Marketed under brand names such as Lipitor and Zocor, the statin drugs help many patients keep their cholesterol in check. But this standard treatment doesn't work for everyone.

"People are still dying [from heart disease] even though they're on cholesterol-lowering drugs," says Helen H. Hobbs, an HHMI investigator at the University of Texas Southwestern Medical Center at Dallas and director of the Dallas Heart Disease Prevention Project, a study of heart disease in a population of 6,000 individuals. "Until Americans decide to give up their hamburgers and French fries, we have to figure out how to interrupt this disease in other ways."

Searching for new approaches, scientists are looking at the problem of cholesterol from a different angle. Rather than focusing on therapies that target production of cholesterol, which is essentially what statins do, researchers are concentrating on the way the body gets rid of surplus cholesterol—and getting promising results.

An important breakthrough came not long ago when researchers identified a unique protein, called LXR (for liver X receptor), which appears to serve as a cellular "master switch" for removing excess cholesterol. A logical next step: Try to develop synthetic compounds that might control this intriguing switch—and in turn control the leading culprit in heart disease.

CHOLESTEROL'S BAD RAP
Despite its rotten reputation in the doctor's office, cholesterol is essential to human health. Cells insert cholesterol into their membranes to help control which substances enter and leave the cells. Cholesterol also is the sole precursor of steroid hormones such as testosterone and estradiol, certain vitamins, and the bile acids in the liver.

How much cholesterol we need varies from person to person. Researchers estimate that a 155-pound male has about 100 grams of cholesterol in his body. Each day, the liver and other tissues produce 600 to 900 milligrams of cholesterol to meet the body's routine daily demands for the substance. We get into trouble when we have an excess of this tough-to-metabolize substance—which the body cannot entirely get rid of.

The liver, being the only organ equipped to break down cholesterol efficiently, serves as a centralized treatment plant. However, it's limited by the countervailing effects of two distinct types of lipoprotein, the form in which cholesterol is carried in the blood. High-density lipoprotein (HDL), the so-called "good" cholesterol, picks up globules of cholesterol that cells have pumped to their outer membranes as if they have dropped off a bag of garbage at the curb. The HDLs then transport the cholesterol to the liver, where it is broken down in the bile. But low-density lipoprotein (LDL), or "bad" cholesterol, functions in an opposite fashion, bringing cholesterol from the liver into the body's cells.

The effects are not surprising. "Studies tell us that high levels of LDL and low levels of HDL are associated with atherosclerosis," says Hobbs. "Conversely, we know that if you lower plasma levels of LDL you have a profound [preventive] effect on the development of heart disease and a profound effect on reducing the incidence of coronary events in people with established heart disease."

REVERSE TRANSPORT
Scientists have long hoped to define precisely how cells pump the excess cholesterol to their membranes—a process that's commonly called "reverse transport"—for pickup and disposal. That knowledge could pave the way for the design of drugs to manipulate the process. (Statins, the current drugs of choice, largely target an enzyme involved in the body's own production of cholesterol, a completely different cellular process than reverse transport.) About four years ago, a team of scientists provided an important first clue to the reverse-transport puzzle when they showed that a protein called ABCA1 transfers cholesterol from the cell membrane to HDL. This raised an obvious next question: What activates ABCA1?

The answer came soon thereafter from the laboratory of David J. Mangelsdorf, an HHMI investigator at the University of Texas Southwestern Medical Center at Dallas. As a postdoc, Mangelsdorf had cloned the genes of a dozen previously unknown protein receptors in the cell nucleus. Because the functions of these assumed hormone-binding proteins were unknown, scientists referred to them as "orphan nuclear receptors." Later, he discovered that one of these orphans—LXR—bound cholesterol metabolites.

Given that most nuclear receptors, once saturated with their activating ligand, signal for the transcription of specific genes, Mangelsdorf followed up with a series of experiments to determine which genes LXR mobilizes; the hope was that this information would lead to genes and proteins directly involved in the cholesterol-transport process. He observed that when mice were given small molecules that stimulated the LXR receptor, their production of ABCA1 was markedly increased. Although the details have yet to be fully worked out, this result suggests that an LXR-stimulating drug would increase cholesterol transport and boost HDL levels, both beneficial effects.

Actually, as Mangelsdorf and colleagues would show, this was just the tip of the LXR iceberg. In these same mice, the scientists noted a prominent decrease in cholesterol absorption. The explanation was that LXR signals ABCA1 to pump out the cholesterol in the intestine, thereby preventing it from being absorbed into the blood. Though this hypothesis has not been confirmed, it suggests that LXR has the potential to limit the absorption of dietary cholesterol, another greatly desired effect.

