Phloridzin improves lipoprotein lipase activity in stress-loaded mice via AMPK phosphorylation

Abstract

Abstract Long-term stress exposure can lead to disturbed homeostasis and cause many life-style diseases. Phloridzin possesses various bioactivities, but the understanding of the effects of phloridzin on stress-related lipid metabolism disorder is limited. Our results demonstrate that phloridzin improved plasma lipoprotein lipase (LPL) activity and triglyceride metabolism in restrained mice. A decrease of angiopoietin-like protein 4 (ANGPTL4) mRNA expression and an increase of AMP-activated protein kinase (AMPK) phosphorylation were observed after phloridzin treatment. After inhibiting AMPK phosphorylation, the effects of phloridzin on the amelioration of plasma LPL activity and suppression of ANGPTL4 expression were blocked. In addition, cardiac AMPK phosphorylation, plasma LPL activity and ANGPTL4 expression were also affected by phloridzin, even if the glucocorticoid receptor was blocked. Taken together, the down-regulation of ANGPTL4 expression by phloridzin was probably via a direct activation of AMPK pathway. This discovery can provide a biochemical and nutritional basis for the use of phloridzin-containing food and beverage in daily life.

Full text

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ISSN: 0963-7486 (print), 1465-3478 (electronic)
Int J Food Sci Nutr, Early Online: 1–7
!2014 Informa UK Ltd. DOI: 10.3109/09637486.2014.925429
RESEARCH ARTICLE
Phloridzin improves lipoprotein lipase activity in stress-loaded mice via
AMPK phosphorylation
Guo-En Wang
1
*, Yi-Fang Li
1
*, Yan-Ping Wu
1
, Bun Tsoi
1
, Shi-Jie Zhang
2
, Ling-Fang Cao
1
, Hiroshi Kurihara
1
, and
Rong-Rong He
1
1
Anti-Stress and Health Research Center, Pharmacy College, Jinan University, Guangzhou, China and
2
Institute of Clinical Pharmacology,
Guangzhou University of Traditional Chinese Medicine, Guangzhou, China
Abstract
Long-term stress exposure can lead to disturbed homeostasis and cause many life-style
diseases. Phloridzin possesses various bioactivities, but the understanding of the effects of
phloridzin on stress-related lipid metabolism disorder is limited. Our results demonstrate that
phloridzin improved plasma lipoprotein lipase (LPL) activity and triglyceride metabolism in
restrained mice. A decrease of angiopoietin-like protein 4 (ANGPTL4) mRNA expression and an
increase of AMP-activated protein kinase (AMPK) phosphorylation were observed after
phloridzin treatment. After inhibiting AMPK phosphorylation, the effects of phloridzin on the
amelioration of plasma LPL activity and suppression of ANGPTL4 expression were blocked.
In addition, cardiac AMPK phosphorylation, plasma LPL activity and ANGPTL4 expression were
also affected by phloridzin, even if the glucocorticoid receptor was blocked. Taken together,
the down-regulation of ANGPTL4 expression by phloridzin was probably via a direct activation
of AMPK pathway. This discovery can provide a biochemical and nutritional basis for the use of
phloridzin-containing food and beverage in daily life.
