Pioglitazone Stimulates Apolipoprotein A-I Production
Without Affecting HDL Removal in HepG2 Cells
Involvement of PPAR-␣
Shucun Qin, Tianjiao Liu, Vaijinath S. Kamanna, Moti L. Kashyap
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Objective—Pioglitazone, an antihyperglycemic drug, increases plasma high-density lipoprotein (HDL)-cholesterol in
patients with type 2 diabetes. The mechanisms by which pioglitazone regulate HDL levels are not clear. This study
examined the effect of pioglitazone on hepatocyte apolipoprotein AI (apoA-I) and apoA-II production and HDL-protein/
cholesterol ester uptake.
Methods and Results—In human hepatoblastoma (HepG2) cells, pioglitazone, dose-dependently (0.5 to 10 ␮mol/L),
increased the de novo synthesis (up to 45%), secretion (up to 44%), and mRNA expression (up to 59%) of apoA-I.
Pioglitazone also increased apoA-II de novo synthesis (up to 73%) and mRNA expression (up to 129%). Pioglitazone
did not affect the uptake of HDL3-protein or HDL3-cholesterol ester in HepG2 cells. The pioglitazone-induced apoA-I
lipoprotein particles increased cholesterol efflux from THP-1 macrophages. The pioglitazone-induced apoA-I secretion
or mRNA expression by the HepG2 cells was abrogated with the suppression of PPAR-␣ by small interfering RNA or
a specific inhibitor of PPAR-␣, MK886.
Conclusions—The data indicate that pioglitazone increases HDL by stimulating the de novo hepatic synthesis of apoA-I
without affecting hepatic HDL-protein or HDL-cholesterol removal. We suggest that pioglitazone-mediated hepatic
activation of PPAR-␣ may be one of the mechanisms of action of pioglitazone to raise hepatic apoA-I and HDL.
(Arterioscler Thromb Vasc Biol. 2007;27:2428-2434.)
Key Words: apolipoproteins 䡲 lipids 䡲 lipoproteins 䡲 high density lipoprotein 䡲 glitazones
A
growing body of evidence indicates that high-density
lipoprotein (HDL) bears an inverse relationship to the
development of atherosclerotic coronary heart disease.1 The
antiatherogenic effects of HDL and apolipoprotein AI
(apoA-I, major protein of HDL) are mainly attributed to its
ability to: facilitate reverse cholesterol transport pathway,2,3
enhance fibrinolysis, and inhibit platelet aggregation,4 LDL
oxidation,5 endothelial transmigration of monocytes,6 and
proinflammatory cytokine-mediated expression of endothelial cell adhesion molecules.7
Glitazones (a class of thiazolidinediones agents such as
pioglitazone and rosiglitazone) are antihyperglycemic drugs
used in the treatment of type 2 diabetes. These oral antidiabetic agents reduce blood glucose levels by sensitizing
peripheral tissues to insulin.8 In addition to improving glycemic control, glitazones also influence plasma lipid and
lipoprotein levels.9 Treatment of type 2 diabetic patients with
pioglitazone consistently increased plasma HDL-cholesterol
and decreased triglycerides with generally minimal to no
effect on total and LDL-cholesterol.10 –13 Clinically, the effect
of pioglitazone on HDL-cholesterol (about 10% to 15%) is
greater than that observed with statins and several fibrates,
and just below that of niacin. Although the mechanisms are
not clearly understood, the binding and activation of nuclear
receptor peroxisome proliferator-activated receptor- ␥
(PPAR-␥) by glitazones has been suggested to explain their
effects on glucose and lipid metabolism.14 –16 PPARs
(PPAR-␣, -␥, -␦) are members of the nuclear receptor family,
and by ligand activation events, modulate transcription of
various genes involved in lipid metabolism and atherosclerosis (reviewed in references 17,18).
PPAR-␣ agonists (such as fibrates), through heterodimerizing with retinoid receptor and binding to PPAR response
element in the promoter region of apoA-I gene, were shown
to increase the transcription of human apoA-I.19 It was also
shown that the activation of PPAR-␣ regulates nuclear factor
kB (NFkB)-induced reduction in apoA-I and HDL-cholesterol.20 Although pioglitazone has been originally identified as
Original received January 7, 2005; final version accepted August 23, 2007.
