FOOD & FUNCTION

Pears www.mnf-journal.com

Pear Extract and Malaxinic Acid Reverse Obesity, Adipose

Tissue Inflammation, and Hepatosteatosis in Mice
Xuan T. Truong, Thuy T. P. Nguyen, Man-Jong Kang, Chang Hwa Jung, Sueun Lee,
Changjong Moon, Jae-Hak Moon, and Tae-Il Jeon*

Scope: Obesity and diabetes are major public health problems and are 1. Introduction
emerging as pandemics. Considerable evidence suggests that pear fruit The increasing prevalence of obesity is a
consumption is associated with a lower risk of obesity-related complications. global threat to public health. It is esti-
Thus, the present study is conducted to investigate the therapeutic potential mated that there were more than 2 bil-
lion obese or overweight people world-
of pear extract (PE) for reversing obesity and associated metabolic
wide in 2013, which represents 30% of
complications in high-fat diet-induced obese mice. the world’s population.[1] The economic
Methods and results: Obesity is induced in male C57BL/6 mice fed a high-fat cost related to obesity and its complica-
diet for 11 weeks. After the first 6 weeks on the diet, obese mice are tions is very high.[2]
administered vehicle or PE for 5 weeks. PE treatment decreases body weight Massive expansion of white adipose
gain, expands white adipose tissue (WAT), and causes hepatic steatosis in tissue (WAT), a hallmark of obesity, is
characterized by adipocyte hypertrophy
obese mice, as well as inhibits adipogenesis and lipogenesis. Impaired and hyperplasia. This phenomenon leads
glucose tolerance and insulin resistance are improved by PE. In addition, PE to dysfunction of adipose tissue accom-
reduces macrophage infiltration and expression of pro-inflammatory genes panied by increased infiltration of im-
and deactivates mitogen-activated protein kinases in WAT. Finally, malaxinic mune cells, particularly macrophages,
acid is identified as an active component responsible for the anti-obesity and the activation of inflammation and
is largely associated with obesity-related
effects of PE in mice.
complications such as insulin resistance
Conclusion: The results demonstrate that PE supplementation ameliorates (IR), type 2 diabetes (T2D), and non-
diet-induced obesity and associated metabolic complications and suggest the alcoholic fatty liver disease (NAFLD).[3]
health-beneficial effects of both pear fruits and malaxinic acid in WAT inflammation in obesity can pro-
counteracting these diseases. mote IR, which enhances the lipoly-
sis of stored fat. Increased delivery of
fatty acids to the liver results in exces-
sive accumulation of intracellular lipids,
particularly triglyceride, and the subsequent development of
X. T. Truong, T. T. P. Nguyen, Prof. M.-J. Kang, Prof. T.-I. Jeon NAFLD.[4] Indeed, several clinical studies have shown that WAT
Department of Animal Science
Chonnam National University inflammation in obese patients with NAFLD was greater than
Gwangju 61186, Republic of Korea that in obese patients with normal hepatic triglyceride levels.[5]
E-mail: tjeon@jnu.ac.kr Therefore, modulating processes that control the expansion or
Dr. C. H. Jung differentiation of WAT may be an effective strategy for prevent-
Research Group of Natural Materials and Metabolism ing or treating metabolic diseases, including obesity.
Korea Food Research Institute
Intensive lifestyle modification including diet and exercise is
Wanju-gun, Jeollabuk-do 55365, Republic of Korea
clinically beneficial for many obese patients. However, changing
S. Lee, Prof. C. Moon
Department of Veterinary Anatomy and Animal Behavior behavioral factors is difficult to achieve and maintain and phar-
College of Veterinary Medicine and BK21 Plus Project Team macological options are often required. Although several anti-
Chonnam National University obesity agents are currently available, long-term consumption of
Gwangju 61186, Republic of Korea these agents may have severe side effects.[6] Increasing evidence
Prof. J.-H. Moon indicates that fruit consumption is associated with a lower risk of
Department of Food Science and Technology and Functional Food
Research Center obesity-related complications, and many national guidelines rec-
Chonnam National University ommend increasing fruit and vegetable consumption to manage
BK21 Plus Program, Gwangju 61186, Republic of Korea overall health and obesity.[7]
Pears (Pyrus spp.) are commonly consumed worldwide in ei-
The ORCID identification number(s) for the author(s) of this article ther fresh or processed forms such as juices and jams in daily
can be found under https://doi.org/10.1002/mnfr.201801347 life and are also traditionally used as herbal medicines to treat
DOI: 10.1002/mnfr.201801347 respiratory diseases in Asia.[8] Our and other research groups

