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Vol. 59, Issue 2, 170-176, February 2001
Department of Integrative Biology and Pharmacology (P.D., S.B., G.S.), University of Texas School of Medicine, Houston, Texas; Department of Medicine (R.E.), University of Colorado School of Medicine, Denver, Colorado; Institut National de la Sante et de la Recherche Medicale (N.H., C.F.), Institut Pasteur, Lille, France; Ligand Pharmaceuticals (D.C., K.O., M.L., R.H.), San Diego, California; Department of Biochemistry and Molecular Biology (J.P.-O.), University of Barcelona, Spain; and Institut de Génétique et Biologie Moleculaire et Cellulaire (J.A.), Institut National de la Sante et de la Recherche Medicale/Centre National de la Recherche Scientifique/ULP, Illkirch, France
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Hypertriglyceridemia is a frequent complication accompanying the treatment of patients with either retinoids or rexinoids, [retinoid X receptor (RXR)-selective retinoids]. To investigate the cellular and molecular basis for this observation, we have studied the effects of rexinoids on triglyceride metabolism in both normal and diabetic rodents. Administration of a rexinoid such as LG100268 (LG268) to normal or diabetic rats results in a rapid increase in serum triglyceride levels. LG268 has no effect on hepatic triglyceride production but suppresses post-heparin plasma lipoprotein lipase (LPL) activity suggesting that the hypertriglyceridemia results from diminished peripheral processing of plasma very low density lipoproteins particles. Treatment of diabetic rats with rexinoids suppresses skeletal and cardiac muscle but not adipose tissue LPL activity. This effect is independent of changes in LPL mRNA. In C2C12 myocytes, LG268 suppresses the level of cell surface (i.e., heparin-releasable) LPL activity without altering LPL mRNA. This effect is very rapid (t1/2 = 2 h) and is blocked by the transcriptional inhibitor actinomycin D. These studies demonstrate that RXR ligands can have dramatic effects on the post-translational processing of LPL and suggest that skeletal muscle may be an important target of rexinoid action. In addition, these data underscore that the metabolic consequences of RXR activation are distinct from either retinoic acid receptor or peroxisome proliferator-activated receptor activation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Retinoids,
naturally occurring and synthetic derivatives of vitamin A, are nuclear
receptor ligands that have therapeutic applications in a variety of
dermatologic and oncologic diseases. Retinoids are morphogens that
regulate the growth and differentiation of embryonic cells. In addition
to these effects on cell differentiation, retinoids also can have
dramatic effects on serum lipid metabolism. Administration of retinoids
to both experimental animals (Gerber and Erdman, 1981b
; McMaster et
al., 1989; Oliver and Rogers, 1993; Standeven et al., 1996) and humans
(Lyons et al., 1982; Bershad et al., 1985) often results in increases
in serum triglyceride levels. Although the modest hypertriglyceridemia
most frequently induced by retinoid therapy may be innocuous, the
extreme elevations occasionally encountered in retinoid-treated
patients can precipitate pancreatitis (McCarter and Chen, 1992). For
this reason understanding of the cellular and molecular basis of
retinoid-induced hypertriglyceridemia will aid in efforts to develop
retinoids suitable for prolonged therapy.
The hypertriglyceridemia induced by retinoic acid isomers (either
all-trans-RA or 13-cis-RA) is due to alterations
in several aspects of serum triglyceride metabolism. In fasted animals,
plasma triglyceride levels represent an equilibrium between the rate of
hepatic secretion of very-low-density lipoproteins (VLDL), and the rate
of their clearance. VLDL particles are cleared by lipoprotein lipase
(LPL) activity present in the tissue vascular beds and hepatic lipase
present in the liver. In rats, retinoic acid-induced
hypertriglyceridemia is due to both increased hepatic production of
VLDL (Gerber and Erdman, 1981a) and a suppression of LPL activity in
peripheral tissues (Gerber and Erdman, 1981a; Oliver and Rogers, 1993).
In humans there are no data on the effects of retinoids on hepatic VLDL
production, however, 13-cis-RA-treated patients have a
reduced capacity to clear infused triglycerides (Vahlquist et al.,
1987), suggesting reduced tissue lipolytic activity. Furthermore, in
humans, 13-cis-RA increases the levels of apolipoprotein
(apo)-CIII a hepatic protein that binds to VLDL and interferes with
their lipolytic processing (Vu-Dac et al., 1998).
