© 1997 American Heart Association, Inc.
Articles |
Regulation of Lipoprotein Metabolism by Thiazolidinediones Occurs through a Distinct but Complementary Mechanism Relative to Fibrates
From the U.325 INSERM, Département d'Athérosclérose, Institut Pasteur, 1 Rue Calmette, Lille, France; Department of Biochemistry and Molecular Biology, University of Barcelona, Spain (J.P.-O.); Ligand Pharmaceuticals Inc., San Diego, Calif (M.R.B., J.R.P.).
Correspondence to Dr. Bart Staels, INSERM U.325, Institut Pasteur, 1, rue du Prof. Calmette, 59019 Lille Cédex, France.
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Abstract |
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Abstract Thiazolidinediones are antidiabetic agents, which not only improve glucose metabolism but also reduce blood triglyceride concentrations. These compounds are synthetic ligands for PPAR
, a transcription factor belonging to the nuclear
receptor subfamily of PPARs, which are important transcriptional
regulators of lipid and lipoprotein metabolism. The goal of
this study was to evaluate the influence of a potent thiazolidinedione,
BRL49653, on serum lipoproteins and to determine whether its
lipid-lowering effects are mediated by changes in the expression of key
genes implicated in lipoprotein metabolism. Treatment of
normal rats for 7 days with BRL49653 decreased serum
triglycerides in a dose-dependent fashion without affecting
serum total and HDL cholesterol and apolipoprotein (apo)
A-I and apo A-II concentrations. The decrease in
triglyceride concentrations after BRL49653 was mainly due
to a reduction of the amount of VLDL particles of unchanged lipid and
apo composition. BRL49653 treatment did not change
triglyceride production in vivo as analyzed
by injection of Triton WR-1339, indicating a primary action on
triglyceride catabolism. Analysis of the influence
of BRL49653 on the expression of LPL and apo C-III, two key players in
triglyceride catabolism, showed a dose-dependent increase
in mRNA levels and activity of LPL in epididymal adipose tissue,
whereas liver apo C-III mRNA levels remained constant. Furthermore,
addition of BRL49653 to primary cultures of differentiated adipocytes
increased LPL mRNA levels, indicating a direct action of the drug on
the adipocyte. Simultaneous administration of BRL49653 and
fenofibrate, a hypolipidemic drug that acts primarily on liver through
activation of PPAR
both decreased liver apo C-III and increased
adipose tissue LPL mRNA levels, resulting in a more pronounced lowering
of serum triglycerides than each drug alone. In conclusion,
both fibrates and thiazolidinediones exert a
hypotriglyceridemic effect. While fibrates act
primarily on the liver by decreasing apo C-III production,
BRL49653 acts primarily on adipose tissue by increasing lipolysis
through the induction of LPL expression. Drugs combining both PPAR
and activation potential should therefore display a more efficient
hypotriglyceridemic activity than either compound
alone and may provide a rationale for improved therapy for elevated
triglycerides.
Key Words: gene regulation • atherosclerosis • PPAR • triglycerides • hypolipidemic drugs
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thiazolidinediones, also termed glitazones, are antidiabetic agents that may be useful in the treatment of type II or non–insulin-dependent diabetes mellitus (NIDDM) (reviewed in 1 ). These compounds improve glucose metabolism by increasing peripheral insulin sensitivity, as demonstrated in animal models of insulin resistance such as the genetically obese ob/ob mice and Zucker fa/fa rats or genetically diabetic yellow KKA mice.2 3 4 Treatment of these animals with different thiazolidinediones, such as ciglitazone, troglitazone (CS-045), pioglitazone, and englitazone, results in markedly improved glucose tolerance.2 3 4 5 6 7 Similarly, administration of troglitazone to humans resulted in reduced insulin and glucose levels and improved insulin sensitivity, as measured by oral glucose tolerance test and euglycemic-hyperinsulinemic clamp studies.8 9 In addition to their effects on plasma glucose metabolism thiazolidinediones are also potent hypolipidemic agents, decreasing plasma triglyceride concentrations both in diabetic and normal rodents, primates, and humans.2 5 6 7 8 10
Although relatively little is known about the molecular mechanism
of action of these compounds, recent data using the thiazolidinedione
BRL49653 suggest that their effects may, at least in part, be mediated
through binding and activation of a specific transcription factor
