Curcumin Prevents High Fat Diet Induced Insulin Resistance and Obesity via Attenuating Lipogenesis in Liver and Inflammatory Pathway in Adipocytes

Abstract

Mechanisms underlying the attenuation of body weight gain and insulin resistance in response to high fat diet (HFD) by the curry compound curcumin need to be further explored. Although the attenuation of the inflammatory pathway is an accepted mechanism, a recent study suggested that curcumin stimulates Wnt signaling pathway and hence suppresses adipogenic differentiation. This is in contrast with the known repressive effect of curcumin on Wnt signaling in other cell lineages.
We conducted the examination on low fat diet, or HFD fed C57BL/6J mice with or without curcumin intervention for 28 weeks. Curcumin significantly attenuated the effect of HFD on glucose disposal, body weight/fat gain, as well as the development of insulin resistance. No stimulatory effect on Wnt activation was observed in the mature fat tissue. In addition, curcumin did not stimulate Wnt signaling in vitro in primary rat adipocytes. Furthermore, curcumin inhibited lipogenic gene expression in the liver and blocked the effects of HFD on macrophage infiltration and the inflammatory pathway in the adipose tissue.
We conclude that the beneficial effect of curcumin during HFD consumption is mediated by attenuating lipogenic gene expression in the liver and the inflammatory response in the adipose tissue, in the absence of stimulation of Wnt signaling in mature adipocytes.

Full text

Curcumin Prevents High Fat Diet Induced Insulin
Resistance and Obesity via Attenuating Lipogenesis in
Liver and Inflammatory Pathway in Adipocytes
Weijuan Shao
1.
, Zhiwen Yu
2.
, Yuting Chiang
1,3
, Yi Yang
1¤
, Tuanyao Chai
1
, Warren Foltz
4
, Huogen
Lu
1,3,5
, I. George Fantus
1,3,5
, Tianru Jin
1,3,5
*
1Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, Toronto, Canada, 2Department of Nutrition, Public Health
Institute, Sun Yat-Sen University, Guangzhou, China, 3Department of Physiology, University of Toronto, Toronto, Canada, 4Radiation Medicine Program, The STTARR
Innovation Centre, Princess Margaret Hospital, University Health Network, Toronto, Canada, 5Banting and Best Diabetes Centre, Faculty of Medicine, University of
Toronto, Toronto, Canada
Abstract
Background:
Mechanisms underlying the attenuation of body weight gain and insulin resistance in response to high fat diet
(HFD) by the curry compound curcumin need to be further explored. Although the attenuation of the inflammatory
pathway is an accepted mechanism, a recent study suggested that curcumin stimulates Wnt signaling pathway and hence
suppresses adipogenic differentiation. This is in contrast with the known repressive effect of curcumin on Wnt signaling in
other cell lineages.
Methodology and Principal Findings:
We conducted the examination on low fat diet, or HFD fed C57BL/6J mice with or
without curcumin intervention for 28 weeks. Curcumin significantly attenuated the effect of HFD on glucose disposal, body
weight/fat gain, as well as the development of insulin resistance. No stimulatory effect on Wnt activation was observed in
the mature fat tissue. In addition, curcumin did not stimulate Wnt signaling in vitro in primary rat adipocytes. Furthermore,
curcumin inhibited lipogenic gene expression in the liver and blocked the effects of HFD on macrophage infiltration and the
inflammatory pathway in the adipose tissue.
Conclusions and Significance:
We conclude that the beneficial effect of curcumin during HFD consumption is mediated by
attenuating lipogenic gene expression in the liver and the inflammatory response in the adipose tissue, in the absence of
stimulation of Wnt signaling in mature adipocytes.
Citation: Shao W, Yu Z, Chiang Y, Yang Y, Chai T, et al. (2012) Curcumin Prevents High Fat Diet Induced Insulin Resistance and Obesity via Attenuating
Lipogenesis in Liver and Inflammatory Pathway in Adipocytes. PLoS ONE 7(1): e28784. doi:10.1371/journal.pone.0028784
Editor: Regine Schneider-Stock, Erlangen University, Germany
Received July 8, 2011; Accepted November 15, 2011; Published January 9, 2012
Copyright: ß2012 Shao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: CIHR, Canadian Institutes of Health Research (http://www.cihr-irsc.gc.ca/e/193.html). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tianru.jin@utoronto.ca
.These authors contributed equally to this work.
¤ Current address: Ningxia Medical University, Yinchuan, People’s Republic of China
Introduction
Type 2 diabetes mellitus (T2D) is increasing at an alarming rate
in both developed and developing countries, associated with a
combined health and economic burden worldwide [1]. The
epidemic of obesity and its related insulin resistance have
contributed significantly to the incidence of diabetes. It is now
generally accepted that both obesity and T2D are associated with
low grade chronic inflammation and that adipose tissue appears to
be the first organ that is affected [2]. The development of
inflammation and oxidative stress in adipose tissue leads to insulin
resistance [3,4]. Furthermore, accelerated hepatic lipogenic gene
expression and reduced liver fat export may also contribute to the
development of obesity [5,6].
