RXR Agonists Activate PPAR-Inducible Genes, Lower Triglycerides, and Raise HDL Levels In Vivo

From the Departments of Pharmacology (R.M., L.J., P.H., J.R.P.), Drug Safety and Disposition (J.S.), and Retinoid Research (R.A.H.), Ligand Pharmaceuticals, Inc, San Diego, Calif.

Correspondence to Ranjan Mukherjee, Department of Pharmacology, Ligand Pharmaceuticals, Inc, 10255 Science Center Drive, San Diego, CA 92121. E-mail rmukherjee{at}ligand.com

	Abstract

Top Abstract Introduction Methods Results and Discussion References

 
Abstract—Peroxisome proliferator–activated receptors (PPARs) and retinoid X receptors (RXRs) are members of the intracellular receptor superfamily. PPARs bind to peroxisome proliferator–response elements (PPREs) as heterodimers with RXR and as such activate gene transcription in response to activators. Fibrates like gemfibrozil are well-known PPAR activators and are used in the treatment of hyperlipidemia. We show that the RXR ligand LGD1069 (Targretin^(TM)), like gemfibrozil, can activate the PPAR/RXR signal-transduction pathway, including transactivation of the bifunctional enzyme or acyl-CoA oxidase response elements in a cotransfection assay. The activation also occurs in vivo, whereby in rats treated with LGD1069 or gemfibrozil, bifunctional enzyme and acyl-CoA oxidase RNA are induced and the combination of LGD1069 and gemfibrozil leads to a greater induction. Importantly, in hypertriglyceridemic db/db mice treated with RXR or PPAR agonists, triglyceride levels are lowered, and the combination again has significantly greater efficacy. RXR agonists also raise HDL cholesterol levels without changing apoA-I RNA expression. This observation suggests the use of RXR-selective agonists, "rexinoids," either alone or in combination with a fibrate as a new therapeutic approach to treating patients with high triglyceride and low HDL cholesterol levels.

Key Words: RXR • PPAR • rexinoids • hypertriglyceridemia • low HDL

	Introduction

Top Abstract Introduction Methods Results and Discussion References

 
Peroxisome proliferator–activated receptors are members of the intracellular receptor superfamily. Three subtypes have been identified, PPAR, PPARß (NUC1 or FAAR or PPAR), and PPAR (see References 1 through 4^{1 2 3 4} and references therein). PPARs bind to PPREs as heterodimers with the RXR and, in response to PPAR ligands,^{5 6} activate gene transcription.

A diverse array of compounds, including plasticizers, fatty acids, eicosanoids, leukotrienes,⁷ indomethacin,⁸ and the fibrate class of lipid-lowering drugs like gemfibrozil and fenofibrate,⁹ activate PPAR, while thiazolidinediones and prostaglandin J2 are PPAR ligands.^{10 11 12} In fibrate-treated animals, there is a rapid increase in the expression of genes that encode enzymes for the ß-oxidation of fatty acids such as AOX and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme).¹³ PPREs have been identified in the promoters of these genes, suggesting that activation of the peroxisomal fatty acid ß-oxidation pathway contributes to the lipid lowering observed with fibrates.

We have recently shown that the RXR/PPAR heterodimer is activated by RXR agonists.⁴ This finding emphasizes the permissive nature of the RXR/PPAR heterodimer, whereby either partner can bind ligand and activate gene expression. RXR agonists have similar effects as thiazolidinediones; they induce adipocyte differentiation,¹⁴ lower elevated glucose and insulin levels, and improve insulin resistance in ob/ob and db/db mice.¹⁵ We refer to these RXR-selective ligands as "rexinoids" because their pharmacology is clearly distinct from "retinoids," which are retinoic acid receptor activators that mimic the action of retinoic acid.¹⁶

We hypothesized that rexinoids would mimic the effects of fibrates via activation of the RXR side of the RXR/PPAR heterodimer. Here we demonstrate for the first time that rexinoids elicit similar responses as PPAR activators in vivo. In particular, expression of the bifunctional enzyme and AOX gene is induced in rat livers by gemfibrozil or an RXR-selective agonist LGD1069 (Targretin^(TM)) treatment.¹⁷ The combination of LGD1069 and gemfibrozil gives a much stronger induction. Further, in db/db mice, RXR activators like LGD1069¹⁷ and LG100268¹⁸ lower triglyceride levels. HDL-C levels are also raised in rexinoid-treated mice. This finding demonstrates a convergence of the RXR and PPAR pathways in vivo and suggests that RXR modulators can be used in the treatment of lipid disorders such as hypertriglyceridemia and low HDL-C levels.