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previously showed that pear fruits, particularly those that are im-
mature, are rich sources of bioactive phenolic compounds with
antioxidant and anti-inflammatory properties.[8b,9] Recently, di-
etary fruit polyphenols have gained recognition as promising
agents for conferring protection against metabolic diseases;[10]
two clinical trials have indeed suggested that increased daily pear
consumption is inversely correlated with weight gain.[11] Thus, in
the current study, we examined whether pear can improve high-
fat diet (HFD)-induced obesity. We found that pear treatment
effectively improved HFD-induced body weight gain, IR, and
hepatic steatosis in mice. Pear predominantly targeted adipose
tissue, thereby reducing adipocyte hypertrophy and WAT inflam-
mation. Moreover, we showed that malaxinic acid (MA) is a po-
tent anti-obesity component in pear.
2. Experimental Section
2.1. Materials and Sample Preparation
DMEM, fetal calf serum (FCS), fetal bovine serum (FBS), and an-
tibiotics were purchased from Hyclone (Logan, UT). 3-Isobutyl-1-
methylxanthine (IBMX), dexamethasone (DEX), insulin, and Oil
red O powder were obtained from Sigma-Aldrich (St. Louis, MO).
The whole immature pear fruits (2.5 kg fresh wt.) were pulverized
using a square cutter, and extracted with 25 L of hot water (2 h,
100 °C), followed by filtration to obtain an extract. The extract
was concentrated in a vacuum at 60 °C, lyophilized for 3 days,
and stored at −20 °C until use. The detailed methods for isola-
tion, purification, and identification of MA from pear extract (PE)
were reported in our previous studies.[9a,12]
2.2. Analysis of Polyphenol Composition in PE
Total phenolic and flavonoid contents in PE were measured using
Folin–Ciocalteu’s method and the aluminum chloride colorimet-
ric assay, respectively[8a] (Figure S1A, Supporting Information).
For polyphenol composition analysis, PE was dissolved in 80%
MeOH and subjected to HPLC using an ODS column (UG120,
4.6 mm I.D. × 250 mm, 5 µm, Shiseido, Tokyo, Japan). The mo-
bile phase was composed of H2 O:acetic acid (98:2, v/v, eluent A)
and 50% MeOH (eluent B). The gradient program began at 100%
A and was increased to 100% B over a period of 40 min. The col-
umn temperature was maintained at 40 °C, and the flow rate was
1 mL min−1 . Phenolic compounds were monitored at 280 and
254 nm, respectively, using a photodiode array detector (SPD-
M20D; Shimadzu, Kyoto, Japan). The contents were quantified
against the chromatographic peak areas of the external standards.
The calibration curves were plotted over a concentration range of
0.01–10 µg (Figure S1, Supporting Information).
2.3. Cell Culture
3T3-L1 cells were purchased from the American Type Culture
Collection (Manassas, VA). Cells were maintained in DMEM sup-
plemented with 10% FCS and 1% antibiotics in an atmosphere
of 5% CO2 at 37 °C. Adipocyte differentiation was induced by
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treating confluent cells for 48 h in medium containing 10% FBS,
0.5 mm IBMX, 1 µm DEX, and 10 µg mL−1 insulin. The medium
was replaced with that supplemented with only 5 µg mL−1 insulin
every other day. The cells were treated with or without PE or MA
for 8 days during adipogenesis.
2.4. Oil Red O Staining
Cells were fixed with 10% formalin solution for 10 min and
stained with freshly prepared Oil Red O solution for 30 min at
RT. Images were acquired using a Leica DM IL LED microscope
(Wetzlar, Germany). Intracellular lipid contents were measured
by extracting Oil Red O with isopropanol, and the absorbance was
measured at 500 nm with a microplate reader (Biotek, Winooski,
VT).
2.5. RNA Analysis
Total RNA from tissues and cells was isolated using TRI-
zol(Invitrogen, Carlsbad, CA) and cDNA was synthesized using a
ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative
PCR was performed with SYBR Green Master Mix (Enzynomics,
Daejeon, South Korea) using a Mx3000P real-time PCR System
(Agilent Technologies, Santa Clara, CA). Primer sequences are
shown in Table S1, Supporting Information. mRNA levels were
normalized for expression to mouse ribosomal protein, Large, P0
(RPLP0) as control and calculated by the comparative threshold
cycle method.
2.6. Immunoblotting
Cells and tissues were lysed in RIPA buffer containing protease
and phosphatase inhibitors. The lysates were sonicated and cen-
trifuged at 20 000 × g for 20 min at 4 °C. The supernatants were
separated by SDS-PAGE and then transferred to nitrocellulose
membranes. The membranes were incubated with primary anti-
bodies (Table S2, Supporting Information) overnight at 4 °C, fol-
lowed by incubation with secondary antibodies (Thermo Fisher
Scientific, Waltham, MA). Blots were developed using an EZ-
Western Lumi Pico or Femto western blot detection kit (DAEIL-
LAB Service, Seoul, South Korea).
2.7. Animals
All animal studies were performed in accordance with accepted
standards of animal welfare and with permission from the
Chonnam National University Institutional Animal Care and
Use Committee (YB-2016-01). Five-week-old male C57BL/6 mice
were obtained from OrientBio (Seongnam, South Korea) and
maintained on a chow diet for 1 week with a 12 h light/dark cycle
for acclimatization. At 6 weeks of age, the mice were randomly
assigned (n = 5 per group) to a group fed regular chow diet (CD,
LabDiet 5L79, OrientBio) or to a group fed HFD (Research Diets,
#D12492, New Brunswick, NJ). The CD provided 3.43 kcal g−1
of energy (21% calories from protein, 14% calories from fat, and