Many naturally occurring dietary polyphenols possess antioxi-
dant and anti-inflammatory properties [7]. This could be achieved
by modulating an inflammatory or oxidative signaling pathway,
including NF-kB, Nrf2, and/or MAPK-dependent signaling
pathways [7,8,9]. Certain dietary polyphenols, such as curcumin,
also possess the anti-carcinogenic effects. One potential mecha-
nism of curcumin to repress tumorigenesis has been suggested to
be the inhibition of Wnt signaling, an essential pathway for
embryogenesis and cell proliferation [10,11].
Curcumin, a low-molecular-weight polyphenol derived from the
herbal remedy and dietary spice turmeric, was found to prevent obesity
and diabetes in mouse models [12]. Mechanistically, curcumin may
exert its beneficial effects via reducing insulin and leptin resistance,
attenuating inflammatory cytokine expression, accelerating fatty acid
oxidation, as well as increasing antioxidant enzyme expression [7]. In
addition, curcumin could also function as an inhibitor of p300 histone
acetyltransferase (HAT), a potential molecular mechanism for cancer
prevention and cardiovascular improvement [13,14].
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The Wnt/b-catenin (b-cat) signaling pathway was initially
discovered in colon cancer and in developmental studies of
Drosophila and frogs [15]. The role of the canonical Wnt signaling
pathway (defined as Wnt pathway hereafter) in metabolic
homeostasis has recently received increasing attention
[15,16,17]. Activation of Wnt pathway increases cellular and
nuclear b-cat level, which represses adipogenesis, while the
inhibition of Wnt signaling is required for PPARcinduction and
preadipocyte differentiation [18]. A very recent study showed that
curcumin stimulates Wnt/b-cat signaling in 3T3-L1 preadipocytes
and hence suppresses adipogenic differentiation [19]. Although
this finding provides a potential molecular mechanism for the
effect of curcumin in attenuating obesity, it is contradictory with
other reports in two ways. First, numerous studies have indicated
that curcumin exerts its anti-cancer effect via repressing Wnt
signaling [10,11]. Second, Wnt activation in mature adipocytes
was shown to induce insulin resistance [20], while curcumin is
known to attenuate insulin resistance [12].
In this study we have examined the effect of dietary curcumin in
a HFD mouse model in which the development of obesity and
insulin insensitivity was relatively slow due to the administration of
45% rather than 60% of calories from fat [8]. In this mouse model
as well as in primary rat adipocytes, we did not observe stimulation
of curcumin on Wnt pathway components or Wnt target gene
expression. However, curcumin attenuated lipogenic gene expres-
sion in hepatocytes, and blocked the effect of HFD on the
inflammatory response in the adipose tissue, associated with
decreased weight/fat gain, and the maintenance of normal glucose
tolerance and insulin sensitivity.
Materials and Methods
Materials
Curcumin was purchased from Sigma (St. Louis, MO).
Antibodies against PKB/Akt, phosphorylated PKB (Ser473),
phosphorylated b-cat (Ser675 b-cat), GSK-3b, phosphorylated
GSK-3a/b, and b-actin were obtained from Cell Signaling
Technology (Beverly, MA). Antibodies against NF-kB, total b-
cat, c-Myc, cyclin D1, phosphorylated JNK, SREBP1-c, F4/80
and HO-1 were from Santa Cruz Biotechnology (Santa Cruz,
CA). Thioredoxin-interacting protein (TxNIP) antibody was
obtained from MBL International Corporation (Woburn, MA).
ChREBP antibody was purchased from Novus Biologicals,
(Littleton, CO). Kits for glucose, cholesterol, free fatty acids
(FFA), and HDL assessment were from Abcam (Cambridge, MA).
Triglyceride (TG) assay kit was from Cayman Chemical (Ann
Arbor, Michigan). Leptin/insulin ELISA kit were from Crystal
Chem. Inc. (Downers Grove, IL). Adiponectin ELISA kit was from
R&D Systems (Minneapolis, MN). GSH/GSSG assay kit was from
Oxford Biomedical Research (Oxford, MI) and the method for
determining plasma GSH/GSSG ratio was described previously
[8].
Animal care and treatment
Male C57BL/6J mice from Jackson Laboratory (Bar Harbor,
Maine) were housed 5 per cage under the conditions of constant
temperature (22uC), a light/dark cycle of 12 h with free access to
food and water. Thirty-six five-week-old mice were randomly
divided into three groups. Group A were fed with the low-fat-diet
(LFD, control diet, 10% Kcal from fat), while group B with the
HFD (45% Kcal from soy bean fat). Group C were fed with HFD
with curcumin (4g/kg diet) added 2 days/week (Mondays and
Thursdays). Diets were prepared by Harlan Tekland (Madison,
WI) [8]. The animal experiments and protocols were approved by
the University Health Network Animal Care Committee and
performed in accordance with the guidelines of the Canadian
Council of Animal Care. The approval ID for this study is
AUP1561.11.
