Gene mutation causes lethally low-fat diet

We are all familiar with the dangers of too much fat in our diet--increased risk of diabetes, heart disease, and obesity are just a few of the most severe consequences. But some rare metabolic diseases, such as hypolipidemia and Tangier disease, seem to work in reverse--they severely limit the amount of fat and cholesterol that makes it into the bloodstream. Researchers from the Carnegie Institution and the University of Pennsylvania have found a specific gene that could be responsible for such conditions; when the gene is disrupted, so is the ability to absorb lipids (fatty substances that include cholesterol) through the intestine.

In their latest research, published in the April 4 issue of the journal Cell Metabolism, Steve Farber of Carnegie's Department of Embryology and Michael Pack, of the University of Pennsylvania School of Medicine describe their efforts to locate a gene called fat free within the genome of the zebrafish. These fish have become popular research organisms because their embryos are transparent, allowing studies that are not possible with traditional model organisms, such as mice and rats. Farber and Pack found that, despite the distant evolutionary relation between humans and zebrafish, the fat free gene in zebrafish is quite similar to a pair of human genes.

The researchers also explore the physical effects of a specific mutation of the gene, seeking to explain why larval fish with the mutation exhibit an impaired ability to absorb cholesterol. These fish die when they are about a one-and-a-half weeks old because of this defect, even though they look normal and swallow properly.

"There is a lot we still don't know about how animals absorb, transport, and otherwise manage lipids," Farber said. "The fact that just one gene can have such a huge effect is encouraging, because it might reveal a means for treatment of human disease."

The scientists began by looking for structural defects in the mutants' digestive organs. Their livers have abnormalities in the cells and ducts that produce bile--a salty, somewhat soapy fluid that helps lipid digestion. Certain pancreatic cells are also flawed, interfering with the production of digestive enzymes necessary for the breakdown of complex lipid molecules.

More importantly, the mutants also have defects in the cells that line the intestine, where fat and cholesterol absorption takes place. Normally, globules of lipid pass into these cells in small sacs called vesicles. These vesicles connect with the Golgi apparatus, a labyrinth of membranes filled with enzymes that modify the fats, and then new vesicles transport the fats out of the cell and into the bloodstream. The researchers found that this process is disrupted in the fat free mutants, preventing fats from reaching the bloodstream, and thereby depriving the animal of needed lipids.

Farber and Pack used a strategy called positional cloning both to locate fat free in the zebrafish genome and to determine its sequence. They found that the gene shares 75 percent of its sequence with a human gene called ANG2 (Another New Gene 2), which up to this time has had no known function. It also shares parts of its sequence with a gene called COG8, which is known to affect the Golgi apparatus. They also found that a change in only one base--one "letter" in the DNA code--results in the lethal mutation in zebrafish.

"This gene is absolutely necessary for cholesterol absorption--without it, the animals die," Farber said. This is encouraging for Pack, a physician-scientist in Penn's Department of Medicine, "If we can understand this process in zebrafish, perhaps we can take what we learn and apply it to similar genes in humans, which could in turn lead to treatment for lipid metabolism disorders."

Image Caption: http://www.carnegieinstitution.org/fatfree/
Zebrafish larvae with a lethal mutation affecting fat metabolism (ffr) look the same as normal larvaes (wt) under normal magnification (left). However, when the embryos ingest lipid molecules labeled with a fluorescent tag (right), differences in fat metabolism become apparent. The normal embryos absorb the labeled lipids into their bloodstream, resulting in a fluorescent gut, while the mutants eliminate them, resulting in a darker gut.

This work was supported by grants from the American Heart Association, National Institutes of Health, The Pew Scholars Fund, and the Carnegie Institution. The Carnegie Institution of Washington has been a pioneering force in basic scientific research since 1902. It is a private, nonprofit organization with six research departments throughout the U.S. Carnegie scientists are leaders in plant biology, developmental biology, astronomy, materials science, global ecology, and Earth and planetary science. See www.carnegieinstitution.org

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