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65% calories from carbohydrate), whereas the HFD provided 5.21
kcal g−1 of energy (20% calories from protein, 60% calories from
fat, and 20% calories from carbohydrate). The ingredients includ-
ing sucrose in CD were not perfectly matched with HFD. After 6
weeks on the diet, HFD-induced obese mice were orally admin-
istered with vehicle (saline) or PE at 200 mg per kg body weight
per day for 5 weeks. In MA analysis, mice were fed a HFD for 9
weeks, followed by oral administration of MA (40 mg per kg body
weight per day) for 7 weeks. Body weight was measured weekly
and food intake was recorded every other day. After the feeding
periods, the mice were sacrificed at 8 a.m. (at the end of the dark
cycle) by CO2 asphyxiation.
2.8. Dosage Information
Mice were orally gavaged with PE (200 mg kg body weight−1 )
or MA (40 mg kg body weight−1 ) once daily for 5 or 9 weeks,
respectively. The dosages were chosen according to the previ-
ous studies.[[13] The dose of PE or MA used in the present study
was roughly equivalent to 16.2 or 3.2 mg kg body weight−1 in
humans,[14] which equates to a 972-mg dose of PE or a 192-mg
dose of MA for a person weighing 60 kg. Based on an average of
0.5 g PE derived from each immature pear fruit (10 g), an indi-
vidual should consume approximately two immature pear fruits
per day.
2.9. Biochemical Analysis of Plasma
Blood was collected by cardiac puncture and the plasma was sepa-
rated by centrifugation. Plasma glucose, triglyceride, total choles-
terol, glutamic oxaloacetic transaminase, and glutamic pyruvic
transaminase were determined using Asan enzymatic kits (Asan,
Seoul, South Korea). Plasma insulin concentrations were mea-
sured with the Ultrasensitive mouse Insulin ELISA Kit (ALPCO,
Salem, NH). The homeostasis model assessment for insulin re-
sistance (HOMA-IR), an indicator of IR, was calculated with a
HOMA2 calculator released by the Diabetes Trials Unit, Univer-
sity of Oxford.[15]
2.10. Glucose and Insulin Tolerance Test
For the glucose tolerance test (GTT), mice were fasted for 16 h
prior to oral gavage of glucose (1 g kg body weight−1 ). In the in-
sulin tolerance test (ITT), mice were intraperitoneally injected
with insulin (0.75 U kg body weight−1 ) after fasting for 4 h.
The glucose concentrations were measured at 0, 15, 30, 60, 90,
and 120 min in the tail blood using a glucometer (Roche, Basel,
Switzerland).
2.11. Histology and Immunohistochemistry
Tissues were fixed with 4% paraformaldehyde, embedded in
paraffin wax, and sectioned sagittally (4 µm thick). Multiple sec-
tions were stained with hematoxylin and eosin to detect mor-
phological changes. The sections were dewaxed and hydrated,
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and then antigen retrieval was performed by heating the sections