From the Atherosclerosis Research Center, Southern California Institute for Research and Education, Department of Veterans Affairs Healthcare
System, Long Beach, and the Department of Medicine, University of California, Irvine.
Part of this work was presented in abstract form at the American Heart Association meeting, November, 2004, New Orleans, La.
V.S.K. and M.L.K. are senior coauthors of this paper.
Correspondence to Moti L. Kashyap, MD or Vaijinath S. Kamanna, PhD, Atherosclerosis Research Center, Department of Veterans Affairs Healthcare
System, University of California, Irvine, 5901 East Seventh Street, Long Beach, California 90822. E-mail moti.kashyap@med.va.gov or
vaijinath.kamanna@med.va.gov
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://atvb.ahajournals.org
2428
DOI: 10.1161/ATVBAHA.107.150193
Qin et al
high affinity ligand for PPAR-␥, an earlier report indicated
that pioglitazone serves as a weak human PPAR-␣ activator.21 Despite consistent beneficial effect of pioglitazone on
HDL levels, the hepatocellular mechanisms by which pioglitazone raise apoA-I and HDL concentrations are not clearly
understood. In this study, we examined the effect of pioglitazone on apoA-I de novo synthesis, secretion, and mRNA
expression, and the participation of PPAR-␣ in apoA-I
production in human hepatoblastoma cells (HepG2). Additional studies were performed to examine the effect of
pioglitazone HDL-apoA-I/cholesterol ester uptake by HepG2
cells to determine whether the rise in apoA-I/HDL observed
clinically with this agent could be a result of decreased
hepatic HDL removal.
Materials and Methods
Materials
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Tissue culture materials, media, and FBS were obtained from Sigma
Chemical Company. L-[4,5-3H]Leucine and [3H]cholesterol were
purchased from Amersham Corporation. HepG2 cells and human
macrophage THP-1 cell line were obtained from American Type
Culture Collection. The polyclonal antibodies for human apoA-I and
apoA-II were purchased from Boehringer Mannheim Biochemicals.
All other chemicals used were of analytical grade.
Studies on Secretion of ApoA-I in HepG2
HepG2 cells were grown in high-glucose DMEM (containing 10%
FBS, 1% glutamine-penicillin-streptomycin, and 1% fungizone) for
3 to 4 days to attain 75% to 80% confluence. Cells were incubated
with various amounts of pioglitazone (0 to 10 ␮mol/L) at 37°C for
48 hours. Culture medium was removed, the cell monolayer washed
with PBS, and collected for cellular protein measurement. A 50-␮L
sample of culture medium was used to measure apoA-I secreted into
the media by an enzyme-linked immunoassay using a human apoA-I
specific monoclonal antibodies, according to our previously described procedures.22 The concentration of apoA-I is expressed as ␮g
per mg of cellular protein.
Studies on De Novo Synthesis of ApoA-I
Studies examining the effect of pioglitazone on the de novo synthesis
of apoA-I by Hep-G2 cells were performed by measuring the
incorporation of radiolabeled leucine into apoprotein secreted into
the media.22 Hep-G2 cells were incubated with varying concentrations of pioglitazone (0 to 10 ␮mol/L) in DMEM media containing
10% FBS for 48 hours. The medium was replaced with leucine-poor
DMEM (5% leucine of normal media) without FBS containing the
corresponding amounts of pioglitazone and 3H-leucine (5 ␮Ci/mL)
and incubated for 18 hours at 37°C. The medium was collected and
used for immunoprecipitation using monospecific antibodies for
apoA-I. The incorporation of 3H-leucine into apoA-I was expressed
as cpm/mg cellular protein.