called peroxisome proliferator activated receptor
(PPAR).11 PPAR belongs to the PPAR subfamily of
nuclear hormone receptors, which are ligand-activated
transcription factors (for review see 12 ). In contrast to
PPAR, which is predominantly expressed in tissues catabolizing high
amounts of fatty acids such as liver, heart, and brown adipose tissue,
PPAR is predominantly expressed in white adipose tissue in
rodents.13 14 15 16 17 PPARs bind as heterodimers with the retinoid
X receptor (RXR) to specific response elements termed PPREs in the
regulatory regions of target genes and subsequently alter their
transcription. The majority of the genes whose expression is under
control of PPARs, code for proteins involved in intra- and
extracellular lipid metabolism, such as the enzymes of the
peroxisomal and mitochondrial ß-oxidation pathways,18 19 20 21 22 23 24
3-hydroxy-3-methylglutaryl-coA synthase,25 adipocyte fatty
acid–binding protein aP2,14 acyl-coA
synthetase,26 and apolipoprotein (apo)(s) A-I, A-II, and
C-III.27 28 29 In fact, recent studies in our laboratory have
indicated that the lipid-lowering effects of the widely used
hypolipidemic drugs, fibrates, are mediated through activation of PPAR
and subsequent changes in the expression of genes involved in
lipoprotein metabolism in rodents as well as in humans (for
review see 12 ). Interestingly, our findings indicate that
fibrates such as fenofibrate have a predominant effect on lipoprotein
gene expression in liver but not in other tissues such as intestine or
adipose tissue. For instance, the effects of fibrates on plasma HDL
concentrations are at least partially due to a PPAR-mediated
transcriptional regulation of the major HDL apolipoproteins apo A-I,
apo A-II, and apo A-IV in liver but not in
intestine.27 28 30 31 Similarly, the
hypotriglyceridemic action of fibrates can be
attributed to alterations in liver gene expression, leading to a
decreased production of the VLDL particles of different
composition, which renders them more susceptible to lipolysis and
subsequent clearance from plasma (reviewed in 12 ). This
latter effect is most likely mediated by lipoprotein lipase (LPL) and
apo C-III, two key players displaying antagonistic
properties in triglyceride metabolism. Indeed
fibrates have been shown to regulate the expression of both genes (LPL
induction, apo C-III reduction) in liver but not in other tissues such
as intestine (apo C-III), heart (LPL), or adipose tissue
(LPL).32 33
In view of the crucial role of PPARs in regulating plasma lipoprotein
metabolism and the recent identification of
thiazolidinediones as synthetic PPAR ligands,11 we
decided to study in more detail the effects of potent thiazolidinedione
BRL49653 on serum lipoprotein and apolipoprotein
metabolism. Since the lipid-lowering activity of BRL49653
is observed both in normal and insulin-resistant
rats,34 we decided to study its effects in normolipidemic
rats, which allows comparison to the effects previously described for
fenofibrate. Our results show that BRL49653 treatment lowers serum
triglyceride-rich lipoproteins without affecting HDL
concentrations. Moreover, simultaneous treatment with
BRL49653 and fenofibrate results in a more pronounced
triglyceride-lowering activity than with either agent used
alone. In contrast to fibrates, which act primarily on the liver by
decreasing apo C-III expression,32 33 the
hypotriglyceridemic action of BRL49653 is mediated
by a specific action on adipose tissue via an enhancement of LPL
expression, whereas no effects are observed on liver apo C-III gene
expression. These findings provide a mechanistic basis for the
hypotriglyceridemic action of BRL49653
treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
BRL49653 was synthesized at Ligand Pharmaceuticals. Fenofibrate and triton WR-1339 (Tyloxapol®) were obtained from Sigma and carboxymethylcellulose from Serva (Heidelberg, Germany).
Animals
Male Sprague-Dawley rats were randomized to treatment groups and
treated daily intragastrically for the indicated periods of time with
BRL49653 and/or fenofibrate suspended in 1% carboxymethylcellulose at
the indicated doses. Control animals received an equal volume (5
ml/kg/d) of carboxymethylcellulose solution. At the end
of the experiments animals were weighed and sacrificed by
exsanguination while ether anesthesia was administered.
Blood was collected and serum separated and used within 1 week for
analysis of lipids, lipoproteins, and apolipoproteins. Liver
and epididymal fat pads were removed immediately, weighed, and frozen
in liquid nitrogen.