with 10 mm citrate buffer, pH 6. Sections were blocked in 5%
normal goat serum and 0.1% Tween 20, reacted with anti-IBA1
(1:400) overnight at 4 °C, and then incubated with biotinylated
goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for 1 h
at RT. The immunoreaction with avidin–biotin peroxidase com-
plex (Vector Laboratories) was conducted for 1 h at RT. The per-
oxidase reaction was developed using a DAB Substrate Kit (Vec-
tor Laboratories). Images were acquired with a BX-40 microscope
(Olympus, Tokyo, Japan) fitted with an eXcope × 3 digital camera
(DIXI Optics, Daejeon, South Korea). Average adipocyte size, fre-
quency distribution of adipocyte sizes, and number of crown-like
structures (CLSs) were counted using Image J software.
2.12. Statistical Analysis
Data were presented as the mean ± SEM. Differences between
the means of individual groups were assessed by one-way analy-
sis of variance followed with Tukey’s multiple comparisons test;
differences were considered significant at p < 0.05. The statisti-
cal software package, Prism 6.0 (GraphPad Software, La Jolla, CA,
USA), was used for these analyses.
3. Results
3.1. PE Reduces Body Weight Gain and Improves Glucose
Homeostasis in HFD-Induced Obese Mice
Six weeks of HFD feeding resulted in significantly increased body
weight and impaired glucose tolerance compared with that in CD
mice (data not shown). To investigate the therapeutic effects of PE
on diet-induced obesity, we orally administered vehicle (saline)
or PE to HFD-induced obese mice for 5 weeks. Although food in-
take was similar as in the vehicle control group, PE supplemen-
tation significantly decreased body weight (Figure 1A,B). Next,
to determine the effect of PE on glucose homeostasis and in-
sulin sensitivity, GTT and ITT were performed after 4 weeks of
PE treatment. As shown in Figure 1C,D, PE-treated HFD-fed
mice exhibited lower glucose levels at all time points up to 120
min after oral glucose challenge or intraperitoneal insulin injec-
tion and reduced areas under the curves compared with vehicle
control HFD-fed mice. Moreover, PE supplementation lowered
the plasma levels of glucose and insulin, thereby decreasing the
HOMA-IR index (Figure 1E). Additionally, PE supplementation
significantly decreased the plasma levels of triglyceride and total
cholesterol, whereas no differences were observed in enzymes
associated with hepatic injury (Figure S2, Supporting Informa-
tion). These results indicate that PE treatment effectively reduced
obesity-associated hyperglycemia, hyperinsulinemia, and hyper-
lipidemia in mice, and these effects did not result from reduced
food consumption or toxicity.
3.2. PE Decreases WAT Expansion by Inhibiting Adipogenesis
In parallel with weight loss caused by PE, WAT weights (Fig-
ure 2A), mean adipocyte size (Figure 2B), and large adipocyte