The multiple effects of retinoids on serum triglyceride metabolism may
be due to the distinct activities of the different retinoid receptors,
all members of the nuclear receptor superfamily (Mangelsdorf et al.,
1993). All-trans-RA is the physiological ligand for the
retinoic acid receptors (RAR
, 
, and 
), which mediate many of
the effects of retinoids on morphogenesis and connective tissue
metabolism. In addition, all-trans-RA readily isomerizes to
9-cis-RA, a ligand for not only the RARs but also for the
retinoid X receptors (RXRs) (Heyman et al., 1992; Levin et al., 1992).
13-cis-RA binds to neither the RARs nor the RXRs, but in
vivo, it spontaneously isomerizes to both all-trans-RA and
9-cis-RA and thereby indirectly activates both RARs and RXRs (Chen and Juchau, 1998). Ligand-dependent activation of the RXRs is
particularly interesting because RXRs form both homodimers and serve as
obligate heterodimeric partners for a number of nuclear receptors, such
as RARs, peroxisome proliferator-activated receptors (PPARs), thyroid
hormone receptors, the vitamin D receptor, and several other
orphan nuclear receptors (Mangelsdorf and Evans, 1995). Thus, the
pharmacologic effects of all-trans-RA, 9-cis-RA, or 13-cis-RA can be due to activation of both RAR- and
RXR-dependent signaling pathways.
Retinoid-induced hypertriglyceridemia was initially encountered in
patients receiving retinoic acid isomers that either directly or
indirectly activate both RARs and RXRs. RAR-specific retinoids induce
hypertriglyceridemia in both experimental animals (Standeven et al.,
1996) and humans (Takeuchi et al., 1998). The situation with
RXR-selective retinoids (rexinoids) is less clear-cut. In humans,
rexinoids cause hypertriglyceridemia (Miller et al., 1997; Rizvi et
al., 1999), but in experimental animals, they have been reported either
to have no effect (Standeven et al., 1996) or to lower serum
triglyceride levels (Mukherjee et al., 1997a, 1998; Lenhard et al.,
1999). In the studies reported here on the acute effects of rexinoids
in diabetic rats, we found that rexinoids markedly increased serum
triglyceride levels by suppressing LPL activity in both skeletal and
cardiac muscle. We have further demonstrated that rexinoids suppress
the expression of LPL activity on the surface of differentiated
myocytes. Taken together these findings suggest that RXR-dependent
signaling pathways are intimately involved in the regulation of
triglyceride metabolism via their specific effects on the LPL activity
of cardiac and skeletal muscle.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals and Treatments.
Harlan Sprague-Dawley rats (8-weeks
old, approximately 220 g) were obtained from Harlan Laboratories,
Inc. (Indianapolis, IN) and Zucker diabetic fatty (ZDF) rats (8-weeks
old, approximately 350 g) were obtained from Genetic Models Inc.
(Indianapolis, IN). Rats were housed individually on a 12:12 light/dark
cycle (lights on at 6:00 AM) with food (Teklad 7012a or Purina 5008, respectively) and tap water continuously available. Blood samples were
obtained via the tail vein, 3 h after dosing on the indicated day.
For chronic treatment, rats were gavaged with vehicle, rosiglitazone (3 mg/kg), LGD1069, or LG268 (3, 10, 30, or 100 mg/kg). At the end of the
experiments, animals were weighed and anesthetized. Blood was collected
by cardiac puncture before euthanization with CO2. Serum was separated and used within 1 week
for analysis of lipids, lipoproteins, and apolipoproteins. The liver,
muscle, and adipose tissue were removed immediately, weighed, frozen in liquid nitrogen, and stored at 
80°C until further analysis.
Cultured Cells.
Control C2C12 cells, stably transfected with
a -galactosidase reporter construct, (C2--gal) and C2C12 cells
transfected with pMCKhLPL (Poirier et al., 2000) were maintained in
100-mm dishes in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 20% fetal calf serum, penicillin, streptomycin, and
G-418 (200 µg/ml). For individual experiments, cells were plated in 12-well plates at 105 cells per well in DMEM + 20% fetal calf serum. After 3 days, when the cell achieved confluency,
the medium was changed to DMEM + 2% horse serum + cytosine arabinoside
(10 µM), and thereafter, the medium was changed every 3 days until
>95% myotubes formed (6 days).