Serum Lipid, Apolipoprotein, and Lipoprotein Measurements
Serum lipoprotein lipid concentrations (total and free
cholesterol, triglycerides, and phospholipids)
were measured colorimetrically using enzymatic test
kits from Boehringer Mannheim (Mannheim, Germany). Serum HDL
cholesterol content was determined after precipitation of
apo B-containing lipoproteins with phosphotungstic acid/Mg
(Boehringer Mannheim). Serum levels of rat apo A-I and apo A-II
were measured by an immunoephelometric assay using specific polyclonal
antibodies.
The lipoprotein fractions (VLDL, d<1.006; IDL+LDL, d=1.006–1.063; HDL, d=1.063–1.21 g/mL) were isolated by sequential ultracentrifugation of pooled rat serum.35 Each fraction was further purified by a second ultracentrifugation at the same density intervals before analysis. After extensive dialysis at 4°C against 10 mmol phosphate-buffered saline (PBS) at pH 7.2 containing 10 µmol/L EDTA, the protein concentration of each lipoprotein fraction was determined by the method of Lowry et al.36
For fast protein liquid chromatography (FPLC) size fractionation of lipoproteins, 300 µg of serum lipoprotein protein (d<1.21 g/mL) isolated from individual rats was injected on a Sepharose 6HR 10/30 prepacked column (Pharmacia, Uppsala, Sweden) and eluted at a constant flow rate of 0.2 mL/min with PBS pH 7.2. The effluent was monitored at 280 nm, collected in 0.3-mL fractions, and cholesterol and triglyceride concentrations were determined in 0.1 mL of each fraction.
The distribution of lipoproteins in serum from individual rats was analyzed by nondenaturing discontinuous gradient polyacrylamide gel electrophoresis (Lipofilm kit, Sebia, Issy-les-Moulineaux, France). Briefly, 5 µL of Sudan Black prestained samples were electrophoresed at 10°C for 45 minutes at a constant voltage of 170 V in a Tris-barbital buffer, pH 8.3. The wet gels were immediately scanned using the Biorad Gel-Doc 1000 system.
The apolipoprotein composition of isolated lipoproteins was analyzed by nonreducing SDS-PAGE as described in an earlier study.37 Protein samples (15 µg) were heat denatured and loaded on 3% to 19% gradient gels, separated by electrophoresis at 150 V for 45 minutes, and visualized by Coomassie brilliant blue staining. The distribution of the apo C-II and apo C-III subspecies was analyzed by isoelectric focusing gel electrophoresis. VLDL proteins (200 µg) were precipitated with a mixture of acetone and ethanol (1:1) and delipidated with diethylether at -20°C.38 The delipidated proteins were then electrophoresed on a polyacrylamide gel at 4°C for 30 minutes at 100 V, 14 hours at 250 V, and 1 hour at 1000 V. Gel preparation, fixing, and protein staining were performed as described by Kane.39
Determination of in vivo Triglyceride Synthesis
Rats (n=3/group) were treated for 7 days with BRL49653 (10
mg/kg/d) or vehicle. At the end of the treatment period
rats were injected in the caudal vein with a 20% w/v Triton WR-1339
solution at 500 mg/kg of body weight. Blood was collected with
the rats under ether anesthesia just prior to injection and
at 1 and 2 hours after injection. Serum triglycerides were
determined subsequently.
Measurement of Tissue LPL Activity
LPL was measured in extracts from epididymal adipose and
gastrocnemius muscle tissue according to the procedure of Ramirez et
al.40 One unit of enzyme activity was defined as the
amount of enzyme that releases 1 µmole of oleate per minute at
25°C.
Isolation of Primary Adipocytes
Primary adipocytes were isolated from epididymal fat pads
of male Sprague-Dawley rats exactly as described by Hajduch et
al.41
RNA Analysis
RNA was isolated from liver and epididymal adipose tissue
by the acid guanidinium thiocyanate/phenol/chloroform
method.42 Northern and dot blot analysis of total
cellular RNA was performed as described by Staels et al.31
Rat apo A-I, apo A-II, apo A-IV, apo C-III, acyl CoA oxidase (ACO), and
human LPL cDNA clones were used as probes.31 33 43 cDNA
clones for ß-actin44 and 36B445 (encoding
the human acidic ribosomal phosphoprotein PO46 ) were used
as control probes. All probes were labeled by random primed labeling
(Boehringer Mannheim). Filters were hybridized to
1.5x106 cpm/mL of each probe as described by Staels et
al.31 They were washed once in 500 mL of 75
mmol/L NaCl, 7.5 mmol/L sodium citrate, and 0.1%
SDS for 10 minutes at room temperature and twice for 30 minutes at
65°C and subsequently exposed to X-ray film (X-OMAT-AR, Kodak).