RT-PCR Analysis for ApoA-I mRNA Expression
Hep-G2 cells were incubated with various amounts of pioglitazone
(0 to 10 ␮mol/L) at 37°C for 24 hours. Culture medium was
removed, the cell monolayer washed with PBS and collected for total
RNA isolation. Cells were collected by trypsinization and total
RNAs were extracted using RNeasy mini kit (Qiagen). cDNA was
synthesized from 850 ng of total RNA in 20 ␮L using random
hexamers and Murine Moloney leukemia virus reverse transcriptase
(Life Technologies Inc). The apoA-I primers used were as follows:
sense, 5⬘-ATG AAA GCT GCG GTG CTG ACC-3⬘ and antisense,
5⬘-GGA GCT CTA CCG CCA GAA GGT G-3⬘. Semiquantitative
RT-PCR analysis was performed starting with first strand cDNA
from reverse transcription with 150 nmol/L of sense and antisense
primer in a final volume of 50 ␮L. Sense and antisense primer for
Pioglitazone Stimulates ApoA-I Production
2429
␤-actin (150 nmol/L) were also included in the reaction mixture.
PCR amplification products at different cycles were separated on
1.2% agarose gel and were visualized with ethidium bromide (cycle
19 was found be in linear phase). The band intensity was measured
using Eagle Eye gel documentation system.
Studies on the Participation of PPAR-␣ in ApoA-I
Expression and Secretion
The involvement of PPAR-␣ in pioglitazone-induced apoA-I mRNA
expression was examined using PPAR-␣ suppression in Hep G2 cells
with either a specific inhibitor of PPAR-␣ or small interference RNA
(siRNA). The same protocols described above in “RT-PCR analysis
for apoA-I mRNA expression” was used for studies with MK886, a
specific inhibitor of PPAR-␣.20 In brief, Hep-G2 cells were preincubated with MK886 (10 ␮mol/L) for 60 minutes. After the
pretreatment, pioglitazone was added to cells and incubated for 24
hours. Total RNA was isolated and used for apoA-I mRNA expression using RT-PCR analysis as described above.
Suppression of PPAR-␣ expression in HepG2 with siRNA was
accomplished as described by Shoda et al23 and according to the
transfection kit instructions. Cells (70% to 80% confluent) were
ransfected with GenEclipse siRNA Vector (Chemicon) using DNA/
Lipofectamine 2000 (Invitrogen) and distributed into 6-well plate.
After 24 hours, cells were treated with vehicle or pioglitazone
(10 ␮mol/L) for 24 hours. Total RNA and cell lysates were then
prepared, and mRNA levels of PPAR-␣ and medium apoA-1 protein
level were determined as described above.
Studies on ApoA-II Synthesis and
mRNA Expression
Studies examining the effect of pioglitazone on the de novo synthesis
of apoA-II by Hep-G2 cells was performed by measuring the
incorporation of radiolabeled leucine into apoprotein secreted into
the media, as described in apoA-I de novo synthesis. ApoA-II
mRNA expression measurement was done by real-time PCR.
Hep-G2 cells were incubated with various amounts of pioglitazone
(0 to 10 ␮mol/L) at 37°C for 24 hours. The cell monolayer was
washed with PBS and collected for total RNA isolation. Total RNAs
were extracted using RNeasy mini kit (Qiagen). cDNA was synthesized from 1.4 ␮g of total RNA in 20 ␮L using random hexamers
and Ommi-script R. Transciptase (Qiagen). The apoA-II primers
were purchased from SuperArray. The real-time PCR was performed
with the use of the PCR Master Mix RT2 SYBR Green /Fluorescein
(SupperArray). Sequence-specific amplification was detected with
an increased fluorescent signal of SYBR during the amplification
cycle. Amplification of the human GAPDH gene was performed in
the same reaction on all samples tested as an internal control for
variations in RNA amounts. ApoA-II mRNA levels were normalized
to GAPDH mRNA levels, and were presented as fold difference of
treated cells against untreated cells.