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Figure 1. PE promotes weight loss and improves glucose homeostasis and insulin sensitivity in HFD-induced obese mice. Mice were fed a HFD for 6
weeks. Subsequently the same mice were administered with vehicle or PE (200 mg kg−1 day−1 ) on the diet for an additional 5 weeks. A) Body weight
gain. B) Food intake. C) GTT and D) ITT were performed at 10 weeks. E) Fasting plasma glucose and insulin levels. HOMA-IR was determined using
both levels. Data are mean ± SEM; n = 5. Data are representative of two independent experiments with five mice per group. *p < 0.05 versus CD; # p <
0.05 versus HFD (vehicle control).

numbers (Figure 2C) were remarkably reduced. Activation of en-
ergy expenditure in brown adipose tissue (BAT) and WAT brown-
ing has recently emerged as a promising strategy for treating
metabolic diseases.[16] However, the expression of thermogenic
gene uncoupling protein 1 (UCP1) was not changed in both
BAT and inguinal WAT from PE-treated mice (Figure S3A, Sup-
porting Information), suggesting that the reduced body weight
gain resulted from inhibition of fat accumulation in WAT. To
evaluate this hypothesis, murine preadipocyte 3T3-L1 cells were
differentiated in the presence or absence of PE. As shown in
Figure 3A,B, PE significantly suppressed fat accumulation as as-
sessed by Oil Red O staining in a dose-dependent manner. Fur-
thermore, the expression of adipogenic transcription factors such
as CCAAT/enhancer binding protein α (C/EBPα) and peroxi-
some proliferator-activated receptor γ (PPARγ ) and lipogenic
genes was markedly inhibited by PE both in vivo (Figure 2D) and
in vitro (Figure 3C,D). Interestingly, PE also restored HFD dys-
regulation of the adipokine leptin and adiponectin expression in
WAT (Figure 2D). These results suggest that reduced adiposity
by PE is associated with decreased adipocyte hypertrophy and in-
hibition of adipogenesis.
3.3. PE Prevents Hepatic Steatosis in HFD-Induced Obese Mice
Adipocyte dysfunction in obesity influences ectopic fat accumu-
lation and IR in the liver, leading to NAFLD.[17] We therefore ex-
amined the effects of PE on hepatic steatosis. Although there
was no significant difference in liver weights between groups,
histological examination revealed an increase in lipid droplets in
the liver of HFD-induced obese mice, whereas PE supplementa-
tion significantly attenuated fat accumulation (Figure 2E). This

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Figure 2. PE reduces adiposity and hepatic steatosis. A) Tissue weights. B) H&E staining in WAT of mice (magnification: ×200, scale bar: 50 µm). C)
Average adipocyte area and frequency distribution of adipocyte sizes were counted using ImageJ. D) mRNA levels in WAT were analyzed by RT-qPCR. E)
H&E staining in liver of mice. F) Liver triglyceride levels. Data are mean ± SEM; n = 5. *p < 0.05 versus CD; # p < 0.05 versus HFD. eWAT, epididymal;
iWAT, inguinal; iBAT, interscapular.