Keywords
Angiopoietin-like protein 4, apple polyphenol
extract, glucocorticoids, lipid metabolism,
restraint stress
History
Received 4 March 2014
Revised 24 April 2014
Accepted 14 May 2014
Published online 16 June 2014
Introduction
A prolonged period of stress can trigger mental and physical
fatigue and induce many life-style diseases due to impaired
homeostasis (Panossian & Wikman, 2009). Mental and physical
fatigue can alter some biochemical parameters, like serum
triglyceride (TG), free fatty acid (FFA) and cortisol (Nozaki
et al., 2009). Imposing restraint stress on mice can result in
tiredness and a poor utilization of TG, along with a decrease in
lipoprotein lipase (LPL) activity in omental adipose tissue
(Kurihara et al., 2006). LPL is responsible for catalyzing the
degradation of TG-rich lipoproteins to FFA in order to maintain
the supply of FFA for physiological utilization (Mead et al.,
2002). LPL is synthesized mainly in the heart, adipose tissue and
skeletal muscle (Mead et al., 2002). The activated dimeric form
of LPL releases into the blood and binds to heparan sulfate
proteoglycans on the luminal surface of the capillary endothelium
(Kim et al., 2012). Decreased LPL activity after restraint stress
can lead to an impaired lipid metabolism (He et al., 2009;
Kurihara et al., 2006). In addition, a large amount of cortico-
sterone is released into the blood after restraint stress (Li et al.,
2012; Tsoi et al., 2011; Zhai et al., 2012) as a deranged feedback
regulation of the hypothalamic–pituitary–adrenal (HPA) axis
(Fediuc et al., 2006). The released rodent glucocorticoid, cor-
ticosterone, binds to glucocorticoid receptor (GR) and induces
genomic and nongenomic effects in the homeostasis of the
cardiovascular system (Lee et al., 2012). A number of down-
stream factors can be influenced after GR activation, such as
AMP-activated protein kinase (AMPK). The activated AMPK
switches on catabolic pathways that generate ATP while switching
off ATP-consuming processes for homeostasis in energy metab-
olism (Hardie, 2003). There have been animal and clinical studies
that demonstrate excess corticosterone could inhibit AMPK
activity in heart and fat tissue (Christ-Crain et al., 2008; Kola
et al., 2008). AMPK phosphorylation can reverse glucocorticoid-
mediated downstream changes (Nader et al., 2010), such as
cardiac LPL activity (An et al., 2005). Therefore, activation of
AMPK can be a promising target for pharmacological interven-
tions to increase LPL activity.
Phloridzin (4,2,4,6-tetrahydroxy dihydrochalcone-20-o-gluco-
side) is a major polyphenol in fresh apples (Lee et al., 2003).
Its content ranges from 11% to 36% of the total phenolic
concentration in apple juice and apple extract (Ehrenkranz et al.,
2005). This demonstrates that phloridzin has been a naturally
occurring constituent of the human diet. Phloridzin possesses
various biomedical activities such as: anti-diabetes mellitus
(Najafian et al., 2012; Zhao et al., 2004), anti-oxidation
(Lee et al., 2003), anti-inflammation (Chang et al., 2012) and
anti-aging (Xiang et al., 2011). However, there are limited reports
about the effect of phloridzin on lipid metabolism. In this study, a
restraint stress mice model (He et al., 2009) was employed to
study the effect of phloridzin on lipid metabolism, and the
possible mechanism was explored.
*These authors contributed equally to this work.
Correspondence: Rong-Rong He, Anti-stress and Health Research Center,
Pharmacy College, Jinan University, 601 Huangpu Avenue West,
Guangzhou 510632, China. Tel: +86-20-85221352. Fax: +86-20-
85221559. E-mail: rongronghe66@163.com
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Materials and methods
Materials
Phloridzin (apple extract, 98%) was generously supplied by
Tianjin Jianfeng Natural Product R&D Co., Ltd. (Tianjin, China).
IntralipidÕ(lipid emulsion including 20% soybean oil, 1.2%
lecithin and 2.2% glycerol) was purchased from Pharmacia AB
Co. (Stockholm, Sweden) and diluted with the same volume of
0.01 M phosphate buffered saline (pH 7.4) before use. Triton
WR1339, compound C, corticosterone and cortisol standards
were purchased from Sigma (St. Louis, MO). RU486 was
purchased from Abcam (Cambridge, UK). Triton WR1339 was
dissolved in normal saline (0.9% NaCl). Compound C and RU486
were dissolved in dimethyl sulfoxide and diluted with normal
saline.
Animals and restraint treatment
Male Kunming mice (20 ± 2 g) were purchased from Guangdong
Provincial Medical Laboratory Animal Center (Guangdong,
China). Kunming mice originated from Swiss mice brought
from the Indian Haffkine Institute to Kunming, China, in 1944
(Shang et al., 2009). Due to high disease resistance and good
adaptive capacity, Kunming mice were widely utilized in the
pharmacological and biological research, such as lipid metabol-
ism study (He et al., 2009; Ma et al., 2009). All mice were
housed in a pathogen-free room under controlled temperature
(24 ± 1 C) and humidity (60 ± 5%) with a 12 h day–night cycle.
