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Agonist and Decreases Glucose Levels in Vivo
Department of Nuclear Receptor Research (R.M.C., K.K., H.R., D.C., R.A.H., D.S.L.) and New Leads (D.R.), Ligand Pharmaceuticals, Inc., San Diego, California 92121
Address all correspondence and request for reprints to: Dr. Deepak S. Lala, Department of Biochemistry and Molecular Biology, Pharmacia Corporation, 700 Chesterfield Parkway North, St. Louis, Missouri 63198.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and
the dimer is a molecular target for insulin sensitizers such as the
thiazolidinediones. Ligands for either receptor can activate
PPAR-dependent pathways via PPAR response elements. Unlike PPAR
agonists, however, RXR agonists like LG100268 are promiscuous and
activate multiple RXR heterodimers. Here, we demonstrate that LG100754,
a RXR:RXR antagonist and RXR:PPAR
agonist, also functions as a
RXR:PPAR agonist. It does not activate other LG100268 responsive
heterodimers like RXR:liver X receptor, RXR:liver X receptorß,
RXR:bile acid receptor/farnesoid X receptor and RXR:nerve growth factor
induced gene B. This unique RXR ligand triggers cellular
RXR:PPAR-dependent pathways including adipocyte differentiation
and inhibition of TNF-mediated hypophosphorylation of the insulin
receptor, but does not activate key farnesoid X receptor and liver X
receptor target genes. Also, LG100754 treatment of db/db animals leads
to an improvement in insulin resistance in vivo.
Interestingly, activation of RXR:PPAR by LG100268 and LG100754
occurs through different mechanisms. Therefore, LG100754 represents a
novel class of insulin sensitizers that functions through RXR but
exhibits greater heterodimer selectivity compared with LG100268. These
results establish an approach to the design of novel RXR-based insulin
sensitizers with greater specificity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, a molecular target for insulin
sensitization, has provided biological evidence that the endogenous
heterodimer is indeed permissive for RXR ligands. Supporting this are
data showing that agonists for both receptors can participate in the
control of common biological pathways such as adipocyte differentiation
and carcinogenesis (7, 8). Moreover, both types of
compounds act to improve insulin resistance in various animal models of
diabetes (9). However, while PPAR agonists such as
thiazolidinediones are selective for the PPAR:RXR heterodimer, the
prototypic rexinoid LG100268 (10, 11) activates all other
permissive heterodimers and RXR homodimers (9) and is
likely to regulate multiple RXR-dependent pathways in vivo.
Thus, in developing rexinoids as therapeutic agents, achieving a
greater degree of dimer selectivity would be highly desirable.
We previously described a novel RXR homodimer antagonist,
LG100754, that functioned as a RXR:RAR and RXR:PPAR activator in
cotransfection assays (5). In this study we have examined
the activity of this compound on permissive RXR heterodimers. We
demonstrate that, in addition to RXR:PPAR, LG100754 is also active
on RXR:PPAR heterodimers. Surprisingly, it is inactive on other
permissive heterodimers such as RXR:LXR, RXR:LXRß, RXR:NGFI-B, and
RXR:BAR/FXR, indicating greater selectivity than LG100268. LG100268 and
LG100754 show differential recruitment of coactivators to the
RXR:PPAR heterodimer, suggesting distinct mechanisms of activation
by the two classes of rexinoids. We further demonstrate
that LG100754 activates endogenous RXR:PPAR
heterodimer-mediated pathways through its ability to induce adipocyte
differentiation of 3T3-L1 cells. Also, like LG100268, LG100754 is able
to block TNF-mediated inhibition of insulin receptor (IR)
phosphorylation in mature adipocytes. However, unlike LG100268,
LG100754 does not activate key FXR and LXR target genes. Finally, we
show that LG100754 treatment of db/db animals prevents the rise in
blood glucose levels, suggestive of an improvement in insulin
resistance.