Lipid, Lipoprotein, and Lipase Measurements.
Serum
cholesterol and triglycerides were determined by enzymatic assay
adapted to microtiter plates using commercially available reagents
(Roche Molecular Biochemicals, Mannheim, Germany). Lipoprotein triglyceride and cholesterol profiles, separating the three major lipoprotein classes (VLDL, LDL, and HDL) were obtained by fast protein
liquid chromatography and analyzed by the Millennium 20/0 program
(Waters, Milford, MA) (Duverger et al., 1993). LPL and hepatic lipase
activity was measured in tissue extracts and serum according to the
procedure of Ramirez and coworkers (Grinberg et al., 1995). The
heparin-releasable LPL activity of differentiated C2C12 cells was
measured by washing the cells once with 0.4 ml of DMEM and then
incubating them at room temperature for 10 min with 0.4 ml of DMEM + heparin (100 µg/ml). An aliquot (100 µl) of the cell extract was
assayed for LPL activity (Nilsson-Ehle and Schotz, 1976). One unit of
enzyme activity is the amount of enzyme that releases 1 µmol of
oleate/min at 25°C.
RNA Analysis.
RNA was isolated from tissues by the acid
guanidinium thiocyanate/phenol/chloroform method and Northern and Dot
blot analysis of total cellular RNA with LPL, and apoCIII cDNA probes
were performed as described previously (Auwerx et al., 1989).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of Rexinoids on Serum Triglyceride Levels.
Rexinoids
have been reported to lower both the hyperglycemia and
hypertriglyceridemia of genetically obese and insulin-resistant (db/db) mice (Mukherjee et al., 1997a, 1998; Lenhard et al.,
1999). We were therefore surprised to find that in ZDF rats, LG268, a prototypic rexinoid (Boehm et al., 1995), lowered serum glucose levels
(Fig. 1A) but raised serum triglycerides
from a baseline value of 4.08 ± 0.5 g/liter to a peak of
14.81 ± 2.0 g/liter (Fig. 1B). The rise is serum triglycerides is
not directly linked to the fall in serum glucose levels since
rosiglitazone (BRL49653), a PPAR agonist that lowers serum glucose
levels in these animals, also lowered triglyceride levels (Fig. 1, A
and B).
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; Mukherjee et al., 1997a,
1998). To determine the kinetics of rexinoid-induced hypertriglyceridemia, a single dose of either vehicle or LG268 was
administered (by gavage) to non-obese, non-diabetic rats. At specific
times following drug or vehicle administration, animals were
sacrificed, and blood was collected (terminal bleeds) for serum
triglyceride determinations. Administration of LG268 (but not the
vehicle) to these normal rats resulted in a rapid but transient
increase in serum triglyceride levels from a baseline value of
1.54 ± 0.3 g/liter to a peak of 3.03 ± 0.5 g/liter (Fig. 1C). After a lag of 60 min, serum triglyceride levels rose rapidly and
remained elevated for at least 6 h. Serum triglyceride levels returned to normal 24 h following administration of the rexinoid. These results demonstrated that normal rats were as sensitive as
diabetic rats to rexinoid-induced hypertriglyceridemia and that the
hypertriglyceridemic effect of the rexinoid was as prominent in the
naïve animals (Fig. 1C) as it was in those that had been subjected to chronic administration (Fig. 1B).
Rexinoids are presumed to produce their biological effects via
RXR-dependent activation of gene expression. The remarkably rapid
effects of rexinoids on triglyceride levels raised the question of
whether this response was due to their transcriptional activity or
whether it might be due to some previously unrecognized
post-transcriptional effect. To address this issue, we administered
LG268 to non-diabetic rats in which transcription had been blocked by
prior administration of the RNA polymerase II inhibitor actinomycin D
(Act D). Terminal blood samples were collected 3 h following
administration of LG268 to either control or Act D-treated animals. Act
D had no effect on basal triglyceride levels, but it completely blocked
the acute hypertriglyceridemic effect of the rexinoid (Fig. 1D).