	Methods

Top Abstract Introduction Methods Results and Discussion References

 
Gemfibrozil was purchased from Sigma Chemical Company, and LGD1069 and LG100268 were synthesized at Ligand Pharmaceuticals, Inc.

Plasmids and Cotransfection Assays
The bifunctional enzyme PPRE-containing reporter was constructed by ligating a 112-base pair oligonucleotide (5'-GATCC CCT TTG ACC TAT TGA ACT ATT ACC TAC ATT AGATCC CCT TTG ACC TAT TGA ACT ATT ACC TAC ATT AGATCC CCT TTG ACC TAT TGA ACT ATT ACC TAC ATT AGATC-3') containing three copies (in bold) of the bifunctional enzyme PPRE¹⁹ into the BamHI site of pBL-tk-Luc.^{20 21}

Cotransfection assays were performed as previously described.²² Control cells received an equivalent amount of vehicle. Transfections were done in triplicate. Each point represents the mean±SD. The experiments were repeated at least twice. A representative experiment is shown.

In Vivo Studies
Male Sprague-Dawley rats (225 to 250 g) were obtained from Harlan Sprague Dawley (San Diego, Calif). Animals (four per treatment group) were fed standard laboratory diets. Animals were treated with compounds via gavage (5 mL · kg^-1 · d^-1) for 7 consecutive days. The vehicle contained Tween 80, PEG 400, and carboxymethylcellulose (0.05:9.95:0.9). Liver RNA was isolated by RNAzol technique (Tel-Test Inc). The rat bifunctional enzyme cDNA and human GAPDH cDNA (Ambion) were labeled with [³²P]dATP by random priming. AOX cDNA (177 base pairs) was inserted into the Pst I and HindIII sites of pGEM-4 vector (Promega). Antisense AOX riboprobe was made by T7 RNA polymerase. Northern blot analysis was performed by standard techniques.

For protein extracts, livers were homogenized in 10% sucrose, 3 mmol/L imidazole (pH 7.4), and clarified by centrifugation. Protein extract (100 µg per lane) was resolved by 10% denaturing polyacrylamide gel electrophoresis and transferred to Immobilon-P transfer membrane (Amersham). The membrane was incubated with 5% powdered milk in PBS for 30 minutes at room temperature. After washing with PBST (PBS with 0.075% Tween 20), the membrane was incubated with a polyclonal anti–bifunctional enzyme antibody in PBST. The membrane was again washed with PBST, incubated with ¹²⁵I-labeled protein A, and the antigen-antibody complex visualized by autoradiography. The band intensities on the Northern and Western blots were quantified by a Phosphorimager (Molecular Dynamics).

Female C57BLKs/J-m+/+db mice (7 weeks old at commencement of study, nine animals per treatment group) were dosed with vehicle or gemfibrozil, LG100268, or the combination of gemfibrozil and LG100268, as indicated in the figure legends, once daily by gavage (0.6 mL/42 g) for 14 days. In a separate study, animals were dosed with vehicle (eight animals) or LGD1069 (seven animals) for 14 days. On the days indicated, animals were fasted for 3 hours and a sample of blood was drawn. The GPO-Trinder kit (Sigma) was used to measure plasma triglycerides. On day 15, the animals were sacrificed and plasma HDL-C levels measured by the phosphotungstic acid magnesium ion precipitation technique (Boehringer Mannheim).

Total liver RNA was prepared and analyzed by Northern blotting, with a ³²P-labeled probe specific to apoA-I. The blot was then stripped and rehybridized to a probe specific to GAPDH. The bands were quantified by a Phosphorimager (Molecular Dynamics). The intensity of the apoA-I signal in each lane was normalized to the intensity of the GAPDH signal of the same lane and the mean of each group calculated.