Our data indicate that the RXR homodimer antagonist LG100754 is a novel
RXR:PPAR agonist that represents a new class of rexinoids that are
more dimer selective. Such compounds may be useful in improving insulin
resistance and offering benefit for the treatment of human metabolic
disease.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) and is a
RXR homodimer antagonist (5). Interestingly, both LG100754
and LG100268 are active on RXR:PPAR (Fig. 1B and Ref. 5). In
addition, as shown in Fig. 1C, both rexinoids are active on the
RXR:PPAR heterodimer with comparable efficacy. Both are also active
on RXR:PPAR
, although not as much as on RXR:PPAR (Fig. 1D).
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, RXR:LXRß,
RXR:BAR/FXR, and RXR:NGFI-B. In each case, the response of a specific
dimer to the rexinoids was determined on its cognate hormone response
element (HRE). Surprisingly, while LG100268 acts as a strong and potent
agonist of RXR:LXR, RXR:LXRß, RXR:FXR, and RXR:NGFI-B
heterodimers, LG100754 is inactive on all four heterodimers (Fig. 2 A–D). Further, while combination of
LG100268 with either FXR or LXR ligands had an additive effect,
LG100754 did not (Fig. 2, E and F). In our studies, in addition to
RXR:PPAR, the only other heterodimer LG100754 is capable of activating
RXR:RAR (5). Thus, while LG100268 acts as a highly
promiscuous agonist of RXR heterodimers, LG100754 exhibits greater
selectivity toward RXR:PPARs. Other RXR antagonists structurally
related to LG100754 also exhibit a similar selectivity for RXR:PPARs
(data not shown). These data suggest that, in general, this class of
RXR homodimer antagonists may be more dimer selective than agonists
such as LG100268.
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Heterodimers heterodimer.
To test this, we employed a modified mammalian two-hybrid system that
was set up to detect coactivator interactions with the RXR:PPAR
heterodimer in the presence of rexinoids. This system consists of a
coactivator receptor interacting domain (RID) fused to the Gal4-DNA
binding domain, a GAL4 response element driving the luciferase cDNA,
and the PPAR LBD fused to the VP16 activation domain. Neither component
by itself or in combination can respond to rexinoids. Introduction of
the RXR LBD minus its DNA-binding domain allows heterodimer formation;
however, no RXR homodimer activity is measured since the RXR-LBD cannot
bind DNA (data not shown). Similar assays have been used to demonstrate
that LG100268 can recruit the coactivator, steroid receptor
coactivator-1 (SRC-1), to the RXR:PPAR heterodimer
(16). We therefore used our assay to first compare the
ability of LG100268 and LG100754 to recruit SRC-1 to RXR:PPAR.
As shown in Fig. 3A, LG100268
induces an interaction of RXR:PPAR with the RID of SRC-1;
surprisingly, LG100754 does not. Similar results were obtained using
SRC-2 or SRC-3 RIDs (data not shown). These data indicate that, in this
assay, LG100268, but not LG100754, can promote interactions with the
SRC family of coactivators. To determine whether other cofactors might
be recruited by LG100754, we compared the ability of both rexinoids to
recruit the coactivator cAMP response element-binding
protein-binding protein (CBP). LG100754 was a weak recruiter of CBP
in this assay (2-fold); LG100268 also recruited CBP much more weakly
than SRC-1 (Fig. 3B). Since LG100754 is clearly active on RXR:PPAR
heterodimers, these data suggest that either the weak recruitment
of CBP in response to LG100754 is sufficient for activation, or other
classes of coactivators are required for its activity. To test the
latter, we examined the ability of both compounds to recruit
TR-associated protein 220 (TRAP220), a coactivator identified as part
of a larger complex recruited to the activation function (AF-2) domain
of the TR (17). In contrast to SRC-1, LG100754 was able to
recruit TRAP220 5-fold to RXR:PPAR (Fig. 3C). Interestingly, the
promiscuous rexinoid LG100268 recruited TRAP220 as well (Fig. 3D),
suggesting it places the heterodimer in a conformation that is more
flexible for coactivator binding compared with LG100754. Therefore,
different rexinoids may induce unique conformational changes within
RXR:PPAR and activate the heterodimer using different
coactivators. Our data suggest that although both LG100268 and LG100754
are active on the RXR:PPAR heterodimer, they activate it via
different mechanisms.
