Flipping the Fat-Sensing Switch

Image: Courtesy of David J. Mangelsdorf YIN AND YANG: Although they play opposite roles in bile acid production, these RXR heterodimers synergistically modulate cholesterol absorption and transport. When the molecular basis of Tangier disease was discovered in 1999,1 researchers lined up to study this orphan genetic disorder. Patients with Tangier have a propensity for heart disease and atherosclerosis, making this rare malady a model for some pressing health problems found in industrialized nations. Following close behind the discovery of the Tangier gene were two studies, now deemed Hot Papers,2,3 that examined some of the molecular mechanisms upstream of the ATP-binding cassette A1 (ABCA1) involved in cholesterol transport and defective in patients with Tangier. One in vitro study demonstrated an orphan nuclear hormone receptor that senses the presence of cholesterol and turns on ABCA1.2 The second paper showed how molecules activating this gene and related pathways can block absorption of dietary cholesterol in mice.3 No one missed the obvious therapeutic applications, but figuring out how safely to flip the fat-sensing switch continues to elude them. Like a Dickens classic, orphans run rampant through the story of cholesterol metabolism: ABCA1 is the genetic source of an orphan disease that afflicts relatively few and remains largely 'unadopted' by pharmaceutical companies. Furthermore, ABCA1's regulation can be traced back to orphan nuclear hormone receptors, named as such because their ligands were initially unknown. For Alan Tall, professor of medicine at Columbia University, the ideas began to coalesce soon after the Tangier discovery. Groups had been defining the role of orphan receptors such as the liver X receptors (LXRand LXRß).4 Known then only to activate cholesterol 7*-hydroxylase (CYP7A1), which catalyzes bile acid production from excess cholesterol, LXRs are stimulated by oxygenated cholesterol esters called oxysterols. In this way, LXRs sense cholesterol in the liver to stimulate the lipid's degradation. Researchers were pondering what other effects LXRs might have in maintaining cholesterol homeostasis, and they began hunting down mechanisms. A GOOD MARRIAGE To work, LXR must dimerize with the retinoid X receptor (RXR), a nuclear hormone receptor that responds to retinoids. Tall performed in vitro experiments that stimulated LXR and RXR with 22-hydroxycholesterol and 9-cis-retinoic acid, respectively. Singly, each chemical caused upregulation in ABCA1. When used together, the effect was additive. "[These results] suggested to us that ABCA1 was in fact the target for LXR/RXR," says Tall. "They work together. ... It's like a good marriage with both partners participating." His group then isolated the promoter element, DR4, upstream of the ABCA1 gene. The element has two nuclear hormone-specific sequences separated by a spacer of four base pairs. Transcription can be activated from either the RXR or the LXR side. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. P. Costet et al., "Sterol-dependent transactivation of the ABC1 promoter by the liver x receptor/retinoid x receptor." Journal of Biological Chemistry, 275:28240-5, Sept. 8, 2000. (Cited in 100 papers) J.J. Repa et al., "Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers," Science, 289:1524-9, Sept. 1, 2000. (Cited in 156 papers) LIKE MINDS David J. Mangelsdorf, professor of pharmacology, University of Texas (UT), Southwestern Medical Center at Dallas, and a Howard Hughes Medical Institute (HHMI) investigator, has been studying transcriptional signaling by nuclear hormone receptors for years. Tall attributes much of his discovery to Mangelsdorf's lead. Soon after Tall's results were published online, Mangelsdorf released his paper with the same finding--that RXR/LXR stimulates ABCA1 production. Mangelsdorf also viewed the mechanism in a living system, showing dramatic effects on cholesterol absorption in mice when treated with RXR agonists called rexinoids. A dose of 1.4 mg/kg reduced cholesterol absorption by one-half in mice; a dose of 14 mg/kg resulted in total inhibition. Despite being fed 10 times the normal amount of cholesterol, mice were still producing it endogenously to maintain homeostasis. Mitchell Lazar, director of the University of Pennsylvania Diabetes Center, says the timing of the Mangelsdorf paper was impeccable. The authors proposed a number of mechanisms by which the RXR agonists could be working. Mangelsdorf posits the farnesoid X receptor (FXR) as one player. FXR, which senses bile acids, also dimerizes with RXR. When activated, it shuts off bile production by spurring transcription of the short heterodimer partner (SHP), which is a strong inhibitor of CYP7A1, a target gene of RXR/LXR. "It's kind of the yin and the yang of bile acid metabolism here in the liver," Mangelsdorf quips. The inhibition of CYP7A1, the rate-limiting enzyme in bile acid production, lowers bile availability in the intestine, hindering the emulsification and absorption of cholesterol and other fats, he adds. Mangelsdorf considered a second reason for the striking results: RXR/LXR activates transcription of the ABCA1 transporter