Mice were randomly divided into normal, restraint stress model,
Triton WR1339 and two phloridzin groups after acclimation for a
week in the laboratory environment. Phloridzin was dissolved
in distilled water and orally administered to animals at dosages of
50 and 100 mg/kg body weight daily for seven days. Mice in the
normal, restraint stress model and Triton WR1339 groups were
administered with an equivalent volume of distilled water. On the
seventh day, animals in restraint stress model and phloridzin
groups were physically immobilized in a 50 ml restraint tube with
holes for 20 h (He et al., 2009), while animals in Triton WR1339
group were intravenously injected with 300 mg/kg Triton
WR1339 (Yu et al., 2011). Triton WR1339, a typical LPL
inhibitor in mammals (Abe et al., 2007), was used as a positive
(model) control to inhibit plasma LPL activity. Twenty hours after
the restraint stress or Triton WR1339 treatment, all mice were
anesthetized by ethyl ether. Blood was drawn by heart puncture
and transferred to centrifuge tubes with heparin (100 U/ml). The
blood was centrifuged at 2300 gfor 5 min to obtain plasma. The
heart was also quickly removed for the following experiments.
Procedures for animal experiments were conducted in accordance
with the Guiding Principles for the Care and Use of Laboratory
Animals as adopted and promulgated by the United States
National Institutes of Health.
Inhibition of AMPK phosphorylation by compound C
Phloridzin (100 mg/kg) or distilled water was orally administered
to mice daily for seven days. On the last day, all mice except the
control group were intraperitoneally injected with the AMPK
inhibitor compound C (20 mg/kg). The control mice were injected
with equivalent vehicle injections (Shen et al., 2008). All mice
then received the restraint treatment as mentioned in the above
section.
Blockade of the GR with RU486
Phloridzin (100 mg/kg) or distilled water was orally administered
to mice daily for seven days. On the seventh day, all mice except
the control group were intraperitoneally injected with the
glucocorticoid antagonist RU486 (25 mg/kg). The control mice
were given equivalent volumes of vehicle solution (Li et al.,
2012). All mice then received the restraint treatment as mentioned
in the above section.
Plasma TG tolerance test
Thirty minutes after the restraint treatment, lipid emulsion was
intravenously injected to restrained mice at 0.1 ml/10 g body
weight. Plasma was obtained at 35 min after the injection
(Kurihara et al., 2006). TG level was enzymatically determined
by the glycerol kinase/glycerol-3-phosphate oxidase method with
a commercial TG kit (Nanjing Jiancheng Bioengineering Co Ltd.,
Nanjing, China).
Measurement of plasma LPL activity
For plasma LPL activity assessment, heparin (10 units/mouse)
was intravenously injected into mice at 10 min before blood
collection (Qi et al., 2008). LPL activity assay was carried out
using a Total Lipase Test kit (Nanjing Jiancheng Bioengineering
Co Ltd.). LPL activity was expressed as units per ml of plasma.
Measurement of plasma corticosterone
Corticosterone was extracted from plasma and quantified by high-
performance liquid chromatography (HPLC) (Li et al., 2012).
Cortisol (0.1 mg) was added to 200 ml of plasma as an internal
standard. Steroids were extracted twice by mixing thoroughly
with 800 ml of acetic ether. The mixture was centrifuged at
200 gfor 5 min. The organic phase was washed with 320 mlof
0.1 mol/l NaOH solution and 320 ml of HPLC-grade water,
respectively. The organic phase was then evaporated at room
temperature under nitrogen. The residue was re-dissolved in 50 ml
of methanol–water (60:40 v/v). The 5-mm Cosmosil 5C18
reversed-phase column (4.6 mm I.D. 250 mm) was equilibrated
using acetonitrile–water (45:55 v/v) at a f low rate of 1 ml/min.