Autoradiograms were analyzed by quantitative
scanning densitometry (Biorad GS670 Densitometer) as described by
Staels et al.31
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Results |
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Treatment with BRL49653 Decreases Serum Triglyceride and VLDL Concentrations without Changing VLDL Composition
In order to study the effects of BRL49653 on serum lipids and lipoproteins, rats were treated with different doses of BRL49653 previously shown to affect lipid metabolism in the rat.34 Administration of BRL49653 for 7 days did not change body or liver weights (Table 1
).
Epidydimal fat pad weights increased in a dose-dependent fashion, an
observation which is consistent with previously described
inductive effects of thiazolidinediones on adipocyte differentiation
and adipose tissue mass.47 48 49 50 51 Serum total, free, and HDL
cholesterol concentrations remained constant, whereas in
fed rats serum triglycerides decreased in a dose-dependent
manner, dropping to less than 50% of that of control rats at the
highest dose tested (5 mg/kg/d) (Table 2). Serum glucose concentrations did not
change after BRL49653 treatment (not shown), which is in agreement with
previous studies showing that thiazolidinediones do not exert a
hypoglycemic action in the normoglycemic, nondiabetic
rat.2 34 Separation of the different lipoprotein fractions
by electrophoresis followed by lipostaining (Fig 1A) and gel filtration
chromatography (Fig 1B) indicated that the changes in
triglycerides after BRL49653 were due to a decrease in VLDL
concentrations in serum. The decrease in VLDL was accompanied by an
increase in IDL-LDL lipoproteins (Fig 1A). By contrast, serum HDL
cholesterol concentrations remained fairly constant after
BRL49653 treatment (Fig 1A and C, Table 2).
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To determine whether BRL49653 treatment affected lipoprotein
composition, rats were treated for 14 days with BRL49653 at doses from
5-20 mg/kg/d. Serum lipoproteins were separated by
sequential ultracentrifugation and their composition
was analyzed next and expressed as a relative percentage (Table 3). Compared with control, VLDL particles
isolated from rats treated with BRL49653 displayed a similar
cholesterol, triglyceride, phospholipid, and
protein content both at a dose of 5 mg/kg/d as well as at
doses up to 20 mg/kg/d (Table 3). In contrast, the
relative triglyceride content of particles flotating in the
density interval between 1.006 and 1.063 (IDL+LDL) decreased by 25%
after BRL49653 at a dose of 5 mg/kg/d and up to 50% at
20 mg/kg/d (Table 3). The relative decrease in
triglyceride content of the IDL+LDL particles was
compensated by an enrichment in phospholipids and, to a lesser extent,
in total cholesterol (Table 3). Finally, although HDL
triglycerides decreased after BRL49653, overall HDL
composition was not greatly affected by BRL49653 treatment (Table 3).
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Next, the apolipoprotein composition of the different lipoprotein
fractions was analyzed by SDS-PAGE. Compared with control rats,
rats treated with BRL49653 did not show major changes in either VLDL,
IDL+LDL, or HDL apolipoprotein distribution (Fig 2). In addition, comparison of the
relative content of apo C-I, apo C-II, and apo C-III in VLDL by
isoelectric focusing analysis did not show major changes
between BRL49653 rats and control rats (data not shown). Altogether,
these results indicate that BRL49653 lowers serum
triglycerides by decreasing the number of VLDL particles
without changing their composition in lipids or apolipoproteins.
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BRL49653 Treatment Does Not Change the Rate of Triglyceride
Secretion
Next, the mechanism behind the
hypotriglyceridemic effect of BRL49653 was studied.