To characterize the effects of rexinoids on serum lipid metabolism, we
examined the dose-dependent effects of a second rexinoid, LGD1069
(Targretin) (Boehm et al., 1994), on triglyceride and cholesterol
levels in diabetic rats. Like LG268, LGD1069 caused marked
dose-dependent hypertriglyceridemia. Over the same dose range, total
serum cholesterol levels remained virtually unchanged (Fig.
2A). The dose-response range is very
broad, doses as low as 0.3 mg/kg/day caused significant elevations in
serum triglyceride levels, and the maximum effect occurred at doses
equal to or higher than the highest dose tested (30 mg/kg/day). To
determine which lipoproteins accounted for the increase in total
triglycerides, we performed lipoprotein profile analysis on pooled
serum from three untreated animals (total cholesterol level in the pool
of 0.92 g/dl) and three animals that received LGD1069 (30 mg/kg/day; total cholesterol level of 1.4 g/dl). The increase in triglyceride levels in LGD1069-treated animals was entirely due to increased levels
of VLDL as evidenced by size exclusion chromatography of lipoproteins
and subsequent analysis of both triglyceride (Fig. 2B) or cholesterol
distribution (Fig. 2C) in the different fractions. LGD1069
administration also resulted in a slight change in HDL particle
characteristics. HDL particles became less heterogeneous and more bulky
relative to control HDL (Fig. 2C). No significant changes in
intermediate density lipoprotein and LDL fractions were observed upon
fast protein liquid chromatography.
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Effect of Rexinoids on VLDL Metabolism and LPL in Vivo.
Increased steady-state plasma VLDL levels can arise from either
increased hepatic production or decreased peripheral clearance or both.
To determine the mechanism of rexinoid-induced hypertriglyceridemia in
rats, we measured the effects of rexinoids on hepatic triglyceride production and heparin-releasable plasma LPL activity. Administration of Triton WR-1339 blocks the clearance of triglyceride-rich
lipoproteins by inhibiting their degradation. The resulting increase in
triglyceride levels provides an indirect measurement of hepatic
triglyceride secretion (Gerber and Erdman, 1981a). The triglyceride
secretion rate of the control and rexinoid-treated (LG268 at 3 mg/kg/day) rats were similar, 410.47 ± 14.9 and 407.29 ± 33.02 mg/dl/h, respectively (Fig. 3A).
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). To determine whether rexinoids
affected the level of expression or activity of LPL in tissues, RNA and
heparin-treated tissue extracts were prepared from white adipose
tissue, skeletal muscle (quadriceps), and cardiac muscle (ventricle) of
ZDF rats that had been treated with LGD1069 for 14 days (Fig.
4). Rexinoid treatment resulted in a
marked decline of LPL activity in both skeletal and cardiac muscle.
Skeletal muscle was the most sensitive, doses of LGD1069 as low as 0.3 mg/kg/day produced a significant decrease in tissue LPL activity, and 3 mg/kg/day was a maximally effective dose. There was also a marked
suppression of cardiac LPL activity with a minimally effective dose of
1 mg/kg/day and a maximal effect at 10 to 30 mg/kg/day. The decrease in
LPL activity in both skeletal and cardiac muscle was not a reflection
of decreased LPL expression since the level of LPL mRNA in cardiac
muscle was unaffected, and the level of LPL mRNA in skeletal muscle was
only slightly depressed by the highest dose of rexinoid. There was no
significant decrease in the LPL activity of adipose tissues taken from
animals treated with LGD1069 in doses ranging from 0.3 to 30 mg/kg/day. There does appear to be a small decrease in the LPL activity of the
animals treated with the highest doses of LGD1069 (10-30 mg/kg/day), but this decrease did not achieve statistical significance. Rexinoids have no effect on the level of LPL mRNA in the adipose tissue.
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Effect of Rexinoids on LPL Metabolism in Cultured Myocytes.
The preceding studies demonstrated that rexinoids could have dramatic
effects on muscle LPL metabolism in vivo. To determine whether these
same compounds could have direct effects on the metabolism of LPL in
vitro, we examined the effects of LG268 on the expression and activity
of the enzyme in differentiated mouse C2C12 myocytes (Fig.
