) The results of oral glucose tolerance test
(OGTT). (B) The area under the curve (AUC) of OGTT. (C) The results of intraperitoneal insulin tolerance test (IPITT). (D) The AUC of IPITT. (E) The serum insulin
level. (F) The graph of HOMA-IR. (G) Protein expression of p-Akt (ser473) and Akt in eWAT. (H) Protein expression of glycometabolism related markers in liver
tissue. Statistical differences within groups were determined by ANOVA and Duncan’s multiple range test. Different lowercase superscripts indicate significant
differences between groups at p < 0.05.

metabolic flux (e.g., exercise) enhances the recycling rate to facilitate content in serum was significantly elevated in the HD group. In addition,
elimination of excessive ATPs (Gauthier et al., 2008; Reshef et al., 2003). the mRNA levels of adiponectin receptors (AdipoR1 and AdipoR2) and
Although there is no alteration of energy level during lipolysis, the Appl1, the link between adiponectin and AMPK activation, were also
requirement of ATP in this cycle is approximately 2.7 ATP/FA, which is increased by HD extract. Thus, HD extract clearly upregulated adipo­
consumed when FAs are acetylated to acyl coenzyme A (acyl-CoA) by nectin/AMPK signaling cascades in the DIO mouse model.
acyl-CoA synthetase, resulting in a waste of ATP through the TG/FA Adiponectin/AMPK signaling mediates versatile downstream mech­
cycle spends (Mottillo et al., 2014; Townsend, Knuth, & Wright, 2017). anisms. First, the vigorous stimulation of AMPK could promote UCP-1-
We found that HD extract activated genes related to lipolysis (Atgl, Hsl) independent energy expenditure through the TG/FA cycle as stated
and re-esterification (Pepck) in the WAT of mice; therefore, we assume above, which can cause loss of calories without exercise. Moreover,
that the elevated AMPK activation by HD extract eliminated redundant AMPK activation is highly associated with relief of insulin resistance, a
ATPs via the TG/FA cycle, and this futile dissipation of energy alleviated representative symptom of metabolic syndrome. Insulin resistance in
obesity in DIO mice. patients with obesity is mainly caused by loss of insulin sensitivity of the
Adiponectin is a hormone that originates from the adipose tissue, and receptor owing to continuous oxidative stress and inflammatory re­
has been identified as a novel metabolic messenger in numerous organs sponses (Shoelson, Lee, & Goldfine, 2006). There is ample evidence
(e.g., the liver, heart, kidney, brain, beta cell, adipose tissue, and mus­ suggesting an association of anti-oxidation and anti-inflammation-
cle). Adiponectin regulates broad biological responses, including anti- related responses with AMPK activation (Huang et al., 2015; Zimmer­
hyperglycemia, anti-inflammatory, anti-apoptotic, and anti-insulin mann et al., 2015); the HD extract also showed anti-oxidative and anti-
resistance, and has therefore been considered a possible therapeutic inflammatory responses in the eWAT. Adiponectin/AMPK signaling
target for several diseases (Ghadge, Khaire, & Kuvalekar, 2018; Ng et al., regulates glucose utilization and FA oxidation throughout the body
2016; Straub & Scherer, 2019). The full length adiponectin comprises (Berg, Combs, & Scherer, 2002; Ceddia, 2013), and APPL1 (an inter­
three main domains: an N-terminal domain, collagen-like fibrous mediate protein of adiponectin/AMPK signaling) can facilitate binding
domain, and a globular domain, and several molecular chaperones are of insulin receptor and insulin receptor substrate proteins (Ryu et al.,
associated with its biosynthesis and secretion (Achari & Jain, 2017). 2014). The interaction between APPL1 and insulin sensitivity was
Adiponectin is mainly secreted from the adipose tissue, although other identified in Appl1 knockout mice and its expression regulating model,
cell types, including skeletal and cardiac cells, have also recently been and the deficiency of APPL1 reduced glucose uptake in adiponectin-
shown to secrete adiponectin for metabolic regulation (Diniz et al., sensitive organs; conversely, overexpression of APPL1 resulted in
2019). Although the precise mechanism is not yet clear, the main factor enhanced activation of Akt in target organs, in turn mitigating insulin
for augmentation of adiponectin secretion appears to be acute aerobic resistance (Cheng et al., 2009; Hosch, Olefsky, & Kim, 2006). Akt is an
exercise leading to an energy-deficient state in the body (Simpson & instrumental mediator for glucose metabolism that is activated by the
Singh, 2008). The metabolic regulation of adiponectin is mainly medi­ insulin signaling cascade; therefore, its irregular lowered activation is a
ated via AMPK phosphorylation, and because of the broad regulatory principal indicator of insulin resistance (Carvalho-Filho et al., 2005).
roles of AMPK, adiponectin can evoke downstream signaling of AMPK in The crucial role of Akt is to enhance glucose uptake by promoting
target organs (Wang, Li, Qiao, Li, & Qiao, 2019). Interestingly, we found GLUT4 translocation to the cell membrane in the major organs, and Akt
that HD extract markedly stimulated the expression of genes related to also stimulates glycogen synthesis by converting GSK-3β to its inactive
the maturation and secretion of adiponectin, and the adiponectin form (phosphorylated form at Ser9) (Hou et al., 2019; Kim, Nikoulina,

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Y.-J. Son et al. Journal of Functional Foods 84 (2021) 104581

Fig. 5. HD ethanol extract and the bergenin, a major compound of HD ethanol extract, impeded lipid accumulation in 3T3-L1 cell line. (A) The lipid accumulation
result and (B) triglyceride contents after HD extract treatment. (C) The lipid accumulation result and (D) triglyceride contents after bergenin treatment. (E) Lipid
metabolism related proteins were analyzed with Western blot. Statistical differences within groups were analyzed by ANOVA and Duncan’s multiple range test.
Different lowercase superscripts indicate significant differences between groups at p < 0.05.