First, we analyzed whether BRL49653 treatment affects the
production of triglycerides in vivo. Injection with
Triton WR-1339 blocks the clearance of triglyceride-rich
lipoproteins by inhibiting their lipolytic degradation52
and thus allows an indirect measurement of triglyceride
secretion rates by the measurement of serum
triglycerides.53 When rats treated for 7 days
with BRL49653 (10 mg/kg/d) or vehicle were injected with
Triton WR-1339 serum triglyceride concentrations increased
in a linear fashion (correlation coefficients of 0.977 and 0.967 for
control and BRL49653-treated rats respectively) (Fig 3). Calculation of the slopes of the
curves indicated that the triglyceride secretion rates were
not significantly different between control and BRL49653-treated rats
(520±46 and 606±49 mg/dL/h, respectively), indicating that
BRL49653 treatment does not act by changing triglyceride
production. Therefore, the changes in triglyceride
levels produced by BRL49653 must result from the alteration of
triglyceride removal rate.
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BRL49653 Treatment Increases Adipose Tissue LPL Expression, without
Changing Liver Apolipoprotein Gene Expression
These results suggested that the
hypotriglyceridemic action of BRL49653 is mediated
by changes in the catabolism of triglyceride-rich
lipoproteins rather than their production. Therefore, the
influence of BRL49653 on the expression of two major genes involved in
plasma triglyceride catabolism, that is, adipose tissue LPL
and liver apo C-III, was studied next. Treatment with BRL49653
increased epididymal adipose tissue LPL mRNA levels more than twofold
at doses of 5 and 10 mg/kg/d (Fig 4A and
B). The increase in LPL gene expression
after BRL49653 was paralleled by a similar increase in LPL activity
in epididymal adipose tissue (Fig 4A). Interestingly, LPL activity in
skeletal muscle, the other major LPL-expressing tissue, did not change
significantly after BRL49653 (10 mg/kg/d for 7 days)
treatment (control 8±1; BRL49653 10±1 mU/g of tissue), indicating
that BRL49653 acts only on tissues expressing PPAR.14
In order to determine whether BRL49653 treatment induces LPL expression
through a direct action on the adipocyte, its effects on LPL gene
expression were studied next in primary cultures of differentiated
epididymal adipocytes. Addition of BRL49653 to the culture medium
resulted in a more than twofold increase in LPL mRNA levels (Fig 4C).
By contrast to LPL, treatment of rats for 7 days with BRL49653 did not
change liver apo C-III mRNA levels even at doses up to 20
mg/kg/d (control 100±29; BRL49653 1
mg/kg/d, 105±24; BRL49653 2 mg/kg/d,
110±16; BRL49653 5 mg/kg/d, 111±21; BRL49653 10
mg/kg/d, 99±13; BRL49653 20 mg/kg/d,
96±12).
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Since other PPAR activators, such as the fibrates
fenofibrate, clofibrate, and gemfibrozil as well as fish oil derived
n-3 polyunsaturated fatty acids, have major effects on HDL
apolipoprotein expression both in rodents30 31 54 and in
humans,27 28 the effects of BRL49653 on HDL apolipoprotein
expression was investigated next. Treatment of adult male rats with
increasing doses of BRL49653 did not result in any significant changes
in liver apo A-I, apo A-II, and apo A-IV or serum apo A-I and apo A-II
concentrations (Fig 5). These results
indicate that, in contrast to fibrates, the thiazolidinedione BRL49653
does not have major effects on any of the hepatic apolipoprotein
parameters tested but increases the expression and activity
of adipose tissue LPL.
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Simultaneous Treatment with BRL49653 and Fenofibrate
Have Combined Effects on Liver Apolipoprotein and Adipose Tissue LPL
Gene Expression
From these data it is evident that although treatment with
BRL49653 and fenofibrate both decrease serum triglyceride
concentrations, the mechanism of action of each compound is different.
Hence it was hypothesized that both compounds may have complementary
effects. Therefore, the effects of BRL49653 and fenofibrate either
alone or together on serum triglyceride concentrations were
analyzed. When compared with the pretreatment
triglyceride concentrations, treatment with BRL49653 or
fenofibrate resulted in a decrease in serum triglycerides,
whereas no changes were observed in the sham-treated rats (Fig 6). Interestingly, treatment with
BRL49653 and fenofibrate together resulted in a significantly more
pronounced lowering of triglyceride concentrations compared
with that of each drug alone (Fig 6).