Studies on the Uptake of HDL-Protein
Studies examining the uptake by HepG2 cells were performed by
using radiolabeled HDL total protein or apoA-I–HDL.22 Radio
iodination of HDL total protein was carried out by incubating freshly
isolated HDL3 with carrier-free 125I.22 After the iodination, unreacted
125
I was removed by gel filtration followed by exhaustive dialysis
against PBS. Uptake studies were initiated by preincubating HepG2
cells with varying concentrations of pioglitazone (0 to 10 ␮mol/L)
for 48 hours at 37°C. The medium was replaced with fresh DMEM
containing fetal bovine albumin (5 mg/mL) and 125I-HDL (50 ␮g
protein/mL). After 16 hours of incubation at 37°C, cell monolayers
were washed thoroughly and digested with 1N sodium hydroxide
solution. An aliquot was used for radioactivity measurement. The
uptake of radiolabeled HDL-protein by HepG2 cells was expressed
in terms of cellular protein.
Uptake of 3H-Cholesterol Esters Labeled HDL
For these studies, radiolabeled HDL-cholesterol ester was prepared
by incubating 4 ␮Ci of [1a,2a(n)-3H] cholesterol with the serum
2430
Arterioscler Thromb Vasc Biol.
November 2007
lipoprotein particles) to efflux free cholesterol was measured using
[3H]cholesterol-labeled human macrophage THP-1 cells. Cholesterol efflux assay were initiated by incubating concentrated culture
medium with [3H]cholesterol-labeled THP-1 cells for 20 hours as
described previously.24 Quantitative analysis of the ability of HepG2
cell culture medium (in the presence or absence of pioglitazone) to
efflux cholesterol was performed by measuring the [3H]cholesterol
radioactivity appearing in the medium per milliliter of incubation
medium per mg of THP-1 cellular protein.
Statistical Analysis
Data presented are the mean⫾SE of 3 separate experiments done in
duplicate. Statistical significance was calculated by using the Student
t test, and a value of P⬍0.05 was considered significant.
Results
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Figure 1. Effect of pioglitazone on apoA-I secretion by HepG2
cells. Cells were incubated with pioglitazone (0 to 10 ␮mol/L) for
48 hours, culture medium was assayed for apoA-I by ELISA.
Statistical significance was compared with results of control.
Data are mean⫾SE of 3 experiments.
HDL fraction for 18 hours at 37°C (through LCAT enzyme reaction
mediated cholesterol-ester formation; 22). The [3H] cholesterol
ester-HDL was isolated by ultracentrifugation at d⫽1.210g/mL and
dialyzed extensively against 0.15 mol/L NaCl. Uptake studies were
performed by preincubating HepG2 cells pioglitazone (0 to
10 ␮mol/L) for 48 hours. Medium was removed, and fresh DMEM
containing 5 mg/mL FBA (fatty acid free) and [3H] cholesterol
ester–labeled HDL (50 ␮g HDL protein/mL) was added. Cells were
harvested 6 hours later, washed thoroughly, and digested with 1 mL
of 1N NaOH. Radioactivity was measured and expressed as cpm per
mg cellular protein.
Measurement of Cholesterol Efflux
Experimental protocols for these studies were exactly same as
described for apoA-I secretion studies. After the incubation of
HepG2 cells with pioglitazone, the medium was collected and used
for cholesterol efflux measurements.24 An aliquot of culture medium
(5 mL) was concentrated to 1 mL by lyophilization and dialyzed
against DMEM to remove excess salt present in the concentrated
sample. The ability of these media (containing secreted apoA-I
As shown in Figure 1, a significant increase in apoA-I secretion
by HepG2 cells was noted at 1.0 ␮mol/L pioglitazone (40% over
control), and pioglitazone at 10 ␮mol/L increased apoA-I
secretion by 44% as compared with control. The percent
increase in apoA-I secretion by pioglitazone (10 ␮mol/L) in
various experiments were 44%, 60%, and 52%. Data from the de
novo synthesis of apoA-I studies show that the incorporation of
radiolabeled leucine into apoA-I increased in a dose-dependent
manner by HepG2 cells incubated with pioglitazone (Figure 2).
Pioglitazone as low as 0.5 ␮mol/L increased apoA-I de novo
synthesis (19% over control), and the maximum effect was
observed at 10 ␮mol/L pioglitazone (45% increase compared
with control level, Figure 2).