MRI assessment of total fat mass and lipid content
MRI was performed using a 7 tesla Biospec 70/30 USR (Bruker
BioSpin MRI GmbH, Ettlingen, Germany) in The STTARR
Innovation Centre, Radiation Medicine Program, Princess
Margaret Hospital, University Health Network, Toronto, Canada,
as previously described [8].
Intraperitoneal (i.p.) glucose, insulin and pyruvate
tolerance tests
Mice were fasted overnight for glucose tolerance tests; or fasted
for 6 h for insulin and pyruvate tolerance tests. Following the
fasting, glucose (2 g/kg), insulin (0.65 U/kg) or pyruvate (2 g/kg)
was i.p. injected. Blood samples collected from tail vein were used
for glucose measurements.
Determination of blood biochemistry and liver TG
content
Ambient levels of plasma glucose, TG, total cholesterol, FFA
and HDL after an overnight fast were measured using kits
following the manufacturers’ instructions. Liver TG content was
determined as described [21].
Quantitative real-time RT-PCR
Real-time PCR was performed using iQ
TM
Sybr Green (Bio-
Rad, Mississauga, On., Canada) using the Rotorgene according to
the protocols provided by the manufacturer. The relative mRNA
transcript levels were calculated according to the 2
2DDCt
method.
Oligonucleotide primers for RT-PCR are summarized in Table 1.
Cell lines and primary cell cultures
HepG2 cells were purchased from ATCC (Manassas, VA).
They were cultured in a-MEM supplemented with 10% fetal
bovine serum (FBS). Epididymal fat pads from male wild-type
Wistar rats fed a normal diet were excised, minced in DMEM
containing 3% BSA and digested with 1 mg/ml collagenase Type
1 (Worthington Biochemical Corporation, Lakewood, NJ) at 37uC
for 60 min. The digested fat tissue was filtered through a mesh
(100 mm) and centrifuged at 500 rpm for 5 min to separate
floating adipocytes from the medium.After washing 4 times, cells
were maintained in DMEM containing 1% BSA at 37uC, with
additions as indicated.
Western blot analysis
Tissue samples were homogenized and equal amounts of
proteins (30 mg) were separated with denaturing SDS 10%
polyacrylamide gels. The method for Western blotting was as
previously described [8,22].
Luciferase (LUC) reporter analysis
The generation of ChREBP promoter constructs, as well as the
method for LUC reporter gene analysis was described previously
[23,24].
Immuno and Histological staining
The sections of epididymal adipose tissue were fixed in 10%
formalin, dehydrated, and embedded in paraffin. Adipose tissue
sections were stained with hematoxylin and eosin (H&E) to
examine the morphology and with the F4/80 antibody to detect
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macrophages. Hepatic Oil O Red staining was conducted with the
routine method.
Statistics
All results are expressed as mean 6SEM. Statistical significance
was assessed by ANOVA. Pvalue less than 0.05 was considered to
be statistically significant.
Results
Long term dietary curcumin administration prevented
HFD-induced body-weight gain and obesity
In this chronic HFD mouse model, body-weight increased
significantly only after 16 weeks of HFD feeding (Fig. 1A). From
this time point to the end of the 28th week, dietary curcumin
significantly blocked the effect of HFD on body-weight gain
(Fig. 1A). HFD also significantly increased the weight of
epididymal fat pads, while curcumin supplementation significantly
blocked this stimulation (Fig. 1B). We then assessed total fat mass
of the animals by MRI and found stimulation by HFD and
inhibition by curcumin supplementation (Fig. 1C).
HFD feeding increased the fasting plasma insulin level
(0.2860.15 ng/ml for LFD, 0.6760.10 ng/ml for HFD,
p,0.05), while dietary curcumin resulted a reduction on its level
(0.1060.06 ng/ml in HFD plus curcumin, p,0.01 versus HFD).
Although HFD reduced and curcumin increased plasma adipo-
nectin level, the differences did not reach statistical significance
(7.2661.08 mg/ml for LFD, 5.9962.14 mg/ml for HFD, and
7.2261.09 mg/ml for HFD/curcumin).