	Results and Discussion

Top Abstract Introduction Methods Results and Discussion References

 
To study the effect of rexinoids and fibrates on the RXR/PPAR heterodimer, we used the cotransfection assay with PPAR and RXR expression vectors and a reporter containing three copies of the PPRE identified in the AOX gene⁵ or the bifunctional enzyme gene. The transcriptional response increases 4-fold with gemfibrozil and 13-fold with LGD1069, an RXR-selective agonist (K_d 21–36 nM for RXRs¹⁷) on the AOX PPRE (Fig 1A). For the bifunctional enzyme PPRE, the transcription response was increased by 3-fold and 5-fold with gemfibrozil and LGD1069, respectively (Fig 1B). This result is consistent with our earlier data showing activation of RXR/PPAR with 9-cis retinoic acid (a retinoic acid receptor and RXR agonist^{5 6} ). However, the greatest response (49-fold with the AOX PPRE and 12-fold with the bifunctional enzyme PPRE) was observed in the presence of both gemfibrozil and LGD1069. This finding shows that a greatly enhanced transcriptional response occurs when both partners of the RXR/PPAR heterodimer are activated by their respective agonists.

View larger version (23K): [in this window] [in a new window]   Figure 1. Activation of the acyl-CoA oxidase and bifunctional enzyme (BE) PPRE by RXR and PPAR agonists. CV-1 cells were transfected with pCMVhPPAR (ref 6) and pRShRXR (ref 23) and a reporter containing three copies of the AOX PPRE (ref 5) (A and C) or three copies of the PPRE identified in the bifunctional enzyme gene (B and D). PPAR and RXR were used in A and B, while PPAR and RXR(L451A) (ref 24) were used in C and D. Gemfibrozil and LGD1069 were added, to a final concentration of 100 µmol/L and 1 µmol/L, respectively.

We hypothesized that the activation of the AOX and bifunctional enzyme PPREs and the synergistic activation by PPAR and RXR ligands occur through activation of the RXR/PPAR heterodimer. If this is the case, a nonfunctional RXR is expected to blunt the transcriptional response. We used such a mutant of RXR, RXR(L451A) (Reference 31³¹ and Schulman et al, unpublished data, 1997). In this mutant RXR, the amino acid at position 451 is changed to alanine. This region has been identified to be important for ligand-induced transcriptional activation by the receptor (the AF-2 domain) and is situated at the extreme carboxy- terminus of the receptor. RXR(L451A) binds RXR ligands with normal affinity but cannot activate transcription in response to ligands.²⁴ We show that when RXR(L451A) is partnered with PPAR, transcriptional activation in reponse to gemfibrozil or LGD1069 drops significantly (Fig 1C and 1D). In particular, activation with gemfibrozil and LGD1069 drops by 88% and 97% on the AOX and bifunctional enzyme PPREs, respectively (Fig 1; compare 1A with 1C and 1B with 1D). The simplest explanation is that the transcriptional activation of the AOX and bifunctional enzyme genes by both compounds is mediated via the PPAR/RXR heterodimer. Our interpretation is also consistent with published data demonstrating an increased transcription rate of both the AOX and bifunctional enzyme genes in response to fibrates.^{25 26}

Because both gemfibrozil and LGD1069 activate the RXR/PPAR heterodimer and the bifunctional enzyme and AOX genes are induced in the livers of rats treated with fibrates,¹³ we determined whether these genes are regulated by RXR agonists in vivo. Bifunctional enzyme and AOX RNA levels in livers of treated animals were determined by Northern blotting (Fig 2A). AOX RNA expression is induced 2-fold by gemfibrozil and 2.5-fold by LGD1069. In agreement with the cotransfection data, the induction is greater in the presence of both inducers (4-fold). Similarly, the bifunctional enzyme RNA was increased 4.4-fold and 4.8-fold by gemfibrozil and LGD1069 treatment, respectively (Fig 2A), and is also significantly greater in the presence of both compounds (11-fold) compared with treatment with a single agent. Hence, PPAR and RXR agonists induce hepatic expression of AOX and bifunctional enzyme RNA in treated animals.

View larger version (70K): [in this window] [in a new window]   Figure 2. Induction of the bifunctional enzyme and AOX gene in rats treated with gemfibrozil and LGD1069. Rats were treated with vehicle (control), gemfibrozil (100 mg · kg-1 · d-1), LGD1069 (30 mg · kg-1 · d-1), or gemfibrozil plus LGD1069 (100 mg · kg-1 · d-1 and 30 mg · kg-1 · d-1, respectively) by gavage for 7 days. A, Northern blot analysis was performed with 10 µg of total liver RNA per lane and a bifunctional enzyme (BE), AOX, or GAPDH probe. B, Western blot analysis was performed with 100 µg of protein extract from livers of treated animals and a polyclonal antibody to the rat bifunctional enzyme. The position of the expected band (78 kD) is denoted by an arrow.