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/FXR/LXR pathways in relevant cells. Adipocyte
differentiation is a well established PPAR-dependent pathway,
and both LG100268 and PPAR-ligands induce differentiation in 3T3-L1
preadipocytes (14, 18, 20, 21). We therefore tested the
ability of LG100754 to promote adipogenesis in this cell line.
Consistent with its ability to activate the RXR:PPAR heterodimer in
transfections, LG100754, like LG100268, induces differentiation in a
dose-dependent manner at concentrations comparable to those at which it
activates RXR:PPAR in vitro, and 100
nM of either rexinoid was equally effective (Fig. 4, A and B). Thus, LG100754 can activate
endogenous RXR:PPAR heterodimers and drive cellular
PPAR-dependent pathways. Next, we looked at the ability of both
rexinoids, at the same concentration, to induce intestinal bile-acid
binding protein (IBABP) gene expression, a FXR target gene in
differentiated Caco-2 cells (22, 23). These cells have low
IBABP, which is strongly induced by FXR ligands (22).
Using a semiquantitative RT-PCR method, we show strong induction of
IBABP with chenodeoxycholic acid (CDCA), a FXR ligand, consistent with
previous results (Ref. 22 and Fig. 4C). LG100268 also induces IBABP
very significantly; in contrast, LG100754 is unable to induce IBABP
expression either alone or in the presence of CDCA (Fig. 4C). ABCA1 was
recently identified as a direct LXR target gene in macrophages and is
induced by LG100268 and LXR ligands in vivo and in
vitro (24). Interestingly, it can also be
up-regulated indirectly but less effectively by BRL (rosiglitazone, a
PPAR ligand) through RXR:PPAR (25). We therefore
speculated that LG100754 would behave like BRL and induce ABCA1
less effectively than LG100268 or 22(R)hydroxycholesterol
[22(R)OHC], a LXR ligand (24). To test this, we
treated RAW264.7 cells, a mouse macrophage cell line, with compounds
and used a highly quantitative real-time PCR method to look
for differences. As predicted, while LG100268 and 22(R)OHC,
RXR:LXR agonists, induce ABCA1 significantly (6- to 13-fold), both
LG100754 and BRL, selective RXR:PPAR agonists, are weak activators
(
2.5 fold, Fig. 4D). No further increase was observed by combination
of 22(R)OHC with LG100754. Similar results were obtained with ApoE,
another LXR target gene (data not shown). Thus, concentrations of
LG100754 that are as effective as LG100268 in inducing adipogenesis, a
PPAR-dependent pathway, are completely ineffective on IBABP, a FXR
target gene, and less effective on ABCA1, a LXR target gene. Moreover,
the weak induction of ABCA1 by LG100754 is most likely via RXR:PPAR
since it is also induced by BRL, and these cells express low levels of
PPAR (data not shown). These data confirm that LG100754, unlike
LG100268, is a dimer-selective agonist of cellular PPAR but not of
FXR or LXR.