observation was clearly confirmed by measuring hepatic triglyc-
eride levels (Figure 2F). We further analyzed the mRNA expres-
sion levels of genes involved in hepatic lipid metabolism. Com-
pared with the vehicle control mice, PE significantly suppressed
lipogenic genes including sterol regulatory element binding
protein-1c, stearoyl-CoA desaturase 1, fatty acid synthase, and
acetyl-CoA carboxylase 1 (Figure S3B, Supporting Information).
However, HFD suppression of carnitine palmitoyltransferase 1α
for fatty acid oxidation was not recovered by PE (Figure S3B, Sup-
porting Information), suggesting that PE ameliorates obesity-
associated hepatic steatosis by inhibiting lipogenesis. Together,
these results demonstrate that PE supplementation greatly re-
duced adiposity, maintained glucose homeostasis, and improved
hepatic steatosis in HFD-induced obese mice.
3.4. PE Suppresses HFD-Induced Macrophage Infiltration and
Inflammation in WAT
As adipocyte hypertrophy is closely associated with WAT in-
flammation, characterized by increased infiltration and polariza-
tion of macrophages to the pro-inflammatory M1 phenotype,[18]
we next examined the effects of PE on HFD-induced WAT in-
flammation. Immunostaining of the macrophage-specific pro-
tein IBA1 revealed abundant CLSs formed from clusters of
macrophages surrounding necrotic adipocytes in the WAT of
HFD-fed mice, whereas PE markedly decreased the recruitment
of macrophages and the number of CLSs (Figure 4A). Consis-
tent with these histological observations, after PE treatment, the
mRNA expression levels of the macrophage-specific markers
F4/80 and Cd68 were significantly reduced (Figure 4B). Simi-
larly, PE-treated mice showed reduced expression levels of sev-
eral markers of pro-inflammatory M1 macrophages including tu-
mor necrosis factor α (Tnfα), monocyte chemoattractant protein
1 (Mcp1), and integrin, alpha X (Itgax/Cd11c), whereas there were
no significant changes in the expression of the anti-inflammatory
M2 macrophage markers between groups (Figure 4C). While
mitogen-activated protein kinases (MAPKs) are essential for reg-
ulating the proliferation and differentiation of adipocytes, ex-
cessive activation of MAPKs is associated with metabolic dys-
functions in obesity and diabetes.[19] Thus, we examined the
phosphorylation (activation) level of three major MAPKs, c-Jun
N-terminal kinase (JNK), extracellular signal-regulated kinase
(ERK), and p38 in WAT. HFD feeding significantly increased the

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Figure 3. PE inhibits adipogenesis in 3T3-L1 cells. 3T3-L1 cells were differentiated for 8 days in the presence or absence of PE. A) Oil Red O staining of
differentiated adipocytes and B) quantification via absorbance measurements. C,D) mRNA and protein expression levels were analyzed by RT-qPCR and
immunoblotting, respectively. Data are mean ± SEM; n = 3 for three separate experiments. *p < 0.05 versus preadipocyte; # p < 0.05 versus adipocyte
(vehicle control); ## p < 0.01 versus adipocyte. MDI, methylisobutylxanthine, dexamethasone, insulin; ORO, Oil Red O.

phosphorylation of all three MAPKs in the WAT, which was sig-
nificantly reversed by PE treatment (Figure 4D). Macrophage-
specific JNK deficiency prevented the accumulation of adipose
tissue macrophages (ATMs) expressing surface markers for M1
polarization in the WAT of HFD-fed mice without affecting M2
polarization.[20] Therefore, our results indicate that PE decreased
WAT inflammation in HFD-fed mice by reducing macrophage
infiltration and the number of ATMs with M1 phenotype in WAT.
This suggests that deactivation of the JNK signaling pathway
contributes to ATM polarization by PE, although the regulatory
mechanism remains unclear.
3.5. MA Improves Obesity-Associated Metabolic Parameters in
HFD-Fed Mice
Because MA (Figure 5A), a type of phenolic acid, was identified
as the major antioxidant component present in PE in our pre-
vious studies,[9a,13b] we investigated whether MA was the active
constituent responsible for the anti-obesity effects of PE in mice.
HFD-induced obese mice were orally administered with vehicle
(saline) or MA. A significant reduction in body weight during the
7 weeks of MA treatment was observed compared with the ve-
hicle control group (Figure 5B). GTT and ITT revealed that MA-
treated mice were significantly more glucose tolerant (Figure 5C)
and sensitive to insulin (Figure 5D), with significantly lower fast-
ing plasma glucose and insulin levels also observed in these mice
(Figure S4, Supporting Information), suggesting that MA amelio-
rated glucose homeostasis and IR in HFD-induced obese mice.
Consistent with the effects of PE on adiposity (Figure 2) and
adipogenesis (Figure 3), MA treatment significantly reduced the
WAT mass (data not shown) and adipocyte differentiation accom-
panied by downregulation of PPARγ and C/EBPα both in vitro
(Figure S5, Supporting Information) and in vivo (Figure 5E). We
also observed a dramatic reduction in most pro-inflammatory
M1 markers (Tnfα, Mcp1, Itgax, Il6, and Nos2) with decreased
macrophage infiltration (F4/80 and Cd68) in MA-treated mice
WAT (Figure 5F). Furthermore, MA supplementation effectively
alleviated hepatic steatosis and the expression of lipogenic genes
(Figure 5G). Collectively, these results indicate that MA was re-
sponsible for the metabolic effects of PE on HFD-induced obesity.
4. Discussion
Our previous studies of the chemical profiles and bioactive prop-
erties of pear and recent clinical observations indicated that pear