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These results with BRL49653 and fenofibrate suggested combined
effects of these drugs together on liver apolipoprotein and adipose
tissue LPL gene expression. Indeed, whereas treatment with BRL49653
alone did not affect the expression of any of the apolipoproteins
analyzed in liver (Fig 7), LPL
mRNA levels increased to a comparable extent (2-fold) as in Fig 4
(data
not shown). In contrast, fenofibrate treatment alone provoked
significant decreases in liver apo A-I, apo A-II, apo A-IV, and apo
C-III mRNA levels (Fig 7) without changing adipose tissue LPL mRNA
levels, thereby confirming previous observations.31 32 33
Treatment with fenofibrate and BRL49653 together resulted in a similar
decrease in liver apolipoprotein mRNA levels (Fig 7) and increase in
adipose tissue LPL mRNA levels (2-fold) as treatment with fenofibrate
and BRL49653 alone, respectively.
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Finally, the effects of BRL49653 treatment were investigated on the
expression of the ACO gene, whose regulation by fibrates is under
control of the PPAR isoform.55 Treatment with
fenofibrate either alone or in combination with BRL49653 resulted in a
more than 10-fold induction of liver ACO mRNA levels (Fig 7), results
which confirm previous observations in rats.33 In
contrast, administration of BRL49653 did not change liver ACO mRNA
levels (Fig 7), even when given for 14 days at doses of 10 and 20
mg/kg/d (control 100%±32, BRL49653 10
mg/kg/d, 103%±46, BRL49653 20 mg/kg/d,
86%±39). These data show that treatment with BRL49653 does not result
in any significant activation of PPAR in the liver, even at doses
higher than required to lower serum triglycerides, further
indicating that the triglyceride-lowering activity of
BRL49653 occurs independently of PPAR activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most frequent lipid abnormality in patients with NIDDM is hypertriglyceridemia, which puts them at increased risk for coronary heart disease. Therefore, pharmacologic treatment of these patients should not only aim at normalizing parameters of glucose metabolism but also at correcting lipid abnormalities. Current treatment of the hyperlipidemia in NIDDM involves the use of classical hypolipidemic drugs such as HMG-CoA reductase inhibitors and fibrates, but these compounds do not have any effect on glucose metabolism.56 In contrast, thiazolidinediones are antidiabetic drugs that not only improve peripheral insulin sensitivity and decrease glucose intolerance in diabetic humans and animals (for review see 1 ) but also lower plasma triglycerides. We decided to study the effects of thiazolidinedione BRL49653 on lipoprotein metabolism in normolipidemic, normoglycemic rats, which allows comparison of its mechanism of action to the well-described effects of fibrates in this animal model (reviewed in 12 ).
Our results show that treatment with BRL49653 decreases serum triglyceride concentrations in normal rats without changing plasma glucose concentrations, thereby confirming previous observations with pioglitazone and BRL49653.2 34 This decrease is mainly reflected by a decrease in triglycerides contained in the VLDL fraction (this study and 57 58 ), due to a reduced number of VLDL particles with unchanged lipid and apolipoprotein composition (this study). By contrast, the composition of particles in the IDL+LDL range has a lowered triglyceride content, which is compensated for by an increased phospholipid and, to a lesser extent cholesterol, content. These changes after BRL49653 treatment are indicative of an increased lipolysis of triglycerides in plasma lipoproteins leading to an accelerated conversion into IDL and LDL particles rather than a reduced hepatic VLDL production. This is corroborated by the fact that in vivo triglyceride secretion rates were similar in BRL49653-treated and control animals. In contrast, the decrease in triglycerides observed after treatment with fibrates and other peroxisome proliferators is associated with a reduced hepatic VLDL secretion.59 60 61 62 63 Furthermore, fenofibrate treatment does not change the lipid composition of VLDL or IDL+LDL lipoproteins (data not shown), suggesting that these drugs have only minor effects on VLDL lipolysis. Therefore, although both BRL49653 and fenofibrate treatment results in reduced plasma triglyceride concentrations, they appear to operate through distinct mechanisms. This is supported by the observation that simultaneous administration of BRL49653 and fenofibrate results in a more pronounced plasma triglyceride lowering than with either drug alone.
