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Introduction
Type 2 diabetes is a chronic disease, often characterized
by insulin resistance, which can lead to several secondary
complications, such as hypertension, atherosclerosis,
coronary artery disease, and
hyperlipidemia[1]. Approximately 150 million people worldwide are afflicted with the disease at
present, and with a projection of 300 million people being
affected by the year 2025, it has become a serious public
health problem, particularly in developed
countries[2]. Research of an effective antidiabetic agent would be of great
interest for the treatment of type 2 diabetes.
Thiazolidinediones (TZD), which are peroxisome proliferator-activated receptor
γ (PPARγ) agonists, have been demonstrated to have a variety of clinical effects, including
improving insulin sensitivity and glucose
tolerance[3,4]. PPAR are ligand-activated transcription factors that belong to the
nuclear receptor superfamily. They have specific tissue
distribution and play a pivotal role in regulating the expression
of a large number of genes involved in glucose and lipid
metabolism[5]. PPARγ is mainly distributed in adipose tissue
and skeletal muscle, and regulates glucose metabolism.
Moreover, PPARγ2 is specific in adipose tissue and essential
for adipocyte differentiation[6].
PPARγ agonists could enhance the differentiation of preadipocytes into adipocytes
which is relative to their antidiabetic
activities[7,8]. Because of the adverse reactions of TZD, such as hepatic toxicity
and fluid retention, the research of a non-thiazolidinedione
insulin sensitizer has attracted much more attention in
recent years[4].
YY20, a novel synthesized non-TZD compound named
2-(3-furan-2-yl-acryloylamino)-3-{4-[2-(5-methyl-2-phenyl-
oxazol-4-yl)-ethoxy]-phenyl}-propionic acid (Figure 1), was
a α-methyl-α-phenoxylpropionate
derivative[9,10]. The activation effects of rosiglitazone and
YY20 on human PPARγ were tested by using a transient transfection assay.
According to the luciferase activity, YY20 activated
PPARγ 14±2.4 times to the control at 10 µmol/L, whereas a TZD
compound rosiglitazone was 8.7±1.4 times to the control at the
same concentration. Although YY20 exhibited potent
PPARγ agonist activity, like rosiglitazone in the report gene system,
it is essential that the insulin sensitizing effects are proved
in the insulin-sensitive cell lines. In this study, the
enhancement effects of YY20 on the differentiation of 3T3-L1
preadipocytes into adipocytes were examined and compared
with that of rosiglitazone. As adipocyte differentiation
markers[6], PPARγ2, glucose transporter-4 (GLUT4), insulin
receptor substrate-1 (IRS-1), and adiponectin (ACRP30)
expressions were studied in 3T3-Ll preadipocytes treated with YY20.
In order to further confirm the insulin-sensitizing effects of
YY20, the enhancement effects of insulin-mediated glucose
consumption were also investigated in the HepG2 human
hepatocellular carcinoma line.
Materials and methods
Materials 3T3-L1 mouse preadipocytes and the HepG2
human hepatocellular carcinoma line were purchased from
the Cell Center of the Chinese Academy of Medical Sciences,
Shanghai, China. Insulin was the product of Eli-Lilly & Co
(Indianapolis, IA, USA); isobutylmethylxanthine (IBMX),
dexamethasone (DEX), SR-202, Oil red O, methylthiotetrazole
(MTT), and bovine serum albumin (BSA) were purchased
from Sigma (St Louis, MO, USA). GLUT4, IRS-1, ACRP30,
and the β-actin primary antibody were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). Trizol, M-MuLV
reverse transcriptase, Taq polymerase, and cell culture
mediums were the products of Gibco BRL (Gaithersburg, MD,
USA). The PCR primers were synthesized by Sangon (shanghai, China). Enhanced chemiluminescent (ECL)
substrate was from Pierce (Rockford, IL, USA). YY20 and
rosiglitazone were synthesized in our laboratory and were
dissolved in dimethyl sulfoxide (DMSO) to prepare the stock
solution.