Curcumin improved glucose disposal and insulin
sensitivity
To evaluate the functional outcome of long-term curcumin
supplementation on glucose homeostasis, we conducted an
intraperitoneal glucose tolerance test (IPGTT). A representative
IPGTT result performed at the end of the 20th week is presented
in Fig. 2A. Blood glucose levels were higher in the HFD animals
while curcumin significantly blocked these increase. To determine
whether liver was implicated in the improvement of glucose
disposal by curcumin, we conducted intraperitoneal pyruvate
tolerance tests (IPPTT) at the end of the 23
rd
week. As shown in
Fig. 2B, glucose production following pyruvate administration was
significantly enhanced in the HFD fed mice, while curcumin
significantly blocked the effect of HFD, indicating that increased
hepatic gluconeogenesis by HFD feeding was inhibited by dietary
curcumin. Finally, we conducted intraperitoneal insulin tolerance
tests (IPITT) at the end of 26 weeks. Insulin was less effective in
lowing glucose level in HFD animals, while curcumin supplemen-
tation efficiently blocked this effect of HFD. These data suggest
that curcumin improves whole body glucose disposal by both
stimulation of insulin sensitivity and inhibition of hepatic
gluconeogenesis.
Curcumin improved insulin signaling in adipose tissue
and hepatocytes
To further explore molecular mechanisms underlying the
protective effects of dietary curcumin, we assessed insulin signaling
by determining PKB/Akt Ser473 phosphorylation in response to
insulin injection in insulin responsive tissues. We did not see the
deleterious effect of our HFD on insulin stimulated PKB Ser473
phosphorylation in soleus and gastrocnemius muscles (Figure S1).
However, in both adipose tissue and liver, HFD impaired insulin-
stimulated PKB phosphorylation and the impairment was
prevented by curcumin supplementation (Fig. 3A and 3B).
Fig. 3C shows that in the human hepatic cell line HepG2, insulin
stimulated PKB Ser473 phosphorylation (lanes 1 and 2), while
glucose oxidase (GO) pre-treatment, which is known to induce
oxidative stress [25], blocked the stimulatory effect of insulin (lane
3). Curcumin, however, was found to dose-dependently restore the
stimulatory effect of insulin, in the presence of GO (lanes 4–8).
Curcumin did not stimulate Wnt signaling in mature
adipocytes
We have observed previously that insulin stimulates b-cat
Ser675 phosphorylation in a gut endocrine L cell line (data not
shown) and non-endocrine intestinal cells [22], which represents a
novel mechanism for the crosstalk between insulin (possibly other
signaling molecules) and the Wnt signaling pathway [26,27]. Here
we tested whether this crosstalk occurs in mature adipocytes. After
the mice received intraperitoneal insulin injection for 30 min, we
took the epididymal fat tissue for Western blotting. As shown in
Fig. 4A, in adipose tissue, there was no stimulation on b-cat
Ser675 phosphorylation in any of the three groups of animals,
although stimulation of GSK3a/bphosphorylation was apprecia-
ble. Furthermore, curcumin reduced the overall levels of both
Ser675 b-cat and total b-cat (Fig. 4A). In addition, HFD increased
total GSK-3blevel, while curcumin reduced the level of total
GSK-3b. Finally, curcumin supplementation showed no stimula-
tion on the expression of c-Myc and cyclin D1 protein, two
downstream targets of the Wnt signaling pathway (Fig. 4A) [28].
Real time RT-PCR also showed that dietary curcumin did not
stimulate the expression of cyclin D1 and LRP5 mRNA (data not
Table 1. List of oligonucleotide primers utilized in this study.
Genbank#DNA Sequence
Anticipated size of the
product (bp)
ChREBP NM_021455.3 Forward:59-ACCGGGGTGCCCATCACACA-39315
Reverse: 59-CTGCCCGTGTGGCTTGCTCA-39
SREBP-1c NM_011480.2 Forward:59-TAGAGCATATCCCCCAGGTG-39244
Reverse:59-GGTACGGGCCACAAGAAGTA-39
L-PK NM_013631.1 Forward:59-GAGTCGGAGGTGGAAATTGT-39173
Reverse:59-CCGCACCACTAAGGAGATGA-39
TxNIP NM_023719.2 Forward:59-AGAGCAGCCTACAGGTGAGA-39260
Reverse:59-TCTCCTTTTTGGCAGACACT-39
doi:10.1371/journal.pone.0028784.t001
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shown). Taken together, these observations indicate that in this
animal model, curcumin did not stimulate Wnt pathway
components or Wnt pathway downstream target genes in mature
adipocytes.
We then conducted further experiments in primary rat
adipocytes. Fig. 4B shows that treating rat adipocytes with insulin,
or curcumin, or insulin plus curcumin for 5 to 60 min generated
no significant effect on total GSK-3bor Ser675 b-cat expression
level. Total b-cat level, however, appeared to be repressed by
curcumin treatment. Furthermore, curcumin did not block the
stimulatory effect of insulin on GSK-3 phosphorylation. Within
60 min, curcumin or insulin had no observable effect on the
expression of c-Myc or cyclin D1. We then extended the treatment
time by curcumin to 4 h. As shown in Fig. 4C, curcumin
moderately repressed the expression cyclin D1 and greatly
repressed the expression of c-Myc, associated with no appreciable
effect on GSK-3 phosphorylation. Taken together, we did not see
a stimulation of curcumin on Wnt pathway components or Wnt
target gene expression in vivo in the cultured mature adipocytes.