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-Mediated Hypophosphorylation of the IR
by LG100754 and LG100268 has been shown to affect this
proximal step of insulin signaling by inhibiting tyrosine
phosphorylation of the IR (26). This TNF-mediated
insulin resistance has previously been shown to be completely blocked
by thiazolidinediones in mature adipocytes treated with TNF
(27). That this effect appears to be mediated through the
RXR:PPAR heterodimer was shown by the observation that a similar
inhibition was achieved with other heterodimer agonists such as
15-deoxy-
12, 14 PGJ2, a PPAR ligand, and LG100268
(27). We therefore examined the ability of the
RXR:PPAR-selective rexinoid LG100754 to overcome TNF inhibition
of IR phosphorylation. For this, we first differentiated 3T3-L1
preadipocytes using a standard mixture of isobutylmethylxanthine
(IBMX), dexamethasone, and insulin as described in
Materials and Methods. Differentiated adipocytes were then
treated with TNF (5ng/ml) for a period of 1 or 2 d. At day 2 we
observed a strong inhibition of insulin-induced IR tyrosine
phosphorylation. Importantly, at the same time, we did not observe any
significant decrease in IR levels in the same experiment (Fig. 5A). Next, we tested the ability of the
RXR:PPAR-selective rexinoid LG100754 to block the inhibitory effects
of TNF on day 2 and compared its activity with LG100268. Consistent
with its ability to activate RXR:PPAR, LG100754 completely blocks
the ability of TNF to inhibit insulin-dependent IR phosphorylation
(Fig. 5B). Incubation of differentiated 3T3-L1 cells with LG100268 also
leads to a complete block of the inhibitory effects of TNF (Fig. 5B). Rosiglitazone, the synthetic PPAR ligand, also blocked the
effects of TNF (Ref. 24 and data not shown). Our data
indicate that, like LG100268 and rosiglitazone, LG100754 can also
improve TNF-mediated insulin resistance in mature adipocytes.
These results establish LG100754 as a bona fide activator of endogenous
RXR:PPAR heterodimers and regulator of insulin-dependent
signaling pathways.
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is a target for insulin-sensitizing drugs, and two
PPAR ligands, rosiglitazone (Avandia) and pioglitazone
(Actos) are used clinically for the treatment of diabetes. It
has been shown previously that treatment of db/db mice with
rosiglitazone or LG100268 leads to a decrease in glucose levels,
indicating an improvement in insulin resistance (9). This
may be due, at least in part, to their ability to block the inhibitory
effects of TNF in these animals. Collectively, our results suggested
that LG100754 would also act as an insulin sensitizer in
vivo. To test this, db/db animals were treated with the RXR:PPAR
selective compound, and its ability to decrease glucose levels was
compared with LG100268. As shown in Fig. 6, treatment with 100 mg/kg LG100754
completely blocks the increase in glucose levels observed in untreated
animals, suggesting that LG100754 can improve insulin resistance
in vivo. Our results indicate that LG100754 is a novel
rexinoid that serves as a RXR:PPAR agonist in vivo, but
exhibits a much more restricted pattern of RXR-heterodimer activation
in vitro compared with LG100268 (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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heterodimers
(5). In this study, we have focused on its activity on
other permissive RXR dimers. Like the promiscuous rexinoid LG100268,
LG100754 activates RXR:PPAR in transfection assays; however, unlike
LG100268, it does not activate other permissive RXR heterodimers. Based
on these data, we predicted that LG100754 would act as a selective
agonist of endogenous PPAR-dependent pathways. This was demonstrated
through its ability to induce PPAR-dependent adipocyte
differentiation, but inability to activate IBABP and ABCA1, key FXR and
LXR target genes, respectively. That LG100754 is a bona fide
RXR:PPAR agonist with insulin-sensitizing activity was shown via its
effects on inhibition of TNF-mediated hypophosphorylation of the IR.
Consistent with its ability to modulate cellular PPAR-dependent
pathways, LG100754 treatment of db/db animals led to a decrease in
blood glucose levels, indicating an improvement in insulin resistance.
Although in vivo LG100754 was less efficacious than
LG100268, this may be due to its poor bioavailability, and circulating
concentrations are low in db/db mice (data not shown). Nevertheless, as
demonstrated by its ability to activate several endogenous
PPAR-dependent pathways, but its inability to induce key FXR and
LXR target genes, LG100754 serves as a novel rexinoid whose activation
profile is markedly distinct from that of LG100268. These data
provide, for the first time, proof for the concept that rexinoid
activity can be restricted to specific permissive RXR heterodimers.
Ligand binding to nuclear receptors has been shown to induce
alterations within the LBD of receptors, including a drastic change in
the AF-2 domain (helix 12) as well as changes within helices 3–5.