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Figure 4. PE reduces macrophage infiltration and pro-inflammatory phenotype in WAT. A) Macrophage-specific IBA1 immunostaining and number of
CLSs (asterisks) per high-power field (HPF) in WAT (10 HPF counted per mouse, n = 3 mice per group, magnification: ×200, scale bar: 50 µm). B)
mRNA levels for macrophage-specific and C) M1 and M2 type markers, and D) phosphorylation levels of JNK, ERK1/2, and p38 in WAT. Phosphorylation
of MAPKs was normalized to total MAPKs and conveyed as fold change compared to CD. Data are mean ± SEM; n = 5. *p < 0.05 versus CD; # p < 0.05
versus HFD.

can improve metabolic compilations in obesity.[8a,9a,9b,11] In the
current study, we comprehensively assessed the therapeutic ef-
fects of PE on HFD-induced obesity, IR, and hepatic steatosis
and explored the underlying molecular mechanism. We found
that PE treatment restored metabolic abnormalities in mice
chronically exposed to HFD. Five-week administration of PE re-
sulted in significantly reduced body weight gain, fat mass, and
hyperlipidemia. Consistent with the results of a prior report
in streptozotocin-induced T2D mice,[13a] PE improved glucose
homeostasis and insulin sensitivity as assessed by GTT, ITT, and
HOMA-IR. Interestingly, a recent meta-analysis of a prospec-
tive cohort also revealed an inverse association between apple
and pear consumption and T2D risk.[21] Thus, our findings sug-
gest that PE has therapeutic effects on T2D and cardiovascular
disease, as obesity-associated IR is a major risk factor for these
diseases.
Furthermore, the expansion of adipose tissue and hypertrophy
of adipocytes in obese mice were markedly inhibited by PE. Lipid
accumulation and adipogenic genes were significantly reduced
in differentiated mature adipocytes and WAT, while UCP1 was
not enhanced in BAT and inguinal WAT, indicating that PE re-
duced adiposity likely by inhibiting adipogenesis rather than via
thermogenic activation for energy expenditure. Although small
adipocytes are more insulin sensitive in obesity, inhibiting adipo-
genesis can lead to IR and ectopic liver fat accumulation because
of a reduced fat storage capacity.[22] However, we found that PE
treatment lowered hepatic fat accumulation and improved glu-
cose homeostasis in HFD-fed mice, suggesting that PE improves
the ability to maintain and expand the pool of adipocytes in obe-
sity, which requires further investigation.
In addition to the anti-adipogenic effect of PE, this extract also
significantly downregulated HFD-induced lipogenic gene expres-
sion in both liver and WAT, although growing evidence suggests
that, unlike hepatic lipogenesis, increased WAT lipogenesis im-
proves metabolic phenotypes.[23] Consistent with our observa-
tion, a number of studies have shown that HFD feeding leads
to an increase in lipogenic gene expression in mice WAT.[24] Con-
sidering the diet composition used in our study and that used in
these previous studies, it is possible that the results of our study
are specific to comparison of the effects of HFD to those of a regu-
lar chow diet instead of to those of an ingredient-matched low-fat
diet.
Numerous studies have demonstrated that hypertrophic ex-
pansion of adipocytes leads to dysfunctional adipose tissue asso-
ciated with increased infiltration and activation of inflammatory
cells, lipolysis, and local and systemic IR both in humans and
in animal models.[25] In the current study, PE attenuated the re-
cruitment of macrophages and increased the expression levels of
the pro-inflammatory M1 markers Tnfα, Mcp1, and Itgax in the
WAT of HFD-fed mice, indicating that PE supplementation nor-
malized the inflammatory phenotype of WAT. Intracellular sig-
naling pathways that regulate inflammation, such as the MAPK
pathway, have been shown to regulate insulin sensitivity, obe-
sity, and pathogenesis of diabetes.[26] For instance, JNK deficiency
attenuated obesity-induced IR by decreasing pro-inflammatory
macrophage polarization in mouse WAT,[20] and ERK1 knockout