Curcumin attenuated the inflammatory and oxidative
pathway in adipocytes
In mature adipocytes, increased oxidative stress in response to
HFD could contribute to increased inflammatory signaling, which
is at least partially responsible for the impairment of whole body
insulin sensitivity [29]. Curcumin was found to attenuate oxidative
Figure 1. Long term dietary curcumin supplementation prevents HFD-induced obesity and fat mass. (A) Comparison of the body
weight change of mice fed with LFD, HFD or HFD plus curcumin for 28 weeks (n = 4 for LFD or HFD, and n = 8 for HFD/curcumin). a, LFD versus HFD;
b, HFD versus HFD/curcumin. *, p,0.05. (B) Weights of the epididymal fat pad (n = 4 for LFD and HFD, n = 8 for HFD/curcumin). **, p,0.01. (C) Total
body fat volume assessed by MRI at 26 weeks (n =4 for all three groups).
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Figure 2. Curcumin supplementation improves glucose disposal and insulin sensitivity. Assessment of glucose metabolism in animals
with LFD, HFD or HFD plus curcumin feeding. (A) IPGTT (n = 4, 20 weeks), (B) IPPTT (n = 4, 23 weeks), and (C) IPITT (n = 4, 26 weeks). AUC, area under
the curve. *, p,0.05; **, p,0.01.
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stress and reduce the inflammatory response [7]. We observed that
HFD reduced the ratio of GSH/GSSG (1.2360.27 for LFD,
compared with 0.960.17 for HFD), but the difference did not
reach statistical significance. Curcumin supplementation, however,
significantly increased GSH/GSSG ratio (1.3960.24), when
compared with the HFD group (p,0.01). In addition, we found
that in rat primary adipocytes, 20 mM curcumin significantly
increased the expression of HO-1, a fundamentally important
enzyme of the endogenous anti-oxidant system (Fig. 5A). Curcu-
min treatment, however, did not generate a substantial effect on
NF kB or pJNK levels (Fig. 5A), possibly due to the absence of an
oxidative stress in this in vitro assay. Furthermore, dietary
curcumin not only attenuated the stimulatory effect of HFD on
the size of adipocytes, but also completely blocked the effect of
HFD on macrophage infiltration, determined by the expression of
F4/80 in epididymal adipocytes (Fig. 5B). Finally, in mice,
curcumin attenuated the stimulation of HFD on the inflammatory
response, indicated by the inhibition of the rise in NF-kB
expression level (both in whole cell lysates and nuclear extract)
and JNK signaling pathway activation (Fig. 5C). These observa-
tions are generally consistent with previous findings by other
groups [12,30], and collectively suggest that the inhibition of
oxidative stress and the inflammatory pathway in adipose tissue
are among the mechanisms underlying the protective effect of
Figure 3. Curcumin improves insulin stimulated PKB phosphorylation in fat tissue and hepatocytes. (A) and (B) The three groups of
mice fed with the diets as indicated for 28 weeks were fasted overnight and injected with PBS or insulin. After 30 min, the mice were sacrificed.
Samples of epididymal fat pad (A) and liver (B) were prepared and immunoblotted with PKB or Ser473 phosphorylated PKB (p-PKB) antibody. (C)
HepG2 cells were pre-treated with or without curcumin at indicated concentration overnight, followed by a 6 h treatment with or without glucose
oxidase (GO). The cells were then further treated with or without insulin (100 nM) for 10 min, followed by PKB and p-PKB immunoblotting. The blots
shown are representative of 3 separated experiments.
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dietary curcumin in improving insulin signaling, attenuating
obesity, and preventing the development of diabetes.
Curcumin reduced hepatic lipogenic gene expression
We have then examined histological changes in the liver of each
group of the mice. As shown in Fig. 6, HFD consumption increased
liver lipid content, demonstrated by both H&E staining (Fig. 6A)
and Oil Red O staining (Fig. 6B). In the curcumin group, however,
the effect of HFD on the elevation of lipid content was blocked. The
effect of HFD on macrophage infiltration (assessed by F4/80
staining) was also blocked by curcumin consumption (Fig. 6C).
In this chronic HFD mouse model, although liver weight was
not significantly increased (Fig. 7A), intra-hepatic lipid content
(assessed by MRI) was increased more than 6 fold (Fig. 7B),
consistent with the observation by H&E and Oil Red O staining.
Curcumin significantly reduced liver weight (Fig. 7A) and blocked
the effect of HFD on increasing intra-hepatic lipid content
(Fig. 7B). Furthermore, curcumin reduced hepatic NF-kB level,
although our HFD did not cause the increase of hepatic NF-kB
level (Figure S2).