These conformational changes together are believed to facilitate
interactions of the receptor with coactivators. Subsequently,
coactivators are able to enhance transcriptional activation through the
receptor via mechanisms that include histone acetylation and contacts
with the basal transcriptional machinery (28, 29). x-Ray
crystallographic studies have demonstrated, that for the ER and
PPAR, different ligands for the two receptors can induce different
conformations within their LBDs (12, 13, 14). Further support
for this comes from biochemical studies with ER and the VDR, which
showed different receptor ligands lead to distinct coactivator
interactions (15, 30). Different PPAR agonists can also
have distinct effects on coactivator recruitment. For example, the TZD
MCC-555, a partial agonist of PPAR, is a weaker recruiter of SRC-1
compared with rosiglitazone, a full agonist (31). These
data suggest that the structure of the ligand-bound LBD region that
recruits cofactors is ligand specific. In this regard we showed that
the ability to recruit coactivators to RXR:PPAR by LG100268 and
LG100754 is very different. LG100268 was a strong recruiter of SRC-1 to
the heterodimer while LG100754 was completely inactive, while both
compounds recruited CBP weakly. In contrast, LG100754 induced a
significant interaction with TRAP220 (as did LG100268). LG100754 thus
appears to have a different coactivator recruitment profile than
LG100268. While SRC-1, CBP, and TRAP220 are all recruited to the AF-2
domains of receptors, TRAP220 belongs to a distinct class of
coactivators. Both CBP and SRC-1 possess histone acetyltransferase
activity (HAT) whereas TRAP220, which is part of a large TR-associated
complex, does not (28). Although the precise mechanism by
which SRC and TRAP complexes contribute to overall nuclear receptor
function is unclear, our data suggest that LG100754 might recruit a
different subset of coactivators to RXR:PPAR. Thus, the mechanism by
which LG100754 activates RXR:PPAR is likely to be different from
that of LG100268. Activation of RXR:PPAR by LG100268 or
rosiglitazone also leads to differential cofactor recruitment with
LG100268 recruiting SRC-1, but rosiglitazone recruiting CBP
(16). Our data with LG100754 suggest a third mechanism for
activating RXR:PPAR.
Another aspect of this study is the elimination of RXR homodimer
agonist activity as a requirement for activating PPAR response elements
via RXR. In the context of the RXR:PPAR pathway, the degree to which
the LG100268 response is PPAR dependent vs. RXR homodimer
dependent was a question that remained to be determined. Since LG100754
lacks the ability to activate RXR:RXR, our data indicate that RXR
homodimer activity is not critical for activating endogenous RXR:PPAR
pathways. An interesting question is why LG100754 exhibits selectivity
toward RXR:PPAR and does not activate other RXR-permissive dimers.
The recent crystal structures of RXR:RAR and RXR:PPAR revealed that
both RAR and PPAR use a similar mechanism for heterodimerization
(32, 33). However, the PPAR dimer with RXR reveals an
asymmetrical interaction between PPAR AF-2 helix and helix 7 of RXR,
which is lacking in RXR:RAR. This interaction could stabilize the
PPAR AF-2 in a position that permits interactions with coactivators
even in the absence of a PPAR agonist and may provide a structural
basis for the permissivity of this heterodimer. However, other factors,
including binding of corepressors, the conformation of RXR, the
specific ligand itself, and the precise nature of the HRE, are also
likely to play a role in regulating permissiveness. Crystal structures
and mutational studies of other permissive and nonpermissive RXR dimers
should shed more light on other mechanisms that may be important in
determining permissivity. It is possible that different permissive RXR
partners mediate distinct allosteric interactions with RXR. Such
differences could be exploited to design novel dimer-selective
rexinoids. Thus, it may be possible to synthesize more specific and
potent RXR:PPAR-selective rexinoids or RXR ligands that activate
exclusively RXR:PPAR (or, if desired, both RXR:PPAR and
RXR:LXR, for example). Regulating multiple but very specific
RXR-dimer combinations by a single compound would be a novel approach
to modulate RXR-dependent pathways in vivo with greater
selectivity. Conceptually, this should be easier to accomplish through
the design of an appropriate ligand for RXR, the common partner, than
to expect a ligand to bind to and activate two or more completely
different partners.