To determine whether there was an increase in protein expression, Western blot analysis was performed with protein extracts from livers of animals similarly treated. Gemfibrozil or LGD1069 induce bifunctional enzyme protein expression (Fig 2B). A much stronger induction was observed in the presence of both compounds, consistent with both the cotransfection data and Northern blot analysis (Figs 1 and 2A). Although Western blot analysis is not the most accurate method to quantify proteins, the signal intensities indicate that the increased level of gene expression is mirrored by increased protein expression.

Clinically gemfibrozil is used for the treatment of hypertriglyceridemia, an independent risk factor for cardiovascular diseases that often correlates with low HDL-C levels. Because rexinoids activate the RXR/PPAR heterodimer and activate transcription of fibrate-responsive genes, we next determined whether RXR agonists also lower triglyceride levels in the hypertriglyceridemic db/db mice. These mice have a deranged leptin signaling pathway due to a mutation in the leptin receptor.²⁷ They are obese, hyperglycemic, and have elevated triglyceride levels that continue to increase over time in the untreated state (Fig 3A). After treatment, triglyceride levels decrease by 32% in LGD1069-treated mice compared with control mice.

View larger version (29K): [in this window] [in a new window]   Figure 3. Triglyceride lowering by RXR agonists and PPAR/RXR combination. RXR agonists lower triglycerides, and the combination of PPAR and RXR activators has greater efficacy in triglyceride lowering than a single agent. A, db/db mice were treated with vehicle or LGD1069 (30 mg · kg-1 · d-1) by gavage for 14 days. B, db/db mice were treated with vehicle (control), gemfibrozil (50 mg · kg-1 · d-1), LG100268 (20 mg · kg-1 · d-1), or a combination of gemfibrozil and LG100268 (50 mg · kg-1 · d-1 and 20 mg · kg-1 · d-1, respectively) by gavage for 15 days. The animals were bled on the days shown and plasma triglyceride levels determined. Each data point represents the mean±SEM. *Significantly different from control group (P<.05) as determined by Student's t test. +Significantly different (P<.05) compared with treatment with a single agent (either gemfibrozil or LG100268).

We next tested a more potent and selective RXR ligand and agonist, LG100268 (K_d <5 nM¹⁸), for its ability to lower triglycerides, either alone or in combination with gemfibrozil. LG100268 also activates the RXR/PPAR heterodimer in a cotransfection assay (Reference 28²⁸ and data not shown). Animals treated with near-maximum effective doses of LG100268 or gemfibrozil show a 41% and 30% decrease in triglyceride levels, respectively, compared with control animals at day 11 of the study (Fig 3B). Importantly, the combination of LG100268 and gemfibrozil is significantly more efficacious in triglyceride lowering than either compound alone, decreasing triglycerides by 64% compared with control animals. Hence, the combination did not just prevent the rise in triglyceride levels observed with gemfibrozil or LG100268 alone but decreased them to the normal level seen in lean littermates.²⁹ No significant difference in body weight was observed in treated versus control animals.

Low HDL-C level is a risk factor for cardiovascular disease. Since elevated triglyceride levels often correlate with low HDL-C levels, we investigated whether LG100268 elevates HDL-C in these mice. LG100268 raises HDL-C levels by 27% compared with vehicle-treated animals (Table). Gemfibrozil alone did not significantly raise HDL-C levels, consistent with published data in rodents.³⁰

To determine whether the rise in HDL-C levels is due to induction of apoA-I gene expression, we performed Northern blot analysis of liver RNA from treated animals. There was no change in apoA-I RNA expression in any of the treated groups compared with control animals (data not shown). Hence, the increase in HDL-C is not due to increased apoA-I gene expression and may be secondary to triglyceride reduction.

This is the first demonstration that RXR agonists mimic PPAR activators in vivo. They induce genes normally induced by fibrates, lower triglycerides, and raise HDL-C in an animal model with dyslipidemia. Further, the combination of both compounds has greater efficacy in triglyceride lowering than either compound alone.