Adipocyte differentiation assay 3T3-L1 cells were
maintained at 37 °C in an atmosphere of 5%
CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal
bovine serum (FBS). The 3T3-L1 preadipocytes were grown in
96-well plates until 2 d postconfluence. The differentiation
was induced by addition of 5 µg/mL insulin, 0.5 mmol/L IBMX
and 1 µmol/L DEX (INS-IBMX-DEX cocktail). The induction
medium was removed 2 d after incubation. After an
additional 2 d of incubation in DMEM supplemented with 10%
FBS and 5 µg/mL insulin, the medium was changed every
other day with DMEM supplemented with 10% FBS. Cells
were challenged during the whole period of differentiation
with different concentrations of YY20, with rosiglitazone as
the positive control and 0.1% DMSO as the vehicle control.
After 7 d differentiation, cells were fixed with 10%
formaldehyde for 1 h and then stained with Oil red O (0.1 mg/mL)
for 2 h at room temperature. The medium in each well was
then removed, and isopropyl alcohol was added to dissolve
the precipitate. The optical density (OD) at a wavelength of
510 nm was determined by ELISA
spectrometry[11]. Moreover, 3T3-L1 adipocytes treated with YY20 or rosiglitazone for 2 d
or for 6 d, together with the vehicle control, were collected
for RT-PCR and Western blot analysis.
PPARγ inhibiting assay Two days after confluence,
3T3-L1 cells were treated with 100 µmol/L SR-202 or vehicle for
0.5 h, then 1 µmol/L YY20 or 1 µmol/L rosiglitazone was added.
The cells were challenged for 4 d. After that, the cells were
cultured in DMEM supplemented with 10% FBS for an
additional 2 d and collected for Western blot analysis.
RT-PCR analysis The total RNA from the 3T3-L1
adipocytes treated with increasing concentrations of YY20
or rosiglitazone for 2 d or untreated was isolated using Trizol
reagent in accordance with the manufacturer's instructions.
After extraction, mRNA was precipitated by recommended
procedures and dissolved in 0.1% diethylpyrocarbonate
solution. The RNA content was quantified by using an
ultraviolet spectrophotometer at 260 nm. To synthesize first
strand cDNA, 7 µL total RNA was incubated with 0.5 µg of
oligo (dT) 6 primer and 5 µL deionized water at 65 °C for 15
min. RT of 20 µL was performed with 200 units of M-MuLV
reverse transcriptase, 4 µL of 5× reaction buffer (250 mmol/L
Tris-HCl; pH 8.3 at 25 °C, 375 mmol/L KCl, 15 mmol/L
MgCl2, and 50 mmol/L dithiothreitol) and 1 mmol/L deoxynucleoside
triphosphate (dNTP) mixture for 1 h at 42 °C. PCR of 50 µL
contained 1 µL of the RT reaction product, 5 µLof 10×PCR
buffer (100 mmol/L Tris-HCl, pH 8.3 at 25 °C, 500 mmol/L KCl,
and 15 mmol/L MgCl2), 25 units of
Taq polymerase, 1 µL of 10 mmol/L dNTP mixture, and 30 pmol of each primer. PCR for
the amplification of PPARγ2 was performed with primers (420
bp product; sense: 5'-ATGGGTGAAACTCTGGGA-3', anti-sense: 5'-TCGGCACTCAATGGCCAT-3'), and GAPDH (560
bp product; sense: 5'-ATCTTCTTGTGCAGTGCCAGCC-3', antisense: 5'-GGTCATGAGCCCTTCCACAATG-3') was used
as the internal control. Each amplification cycle consisted of
10 s of denaturation at 94 °C, 20 s of annealing at 55 °C, and
30 s of extension at 72 °C. Following amplification, the PCR
products were separated by 1.5% agarose gel electrophoresis,
stained with ethidium bromide, and visualized under
ultraviolet light with a bio-imaging analyzer (Bio-Rad, Hercules,
CA, USA).