Plasma corticosterone level was measured by HPLC system
(Hitachi, Tokyo, Japan) with an UV detector at 254 nm.
Measurement of plasma malondialdehyde
Plasma malondialdehyde (MDA) assay was carried out using a
commercial MDA kit (Nanjing Jiancheng Bioengineering Co
Ltd.). In brief, MDA in the sample would react with thiobarbituric
acid (TBA) at 95 C for 40 min. The pink adducts of MDA–TBA
were quantified by a MK3 microplate reader (Labsystems,
Vantaa, Finland) at 532 nm.
Measurement of oxygen radical absorbance capacity of
plasma
Automated oxygen radical absorbance capacity (ORAC) assays
were carried out by the method described previously (Li et al.,
2013). The plasma samples were mixed with fluorescein, and the
reaction was initiated with the addition of 2,20-azobis(2-
amidinopropane) dihydrochloride. The analysis was performed
using a GENios luciferase-based microplate reader (Tecan,
Ma
¨nnedorf, Switzerland) with excitation/emission filter pair of
485/527 nm. The results were calculated as the net area under the
fluorescence decay curve using Trolox as a standard.
Measurement of mRNA expression of LPL,
angiopoietin-like protein 4 and
glycosylphosphatidylinositol-anchored high-density
lipoprotein (HDL)-binding protein 1 in the heart
Cardiac gene expression was determined by quantitative real-time
reverse transcription-polymerase chain reaction (RT-PCR). Total
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RNA was isolated using Trizol (TaRaKa, Dalian, China), and a
3mg of total RNA was reverse transcribed to a first-strand
complementary DNA (cDNA) with TIANScript RT Kit
(Tiangen, Beijing, China), followed by PCR amplification.
Thereafter, the cDNA samples were amplified on an IQÔ5 real-
time PCR detection system (Bio-Rad, Hercules, CA) in the
presence of SYBR Green/Fluorescein qPCR Master Mix
(Fermentas, Amherst, NY) and specific primers (Invitrogen,
Carlsbad, CA). The sequences of primers for mouse 18S
(GenBank accession no. K01364, (F) 50-
GGGAGAGCGGGTAAGAGA-30, (R) 50-ACA
GGACTAGGCGGAACA-30, bp: 241), angiopoietin-like protein
4 (ANGPTL4) (GenBank accession no. NM_020581, (F)
50-CCAACGCCACCCACTTAC-30, (R) 50-CTCGGTTCCCTGT
GATGC-30, bp: 282), LPL (GenBank accession no. NM_008509,
(F) 50-CTAACTGCCACTTCAACC-30, (R) 50-CAGACTTCC
TGCTACGC-30, bp: 320) and glycosylphosphatidylinositol-
anchored HDL-binding protein 1 (GPIHBP1) (GenBank accession
no. NM_026730, (F) 50-GAGTGGCTGGGCACAAGA-30, (R) 50-
TGATGGGCTGGCAGGTAT-30, bp: 296) were used. The ampli-
fication of the 18S sequence was performed in parallel and was
used to normalize the values obtained for the target genes. The
results were expressed as fold changes of the comparative threshold
cycle (Ct) values relative to the controls by the 2
DDCt
method.
Measurement of cardiac AMPK phosphorylation
AMPK phosphorylation assay was carried out using western blot
analysis. Protein extracts from cardiac tissue (30 mg) were
separated by 12% sodium dodecyl sulfate–polyacrylamide gel
and transferred to polyvinyldifluoridine membranes (Millipore,
Bedford, MA). Probing of the membranes was performed with
primary antibodies against phosphorylated AMPKa(pAMPKa,
Thr172) and AMPKa(CST, Danvers, MA). The membranes were
incubated with goat anti-rabbit IgG as secondary antibody
(Lianke, Hangzhou, China). Immunoreactivity was detected
with a chemiluminescence detection kit (Lianke). Band intensity
was acquired and then quantified by calculating the average
optical density in each field using Quantity One (Bio-Rad). The
results were expressed as the ratios of pAMPKato AMPKa.