In the liver, the data are equally striking. Mangelsdorf reported that LXR activates a gene whose protein plays a key role in the synthesis of bile acids, meaning a ramping up of cholesterol degradation—another positive effect. There is also evidence that LXR activates other genes that transport cholesterol into the bile.

Then there is LXR's beneficial effect on macrophages, a type of white blood cell that ingests foreign material. Peter Tontonoz, an HHMI investigator at the University of California, Los Angeles, and colleagues have published a series of articles showing that LXR aids in the efflux of cholesterol from macrophages, presumably by activating ABCA1 proteins. This discovery has major implications because cholesterol-laden macrophages contribute to the formation of foam cells, a fundamental component of artery-clogging plaques. In addition, the group has reported that LXR induces macrophages to produce apolipoprotein E, a plasma protein that has a protective effect against atherosclerosis.

COMPLEX INTERPLAY
These and additional studies show that LXR plays a key role in reverse transport and in ancillary processes as well. They also suggest that the pharmaceutical company that corners the LXR market could stand to benefit handsomely.

There seems to be a big obstacle, however. Mangelsdorf has shown that LXR wears a second metabolic hat: It regulates the metabolism of triglycerides, the body's store of fatty acids that constitute a major energy source. This has raised fears that although LXR-targeted synthetic drugs might flush out the extra cholesterol, they might also cause blood fatty acid levels to spike, which can lead to health problems such as pancreatitis.

"Perhaps the LXR linkage of the two is a way to coordinate the balance between triglycerides and cholesterol," speculates Ronald M. Evans, an HHMI investigator at The Salk Institute for Biological Studies. "They are both packaged and trafficked around in the LDL and VLDL [very low density lipoprotein] particles, which constitute the major delivery systems for lipids throughout the body. So it may not be inappropriate, biologically, for them to be linked. Interestingly, internalization of these particles and their lipids is controlled by another set of nuclear receptors, termed PPARs" (see "Searching for the Fat Switch"). A mentor to both Mangelsdorf and Tontonoz, Evans is the father of "reverse endocrinology," an approach that led to Mangelsdorf's discovery of LXR.

Many scientists say they are cautiously optimistic that the problem of balance will one day be solved. But first they have to fill in the blanks in the molecular chain of events that activate LXR. "Cholesterol homeostasis does not occur in a vacuum," says Evans. "The physical links between cholesterol and triglycerides in lipoprotein particles reflect a more global coordination in which other nuclear receptors, along with LXR, control the ebb and flow of metabolic energy. Understanding the logic of this molecular circuit is key."

Referring to the many cellular signals, or pathways, activated by LXR, Mangelsdorf notes that "a signaling network resembles the arms of an octopus, all branching out in various directions but all feeding back in some way to one central thing. The question is: What is that central thing? If answered, simplicity will emerge from the complexity."

Meanwhile, researchers have learned the identity of the target genes that LXRs regulate and that are responsible for the undesired increase in fatty acids and triglycerides. According to Mangelsdorf, the ability of LXRs to target both "good" genes (that lower cholesterol) and "bad" genes (that raise fatty acids and triglycerides) may be exploited by the development of new drugs that selectively activate expression of only the good genes.

He notes, moreover, that the idea of selectively activating a receptor is not without precedent. Cancer researchers have done it for years with tamoxifen, which selectively binds to the estrogen receptor to treat or prevent breast cancer. "The fact that tamoxifen, raloxifene, and estrogen have different activities in different tissues is important," Mangelsdorf says. "It suggests that by giving a selective modulator that's tissue-specific, it might be possible to dial out the bad effects and keep the good effects."

Tontonoz adds that because LXR-modulating drugs and statins target different cellular pathways, they conceivably could be combined to provide a one-two punch to clear out excess cholesterol. He acknowledges, though, that work has only just begun on LXR and the application of its cholesterol-lowering capabilities. "There's a complex interplay between diet, environment, immune responses, and genetic predispositions that will require a lot of investigation," Tontonoz says. "Right now, we're at the point where we can look at individual pathways, but the bigger picture is how all of the pieces fit together—why different people exposed to those same pathways have different responses."

Using the metaphor that the body is a factory, it could be said that the statin drugs fight cholesterol on the assembly line, while LXR helps take it out with the trash. If researchers can ultimately craft therapeutics that adapt LXR's approach, this different angle on controlling cholesterol may be a key to solving the pervasive problem of heart disease.

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Photos: Doug Handel, Tom Keller

Reprinted from the HHMI Bulletin,
September 2003, pages 10-19.
©2003 Howard Hughes Medical Institute