Our results with the RXR (AF-2) mutant indicate that the ligand-inducible transcription-activating domain of RXR contributes not only to the RXR but also to the PPAR agonist response. Similarly, preliminary experiments with PPAR (AF-2) and RXR (AF-2) mutants indicate that mutating either receptor severely compromises the total activity of the heterodimer (data not shown). This observation suggests that both PPAR and RXR activating domains contribute to the total transcriptional activity induced by PPAR and RXR agonists.

PPAR and RXRs bind as a heterodimer to PPREs identified in promoters of several genes. Some of these genes are involved in lipid metabolism.³¹ Further, disruption of the PPAR gene by homologous recombination prevents peroxisome proliferation and induction of target genes in response to fibrates.³² PPAR knockout mice also have abnormal lipid metabolism. Hence, the RXR/PPAR heterodimer is the likely target for fibrates. In support of this hypothesis, clofibric acid and clofibrate induce a conformational change in PPAR³³ and ciprofibrate enhances binding of the heterodimer to DNA,³⁴ suggesting a direct interaction between fibrates and PPAR. Our results suggest that the RXR/PPAR heterodimer is also the target for rexinoids.

We speculate that hypoalphalipoproteinemia (low HDL-C) and hypertriglyceridemia (a risk factor for cardiovascular disease ³⁵ ) are treatable with rexinoids, or even better, with the combination of a rexinoid and a fibrate. It may be possible to use lower doses of a PPAR and RXR activator to obtain the same or increased efficacy. Combining these two classes of compounds may improve lipid profiles and have reduced side effects and toxicity. The combination may offer a novel and potentially important paradigm for the treatment of atherosclerotic cardiovascular diseases.

	Selected Abbreviations and Acronyms

 

AOX = acyl-CoA oxidase HDL-C = HDL cholesterol PPAR = peroxisome proliferator–activated receptor PPRE = peroxisome proliferator–response element RXR = retinoid X receptor

	Acknowledgments

 
We thank Jim Fraser and Regis Saladin for critical comments on the manuscript. We are grateful to Ruiyin Chu and Janardan K. Reddy for the bifunctional enzyme cDNA, Ski Krisans for the bifunctional enzyme antibody and Ira Schulman for RXR(L451A). We also thank Marcus Boehm, Charles Pathirana, and Alex Nadzan for compounds; Jon Rosen and Ed Ulm for support; Glenn Croston, Veronica Martinez and Peter Syka for help and reagents; and the staff at Metabolex Inc for performing the experiments with db/db mice.

Received April 23, 1997; accepted October 25, 1997.

	References

Top Abstract Introduction Methods Results and Discussion References

 
1. Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature. 1990;347:645–650.[Medline] [Order article via Infotrieve]

2. Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W. Control of the peroxisome ß-oxidation pathway by a novel family of nuclear hormone receptors. Cell. 1992;68:879–887.[Medline] [Order article via Infotrieve]

3. Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K, Evans RM. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci U S A.. 1994;91:7355–7359.[Abstract/Free Full Text]

4. Mukherjee R, Jow L, Croston GE, Paterniti JR. Identification, characterization, and tissue distribution of human peroxisome proliferator-activated receptor (PPAR) isoforms PPAR2 versus PPAR1 and activation with retinoid X receptor agonists and antagonists. J Biol Chem. 1997;272:8071–8076.[Abstract/Free Full Text]

5. Kliewer SA, Umesono K, Noonan DJ, Heyman RA, Evans RM. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature. 1992;358:771–774.[Medline] [Order article via Infotrieve]

6. Mukherjee R, Jow L, Noonan D, McDonnell DP. Human and rat peroxisome proliferator activated receptors (PPARs) demonstrate similar tissue distribution but different responsiveness to PPAR activators. J Steroid Biochem Mol Biol. 1994;51:157–166.[Medline] [Order article via Infotrieve]

7. Devchand PR, Keller H, Peters JM, Vasquez M, Gonzales FJ, Wahli W. The PPAR-leukotriene B4 pathway to inflammation control. Nature. 1996;384:39–43.[Medline] [Order article via Infotrieve]

8. Lehman JM, Lenhard JM, Oliver BB, Ringold GM, Kliewer SA. Peroxisome proliferator-activated receptors and are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J Biol Chem. 1997;272:3406–3410.[Abstract/Free Full Text]

9. Todd PA, Ward A. Gemfibrozil: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in dyslipidaemia. Drugs. 1988;36:314–339.[Medline] [Order article via Infotrieve]

10. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor (PPAR). J Biol Chem. 1995;270:12953–12956.[Abstract/Free Full Text]

11. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor and promotes adipocyte differentiation. Cell. 1995;83:813–819.[Medline] [Order article via Infotrieve]

12. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM. 15-Deoxy ^12–14 -prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell. 1995;83:803–812.[Medline] [Order article via Infotrieve]

13. Reddy JK, Goel SK, Nemali MR, Carrino JJ, Laffler TG, Reddy MK, Sperbeck SJ, Osumi T, Hashimoto T, Lalwani ND, Rao SM. Transcriptional regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators. Proc Natl Acad Sci U S A.. 1986;83:1747–1751.[Abstract/Free Full Text]

14. Cesario RM, Mukherjee R, Paterniti JR, Crombie DL, Heyman RA. RXR agonists function as insulin sensitizers in adipogenesis and in mouse models for NIDDM. In: Abstracts of the Keystone Symposia on the Adipose Cell; January 15–21, 1997; Park City, Utah. Abstract 102.

15. Mukherjee R, Davies PJA, Crombie D, Bischoff E, Cesario R, Jow L, Hamann LG, Boehm MF, Mondon CE, Nadzan AM, Paterniti JR, Heyman RA. Sensitization of diabetic and obese mice to insulin by retinoid X receptor agonists. Nature. 1997;386:407–410.[Medline] [Order article via Infotrieve]

16. Sporn MB, Dunlop NM, Newton DL, Smith JM. Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Fed Proc. 1976;35:1332–1338.[Medline] [Order article via Infotrieve]

17. Boehm MF, Zhang L, Badea BA, White SK, Mais DE, Berger E, Suto CM, Goldman ME, Heyman RA. Synthesis and structure activity relationships of novel retinoid X receptor selective retinoids. J Med Chem. 1994;37:2930–2941.[Medline] [Order article via Infotrieve]

18. Boehm MF, Zhang L, Zhi L, McClurg MR, Berger E, Wagoner M, Mais DE, Suto CM, Davies PJA, Heyman RA, Nadzan AM. Design and synthesis of potent retinoid X receptor selective ligands that induce apoptosis in leukemia cells. J Med Chem. 1995;38:3146–3155.[Medline] [Order article via Infotrieve]

19. Zhang B, Marcus SL, Sajjadi FG, Alvares K, Reddy JK, Subramani S, Rachubinski RA, Capone JP. Identification of a peroxisome proliferator-responsive element upstream of the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. Proc Natl Acad Sci U S A.. 1992;89:7541–7545.[Abstract/Free Full Text]

20. Hollenberg SM, Evans RM. Multiple and cooperative trans-activation domains of the human glucocorticoid receptor. Cell. 1988;55:899–906.[Medline] [Order article via Infotrieve]

21. Luckow B, Schutz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987;15:5490.[Free Full Text]

22. Jow L, Mukherjee R. The human peroxisome proliferator-activated receptor (PPAR) subtype NUC1 represses the activation of hPPAR and thyroid hormone receptors. J Biol Chem. 1995;270:3836–3840.[Abstract/Free Full Text]

23. Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature. 1990;345:224–229.[Medline] [Order article via Infotrieve]

24. Willy PJ, Mangelsdorf DJ. Unique requirements for retinoid-dependent transcriptional activation by the orphan receptor LXR. Genes Dev. 1997;11:289–298.[Abstract/Free Full Text]

25. Hertz R, Bishara-Shieban J, Bar-Tana J. Mode of action of peroxisome proliferators as hypolipidemic drugs. J Biol Chem. 1995;270:13470–13475.[Abstract/Free Full Text]

26. Reddy JK, Goel SK, Nemali MR, Carrino JJ, Laffler TG, Reddy MK, Sperbeck SJ, Osumi T, Hashimoto T, Lalwani ND, Rao MS. Transcriptional regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators. Proc Natl Acad Sci U S A.. 1986;83:1747–1751.

27. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis S, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell. 1996;84:491–495.[Medline] [Order article via Infotrieve]

28. Lala DS, Mukherjee R, Schulman IG, Canan-Koch SS, Dardashti LJ, Nadzan AM, Croston GE, Evans RM, Heyman RA. Activation of specific RXR heterodimers by an antagonist of RXR homodimers. Nature. 1996;383:450–453.[Medline] [Order article via Infotrieve]

29. Zhang B, Graziano MP, Doebber TW, Leibowitz MD, Carrington SW, Szalkowski DM, Hey PJ, Wu M, Cullinan CA, Bailey P, Lollman B, Frederich R, Flier JF, Strader CD, Smith RG. Down-regulation of the expression of the obese gene by an antidiabetic thiazolidinedione in Zucker diabetic fatty rats and db/db mice. J Biol Chem. 1996;271:9455–9459.[Abstract/Free Full Text]

30. Haubenwallner S, Essenberg AD, Barnett BC, Pape ME, DeMattos RB, Krause BR, Minton LL, Auerbach BJ, Newton RS, Leff T, Bisgaier CL. Hypolipidemic activity of select fibrates correlates to changes in hepatic apolipoprotein C-III expression: a potential physiologic basis for their mode of action. J Lipid Res. 1995;36:2541–2551.[Abstract]

31. Schoojans K, Staels B, Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res. 1996;37:907–925.[Abstract]

32. Lee SST, Pineau T, Drago J, Lee EI, Owens JW, Kroetz DL, Fernandez-Salguero PM, Westpahl H, Gonzales FJ. Targeted disruption of the isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol. 1995;15:3012–3022.[Abstract]

33. Dowell P, Peterson VJ, Zabriskie M, Leid M. Ligand-induced peroxisome proliferator-activated receptor conformational change. J Biol Chem. 1997;272:2013–2020.[Abstract/Free Full Text]

34. Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors and . Proc Natl Acad Sci U S A.. 1997;94:4312–4317.[Abstract/Free Full Text]

35. Castelli WP. Epidemiology of triglycerides: a view from Framingham. Am J Cardiol. 1992;70:3H–9H.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				J. de Vries-van der Weij, W. de Haan, L. Hu, M. Kuif, H. L. D. W. Oei, J. W. A. van der Hoorn, L. M. Havekes, H. M. G. Princen, J. A. Romijn, J. W. A. Smit, et al. Bexarotene Induces Dyslipidemia by Increased Very Low-Density Lipoprotein Production and Cholesteryl Ester Transfer Protein-Mediated Reduction of High-Density Lipoprotein Endocrinology, May 1, 2009; 150(5): 2368 - 2375. [Abstract] [Full Text] [PDF]

				B. Desvergne, L. Michalik, and W. Wahli Transcriptional Regulation of Metabolism Physiol Rev, April 1, 2006; 86(2): 465 - 514. [Abstract] [Full Text] [PDF]

				T. Harrity, D. Farrelly, A. Tieman, C. Chu, L. Kunselman, L. Gu, R. Ponticiello, M. Cap, F. Qu, C. Shao, et al. Muraglitazar, a Novel Dual ({alpha}/{gamma}) Peroxisome Proliferator-Activated Receptor Activator, Improves Diabetes and Other Metabolic Abnormalities and Preserves {beta}-Cell Function in db/db Mice Diabetes, January 1, 2006; 55(1): 240 - 248. [Abstract] [Full Text] [PDF]

				S. P. Anderson, C. Dunn, A. Laughter, L. Yoon, C. Swanson, T. M. Stulnig, K. R. Steffensen, R. A.S. Chandraratna, J.-A. Gustafsson, and J. C. Corton Overlapping Transcriptional Programs Regulated by the Nuclear Receptors Peroxisome Proliferator-Activated Receptor {alpha}, Retinoid X Receptor, and Liver X Receptor in Mouse Liver Mol. Pharmacol., December 1, 2004; 66(6): 1440 - 1452. [Abstract] [Full Text] [PDF]

				H. Fischer, S. M. G. Dias, M. A. M. Santos, A. C. Alves, N. Zanchin, A. F. Craievich, J. W. Apriletti, J. D. Baxter, P. Webb, F. A. R. Neves, et al. Low Resolution Structures of the Retinoid X Receptor DNA-binding and Ligand-binding Domains Revealed by Synchrotron X-ray Solution Scattering J. Biol. Chem., April 25, 2003; 278(18): 16030 - 16038. [Abstract] [Full Text] [PDF]

				J. J. Repa, K. E. Berge, C. Pomajzl, J. A. Richardson, H. Hobbs, and D. J. Mangelsdorf Regulation of ATP-binding Cassette Sterol Transporters ABCG5 and ABCG8 by the Liver X Receptors alpha and beta J. Biol. Chem., May 17, 2002; 277(21): 18793 - 18800. [Abstract] [Full Text] [PDF]