Statistical analysis
The data are presented as means ± SD and analyzed by SPSS 17.0
statistical software (SPSS Inc., Chicago, IL) statistical software.
One-way analysis of variance is applied to analyze for difference in
data of biochemical parameters among different groups, followed
by Dunnett’s significant post-hoc test for pair-wise multiple
comparisons. The statistical significance is set at p50.05.
Results
Phloridzin increased plasma LPL activity in restrained
mice
As shown in Figure 1, plasma LPL activity was decreased in
stress-loaded mice compared with the normal mice (p50.01).
Triton WR1339 was used as a positive (model) control for
inhibiting plasma LPL activity. There was no difference of plasma
LPL activity in either the Triton WR1339 or restraint stress group.
However, phloridzin treatment (50 and 100 mg/kg) reversed the
plasma LPL activities (p50.01).
Phloridzin ameliorated plasma TG metabolism and
oxidative status in restrained mice
Thirty-five minutes after intravenous injections of lipid emulsion,
the average level of TG in restrained mice was higher than normal
mice (p50.01). The two doses of phloridzin treatment (50 and
100 mg/kg) had respectively decreased the TG levels (p50.05).
This demonstrated that phloridzin had dose-dependently
improved plasma TG metabolism in restrained mice. In addition,
MDA is a major end-product of lipid peroxidation, and ORAC
values can reflect total antioxidative capacity. Results showed that
restraint stress induced an increase in plasma MDA and a
decrease in ORAC value when compared with the normal group
(p50.01). However, phloridzin treatment (100 mg/kg) reduced
plasma MDA (p50.05) and augmented the ORAC level of
plasma (p50.05, Table 1).
Phloridzin up-regulated the mRNA expression of cardiac
ANGPTL4
In this study, the mRNA expressions of LPL, ANGPTL4 and
GPIHBP1 in the heart were determined. There was no difference
in cardiac LPL and GPIHBP1 expression among all groups.
Figure 1. Effects of phloridzin on plasma LPL activity in stress-loaded
mice (n¼7). Mice were treated with phloridzin or distilled water for
seven days. On the last day, mice except for the normal group received
either the 20-h restraint treatment or the Triton WR1339 treatment. Data
are expressed as means ± SD. Significant differences were observed
relative to the normal group (**p50.01) and from the model group
(##p50.01).
Table 1. Effects of phloridzin on plasma triglyceride (TG) elimination,
MDA and ORAC levels in stress-loaded mice.
Group TG (mmol/l) MDA (mmol/l) ORAC (U/ml)
Normal 3.10 ± 0.23 2.70 ± 0.7 137.2 ± 7.7
Model (restraint) 4.85 ± 0.47
a
7.41 ± 0.8
a
99.6 ± 7.0
a
Restraint + phloridzin,
50 mg/kg
4.23 ± 0.30
b
5.40 ± 1.3 108.4 ± 13.3
Restraint + phloridzin,
100 mg/kg
4.05 ± 0.29
b
4.60 ± 0.8
b
117.3 ± 11.1
b
Mice were treated with phloridzin or distilled water for seven days. On the
last day, mice except for the normal group received the 20-h restraint
treatment. For the plasma TG tolerance test, the concentration of plasma
TG in experimental mice was measured at 35 min after the injection of
lipid emulsion. The ORAC value was calculated as the net area under
the f luorescence decay cur ve using 20 mmol/l Trolox as a standard. Data
are expressed as means ± SD from seven mice in each group.
Significant differences were observed relative to the normal group
(
a
p50.01) and the model group (
b
p50.05).
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However, restraint stress induced an increase in cardiac
ANGPTL4 expression (p50.01), while phloridzin decreased the
ANGPTL4 mRNA expression (p50.05, Figure 2).
Phloridzin enhanced LPL activity via AMPK
phosphorylation
Twenty hours of restraint stress was found to remarkably
decrease cardiac AMPK phosphorylation (p50.01). The
administration of phloridzin enhanced AMPK phosphorylation
in a dosage-dependent manner (p50.05 and p50.01, Figure 3A).
To confirm the activation of AMPK pathway by phloridzin,
compound C was used to inhibit AMPK phosphorylation.