ChREBP and SREBP1-c are two well known transcription
factors which stimulate lipogenic gene expression, while L-PK is a
downstream target of ChREBP [31]. We found that in this HFD
mouse model, hepatic ChREBP protein level but not SREBP-1c
protein level, was increased (Fig. 7C). However, dietary curcumin
reduced the levels of these two transcription factors (Fig. 7C). At
mRNA level, dietary curcumin significantly reduced the amounts
of SREBP-1c, ChREBP and L-PK (Fig. 7D). The mRNA level of
Stearoyl-coenzyme A desaturase 1 (SCD1) in our HFD mice,
however, was not increased. Curcumin treatment did not generate
a significant change on its expression (Figure S3).
TxNIP is a sensor of glucose and oxidative phosphorylation
status [32]. A recent study indicated that TxNIP transcription can
be stimulated by ChREBP [33]. We found that TxNIP protein
level in the liver was higher in the animals of the HFD group,
while curcumin supplementation reduced its level (Fig. 7C).
Curcumin supplementation also reduced TxNIP mRNA level
(Fig. 7D). Furthermore, we conducted LUC reporter analysis,
showing that in the HepG2 cell line, ChREBP promoter activity
was repressed by curcumin treatment (Fig. 7E). Finally, we found
that HFD feeding increased the phosphorylation of S6K1, a
downstream target of mTOR signaling (Fig. 7F).
Discussion
Curcumin is the principal curcuminoid of the popular spice
turmeric utilized in Indian and other South Asia countries, which
is a member of the ginger family. This plant polyphenolic
compound has anti-tumor, anti-proliferative, anti-oxidant, and
anti-inflammatory properties [7]. Since the last decade, a few
clinical trials have been conducted, showing the therapeutic effects
of curcumin on various cancers and Alzheimer’s disease [34].
In C57BL/6J HFD fed mice, oral curcumin supplementation
was shown to prevent the development of obesity-associated
inflammation, insulin resistance, as well as diabetes [12]. The
beneficial effect of curcumin in that study was mainly attributed to
the reduction of macrophage infiltration of the adipose tissue, the
increase of adiponectin production, as well as the decrease of
hepatic NF-kB activity [7,12]. The anti-adipogenic effect of
curcumin was then demonstrated in the 3T3-L1 cell model by
other groups [35,36]. The stimulation of HFD on hepatic NF-kB
level, however, was not observed in the current study with our
chronic HFD model, although curcumin supplementation de-
creased NF-kB level in the liver (Figure S2).
Insulin resistance and obesity in C57BL/6J mice are often
induced in the short-term by feeding a diet containing saturated
fatty acids (45%) or with a mixed fatty acid diet with 60% energy
from fat. In the current study we utilized a chronic HFD feeding
model, in which mice did not develop obesity before 16 weeks, and
the deleterious effect of HFD on both the morphology of the liver
and plasma metabolic profiles were not as severe as the utilization
of regular HFD [8] (data not shown). As presented, although our
HFD reduced plasma adiponectin level, it did not reach statistical
significance. Nevertheless, curcumin consumption generated a
significant increase of plasma adiponectin. Furthermore, instead of
routinely providing curcumin-containing HFD with every meal
[12], we provided the curcumin-supplemented diet only two days
per week. This model may more closely mimic the natural
development of insulin resistance, associated with modest dietary
changes along with intermittent curcumin consumption in human
subjects that we can expect. We show in this model that curcumin
supplementation blocked the effect of HFD on fat gain, improved
insulin sensitivity and glucose disposal, and reduced intra-hepatic
lipid content. In addition to the confirmation of the effect of
curcumin in stimulating anti-oxidative signaling and attenuating
inflammatory signaling in hepatocytes, we found that curcumin
reduces mRNA levels of ChREBP and SREBP1-c, two key
transcription factors for hepatic lipogenesis, as well as L-PK, an
important downstream target of ChREBP [31,37]. However, we
did not observe that in mature adipocytes curcumin stimulates
Wnt signaling components or Wnt target genes. We therefore
suggest that curcumin exerts its beneficial effect in our HFD fed
mouse model via attenuating oxidative stress and inflammatory
response in the adipose tissue, and reducing lipogenesis in the liver,
without the stimulation Wnt activity in mature adipocytes.
It should be noted that the activation of Wnt signaling is strongly
associated with the development and progression of colon cancer
and other tumors. The chemotherapeutic effect of curcumin has
been partially attributed to the repression of Wnt activity [38]. For
example, in the LNCaP prostate cancer cells, curcumin represses
total b-cat level, as well as GSK-3 phosphorylation, associated with
reduced c-Myc and cyclin D1 expression [38]. In addition, a recent
study indicated that curcumin disrupts the mammalian target of
rapamycine complex (mTOR) [39]. mTOR is not only a
downstream target of insulin signaling, but also serves as an effector
of the Wnt signaling pathway [27,40]. Thus, the repressive effect of
curcumin on mTOR further supports the notion that curcumin
might repress Wnt activity in cancer cells. In the current study, we
show that HFD induced hepatic expression of phosphorylated
S6K1, a downstream target of mTOR. Curcumin consumption
suppressed S6K1 phosphorylation.