				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 Differential Effects of Rexinoids and Thiazolidinediones on Metabolic Gene Expression in Diabetic Rodents Mol. Pharmacol., April 1, 2001; 59(4): 765 - 773. [Abstract] [Full Text]

				P. J. A. Davies, S. A. Berry, G. L. Shipley, R. H. Eckel, N. Hennuyer, D. L. Crombie, K. M. Ogilvie, J. Peinado-Onsurbe, C. Fievet, M. D. Leibowitz, et al. Metabolic Effects of Rexinoids: Tissue-Specific Regulation of Lipoprotein Lipase Activity Mol. Pharmacol., February 1, 2001; 59(2): 170 - 176. [Abstract] [Full Text]

				J. J. Repa, S. D. Turley, J.-M. A. Lobaccaro, J. Medina, L. Li, K. Lustig, B. Shan, R. A. Heyman, J. M. Dietschy, and D. J. Mangelsdorf Regulation of Absorption and ABC1-Mediated Efflux of Cholesterol by RXR Heterodimers Science, September 1, 2000; 289(5484): 1524 - 1529. [Abstract] [Full Text]

				G. Shao, R. A. Heyman, and I. G. Schulman Three Amino Acids Specify Coactivator Choice By Retinoid X Receptors Mol. Endocrinol., August 1, 2000; 14(8): 1198 - 1209. [Abstract] [Full Text]

				S. M. Lippman and R. Lotan Advances in the Development of Retinoids as Chemopreventive Agents J. Nutr., February 1, 2000; 130(2): 479 - 479. [Abstract] [Full Text]

				E. D. Bischoff, R. A. Heyman, and W. W. Lamph Effect of the Retinoid X Receptor-Selective Ligand LGD1069 on Mammary Carcinoma After Tamoxifen Failure J Natl Cancer Inst, December 15, 1999; 91(24): 2118 - 2118. [Abstract] [Full Text] [PDF]

				H. Knoblauch, A. Busjahn, B. Muller-Myhsok, H.-D. Faulhaber, H. Schuster, R. Uhlmann, and F. C. Luft Peroxisome Proliferator-Activated Receptor {gamma} Gene Locus Is Related to Body Mass Index and Lipid Values in Healthy Nonobese Subjects Arterioscler. Thromb. Vasc. Biol., December 1, 1999; 19(12): 2940 - 2944. [Abstract] [Full Text] [PDF]

				B. Desvergne and W. Wahli Peroxisome Proliferator-Activated Receptors: Nuclear Control of Metabolism Endocr. Rev., October 1, 1999; 20(5): 649 - 688. [Abstract] [Full Text]

				S. I. Sherman, J. Gopal, B. R. Haugen, A. C. Chiu, K. Whaley, P. Nowlakha, and M. Duvic Central Hypothyroidism Associated with Retinoid X Receptor-Selective Ligands N. Engl. J. Med., April 8, 1999; 340(14): 1075 - 1079. [Abstract] [Full Text] [PDF]

				J. Berger, M. D. Leibowitz, T. W. Doebber, A. Elbrecht, B. Zhang, G. Zhou, C. Biswas, C. A. Cullinan, N. S. Hayes, Y. Li, et al. Novel Peroxisome Proliferator-activated Receptor (PPAR) gamma  and PPARdelta Ligands Produce Distinct Biological Effects J. Biol. Chem., March 5, 1999; 274(10): 6718 - 6725. [Abstract] [Full Text] [PDF]

				J. Ribalta, J. Girona, J. C. Vallvé, A. E. La Ville, M. Heras, and L. Masana Vitamin A is linked to the expression of the AI-CIII-AIV gene cluster in familial combined hyperlipidemia J. Lipid Res., March 1, 1999; 40(3): 426 - 431. [Abstract] [Full Text]

				P. Costet, C. Legendre, J. More, A. Edgar, P. Galtier, and T. Pineau Peroxisome Proliferator-activated Receptor alpha -Isoform Deficiency Leads to Progressive Dyslipidemia with Sexually Dimorphic Obesity and Steatosis J. Biol. Chem., November 6, 1998; 273(45): 29577 - 29585. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mukherjee, R. Articles by Heyman, R. A. Search for Related Content PubMed PubMed Citation Articles by Mukherjee, R. Articles by Heyman, R. A.