5). LG268 induced a dose-dependent
suppression in the heparin-releasable LPL activity of the C2C12 cells
with an EC50 of 1 × 10
9 M (Fig. 5A). To further characterize the
effects of the rexinoid on the expression of LPL activity, we used
C2C12 cells stably transfected with an LPL expression vector, pMCKhLPL
(Duverger et al., 1993) that contains the human LPL cDNA under the
control of a creatine kinase promoter. The basal LPL activity of these transfected cells is 10-fold higher than either non-transfected cells
(data not shown) or cells transfected with an irrelevant -galactosidase expression vector. LG268
(109-106 M) produces
an equivalent suppression in the LPL activity of both the
mock-transfected (C2--gal) and LPL-transfected (C2-LPL) cells (Fig.
5A). The onset of the inhibitory effect of LG268 on LPL activity in the
transfected cells is very rapid. Following a reproducible 1 to 2 h
lag, there is a precipitous decline in heparin-releasable LPL activity
such that within 4 to 6 h the activity had decreased to less than
10% of that present in control cells (Fig. 5B).
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ligand), WY14,643 (a PPAR ligand), 4-hydroxycholesterol (a liver X receptor ligand),
1,25-dihydroxycholecalciferol (a vitamin D receptor ligand), or
triiodothyronine (a thyroid hormone receptor ligand) (Fig. 5D). The
effect of the rexinoid was very specific. LG268 induced a reproducible
suppression in heparin-releasable LPL activity, whereas none of the
other nuclear receptor ligands tested had any inhibitory effect on the
level of enzyme activity. Thus, none of the ligands for receptors that can partner with RXR replicated the activity of the RXR ligand.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Retinoid- and Rexinoid-Induced Hypertriglyceridemia.
Activation of RXR is associated with insulin sensitization and has
therefore been proposed as a novel strategy for the treatment of type
II diabetes (Mukherjee et al., 1997a; Lenhard et al., 1999). In human
subjects, however, rexinoid therapy is associated with
hypertriglyceridemia (Miller et al., 1997; Rizvi et al., 1999), and the
results presented here show that the same effects can be observed in
both normal and diabetic rodents. Rexinoid-induced hypertriglyceridemia
developed rapidly after administration of LG268 to normal rats; it
reached peak levels in 2 to 3 h and was resolved in 24 h.
This transient effect is compatible with the short half-life of both
LG268 and LG1069 in rodents (E. Ulm, personal communication). To
develop RXR agonists with a favorable therapeutic index for long-term
treatment of impaired glucose homeostasis, it is important to
understand the molecular basis of this unwanted side effect.
)
and peripheral lipoprotein catabolism (Gerber and Erdman, 1981a; Oliver and Rogers, 1993). In contrast, the rexinoid effect on
triglyceride metabolism is restricted to effects on clearance. The
studies with Triton WR-1339 demonstrated that hypertriglyceridemia
develops in the absence of alterations in the rate of hepatic
triglyceride secretion. Although our studies did not measure
triglyceride clearance rates directly, elevations in the steady-state
triglyceride levels in the absence of alterations in production
strongly suggest a primary defect in lipoprotein catabolism.
Triglyceride-rich lipoproteins are catabolized by two lipases, LPL and
hepatic lipase. Defects in hepatic lipase activity lead to the
accumulation of intermediate density lipoprotein and HDL particles
(Gehrisch et al., 1999), whereas defects in LPL leads to increased
levels of VLDL (Merkel et al., 1998). The hypertriglyceridemia of the
rexinoid-treated animals consisted entirely of elevated levels of VLDL
suggesting a primary defect in LPL-dependent catabolism.
Alterations in LPL-dependent catabolism of VLDL can be due either to
changes in the level of the enzyme in tissues or to changes in the
level of regulatory apoproteins in the plasma. In experimental animals,
all-trans-RA and 13-cis-RA lower LPL activity in
both adipose tissue and skeletal muscle but have no effect on LPL
activity in the heart (Gerber and Erdman, 1981a; Oliver and Rogers,
1993). Rexinoids also suppress LPL activity in skeletal muscle but they differ from retinoids in their effects on LPL activity in adipose tissue and cardiac muscle. Rexinoids have no effect on adipose tissue
LPL activity whereas retinoids do, and reciprocally, while rexinoids
suppress cardiac LPL activity, retinoids have no effect. The results we
have obtained suggest skeletal muscle LPL activity can be affected by
both RAR- and RXR-specific ligands, whereas RAR ligands also affect LPL
metabolism in adipose tissue and RXR ligands affect it in the heart. It
is possible that the lack of effect of rexinoids on LPL activity in
adipose tissue reflects an unusual pattern of tissue distribution for
the drug but we do not think this is the case. The synthetic rexinoids
used in these studies, LG1069 and LG268, are very lipophilic drugs that have marked effects on gene expression in muscle, liver, and adipose tissue in both normal and diabetic rodents (H. S. Ahuja, S. Liu, D. L. Crombie, M. Boehm, M. D. Leibowitz, R. A. Heyman, C. Depre, L. Nagy, P. Tontonoz, and P. J. A. Davies, submitted for publication). It is
more likely that the tissue-specific effects of retinoids and rexinoids
on LPL metabolism reflect differences in the types and amounts of
receptors and their various heterodimeric partners or in the
coactivator and bridging factors present in the different tissues.