The importance of Wnt signaling pathway in metabolic
homeostasis has been broadly recognized recently [41]. Wnt10b
is abundantly expressed in mesenchymal precursor cells. Wnt10b
Figure 4. Curcumin does not stimulate the Wnt signaling pathway in mature adipocytes. (A) Samples from epididymal fat pad of the
three groups of mice were prepared for Western blotting. b-cat and GSK-3 represent Wnt pathway effectors, while cyclin D1 and c-Myc are two
known Wnt target genes. (B) Rat primary adipocytes were prepared and treated with insulin (100 nM), or curcumin (20 mM), or insulin plus curcumin
for 0, 5, 30, and 60 min. Samples were collected for Western blotting, with indicated antibody. (C) Rat primary adipocytes were prepared and treated
with 10 or 20 mM curcumin for 4 h, followed by Western blotting with indicated antibody. All panels show the representative blot (n = 3).
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mediated Wnt activation stimulates the expression of osetogenic
genes at the expense of adipogenic genes [18]. Furthermore,
ectopic expression of Wnt10b in transgenic mice impairs the
development of the adipose tissue and these mice are resistant to
HFD induced obesity [42,43]. Very recently, a study demonstrat-
ed that in the 3T3-L1 cell model, the repression of adipogenic
differentiation was accompanied by Wnt/b-cat activation [19].
The authors found that during adipocyte differentiation, curcumin
reduced the expression of the components of the destructive
complex that are responsible for b-cat degradation, including
Figure 5. Curcumin increases HO-1 expression and reduces inflammatory markers in mature adipocytes. (A) Rat primary adipocytes
were prepared and treated with 10 or 20 mM curcumin for 4 h. Western blotting was performed with the indicated antibodies. (B) Immunostaining
was performed for the detection of macrophage infiltration marker F4/80 in the fat tissue of the three groups of mice. Arrows show the positive
staining. No macrophage infiltration was observed in LFD or HFD/Curcumin animals. (C) Samples from epididymal fat pad of the three groups of mice
were prepared for Western blotting. WSL, whole cell lysates; Nuclear, nuclear extracts. Panel A and C show representative blots (n = 3).
doi:10.1371/journal.pone.0028784.g005
Curcumin Prevents Insulin Resistance and Obesity
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CK1a, GSK-3band Axin, accompanied by increased expression
of total b-cat, Wnt10b, the Wnt pathway receptor Fz2, the co-
receptor LRP5, as well as the Wnt targets c-Myc and cyclin D1
[19]. This study provides a potential novel molecular mechanism
to explain the repressive effect of curcumin on adipogenesis. In
contrast, we found in the current study that the stimulatory effect
of curcumin on Wnt signaling does not occur in mature
adipocytes. How this plant dietary compound exerts opposite
Figure 6. Histological assessment of mouse liver. Representative histological slides show lipid content (A, H&E staining; B, Oil Red O staining)
and macrophage infiltration (C) in the three groups of mice.
doi:10.1371/journal.pone.0028784.g006
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effects on Wnt signaling pathway in pre-adipocytes versus mature
adipocytes deserves further investigations. Nevertheless, we have
previously noted cell-type specific effects of Wnt and/or insulin
signaling. For example, both insulin and lithium chloride, the
latter mimics Wnt activation, stimulate proglucagon gene (gcg)
transcription in gut endocrine L cells, but repress the same gcg
gene in pancreatic islets [44,45,46]. Furthermore, we found the
stimulatory effect of insulin on b-cat Ser675 phosphorylation in
the gut [22], but not in adipocytes (this study). Finally, one study
has shown that in skeletal muscle cells, Wnt activation increases
insulin sensitivity through reciprocal regulation of Wnt10b and
SREBP-1c [47], while another group showed that Wnt activation
in mature adipocytes leads to adipocyte dedifferentiation and
insulin resistance [20].
In this study, curcumin blocked the effect of HFD on
macrophage infiltration in adipose tissue, associated with the
Figure 7. Curcumin reduces intra-hepatic lipid content and lipogenic gene expression. (A) Curcumin supplementation reduced liver
weight in HFD fed mice (n = 4 for all 3 groups). (B) MRI shows that curcumin supplementation reduced intra-hepatic lipid content (n = 4 for all 3
groups). (C) Curcumin supplementation reduced the expression of TxNIP, ChREBP and SREBP-1c in liver of HFD fed mice (A representative blot, n = 3).