Western blot analysis 3T3-L1 adipocytes treated with
different concentration of YY20 for 6 d or untreated were
washed with PBS, collected in RIPA buffer (50 mmol/L
Tris-HCl, 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 25
mmol/L NaF, 25 mmol/L β-glycerolphosphate, pH 7.5, 0.2%
Triton X-100, 5 mmol/L EDTA, 1 mmol/L PMSF, 10 µg/mL
leupeptin, and 10 µg/mL aprotinin) and lysed for 30 min on
ice. Samples were clarified by centrifugation for 30 min at
13 000×g at 4 °C. An aliquot of 40 µg of the supernatant
protein from each sample was heated with 4×SDS sample
buffer at 95 °C for 5 min, and separated electrophoretically
on a 7.5% or 12% SDS-PAGE. Subsequently, proteins were
transferred onto a 0.45 µm pore size nitrocellulose membranes
for 1 h and blocked for 1 h. Nitrocellulose membranes were
then exposed to GLUT4, IRS-1, ACRP30, or the β-actin
primary antibody in blocking buffer at 1:500 dilution overnight
at 4 °C. Then the membranes were incubated with the
anti-rabbit IgG or anti-goat IgG secondary antibody conjugated
with horseradish at 1:10 000 dilution for 1 h. The proteins
were visualized autoradiographically with ECL, and scanned
using a bio-imaging analyzer (Bio-Rad, Hercules, CA,USA).
Glucose consumption assay[12] HepG2 cells were grown
in RPMI-1640 (11.1 mmol/L glucose) containing 10% FBS.
Two days before the experiments, the cells were plated into
96-well, tissue culture plates with some wells left blank.
After the cells reached 80%_90% confluence, the medium was
replaced by RPMI-1640 supplemented with 0.2% BSA. Two
hours later, the medium was removed and the same culture
medium containing YY20, metformin or rosiglitazone, with or
without insulin, was added to all wells, including the blank
wells. After 24 h of treatment, the medium was removed and
its glucose concentrations was determined by the glucose
oxidase method[13]. The amount of glucose consumption
was calculated by the glucose concentrations of blank wells
subtracting the remaining glucose in the cell plated wells.
Following the glucose measuring in the medium, a MTT
assay was used to monitor the cell proliferation and to
adjust the glucose consumption
values[14].
Statistical analysis Data were shown as mean±SD.
Differences between individual groups were analyzed by using
the t-test. A difference with a P value of <0.05 was
considered to be significant.
Results
Effect of YY20 on adipocyte differentiation
After treatment with YY20 and rosiglitazone at 0.1, 1, and 10 µmol/L in
the 3T3-L1 cells during 7 d differentiation, the cells full of
lipid droplets increased significantly compared with that of
the vehicle control (Figure 2A_2D). By Oil red O staining
and OD value determination, it was found that YY20 and
rosiglitazone could promote adipocyte differentiation of
3T3-L1 cells significantly (P<0.05,
P<0.01; Figure 2E).
Effect of YY20 on PPARγ2 gene expression
To elucidate the pattern of PPARγ2 gene expression after 2 d induction in
the 3T3-L1 cells, RT-PCR was analyzed. The results
suggested that the expression of PPARγ2
mRNA increased in the cells challenged with YY20, as well as rosiglitazone (Figure
3).
Effect of YY20 on IRS-1 and GLUT4 protein expression
The regulation of IRS-1 and GLUT4 expression by
increasing concentrations of YY20 was investigated after 6 d of
differentiation in the 3T3-L1 cells. Western blot analysis
revealed that YY20 could obviously increase the expression
levels of IRS-1 and GLUT4 in a concentration-dependent
manner (Figure 4A).
Effect of YY20 on ACRP30 and GLUT4 protein expression, and the influences of the
PPARγ inhibitor After 4 d treatment and 6 d differentiation, YY20, as well as
rosiglitazone at a concentration of 1 mmol/L, could upregulate
the expression of the ACRP30 and GLUT4 protein; this effect
could be reversed by the PPARγ specific inhibitor, SR-202.