Alteration in the level of LPL activity is not the only factor
controlling the rate of VLDL catabolism in vivo. ApoCII and apoCIII are
serum proteins that bind to the VLDL and either enhance (apoCII) or
depress (apoCIII) its susceptibility to hydrolysis by LPL (Jong et al.,
1999). Retinoids have been reported to increase apoCIII expression and
secretion by hepatocytes both in vitro and in vivo via an RXR-dependent
pathway (Vu-Dac et al., 1998). In our studies, LGD1069 induced a minor
increase in hepatic apoCIII mRNA; there was no increase in plasma
apoCIII levels 3 h after LG268 administration at a time when there
was both marked suppression of muscle LPL activity and marked
hypertriglyceridemia (data not shown).
Mechanisms of Rexinoid-Induced Hypertriglyceridemia.
It has
been suggested that the metabolic effects of rexinoids are attributable
to their ability to activate RXR/PPAR heterodimers (Mukherjee et al.,
1997a, 1998; Lenhard et al., 1999). The RXR serves as an obligate
partner for all three members of the PPAR family of receptors
(Mukherjee et al., 1998) and in transfection studies, rexinoids
transactivate PPAR heterodimers as effectively as PPAR ligands
(Mukherjee et al., 1997a,b, 1998). In diabetic mice, rexinoids
replicate the ability of PPAR ligands such as thiazolidinediones to
lower serum glucose levels (Mukherjee et al., 1997a; Lenhard et al.,
1999). The overlapping pharmacological activities of rexinoids,
fibrates, and thiazolidinediones has led to the suggestion that
RXR/PPAR heterodimers are capable of being equivalently activated by
ligands of either of the heterodimeric partners (Mukherjee et al.,
1998). In spite of these overlapping activities in some metabolic
responses, we do not believe that the hyperlipidemic effects of
rexinoids reflect the activation of PPARs. In contrast to rexinoids,
PPAR and PPAR ligands lower triglyceride levels (Auwerx et al.,
1996). Unlike PPAR ligands, which increase LPL expression in adipose
tissue (Schoonjans et al., 1996; Lefebvre et al., 1997), rexinoids have
no effect on either the expression or activity of LPL in adipose
tissue. PPAR ligands can suppress heparin-releasable LPL activity in
cultured 3T3L-1 adipocytes (Ranganathan and Kern, 1998) but they do not inhibit myocyte LPL activity whereas rexinoids do. Thus, as far as we
can tell, the hypertriglyceridemic activity of rexinoids is independent
of their ability to activate PPARs.
). However, in cultured myocytes, rexinoids suppress LPL
activity whereas a potent pan-RAR agonist such as TTNPB does not.
Furthermore, as discussed above, the profile of tissues in which LPL
activity is suppressed by the rexinoid is different from the tissues
affected by RAR agonists. These results suggest that rexinoids and
retinoids share the ability to induce hypertriglyceridemia in vivo but
that they do so via the activation of distinct and different receptor complexes.
Having excluded PPARs and RARs as RXR partners responsible for the
hyperlipidemic activity of rexinoids, the question of which partner
receptors are responsible remains. Ligands for the thyroid hormone and
vitamin D receptors neither produce acute hypertriglyceridemia in vivo
nor do they suppress LPL activity in cultured myocytes. The
hyperlipidemic activity of rexinoids could be due either to the
activation of an unrecognized RXR-containing heterodimeric complex or
to activation of RXR homodimers. RXR receptors form stable homodimers
on DNA (Holmbeck et al., 1998) that are effective activators of
transcription (Mangelsdorf et al., 1991; Vu-Dac et al., 1996). It is
possible that the unique ability of rexinoids, among nuclear receptor
ligands, to induce hypertriglyceridemia is due to their unique ability
to serve as ligands for RXR homodimers.