Phloridzin induced no change in plasma LPL activity and
ANGPTL4 expression in compound C-treated restrained mice
(Figure 3B and C), suggesting a possible relationship between
phloridzin and AMPK activation.
Glucocorticoid-bound GR was not involved in the
enhancement of LPL activity by phloridzin
Restraint stress led to an increase of plasma corticosterone
(p50.01, Figure 4A), as consistent with the previous study
(Li et al., 2012). However, phloridzin was not effective in
lowering plasma corticosterone in restrained mice. In order to
determine if the involvement of glucocorticoid-bound GR was
the mechanism of phloridzin, RU486 was used to block GR to
inhibit glucocorticoid-mediated downstream effects. Results
demonstrated that RU486 treatment restored pAMPK level in
restrained mice (p50.01). Phloridzin even enhanced AMPK
phosphorylation to a higher level than RU486 treatment only
(p50.01, Figure 4B). Plasma LPL activity was 12.3 ± 1.0 U/ml
in RU486-treated restrained mice, while it was 11.3 ± 1.4 U/ml
in restrained mice. The treatment with phloridzin further
increased plasma LPL activity (p50.05, Figure 4C).
In addition, blockade of GR induced a slight decrease of
ANGPTL4 mRNA expression in restrained mice, while phloridzin
treatment significantly reduced ANGPTL4 expression (p50.01,
Figure 4D).
Figure 3. Effects of phloridzin on activation of AMPK. Phloridzin dosage-dependently enhanced AMPK phosphorylation in stress-loaded mice (A),
but induced no change of plasma LPL activity (B) and cardiac ANGPTL4 mRNA expression (C) in stressed mice injected with compound C
(20 mg/kg). Data are expressed as means ±SD. Significant differences were observed relative to the normal group at p50.01 (**) and from the model
group at p50.05 (#) and p50.01 (##).
Figure 2. Effects of phloridzin on mRNA expression of cardiac LPL,
ANGPTL4 and GPIHBP1 in restrained mice. Data are expressed as
means ± SD. Signif icant differences were observed relative to the normal
group (**p50.01) and from the model group (#p50.05).
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Discussion
Fatigue resulting from restraint stress in mice is widely used to
evaluate the effects of numerous compounds on glucose and lipid
metabolism (He et al., 2009; Tsoi et al., 2011). A previous report
had found that restraint stress could induce a decrease of LPL
activity in omental adipose tissue (Kurihara et al., 2006).
Therefore, this study aimed to reveal the mechanism of restraint
stress-induced decrease in plasma LPL activity and to discuss the
effect of phloridzin, a commonly found polyphenol in apple, on
lipid metabolism. Our results demonstrated that restraint stress
decreased plasma LPL activity, along with a suppressed clearance
rate of plasma TG. However, LPL gene expression was not
affected by restraint stress. Therefore, the transportation of LPL
was further studied. LPL transportation is mediated by ANGPTL4
and GPIHBP1. ANGPTL4 is involved in the conversion of LPL
from the active dimeric form to the inactive monomeric form,
while GPIHBP1 acts as a platform for binding of both LPL and
TG-rich lipoproteins in blood vessels (Sonnenburg et al., 2009).
Our data demonstrated that restraint stress decreased ANGPTL4
mRNA expression, but did not affect GPIHBP1 expression. Given
that the regulation of ANGPTL4 expression is related to the
change of AMPK phosphorylation (Daniels et al., 2010; Kim
et al., 2010), we decided to explore possible relation between
restraint stress and AMPK activity. Our results demonstrated that
restraint stress induced an increase in plasma corticosterone and a
decrease in cardiac AMPK phosphorylation. Several studies
suggest that binding of glucocorticoid to GR would induce a
series of transduction signals that will inhibit AMPK phosphor-
ylation (Christ-Crain et al., 2008; Kola et al., 2008). Hyper-
activation of the HPA axis from restraint stress induces a massive
production of corticosterone: this could influence AMPK activa-
tion, which in turn affects the LPL activity.