(D) Curcumin supplementation reduced liver ChREBP, SREBP-1c, L-PK and TxNIP mRNA expression in HFD fed mice (n = 3). (E) The ChREBP-LUC
reporter plasmid construct (3 mg) were transfected into HepG2 cells for 24 h, followed by a 20 h serum starvation and a 4 h curcumin (20 mM)
treatment. The data are presented as mean 6SEM (n = 3). *; p,0.05; **, p,0.01. F. Curcumin supplementation blocked the stimulatory effect of HFD
on S6K1 phosphorylation in the liver.
doi:10.1371/journal.pone.0028784.g007
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repression of NF-kB level and JNK activity, the improvement of
insulin stimulated PKB phosphorylation in adipose tissue and
liver, as well as glucose disposal. These observations are consistent
with current concepts that the activation of endogenous anti-
oxidative system and the repression of inflammatory signaling in
adipocytes improve insulin resistance [48]. Whether there are
additional mechanisms underlying the improvement of insulin
signaling by curcumin supplementation deserves further investi-
gations. For example, mTOR is involved in the development of
insulin resistance via a negative feedback loop, i.e. the inhibition of
IRS-1 tyrosine phosphorylation [49], while the inhibitory effect of
curcumin on mTOR has been demonstrated in certain cancer
cells [39,50]. Since Wnt activation in adipocytes may lead to
insulin resistance [20], the inhibition of mTOR by curcumin may
result in increased insulin sensitization, because mTOR is also
among the effectors of the Wnt signaling pathway [27,40],
Accelerated hepatic lipogenesis is commonly observed in a
number of metabolic disorders, including insulin resistance,
metabolic syndrome and T2D [51]. The increase in lipogenesis
is another mechanism by which HFD consumption leads to
obesity and diabetes. ChREBP and SREBP-1c are two key
transcription factors for genes that encode lipogenic enzymes
[31,37]. Very little is known about the effect of curcumin on
hepatic lipogenesis, although a recent study showed that in
adipocytes curcumin inhibits fatty acid synthase (FAS) [52]. We
found that curcumin reduced liver weight and intra-hepatic lipid
content. More importantly, dietary curcumin was shown to repress
SREBP-1c and ChREBP expression. This, along with the
repression of L-PK by curcumin and the in vitro ChREBP LUC
reporter analysis, suggest that inhibition of ChREBP expression
and function are among the mechanisms by which this dietary
component prevents obesity and its associated metabolic defects. It
should also be pointed out that curcumin can block cardiac
hypertrophy and the implicated underlying mechanism was the
repression of p300-HAT activity and hence the inhibition of p300
acetylation of certain transcription factors [13]. ChREBP was
found to require p300 or CBP as a co-factor in stimulating the
expression of TxNIP [33]. We found here that TxNIP protein and
mRNA expression in curcumin fed mice was also reduced. It
remains to be determined whether p300 inhibition is among the
mechanisms by which curcumin reduces the expression of L-PK
and other targets of ChREBP.
Together, our observations confirm that curcumin improves
insulin signaling, glucose disposal, and blocks obesity during HFD
consumption. Our data confirm that in this chronic HFD feeding
model, the function of curcumin is mediated via increasing the
capability of the animals in anti-oxidative stress and attenuating
inflammatory response in adipocytes. Furthermore, in mature
adipocytes, this appears occur independent of Wnt activation as
curcumin did not activate Wnt pathway components or Wnt
downstream target genes. Finally, we revealed the repressive effect
of curcumin on hepatic lipogeneis, associated with the inhibition of
ChREBP and SREBP-1c expression. Further investigation is
required to determine whether the repressive property of
curcumin on p300/CBP is additionally involved in reducing
hepatic lipogenesis. Thus, the development of curcumin as a
therapy for obesity, insulin resistance and T2D is supported.
Supporting Information
Figure S1 No detectable defect by HFD and no appre-
ciable improvement by curcumin on insulin stimulated
PKA Ser473 phosphorylation in muscles- The three groups
of mice fed with indicated diet for 28 weeks were fasted over night
and injected with PBS or insulin. After 30 min, samples of soleus
(A) and gastrocnemius (B) were prepared and immunoblotted with
PKB or Ser473 phosphorylated PKB (p-PKB) antibody.
(TIF)
Figure S2 Curcumin moderately reduced hepatic NF-kB
activity in HFD fed mice although our HFD did not cause
an appreciable elevation of NF-kB activity- Samples from
liver of the three groups of mice were prepared for Western
blotting with indicated antibody.
(TIF)
Figure S3 No significance difference was observed on
hepatic SCD-1 expression in our animal model. RT-PCR
were conducted with the following SCD-1 primes.
Forward:59-CTACAAGCCTGGCCTCCTGC-39Reverse:59-G-
GCACCCAGGGAAACCAGGA-39. N = 3 for each group.
(TIF)
Acknowledgments
The authors would like to thank Mr. Qiang Xu for providing assistance in
histological studies.
Author Contributions
Analyzed the data: WS ZY YC YY TC WF. Directed and planned
research: IGF TJ. Generated data, discussion and manuscript writing: WS
ZY TC. Provided research data: YC YY WF. Did rat mature adipocytes
preparation: HL. Contributed to manuscript writing: WS ZY IGF TJ.
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