Extract
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differentiation medium (DMEM supplemented with 2% horse
serum).
For drug assays, LO2 cells were treated with 10 µmol/L
C333H for 24 h, and 3T3-L1 cells were treated with 10 µmol/L
C333H in the presence of 10 mg/mL insulin for 72 h. C2C12
cells were differentiated into myotubes for 4 d, and the
medium was replaced with phenol red-free differentiation
medium supplemented with 10 µmol/L C333H for 24 h.
RT-PCR Total RNA from cells was isolated using Trizol
reagent following the manufacturer¡¯s instructions. The RNA
content was quantified by using an ultraviolet
spectrophotometer at 260 nm. For RT-PCR analysis of hACO, mLPL,
maP2 and mGluT4 expression, total RNA was reverse
transcribed and subsequently amplified by PCR using the
BcaBEST RNA PCR kit (version 1.1; TaKaRa Biotechnology,
Dalian, China). The primers for hACO (362 bp product; sense
5¡¯-gggcatggctattctcattgc-3¡¯, antisense
5¡¯-cgaa-caaggtcaacagaagttaggttc-3¡¯); mLPL (403 bp
product; sense 5¡¯-CTTTG AGAAAGGGCTCTGCC-3¡¯, anti-sense
5¡¯-CCTCTCGATGACGAAGCTGG-3¡¯); maP2 (160 bp product; sense 5¡¯-AAGACAGCTCCTCCTCGAAGGTT-3¡¯,
antisense 5¡¯-TGACCAAATCCCCATTTACGC-3¡¯); mGluT4 (504 bp product; sense 5¡¯-AACGAGCTGGACGACGGACA-3¡¯,
antisense 5¡¯-TTGCCCCTC AGTCATTCTCA-3¡¯) and the internal control hGAPDH (176 bp product; sense
5¡¯-ACCCA-CTCCTCCACCTTT G-3¡¯, antisense
5¡¯-CTCTTGTGCTCTT-GCTGGG-3¡¯); mGAPDH (505 bp product; sense 5¡¯-CCCTGGCC
AAGGTCATCCAT-3¡¯, antisense 5¡¯- AGGTCCACCACCCTG-TTGCT-3¡¯). The PCR conditions were as follows: 25 (mLPL,
mGAPDH, and hGAPDH), 28 (maP2), or 30 (mGluT4 and hACO) cycles of 94
°C for 20 s, 52 °C (mGluT4) or 56 °C
(mLPL, maP2, mGAPDH, hGAPDH, and hACO) for 30 s, and
72 °C for 1 min. Following amplification, 5 µL of each PCR
product was separated on a 1% agarose gel, stained with
ethidium bromide and visualized under ultraviolet light with
a MultiImage light cabinet (AlphaImager 2200, USA).
Animal assays Eight-week-old male homozygous
db/db mice were treated once daily with 10 mg/kg C333H or 0.5%
sodium carboxymethylcellulose (control) by intragastric
gavage. C333H was suspended in 0.5% sodium
carboxy-methylcellulose. Blood was taken from the retroorbital
sinuses at d 0 and d 14 from fasting mice. The various serum
parameters were determined by using commercial kits
(Rongsheng Biotech, Shanghai, China).
Statistical analysis Data are shown as mean±SD.
Differences between individual groups were analyzed by using
the t-test for adipocyte differentiation or ANOVA for the
animal assay.
Results
Activation effect of C333H on human PPARa and
PPARg We tested the activation effects of rosiglitazone, fenofibrate,
and C333H on human PPARa and PPARg by using a transient transfection assay. To minimize background
noise caused by endogenous PPAR ligands, an established
chimera system was used, which contained the yeast GAL4
DBD linked to the LBD of PPARa or
PPARg[9]. Interestingly, rosiglitazone was
the only strong activator of PPARg, and fenofibrate did not have a significant effect on
PPARa. C333H was a potent activator of both PPARa and
PPARg. The addition of 10 µmol/L C333H strongly upregulated luciferase
activity 22.2-fold for pM-PPARa, whereas 10 µmol/L
rosiglitazone (PPARg agonist) and 100 µmol/L fenofibrate
(PPARa agonist) produced only a 3.6-fold (data not shown)
and a 8.5-fold increase, respectively. For
pM-PPARg, C333H and rosiglitazone upregulated luciferase activity 8.3- and
8.4-fold, respectively (Figure 2).