Phloridzin is a naturally occurring polyphenol that can be
found in apple fruits. It composes about 11–36% of major
polyphenols in apple juice and apple extract (Ehrenkranz et al.,
2005). The bioactivity of phloridzin is wide-ranging: it
possesses anti-diabetes (Zhao et al., 2004), hepato-protection
Figure 4. The role of glucocorticoid-bound GR in the enhancement of LPL activity by phloridzin. Phloridzin was not effective in lowering plasma
corticosterone levels in restrained mice (A). When the GR of stressed mice was blocked by RU486 (25 mg/kg), cardiac AMPK phosphorylation (B),
plasma LPL activity (C) and cardiac ANGPTL4 mRNA expression (D) were reversed and further regulated by phloridzin treatment. Data are expressed
as means ± SD. Significant differences were obser ved relative to the normal group at p50.01 (**) and from the RU486 group at p50.05 (#) and
p50.01 (##).
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(Deng et al., 2012) and anti-bacterial effects (Muthuswamy &
Rupasinghe, 2007). In this study, we found that phloridzin was
effective in improving LPL activity and TG metabolism in stress-
loaded mice. A decrease of ANGPTL4 mRNA expression and an
increase of AMPK phosphorylation were also noticed by
phloridzin treatment. To further confirm the activation of
AMPK by phloridzin, compound C was used to inhibit AMPK
phosphorylation in restrained mice (Shen et al., 2008). When
AMPK phosphorylation was blocked, phloridzin did not enhance
plasma LPL activity or decrease ANGPTL4 expression. The
content of corticosterone was also not affected. This data
suggested that phloridzin could not affect the secretion or transfer
of glucocorticoids in restraint stressed mice. Since AMPK can
also be regulated by the binding of glucocorticoid to GR, a
glucocorticoid antagonist RU486 was used to examine the effect
of phloridzin under GR inhibition (Fleseriu et al., 2012). Our
results demonstrated that blocking GR restored cardiac AMPK
phosphorylation in restrained mice. However, there was only a
slight decrease in cardiac ANGPTL4 expression and a slight
increase of plasma LPL activity. As the results of the MDA and
ORAC assays showed that restraint stress had lowered the
antioxidative ability of mice, there could be a large amount of
inflammatory cytokines being produced, causing systemic
inflammation (Li et al., 2013; Zhai et al., 2012). This inflam-
mation could stimulate ANGPTL4 expression (Lu et al., 2010)
and suppress LPL activity (Hara et al., 2011). This implied that
stress-related inhibition of LPL activity could probably be evoked
by glucocorticoid-mediated and inflammation-mediated increases
of ANGPTL4 expression. Administration of phloridzin to stressed
mice injected with RU486 promoted both the content of
phosphorylated AMPK and plasma LPL activity while decreasing
cardiac ANGPTL4 expression. The strong antioxidant ability of
phloridzin might also contribute to the enhancement of LPL
activity through the improvement of inflammation.
Conclusions
To summarize the results of this study, phloridzin increased LPL
activity, improving TG metabolism in stress-loaded mice. The
down-regulation of ANGPTL4 expression by phloridzin was
through a direct activation on AMPK, which did not involve the
glucocorticoid-bound GR. These discoveries broaden our under-
standing about the mechanism of phloridzin in improving TG
metabolism. They will provide a biochemical and nutritional basis
for long-term dietary supplement of phloridzin on the amelior-
ation of stress-related TG metabolism disorder.
Acknowledgements
We thank Dr. L. J. Sparvero for editing proper usage of scientific English.
Declaration of interest
All authors have no conflicts of interests and have no direct financial
relationship with the commercial identities mentioned in this article.
This work was supported, in part, by Natural Science Foundation of
China (NO. 81102485), Trans-Century Training Program Foundation for
the Talents of the State Education Commission (NCET-12-0678), Natural
Science Foundation of Guangdong Province (S20120011316) and
Science and Technology Program of Guangzhou (2012J22000073 &
2013J4501037).
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DOI: 10.3109/09637486.2014.925429 Phloridzin improves LPL activity in restrained mice 7
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