In conclusion, our data indicate that LG100754, a RXR:RXR antagonist, represents a novel class of rexinoids that, unlike prototypic agonists such as LG100268, is more selective for RXR:PPAR heterodimers in vitro and can function as an insulin sensitizer in vivo. A number of RXR-containing heterodimers (e.g. the PPARs, the LXRs, and BAR/FXR) either are or likely will be important drug discovery targets for the treatment of diabetes, cardiovascular disease, obesity, and other lipid disorders. RXR homodimer antagonists that exhibit unique heterodimer selectivity may be beneficial for the treatment of human metabolic disease. Our results establish an approach to the design of novel RXR-based insulin sensitizers with greater dimer selectivity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,
pCMVSPORT-LXRß, pCMX-LXR, pCMXPPAR, pCMXPPAR
alone, or with pCMXNGFI-B, or pCMXBAR/FXR in combination with
pCMXRXR. The luciferase reporter constructs contained three to five
copies of either a LXR response element (for LXR/ß), an ecdysone
receptor response element (for BAR/FXR), a NGFIB response element (for
NGFI-B) or acyl coenzyme A oxidase-PPAR response elements
(for PPAR/) (5, 9). Cofactor recruitment experiments
were done by transfecting a Gal4Coactivator RID [SRC-1 (amino acids
380–851), CBP (1–356 amino acids), or TRAP220 (amino acids
442–693)], a VP16-PPARLBD fusion construct, a RXRLBD expression
plasmid, and a Gal4 response element linked to a minimal
promoter-luciferase construct. ß-Galactosidase was included as an
internal control. Experiments were carried out at least three times
with comparable results.
Cell Culture and Differentiation
3T3-L1 cells were obtained from ATCC (Manassas, VA)
and maintained in DMEM supplemented with 10% calf serum and gentamicin
sulfate. For differentiation studies, cells were plated at
approximately 80% confluency and treated 2 d after cells were
100% confluent. Solvent or LG100268 or LG100754 was added to cells in
the presence of insulin (10 µg/ml) for 7 d with a media and
compound change on day 3. During the differentiation assay, cells were
treated in DMEM with 10% FBS medium. Differentiation was monitored via
oil red O staining and triglyceride accumulation.
Oil Red O Staining
Oil red O staining of adipocytes was performed by fixing the
cells with 4% formalin in PBS, followed by a 5-min incubation with
0.5% oil red O stain in propylene glycol, and destaining for 2 min in
85% propylene glycol. Stained cells were covered with PBS and stored
at 4 C.
Triglyceride Accumulation
The triglyceride accumulation assay used a modified method from
Sigma. 3T3-LI cells were plated at 3,500 cells per well in
a 96-well plate. After 7 d of treatment, cells were rinsed two
times with PBS and 50 µl of 0.1% NP-40/well was added to lyse cells.
Triglyceride assay solution (100 µl/well, Sigma
Diagnostics) was added and the plates were incubated for 1
h at 37 C and read at 540 nm. All treatments were done in
triplicate.
Gene Induction and Semiquantitative and Quantitative PCR
Caco-2 cells were differentiated as described previously
(22) and treated with compound at appropriate
concentrations for 24 h; RNA was harvested using TRI Reagent
(Sigma no. T9424) and processed with DNase. RT-PCR
was done using the SuperScript One-step RT-PCR with Platinum
Taq (Life Technologies, Inc., Gaithersburg, MD;
no. 10928–034). IBABP primers were as described previously
(22); GAPDH was used as a control. For ABCA1, RAW 264.7
cells were treated overnight with receptor ligands. RNA was prepared
and subjected to real-time PCR analysis on an ABI Prism 7700 Sequence
Detector (ABI Advanced Technologies, Inc., Columbia, MD). ABCA1
primer and probes were: 5' CAT CGA CAT GGT GAA GAA CCA (forward), 5'GAA
GCG GTT CTC CCC AAA C (reverse), CAT GGC CGA TGC CCT GGA G (probe).