These observations prompted us to study the molecular mechanism of BRL49653 action and to compare it with fenofibrate, the most potent fibrate currently used for the treatment of hyperlipidemia. Since treatment of normal rats with BRL49653 appeared to affect triglyceride catabolism rather than triglyceride production, we analyzed its effects on the expression of two key genes in triglyceride catabolism, LPL and apo C-III. Whereas LPL, after activation by its co-factor apo C-II, promotes the hydrolysis and removal of triglyceride-rich lipoproteins from plasma, apo C-III antagonizes both these activities (for review see 64 ). In contrast to treatment with fibrates, which markedly decrease liver apo C-III mRNA levels,29 33 treatment with BRL49653 did not change liver apo C-III gene expression nor did it change the relative content of apo C-III and apo C-II in VLDL. By contrast, LPL mRNA levels and activity increased in adipose tissue after BRL49653 treatment, whereas previous studies with fibrates showed lack of regulation of LPL adipose tissue in rats as well as in humans.32 63 Altogether, the increased LPL activity observed in this study most likely explains the enhanced triglyceride removal that occurs after thiazolidinedione treatment such as that previously described for troglitazone.58
Interestingly, the induction of adipose tissue LPL expression is accompanied by an increase in adipose tissue mass. Several studies using cell model systems of adipocyte differentiation such as the 3T3-L1, 3T3-F442A, and Ob 1771 cell lines showed that different thiazolidinediones enhance adipocyte differentiation in vitro.47 48 49 50 51 Furthermore, differentiation is accompanied by the induction of the expression of adipocyte-specific genes such as adipsin and aP2, as well as genes involved in glucose transport such as GLUT-1 and GLUT-4, and fatty acid metabolism such as LPL and acyl-CoA synthetase.48 50 However, the induction of LPL mRNA levels by BRL49653 in fully differentiated primary adipocytes indicates that the increase in LPL expression after thiazolidinedione treatment is a primary action of these drugs and not merely secondary to its effects on adipocyte differentiation.
Although thiazolidinediones may influence protein
phosphorylation activities as well as cellular
Ca++-uptake,9 65 66 67 it is likely that most if
not all of their antidiabetic actions at the molecular level are
mediated via activation of the transcription factor PPAR. More
specifically, BRL49653 has been shown to be a high-affinity synthetic
ligand for PPAR.11 Furthermore, the adipose-specific
PPAR2 isoform has been shown to play a crucial role in
adipogenesis.15 PPAR2 furthermore regulates the
expression of adipose genes such as the aP2 and phosphoenolpyruvate
carboxykinase (PEPCK) genes via a PPRE in its
promoter.14 68 Similarly, we recently identified a PPRE in
the human LPL gene promoter suggesting that the induction of adipose
tissue LPL gene expression after BRL49653 is mediated via PPAR. In
addition to a direct effect on LPL gene expression, BRL49653 treatment
may also result in an increased insulin responsiveness of adipose
tissue, which may potentiate its effects on LPL gene expression in fed
animals.
In contrast to fenofibrate, treatment with BRL49653 does not increase
liver weight. In addition, at doses exceeding those needed to decrease
serum triglycerides (up to 20 mg/kg/d during
14 days; data not shown; Fig 7), BRL49653 has no effect on liver apo
C-III mRNA levels, nor on the expression of the ACO gene, whose
regulation is under strict control of PPAR.55 These
data suggest that, in contrast to fenofibrate, BRL49653 has no or only
very little PPAR-activating potential in vivo, and consequently does
not provoke peroxisome proliferation in rodents.
In addition, whereas fenofibrate is a very potent hypocholesterolemic drug in rodents,31 total and HDL cholesterol concentrations remain unchanged with BRL49653 treatment, data which are in line with previous studies using pioglitazone in the KKAy mouse, a model for NIDDM, as well as in normal rats.57 69 These data indicate that, at least in the normal rat, lowering of serum triglycerides due to increased LPL expression in adipose tissue, does not have major effects on HDL metabolism. Since in rodents serum cholesterol is transported mainly in the HDL fraction, the unchanged serum cholesterol levels after BRL49653 can be explained by the unaltered expression of its major apolipoproteins, apo A-I, apo A-II, or apo A-IV, in rat liver. Again, these effects of BRL49653 are in contrast to those of fibrates, which have important effects on the expression of these genes in liver.30 31
In conclusion, the results of this study show that treatment with
thiazolidinedione BRL49653 decreases serum triglyceride
concentrations by enhancing serum triglyceride removal. In
contrast to fibrates, which decrease liver VLDL and apo C-III
production, enhanced lipolysis after BRL49653 is due at least
in part to the induction of LPL expression in adipose tissue. BRL49653,
which activates PPAR, acts therefore through a mechanism
distinct from that of fibrates, which primarily activate the
PPAR isoform and regulate apo C-III and HDL apolipoprotein gene
expression in liver. The additional effects of fenofibrate and BRL49653
on plasma triglycerides indicate that drugs that combine
PPAR and activation are extremely efficient
triglyceride-lowering agents and may be useful in the
treatment of different forms of
hypertriglyceridemia such as that found in
type II diabetes and familial combined
hypertriglyceridemia. In addition, because
of the combined action mechanisms of such compounds, it is conceivable
that lower doses can be used, thereby limiting potential undesirable
side effects such as increased liver (seen after fenofibrate treatment)
and adipose tissue (seen after BRL49653 treatment) weights.