And the increase of ACRP30 expression caused by YY20
was completely reversed by SR-202 (Figure 4B).
Effect of YY20 on glucose consumption After treatment
in medium with 11.1 mmol/L glucose for 24 h, a
glucose-lowering effect of YY20 was observed. With different levels
of insulin added in the culture medium, YY20 exhibited
different efficacy of the glucose-lowering effect (Figure 5).
When the medium was absent of insulin, the glucose
consumption of metformin at 1 mmol/L increased by 59%
compared to the vehicle control (P<0.05), whereas there were
only 16% and 21% increases by 1 mmol/L of rosiglitazone
and YY20, respectively (P>0.05). However, with 1 nmol/L
insulin present in the medium, the glucose consumption of
cells treated with metformin, rosiglitazone, and YY20 were
75%, 45%, and 63%, respectively (P<0.05); the effect of YY20
was related to the concentration (Figure 6). The effects
under 100 nmol/L insulin were similar to that of under 1
nmol/L insulin. According to the MTT results, we found that in
the presence of 1 mmol/L metformin and less than 1 mmol/L
YY20 or rosiglitazone could not affect cell proliferation after
24 h culture (data not shown). When the glucose
consumption values were adjusted with the MTT OD values, the
results changed insignificantly.
Discussion
It has been widely known that PPARγ agonists are
dominant regulators of adipocyte development, and
PPARγ activation improves insulin
resistance[15_17]. As a TZD,
rosiglita-zone is a potent agonist of PPARγ and could significantly
improve the adipocyte differentiation in 3T3-L1 cells.
Activation of PPARγ can increase the number of small
adipocytes, but reduce the number of large adipocytes in white adipose
tissues. Because small adipocytes are more sensitive to
insulin, an increased number of small adipocytes and a
decreased number of large adipocytes in white adipose
tissues can alleviate insulin
resistance[18]. Furthermore, adipocyte differentiation leads to the expression of
adipocyte-specific genes, such as GLUT4 and IRS-1, which are
important components of the insulin receptor signal transduction
pathway[19_21]. In this study, we found that the novel
non-TZD compound YY20 could enhance 3T3-L1 cell
differentiation as effectively as rosiglitazone. YY20 could increase
lipid accumulation in differentiated cells and upregulate
PPARγ mRNA, IRS-1, GLUT4, and ACRP30 protein
expression in 3T3-L1 adipocytes, indicating that it might activate
PPARγ and enhance insulin sensitivity. Besides an adipocyte
diffrentiation mark, ACRP30 is also deemed as a adipocytokine
which can improve insulin
resistance[22]. In our study, the
PPARγ inhibitor could completely reverse the ACRP30
expression upregulated by YY20, but not rosiglitazone,
suggesting that the effect of YY20 on ACRP30 expression is
more dependent on PPARγ activation than rosiglitazone.
What this means to the therapy is still unknown.
Liver, adipose tissue, and skeletal muscles are target
tissues of insulin and major sites of glucose and lipid
metabolism. In our study, the HepG2 human hepatocellular
carcinoma line was used to elucidate the glucose-lowering
effect of YY20 in vitro. According to the results, metformin
could elevate the glucose consumption of cells regardless
of the insulin levels, whereas PPARγ agonist rosiglitazone
and YY20 were somewhat dependent on insulin, suggesting
the different mechanisms of the glucose-lowering effect
between these two kinds of antidiabetic drugs. Since
PPARα, not PPARγ, is predominantly expressed in the
liver[23], we wonder if the mechanism of the glucose-lowering effect in
HepG2 hepocytes is related to the activation of
PPARα.
In summary, as a potential activator of PPARγ, YY20 could
enhance preadipocyte differentiation and upregulate vital
molecules in the insulin signaling pathway, and enhance
glucose consumption in HepG2 cells in a concentration- and
insulin-dependent manner. These results suggest that
further studies should be carried out to develop YY20 as a
substitute of TZD for diseases with insulin resistance, such
as type 2 diabetes.
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