in macrophages and peripheral cells. The protein pumps cholesterol out of cells, processing it into high-density lipoprotein (HDL, the good cholesterol), which the liver can remove from the bloodstream. Defective ABCA1 in patients with Tangier disease results in very low HDL levels and cholesterol-stuffed macrophages that form foam cells, which can cause atherogenesis. Using LXR-specific agonists alone brought about a 50% reduction in cholesterol absorption. Yet questions remained about the role of ABCA1 in stemming cholesterol absorption. Says Tall, "It may be in part true, but it's not yet been proven that ABCA1 is the target responsible for the effect on cholesterol absorption." ABCA1 can definitely flush cholesterol out of macrophages, but the case for its role in the gut and liver is not quite as strong. A more clear-cut reason for the RXR/LXR mechanism emerged following an investigation headed by Helen Hobbs, also at UT Southwestern and a Mangelsdorf collaborator, who looked at other genes upregulated by LXR agonists. They first discovered ABCG5, and found, surprisingly, that it mapped to the same locus as the rare genetic disorder sitosterolemia, which affects fat metabolism.5 Hobbs, professor of internal medicine and molecular genetics and an HHMI investigator, says the group worried when it found that some patients with sitosterolemia did not have mutations in ABCG5. But, with the help of the Celera human genome database, Hobbs says, the group found ABCG8, a G5 partner located directly upstream. Bei Shan, research director at Tularik Pharmaceutical in San Francisco, says he believes ABCG5 and G8 are the main transporters involved in lowering cholesterol absorption, "because they're selectively expressed in liver and in intestine." KNOW YOUR DANCE PARTNERS The research linking these ABC genes to LXR has stirred interest in the biotechs and pharmaceuticals. Shan has collaborated with Hobbs, Mangelsdorf, and others to develop LXR agonists. But even with results as promising as Mangelsdorf's mice, scientists must proceed with caution. The rexinoids used in the mice affected at least two RXR heterodimeric nuclear hormone receptors, but Mangelsdorf says that RXR is a heterodimer to some other well known receptors, such as the vitamin D receptor, and the thyroid hormone receptor. "The consequence of RXR being a partner to so many different receptors is that if you give an RXR ligand ... you have the potential to induce several different nuclear receptor pathways simultaneously." Since 2000, more LXR-specific ligands have been developed, but these also come with a similar warning. LXR is now known to target more than a dozen genes. Most of them are involved in cholesterol homeostasis--preventing cholesterol absorption, metabolizing cholesterol, buffering it inside cells, or transporting it outside cells. But a small percentage of genes have other confounding tasks. The gene for the sterol regulatory element binding protein (SREBP-1c) is one such molecule. First discovered by the Nobel Prize-winning team of Michael Brown and Joseph Goldstein6 at UT Southwestern, and by Bruce Spiegelman at Harvard Medical School,7 SREBP-1c is a master regulator for fatty acid synthesis.8 This can lead to troubles when activating LXR, Mangelsdorf ex-plains. "You see a really remarkable increase in triglyceride in the blood of animals given an LXR-specific agon-ist. That's bad." The condition, hypertriglyceridemia, would essentially cancel out all the good that could be done by lowering cholesterol absorption. The answer, says Shan and others, is an LXR modulator that would induce just the cholesterol-metabolizing pathways, not the fatty acid pathway. There is precedent in selective estrogen receptor modulators, agonists that are selective for certain benefits of estrogen, but without the unwanted effects. Says Tall, "It's going to require a very thoughtful, diligent approach." Brendan A. Maher can be contacted at bmaher@the-scientist.com. References 1. B.A. Maher, "The race to find the Tangier disease gene," The Scientist, 15[19]:21, Oct. 1, 2001. 2. P. Costet et al., "Sterol-dependent transactivation of the ABC1 promoter by the liver x receptor/retinoid x receptor." Journal of Biological Chemistry, 275:28240-5, Sept. 8, 2000. (Cited in 100 papers) 3. J.J. Repa et al., "Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers," Science, 289:1524-9, Sept. 1, 2000. (Cited in 156 papers) 4. D.W. Russell, "Nuclear orphan receptors control cholesterol catabolism," Cell, 97:539-42, 1999. 5. K.E. Berge et al., "Accumulation of dietary cholesterol in sitosteolemia caused by mutations in adjacent ABC transporters," Science, 290:1771-5, Dec. 1, 2000. (Cited in 94 papers) 6. S. Benowitz, "Molecular genetics," The Scientist, 10[8]:14, April 15, 1996. 7. P. Tontonoz et al., "ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation," Molecular and Cellular Biology, 13:4753-9, 1993. 8. J.J. Repa et al., "Regulation of mouse sterol regulatory element-binding protein1c gene (SREBP-1c) by oxysterol receptors, LXR and LXR ß," Genes & Development, 14:2819-30, 2000. (Cited in 70 papers)

Flipping the Fat-Sensing Switch

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