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Acknowledgments |
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This work was supported by grants from INSERM, CNRS, Fondation pour le Recherche Médicale and the European Community (BIOMED PL-921243), and a grant (PB92-9548) from DGICYT, Ministeria de Educacion y Ciencia, Spain. We thank D. Cayet, O. Vidal, B. Derudas, P. Poulain, and R. Saladin for excellent technical assistance and M. Guerre-Millo for helping us to establish the primary adipocyte cultures. J.A. is a Directeur de Recherche and B.S. a Chargé de Recherche of the CNRS.
Received November 10, 1996; accepted March 1, 1997.
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A. Soria, C. Bocos, and E. Herrera Opposite metabolic response to fenofibrate treatment in pregnant and virgin rats J. Lipid Res., January 1, 2002; 43(1): 74 - 81. [Abstract] [Full Text] [PDF]
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N. D. Oakes, P. G. Thalén, S. M. Jacinto, and B. Ljung Thiazolidinediones Increase Plasma-Adipose Tissue FFA Exchange Capacity and Enhance Insulin-Mediated Control of Systemic FFA Availability Diabetes, May 1, 2001; 50(5): 1158 - 1165. [Abstract] [Full Text]
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Z. Chen, S. Ishibashi, S. Perrey, J.-i. Osuga, T. Gotoda, T. Kitamine, Y. Tamura, H. Okazaki, N. Yahagi, Y. Iizuka, et al. Troglitazone Inhibits Atherosclerosis in Apolipoprotein E-Knockout Mice : Pleiotropic Effects on CD36 Expression and HDL Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 372 - 377. [Abstract] [Full Text] [PDF]
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E. Raspé, L. Madsen, A-M. Lefebvre, I. Leitersdorf, L. Gelman, J. Peinado-Onsurbe, J. Dallongeville, J-C. Fruchart, R. Berge, and B. Staels Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPAR{alpha} activation J. Lipid Res., November 1, 1999; 40(11): 2099 - 2110. [Abstract] [Full Text]
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S. Sreenan, S. Keck, T. Fuller, B. Cockburn, and C. F. Burant Effects of troglitazone on substrate storage and utilization in insulin-resistant rats Am J Physiol Endocrinol Metab, June 1, 1999; 276(6): E1119 - E1129. [Abstract] [Full Text] [PDF]
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Y. Andersson, Z. Majd, A.-M. Lefebvre, G. Martin, A. V. Sechkin, V. Kosykh, J.-C. Fruchart, J. Najib, and B. Staels Developmental and Pharmacological Regulation of Apolipoprotein C-II Gene Expression : Comparison With Apo C-I and Apo C-III Gene Regulation Arterioscler Thromb Vasc Biol, January 1, 1999; 19(1): 115 - 121. [Abstract] [Full Text] [PDF]
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S. Ranganathan and P. A. Kern Thiazolidinediones Inhibit Lipoprotein Lipase Activity in Adipocytes J. Biol. Chem., October 2, 1998; 273(40): 26117 - 26122. [Abstract] [Full Text] [PDF]
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H. Nishizawa, K. Yamagata, I. Shimomura, M. Takahashi, H. Kuriyama, K. Kishida, K. Hotta, H. Nagaretani, N. Maeda, M. Matsuda, et al. Small Heterodimer Partner, an Orphan Nuclear Receptor, Augments Peroxisome Proliferator-activated Receptor gamma Transactivation J. Biol. Chem., January 4, 2002; 277(2): 1586 - 1592. [Abstract] [Full Text] [PDF]
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C. Wolfrum, C. M. Borrmann, T. Borchers, and F. Spener Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha - and gamma -mediated gene expression via liver fatty acid binding protein: A signaling path to the nucleus PNAS, February 27, 2001; 98(5): 2323 - 2328. [Abstract] [Full Text] [PDF]
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