Effect of C333H on adipocyte differentiation
It has previously been shown that PPARg agonists are dominant
regulators of adipocyte development[10]. In the present study we
found that C333H and rosiglitazone could promote the
adipocyte differentiation of 3T3-L1 cells. At the same
concentration (10 µmol/L), C333H was a markedly more potent
and efficacious inducer of adipogenesis than rosiglitazone
(Figure 3).
Effect of C333H on gene expression in several cell lines
We investigated the regulation of ACO, LPL, aP2, and GluT4
gene expression by C333H in hepatocytes, adipocytes, and
skeletal muscle cells. We found that C333H increased the
expression levels of ACO mRNA in human LO2 normal hepatocytes, GluT4 in C2C12 skeletal muscle cells, and LPL
and aP2 in 3T3-L1 preadipocytes (Figure 4).
Effect of C333H on circulating lipid and glucose levels
in db/db mice We investigated the pharmacological effect
of C333H in db/db mice. The triglyceride (TG), total
cholesterol (T-CHO), free fatty acid (FFA) and glucose serum con
centrations were measured at d 0 and d 14 . After 14-d
treat-ment with C333H, serum TG, T-CHO, FFA, and glucose were
markedly reduced (Table 1).
Discussion
It is known that PPARg activation improves insulin
resistance, and that PPARa activation induces a decrease in
circulating lipid levels[11_13]. Rosiglitazone is a potent
agonist of PPARg, whereas its activity with respect to
PPARa is weak (data not shown). In vitro reporter gene assays
established that C333H was an effective activator of both
PPARa and PPARg. C333H was a more potent agonist of
PPARa than fenofibrate, and it was found to have a similar
PPARg activation effect to rosiglitazone. In
vivo, we found that C333H significantly reduced the circulating levels of TG,
T-CHO, FFA, and glucose in db/db mice, indicating that it
might enhance insulin sensitivity and improve lipid
metabolic disorders by activating both PPARa and
PPARg.
In an adipocyte differentiation assay, C333H had the
highest lipogenic activity of the compounds tested, which
indicates that C333H could improve insulin resistance by
PPARg activation. Because PPARg plays an important role in the
regulation of adipocyte
differentiation[14], PPARg agonists can promote preadipocyte
differentiation to adipocytes. However, activation of
PPARg can increase the number of small adipocytes and 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[15]. Furthermore, adipocyte differentiation leads to the expression of
adipocyte-specific genes, such as
aP2[16], LPL[17], and
GluT4[18], which indicates that PPARg agonists have good antihyperglycemic
and antihyperlipidemic activity.
Liver, adipose tissue, and skeletal muscle are major sites
of glucose and lipid metabolism. PPARa is predominantly
expressed in the liver, whereas PPARg is most abundant in
adipose tissue, and is also expressed in small amounts in
skeletal muscle[19,20]. In the present study, the ability of C333H
to regulate ACO, LPL, aP2, and GluT4 gene expression was
investigated in various cell lines. Hepatic
PPARa-dependent ACO mainly regulates fatty acid b-oxidation. LPL and
aP2 are regulated by PPARg, and mainly function in
triglyceride and fatty acid
metabolism[21]. Moreover, the insulin-dependent glucose transporter GluT4 functions to regulate
glucose uptake into adipose tissue and skeletal muscle in
response to elevated levels of insulin in the circulation. Our
results showed that C333H upregulated the expression lev
els of these genes, indicating that it could lower blood
glucose and lipid concentrations in type 2 diabetic patients.
In summary, we demonstrated that C333H was a dual
activator of PPARa and PPARg. It not only controlled
glucose and lipid metabolism, but also promoted preadipocyte
differentiation and improved insulin resistance. These
results suggest that further studies should be carried out to
develop C333H as a novel therapy for metabolic disease such
as obesity, hyperlipidemia and type 2 diabetes.
References
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