As shown in representative RT-PCR, the incubation of
varying amounts of pioglitazone with HepG2 cells induced
dose-dependently the mRNA expression of apoA-I (Figure 3,
top panel). Quantitative analysis of apoA-I mRNA message
indicated that the treatment of HepG2 cells with pioglitazone
as low as 0.1 ␮mol/L concentration stimulated apoA-I mRNA
levels by 36% compared with control, the maximal effect
noted at 10 ␮mol/L by 59% compared with control (Figure 3,
lower panel). Beacuse PPAR␣ also regulates apoA-II expression, we examined whether pioglitazone affects apoA-II
synthesis and mRNA expression in HepG2 cells. The data
Figure 2. Effect of pioglitazone on the incorporation of [3H]leucine into newly synthesized apoA-I (A) and apoA-II (B) by HepG2 cells.
Cells were incubated with pioglitazone (0 to 10 ␮mol/L) for 48 hours, and 3H-leucine incorporation was done as described in Methods.
Data are mean⫾SE of 3 experiments.
Qin et al
Pioglitazone Stimulates ApoA-I Production
2431
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Figure 3. Effect of pioglitazone on apoA-I and A-II mRNA expression in HepG2 cells. Cells were incubated with pioglitazone (0 to
10 ␮mol/L) for 24 hours. A, ApoA-I mRNA by RT-PCR (upper panel, representative gel photograph; lower panel, quantitative analysis of
apoA-I mRNA. B, ApoA-II expression was measured by real-time PCR.
indicated that pioglitazone (0.5 to 10 ␮mol) also dosagedependently stimulated apoA-II de novo synthesis by 28% to
73% (Figure 2B) and apoA-II mRNA levels by 31% to 129%
(Figure 3B).
To determine whether pioglitazone increases HDL-cholesterol by decreasing HDL clearance, additional studies were
performed to assess the effect of pioglitazone on uptake of
HDL and its components by HepG2 cells. As shown in Figure
4, the preincubation of HepG2 cells with pioglitazone at
varying concentrations (5 to 10 ␮mol/L) did not alter the
uptake of either HDL-protein or HDL-CE.
The ability of pioglitazone-induced apoA-I– containing lipoprotein particles to efflux cholesterol was examined by using
[3H]cholesterol-labeled human macrophage cell line (THP-1
cells). Cholesterol efflux studies using conditioned medium
obtained from HepG2 cells treated with varying amounts of
pioglitazone (0.5 to 10 mmol/L) showed a dose-dependent
increase in cholesterol efflux by 10% to 39% compared with
control, as measured by the release of [3H]cholesterol from
THP-1 cells into the culture medium (Figure 5).
Activation of PPAR-␣ by agonists (such as fibrates) has
been previously shown to participate in increasing apoA-I
mRNA expression.19 In the present study, we have also
shown that fenofibrate increased apoA-I secretion by 68%
(apoA-I secretion as ␮ g/mg cell protein data: control⫽0.25⫾0.02, 10 ␮mol/L fenofibrate⫽0.42⫾0.05) and
mRNA expression by 73%. This served as a positive control
for PPAR␣-mediated effects on apoA-I expression relevant to
the current study. In addition to strong activation of PPAR-␥,
pioglitazone was also shown to activate human PPAR-␣.21
Using MK-886 (a specific inhibitor of PPAR-␣ activation),
we have examined whether pioglitazone-induced apoA-I
mRNA expression was mediated by activation of PPAR-␣.
As shown in Figure 6A and 6C, preincubation of HepG2 cells
with MK-886 almost completely blocked apoA-I mRNA
expression (92%) and secretion (80%) induced by pioglitazone. MK-886 in control cells did not alter apoA-I mRNA
levels (Figure 6A) or apoA-I secretion in the medium (Figure
6C). In additional experiments, we used gene silencing with
siRNA transfection to further confirm the participation of
PPAR-␣ in pioglitazone-induced apoA-I production. Using
cotransfection with GFP, we estimated that the transfection
efficiency was about 54%. As shown in Figure 6B, siRNA
transfection markedly reduced PPAR-␣ mRNA levels by
Figure 4. Effect of pioglitazone on 125IHDL3-protein and [3H]CE-labeled HDL
uptake by HepG2 cells. Cells were
preincubated with pioglitazone (0 to
10 ␮mol/L) for 48 hours, and uptake
studies were done as described in
Methods. A, 125I-HDL protein uptake.