Each data point was the average of 12 values; amplification
efficiencies were normalized against cyclophilin.
TNF-Induced Insulin Resistance
Two-day postconfluent 3T3-L1 cells were treated with IBMX (0.52
mM), dexamethasone (1 µM), and insulin (10
µg/ml) for 3 d. On day 3, media containing 10 µg/ml insulin
was added and cells were incubated an additional 4 d. To induce
insulin resistance, differentiated cells were treated with TNF (5
ng/ml) for 1–2 d. Protein collection for tyrosine phosphorylation
studies was done as described (23). Rexinoids were added
at the same time TNF treatment was started. Anti-IR antibody
(Oncogene Science, Inc., Manhasset, NY) was used for
overnight immunoprecipitation in RIPA buffer containing aprotinin (2
µg/ml), pepstatin (1 µM), leupeptin (10
µM), sodium orthovanadate (1 mM), and
glycerol-2-phosphate (25 mM) at 4 C. Western blot detection
of phosphotyrosine was performed using antiphosphotyrosine antibody
clone 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY)
at a 1 µg/ml concentration.
db/db Glucose Studies
Female diabetic, 47-d-old, C57BLKS/J-m+/+db mice were dosed with
vehicle, LG100268 (30 mg/kg) or LG100754 (100 mg/kg) once daily for
14 d. Blood was drawn after a 3-h fast from the tip of the tail on
indicated days, and plasma glucose levels were measured by adoption of
the glucose oxidase Trinder protocol (Sigma).
Reagents, RXR, and PPAR Compounds
IBMX, dexamethasone, and insulin were purchased from
Sigma. Recombinant human TNF was purchased from R & D Systems (Minneapolis, MN). LG100268 and LG100754 were
synthesized and solvated (90% ethanol and 10% DMSO) at Ligand Pharmaceuticals, Inc. (San Diego, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Department of Biochemistry and Molecular Biology,
Mail Zone AA4E, Pharmacia Corporation, 700 Chesterfield Parkway North,
St. Louis, Missouri 63198.
Abbreviations: AF-2, Activation function 2; BAR, bile acid receptor; BRL, BRL49653; CBP, cAMP response element-binding protein; CDCA, chenodeoxycholic acid; FXR, farnesoid X receptor; HRE, hormone response element; IBABP, intestinal bile acid binding protein; IBMX, isobutylmethylxanthine; IR, insulin receptor; LBD, ligand binding domain; LXR, liver X receptor; NGFI-B, nerve growth factor-induced gene B; RID, receptor interacting domain; 22(R)OHC, 22(R)hydroxy cholesterol; SRC-1, steroid receptor coactivator 1; TRAP220, TR-associated protein 220.
Received for publication September 6, 2000. Accepted for publication April 23, 2001.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and
. Proc Natl Acad Sci USA 94:4318–4323 associated with human colon
cancer. Mol Cell 3:799–804[CrossRef][Medline]
ligand inhibits
adipocyte differentiation. Proc Natl Acad Sci USA 96:6102–6106
(PPAR) heterodimers: intermolecular synergy requires only the
PPAR hormone-dependent activation function. Mol Cell Biol 186:3483–3494
and retinoid X receptor.
Proc Natl Acad Sci USA 94:237–241 2, a lipid-activated
transcription factor. Cell 79:1147–1156[CrossRef][Medline]
is
required for the differentiation of adipose tissue in vivo and in
vitro. Mol Cell 4:611–617[CrossRef][Medline]
inhibits adipocyte differentiation. J Biol Chem 275:1873–1877-LXR-ABCA1 pathway in macrophages is involved in cholesterol
efflux and atherogenesis. Mol Cell 7:161–171[CrossRef][Medline]
inhibits signaling from the insulin receptor.