Ciaraldi, Henry, & Kahn, 1999). HD extract treatment resulted in the the eWAT, the increased number of mitochondria and their capacity in
phosphorylation of Akt in the eWAT of DIO mice, and also found the WAT may cause UCP1-independent, but AMPK-dependent, energy
changes in protein expression in the liver due to its central role in expenditure via the TG/FA cycle. As a stimulator of AMPK phosphory­
glucose metabolism in the body; Akt and GLUT4 were significantly lation, we revealed upregulation of adiponectin related signaling from
stimulated by HD treatment, which led to inactivation of GSK-3β. In HD extract treatment. Furthermore, the promotion of adiponectin/
turn, HD treatment improved insulin tolerance, and lowered fasting AMPK signaling by HD treatment also alleviated insulin resistance and
glucose and HOMA-IR levels in DIO mice. reinforced glycometabolism in the eWAT and liver. Therefore, daily
In conclusion, we verified the versatile effectiveness of HD extract, intake of HD as part of a regular diet could mitigate the symptoms of
suggesting its use as a potent functional food resource that can relieve metabolic syndrome with multi-targeting effects.
metabolic syndrome, and propose the underlying molecular mechanism
(Fig. 6). HD extract administration in DIO mice resulted in the loss of 5. Ethics statements
body weight and fat tissue weight, and ameliorated lipid status in the
plasma. HD treatment highly activated AMPK, as well as the related The animal study was planned and executed as per the National In­
mechanisms, including anti-oxidation and anti-inflammation processes. stitutes of Health guidelines. The animal experiments were approved by
Although HD did not evoke UCP1-dependent thermogenic signaling in the Institutional Animal Care and Use Committee of Korea Institute of

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Y.-J. Son et al. Journal of Functional Foods 84 (2021) 104581

Fig. 6. The proposed mechanism mediated by HD ethanol extract administration in DIO mice.

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odology, Writing - review & editing, Supervision, Project administra­ receptors and AMPK activity in tissues of diet-induced diabetic mice. Diabetes &
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Declaration of Competing Interest phosphoenolpyruvate carboxykinase in the metabolic syndrome. Biochimie, 87(1),
27–32. https://doi.org/10.1016/j.biochi.2004.12.005.
The authors declare that they have no known competing financial Carvalho-Filho, M. A., Ueno, M., Hirabara, S. M., Seabra, A. B., Carvalheira, J. B., De
Oliveira, M. G., … Saad, M. J. (2005). S-nitrosation of the insulin receptor, insulin
interests or personal relationships that could have appeared to influence receptor substrate 1, and protein kinase B/Akt: A novel mechanism of insulin
the work reported in this paper. resistance. Diabetes, 54(4), 959–967.
Ceddia, R. B. (2013). The role of AMP-activated protein kinase in regulating white
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Acknowledgements https://doi.org/10.1016/j.mce.2012.06.014.
Chang, S. H., Song, N. J., Choi, J. H., Yun, U. J., & Park, K. W. (2019). Mechanisms
This work was supported by the Korea-Mongolia Cooperation Project underlying UCP1 dependent and independent adipocyte thermogenesis. Obesity
Reviews, 20(2), 241–251. https://doi.org/10.1111/obr.12796.
from the National Research Foundation of Korea (NRF) (grant number
Chen, Z., Tao, S., Li, X., & Yao, Q. (2018). Resistin destroys mitochondrial biogenesis by
2008-00592). This work was also supported by the integration project of inhibiting the PGC-1α/ NRF1/TFAM signaling pathway. Biochemical and Biophysical
Korea Institute of Science and Technology (Grant number 2Z05630). Research Communications, 504(1), 13–18. https://doi.org/10.1016/j.
The funders had no role in the study design; collection, analysis and bbrc.2018.08.027.
Cheng, K. K. Y., Iglesias, M. A., Lam, K. S. L., Wang, Y., Sweeney, G., Zhu, W., … Xu, A.
interpretation of data; writing of the report; and in the decision to (2009). APPL1 Potentiates Insulin-Mediated Inhibition of Hepatic Glucose
submit the article for publication. Production and Alleviates Diabetes via Akt Activation in Mice. Cell Metabolism, 9(5),
417–427. https://doi.org/10.1016/j.cmet.2009.03.013.
Cruz, M. M., Lopes, A. B., Crisma, A. R., de Sá, R. C. C., Kuwabara, W. M. T., Curi, R., …
Appendix A. Supplementary data Alonso-Vale, M. I. C. (2018). Palmitoleic acid (16:1n7) increases oxygen
consumption, fatty acid oxidation and ATP content in white adipocytes. Lipids in
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de Freitas, E. V., Brandão, A. A., Pozzan, R., Magalhães, M. E., Fonseca, F., Pizzi, O., …
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