Even though we are not certain of the receptor complexes that mediate
the effects of rexinoids on triglyceride metabolism, these effects
require the induction of new gene expression. Both the LPL-suppressive
effects of the rexinoids in cultured cells and rexinoid-induced
hypertriglyceridemia are blocked by actinomycin D. The time courses for
the effects of the rexinoids in vitro and in vivo are also compatible
with the induction of gene expression and the accumulation of a
specific gene product (or products) that subsequently perturb LPL
metabolism. Our data suggest that the effects of rexinoids on LPL
metabolism occur primarily at a post-translational level. Treatment of
either cells or animals with rexinoids has no effect on the levels of
LPL mRNA even though there are large changes in LPL activity.
Furthermore rexinoids are as effective in suppressing the LPL activity
of cells transfected with an LPL expression construct as they are in
suppressing the activity of endogenous LPL. Chronic administration of
thyroid hormone suppresses LPL activity via regulatory elements encoded in the 3'-untranslated region of the LPL transcript (Kern et al., 1996). However, rexinoids are fully effective in suppressing LPL actvity in cells transfected with an LPL construct that contains a
heterologous 3'-untranslated region so it is unlikely that this type of
mechanism accounts for these inhibitory effects. The abolition of the
hypertriglyceridemic effect of rexinoids by actinomycin D suggests that
their effect on LPL metabolism depends on the induction of
RXR-regulated genes, which affect the complex post-translational processing, dimerization, and export pathways that are necessary for
the expression of cell surface LPL activity.
Physiological Implications.
Rexinoid-induced suppression of
the lipolytic activity of skeletal muscle and heart are likely to have
physiological consequences beyond the induction of
hypertriglyceridemia. Since LPL is a gatekeeper enzyme, controlling the
delivery of fatty acids to tissues, decreased LPL activity will, in the
long-run, result in a depletion of lipid stores in muscle and heart,
which in turn will lead to improved muscle insulin sensitivity. PPAR
agonists induce a preferential delivery of fatty acids to adipose
tissue via the induction of LPL and enzymes and transport proteins that
facilitate fatty acid uptake into adipose tissue (Schoonjans et al.,
1996). The resulting "fatty acid steal" by adipose tissue,
diminishes the quantity of fatty acids taken up by the muscle and may
contribute to the insulin-sensitizing effect of PPAR agonists in
muscle. Rexinoids may cause a depletion of muscle lipid stores not by
increasing the delivery of fatty acids to adipose tissue but by
directly reducing their delivery to muscle. It is possible that this
modulation of fatty acid metabolism in skeletal muscle cells is an
important contributor to the therapeutic effects of these compounds in
animal models of type II diabetes.
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Acknowledgments |
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Appreciation is expressed to Mary Sobieski, Celine Haby, and Nancy Shipley for technical contributions and to Harleen Ahuja, Ph.D., for the helpful discussions of results.
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Footnotes |
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Received July 18, 2000; Accepted October 10, 2000
This work was supported by grants from Ligand Pharmaceuticals, the National Institutes of Health (DK26356), the Fondation de France, the European Union (Grant QLG1-CT-1999-00674), CNRS, INSERM, and Hôpitaux Universitaires de Strasbourg. J.A. and C.F. are research directors with CNRS and INSERM, respectively.
Send reprint requests to: Dr. Peter J. A. Davies, Dept. of Integrative Biology and Pharmacology, University of Texas School of Medicine, P.O. Box 20708, Houston, TX 77225. E-mail: peter.j.davies{at}uth.tmc.edu
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RA, retinoic acid; LDL, low-density lipoprotein; VLDL, very-low-density lipoprotein; LPL, lipoprotein lipase; RAR, retinoic acid receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; ZDF, Zucker diabetic fatty; DMEM, Dulbecco's modified Eagle's medium; HDL, high-density lipoprotein; TTNPB, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid; Act D, actinomycin D.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nature (Lond)
355:
359-361[Medline].This article has been cited by other articles:
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