Proc Natl Acad Sci USA 91:4854–4858-induced inhibition of insulin
signaling. J Clin Invest 100:1863–1869[Medline]
-activating properties. J Biol
Chem 4:32679–32684
/RXR crystal structure reveals the molecular basis of
heterodimerization among nuclear receptors. Mol Cell 5:545–555[CrossRef][Medline]
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 O. Ziouzenkova, G. Orasanu, G. Sukhova, E. Lau, J. P. Berger, G. Tang, N. I. Krinsky, G. G. Dolnikowski, and J. Plutzky Asymmetric Cleavage of {beta}-Carotene Yields a Transcriptional Repressor of Retinoid X Receptor and Peroxisome Proliferator-Activated Receptor Responses Mol. Endocrinol., January 1, 2007; 21(1): 77 - 88. [Abstract] [Full Text] [PDF]
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 W. M. Golden, K. B. Weber, T. L. Hernandez, S. I. Sherman, W. W. Woodmansee, and B. R. Haugen Single-Dose Rexinoid Rapidly and Specifically Suppresses Serum Thyrotropin in Normal Subjects J. Clin. Endocrinol. Metab., January 1, 2007; 92(1): 124 - 130. [Abstract] [Full Text] [PDF]
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 L. Michalik, J. Auwerx, J. P. Berger, V. K. Chatterjee, C. K. Glass, F. J. Gonzalez, P. A. Grimaldi, T. Kadowaki, M. A. Lazar, S. O'Rahilly, et al. International Union of Pharmacology. LXI. Peroxisome Proliferator-Activated Receptors Pharmacol. Rev., December 1, 2006; 58(4): 726 - 741. [Abstract] [Full Text] [PDF]
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P. Germain, P. Chambon, G. Eichele, R. M. Evans, M. A. Lazar, M. Leid, A. R. De Lera, R. Lotan, D. J. Mangelsdorf, and H. Gronemeyer International Union of Pharmacology. LXIII. Retinoid X Receptors Pharmacol. Rev., December 1, 2006; 58(4): 760 - 772. [Abstract] [Full Text] [PDF]
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 S. K. Kolluri, M. Corr, S. Y. James, M. Bernasconi, D. Lu, W. Liu, H. B. Cottam, L. M. Leoni, D. A. Carson, and X.-k. Zhang The R-enantiomer of the nonsteroidal antiinflammatory drug etodolac binds retinoid X receptor and induces tumor-selective apoptosis PNAS, February 15, 2005; 102(7): 2525 - 2530. [Abstract] [Full Text] [PDF]
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 K. M. Ogilvie, R. Saladin, T. R. Nagy, M. S. Urcan, R. A. Heyman, and M. D. Leibowitz Activation of the Retinoid X Receptor Suppresses Appetite in the Rat Endocrinology, February 1, 2004; 145(2): 565 - 573. [Abstract] [Full Text] [PDF]
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 A. Kassam, B. Miao, P. R. Young, and R. Mukherjee Retinoid X Receptor (RXR) Agonist-induced Antagonism of Farnesoid X Receptor (FXR) Activity due to Absence of Coactivator Recruitment and Decreased DNA Binding J. Biol. Chem., March 14, 2003; 278(12): 10028 - 10032. [Abstract] [Full Text] [PDF]
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J. Wu, C. Xia, J. Meier, S. Li, X. Hu, and D. S. Lala The Hypolipidemic Natural Product Guggulsterone Acts as an Antagonist of the Bile Acid Receptor Mol. Endocrinol., July 1, 2002; 16(7): 1590 - 1597. [Abstract] [Full Text] [PDF]
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B. M. Forman The Antidiabetic Agent LG100754 Sensitizes Cells to Low Concentrations of Peroxisome Proliferator-activated Receptor gamma Ligands J. Biol. Chem., April 5, 2002; 277(15): 12503 - 12506. [Abstract] [Full Text] [PDF]
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