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Figure 5. MA improves HFD-induced obesity and associated metabolic complications in mice. Mice were fed a HFD for 9 weeks, followed by MA (40
mg kg−1 day−1 ) was orally administered for 7 weeks. A) Structure of MA. B) Body weight gain. C) GTT and D) ITT were performed at 15 weeks. E) mRNA
levels for adipogenic and adipokine genes and F) macrophage-specific and M1 type markers in WAT. G) H&E staining and mRNA levels for lipogenic
genes in liver (magnification: ×200, scale bar: 50 µm). Data are mean ± SEM; n = 5. Data are representative of two independent experiments with five
mice per group. *p < 0.05 versus CD; # p < 0.05 versus HFD.

mice were protected from HFD-induced obesity and IR because
of inhibited adipogenesis.[27] Although the role of p38 in obe-
sity in vivo remains unclear, a recent study showed that genetic
or pharmacological inhibition of p38α enhanced WAT brown-
ing, thereby preventing obesity in mice.[28] Here, we found that
PE treatment significantly reduced the hyperactivation of JNK,
ERK1/2, and p38 MAPK in WAT of HFD-fed mice, suggesting
that reduced adipogenesis, hypertrophy, and inflammation by PE
are associated with deactivation of the MAPK signaling pathway
in WAT.
We previously reported that MA is present mainly in pear fruits
(Pyrus pyrifolia)[12] and determined its antioxidative activity, ab-
sorption, and metabolism in vivo.[13b] However, no studies have
examined its anti-obesity effects. Although PE contains potent
antioxidants, such as chlorogenic acid and protocatechuic acid,
these compounds did not exert significant effects on 3T3-L1 dif-
ferentiation (data not shown),[29] whereas MA markedly inhibited
adipogenesis and adipogenic gene expression levels. Thus, we
further analyzed the effects of MA on HFD-induced metabolic
dysregulation in mice. As observed during PE treatment, we
found that 7-week administration of MA reversed HFD-induced
obesity and hepatic steatosis and improved glucose tolerance and
insulin sensitivity. Moreover, WAT inflammation in obese mice
was significantly inhibited by MA, suggesting that MA is the anti-
obesity component present in pear fruits.
Taken together, our results demonstrate that PE and MA re-
duce body weight gain, hepatic steatosis, WAT inflammation, and
IR in HFD-induced obese mice and suggest the potential for di-
etary supplementation with pear fruits to improve obesity and
obesity-related complications, including NAFLD and T2D. Fur-
ther studies are needed to determine the molecular targets of PE
and its efficacy and safety in obese individuals.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgments
X.T.T. performed most experiments, analyzed data, and co-wrote the
manuscript. T.T.P.N. and C.H.J. performed in vitro experiments. M.J.K

Mol. Nutr. Food Res. 2019, 63, 1801347 1801347 (8 of 9) 

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www.advancedsciencenews.com
reviewed the manuscript and contributed to the discussion. S.L. and C.M
performed histological analysis. J.H.M. performed PE and MA prepara-
tion and contributed the discussion section. T.-I.J. designed the study and
co-wrote the manuscript. All authors read and reviewed the manuscript.
This work was supported by High Value-Added Food Technology Devel-
opment Program through Korea Institute of Planning and Evaluation for
Technology in Food, Agriculture, Forestry and Fisheries (IPET) funded by
Ministry of Agriculture, Food and Rural Affairs (MAFRA) (315069-3) and
NRF-2018R1D1A1B07041857.
Conflicts of interest
The authors declare no conflict of interest.
Keywords
hepatic steatosis, inflammation, malaxinic acid, obesity, pears
Received: December 10, 2018
Revised: March 30, 2019
Published online: May 9, 2019
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