B, [3H]CE-labeled HDL uptake. Data are
mean⫾SE of 3 experiments.
2432
Arterioscler Thromb Vasc Biol.
November 2007
pioglitazone treatment) transfected with sRNA. Furthermore,
pioglitazone-induced increase in the apoA-I secretion was
significantly abrogated in cells transfected with the PPAR-␣
siRNA complex (P⫽0.014; Figure 6D).
Discussion
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Figure 5. Effect of pioglitazone-induced apoA-I– containing particles from HepG2 cells to efflux cholesterol from THP1 macrophages. HepG2 cells were incubated with pioglitazone (0 to
10 ␮mol/L) for 48 hours, and medium was collected and used
for cholesterol efflux from THP1 cells as described in Methods.
Data are mean⫾SE of 3 experiments.
66% as compared with control vector transfection. PPAR-␣
mRNA levels were reduced by 69% when cells were transfected with PPAR␣ siRNA and subsequently treated with
pioglitazone, an effect similar to that of control cells (without
Several studies clearly established that pioglitazone significantly increased serum HDL-cholesterol and decreased triglycerides, and pioglitazone produced more favorable lipid
profiles than rosiglitazone in patients with type 2 diabetes.25–27
However, the effect of pioglitazone on apo-AI level is not clear.
In a small number of nondiabetic patients with low HDL-cholesterol and metabolic syndrome, Szapary et al have recently
shown that pioglitazone treatment for 6 weeks significantly
increased mean apo-AI by 6.8% and apo-AII by 7.7%; however,
by 12 weeks of treatment, only the change in apo-AII remained
significant.26 Further studies are required in large number of
patients to clearly understand the effect of pioglitazone on
apo-AI levels.
In this study, using HepG2 cell system, we have delineated
the effect of pioglitazone on various cellular processes
involved in HDL metabolism that in turn determine the
overall concentration of apoA-I/HDL mass. The data indicate
that pioglitazone by increasing apoA-I mRNA levels increased the de novo synthesis and secretion of apoA-I
particles in the culture medium. Additionally, we have shown
that the apoA-I– containing lipoprotein particles secreted in
the culture medium of HepG2 cells treated with pioglitazone
Figure 6. Involvement of PPAR-␣ in Pioglitazone-induced apoA-I production. A, Gel and quantitation of apoA-I mRNA in cells pretreated with MK-886. B, Gel and quantitation of PPAR-␣ mRNA in cells transfected with siRNA. C, ApoA-I secretion in cells preincubated with MK-886. D, ApoA-I secretion in cells transfected with siRNA.
Qin et al
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were able to significantly increase cholesterol efflux from
THP-1 macrophages, suggesting that these particles are
functionally active in initiating reverse cholesterol transport.
We have also shown that pioglitazone increases apoA-II
synthesis and mRNA expression in HepG2 cells. Pioglitazone
had no effect on HDL uptake, suggesting that pioglitazone
increases apoA-I/HDL by increasing apoA-I synthesis but not
HDL catabolism in HepG2 cells. These findings, at least in
part, define hepatic mechanisms of action of pioglitazone to
increase apoAI, apoA-II, and HDL seen in humans.26 However, as the findings of this study were derived from in vitro
studies using HepG2 cells (a hepatocarcinoma cell line but
not primary hepatocytes), real limitations exist in extrapolating in vitro findings of this study to in vivo conditions in
humans.
An increasing number of transcription factors have been
reported to be involved in the regulation of apoA-I gene
expression.28,29 These transcription factors include members
of a steroid/thyroid nuclear receptor superfamily, such as
hepatocyte nuclear factor-4␣ (HNF-4␣), apoA-I regulatory
protein-1 (ARP-1), and retinoid X receptor (RXR␣); the
HNF-3/forkhead family of transcription factors, such as
HNF-3␣/␤, early growth response factor (Egr-1), and transcription factor Sp1. PPAR-␣ activators (such as fibrates),
through heterodimerizing with retinoid receptor and binding
to PPAR response element in the promoter region of apoA-I
gene, were shown to increase the transcription of human
apoA-I.19 Recent data also indicated that inactivation of
nuclear factor-kB enhances the expression and secretion of
apoA-I from HepG2 cells through activation of PPAR-␣.20
However, the role of PPAR-␥ in apoA-I transcription is not
clearly understood. Troglitazone, a PPAR-␥ agonist, had no
effect on apoA-I mRNA transcription in Hep-G2 cells.30 Our
data also indicate that 15-dPGJ2, a natural ligand and activator of PPAR-␥, had no effect on apoA-I mRNA expression in
HepG2 cells (data not shown). Our findings and the current
literature point that PPAR-␣, but not PPAR-␥, regulates
apoA-I mRNA levels and subsequent apoA-I production.
Although pioglitazone is known to exhibit high-affinity
binding and activation of PPAR-␥, it is interesting to note that
pioglitazone also acts as a weaker activator for PPAR-␣.21
Sakamoto et al have shown that both pioglitazone and
rosiglitazone (but not troglitazone) transactivated PPAR-␣ by
5.4-fold and 4.2-fold over control, respectively, in COS-1
cells.21 Using competition-binding assays with radioligand, it
was shown that the activation of PPAR-␣ by pioglitazone was
attributable to direct binding of pioglitazone to PPAR-␣.21
Because PPAR-␣ regulates apoA-I transcription, we investigated whether the effect of pioglitazone on apoA-I mRNA
expression is mediated through PPAR-␣. Using specific
inhibitor and siRNA approaches, we have shown that inhibition of PPAR-␣ in Hep G2 cells significantly inhibited
pioglitazone-induced apoA-I production. These findings suggest that the activation of hepatic PPAR-␣ by pioglitazone
regulates apoA-I mRNA expression and subsequent apoA-I
protein synthesis and secretion. It is also important to note
that the effective concentrations of pioglitazone used in our
studies (0.1 to 10 ␮mol/L) are comparable to the plasma
Pioglitazone Stimulates ApoA-I Production
2433
levels observed in patients treated with pioglitazone,31 suggesting clinical relevance to the in vivo situation.
Although used for glycemic control, thiazolidinediones
should be considered also as part of dyslipidemia treatment in
diabetes, in combination with lipid regulating drugs including
statins, niacin, and fibrates. Whereas fibrates and statins act
via PPAR-␣ activation, niacin decreases apoA-I catabolism.22,32 This information is important in using combination
therapy using drugs with complementary mechanisms of
action to achieve aggressive HDL goals. Additional research
is needed to assess the additive efficacy of such combinations
not only on lipids, but also on reducing cardiovascular events
in the high risk population of diabetics beyond monotherapy.
In summary, we suggest that pioglitazone, at least in part
through PPAR-␣–mediated events, increases hepatic apoA-I
mRNA expression resulting in increased plasma levels of
physiologically active apoA-I/HDL particles. Pioglitazone,
by activating both PPAR-␥ and PPAR-␣, beneficially regulates both glucose and HDL levels. Additionally, our findings
may be useful in forming the rationale for combination
therapy using pioglitazone and other HDL raising agents with
different mechanisms to additively increase HDL levels in
diabetic patients.
Sources of Funding
This study was supported, in part, by Takeda Pharmaceuticals North
America Inc., and the Southern California Institute for Research and
Education.
Disclosures
None.
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Pioglitazone Stimulates Apolipoprotein A-I Production Without Affecting HDL Removal
in HepG2 Cells: Involvement of PPAR- α
Shucun Qin, Tianjiao Liu, Vaijinath S. Kamanna and Moti L. Kashyap
Arterioscler Thromb Vasc Biol. 2007;27:2428-2434; originally published online September 13,
2007;
doi: 10.1161/ATVBAHA.107.150193
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