In mice, drug protects against diabetes and atherosclerosis

Blocking a single protein with an experimental drug prevented and treated both type 2 diabetes and atherosclerosis in laboratory mice that had been fed unhealthy diets and were genetically predisposed to these common killers, according to an article published online in Nature.

The team was led by senior author G'khan Hotamisligil, chair of the Department of Genetics and Complex Diseases at the Harvard School of Public Health (HSPH). Lead author was Masato Furuhashi, research fellow in the department.

In earlier studies, Hotamisligil's lab members researched mice lacking two lipid-binding proteins, aP2 and mal1. When these mice were fed a high-cholesterol or high-fat diet, the expected signs of metabolic diseases such as atherosclerosis, type 2 diabetes, and fatty liver disease never developed. Recently, researchers in Hotamisligil's lab and at the Garvan Institute of Medical Research in Australia also demonstrated that these genes were critical in the development of asthma, another disease associated with obesity.

In this new paper, researchers from HSPH, Bristol-Myers Squibb, and elsewhere describe how a designer compound mimics many of these protective effects in mice, conferring substantial immunity to diabetes, heart disease, and other metabolic problems. This immunity takes place even if the animals are severely obese or have high amounts of cholesterol and consume dangerously fatty foods. Use of the compound not only appears to prevent the development of these diseases, but also to reverse the symptoms of these illnesses in mice.

"To have this chemical in hand and to replicate the effects of genetic manipulation is a huge milestone and an incredible source of excitement," said Hotamisligil, Professor of Genetics and Complex Diseases. "This drug is very effective in treating diabetes and heart disease in mice at the same time, and we believe it may turn out to be protective against asthma and other metabolic disorders as well."

The laboratory animals showed no harmful effects from the treatment, though Hotamisligil cautioned that experiments were not designed to look for such effects rigorously.

While it is not certain that the experimental compound or others like it can be developed into successful drugs for humans, Hotamisligil described the animal results as a long-sought success after a decade of intense efforts to achieve with a chemical what has been observed in mice and humans carrying the gene variant.

The lipid chaperone protein aP2 acts as a signal in cells, setting off a chain of inflammatory and metabolic responses to consumption of fatty foods. Some of those effects make the body less sensitive to the sugar-lowering action of insulin, raising the risk of diabetes. For many decades, aP2 was believed to act only in fat cells. In 2001, Hotamisligil and a collaborator discovered that aP2 is also expressed in macrophages, which scavenge and digest cellular debris and microbes in the blood stream. The aP2 signals encourage macrophages to load up on cholesterol and become "foam cells" that adhere to artery walls, creating dangerous plaques that eventually may rupture and trigger a heart attack. In mice lacking these lipid chaperones, there was no sign of heart disease.

Next, the researchers turned to humans. While the scientists could not knock out the aP2 gene in humans, they could focus on subjects in the large Nurses' Health Study and the Health Professionals Follow-Up study who were overweight and who consumed high-fat diets, yet remained healthy. Hotamisligil's team, in collaboration with HSPH Associate Professor Eric Rimm, found a mutation in the aP2 gene. Tissue tests revealed that many of the subjects had a variant of the aP2 gene that sharply reduced production of the aP2 protein. In 2006, the scientists published their findings that individuals with a genetic variation of aP2 helps to protect humans against type 2 diabetes, heart disease, and hypertriglyceridemia. See here for more information.

Now that the importance of this gene in diabetes and heart disease was clear in both mice and humans, the researchers sought to reproduce the effects of these genetic mutations through the development of drugs. Hotamisligil worked with Dr. Rex Parker at the Bristol-Myers Squibb Research Institute in Princeton, NJ, to develop an inhibitor of the aP2 protein.

"There was no traditional model of making a drug to inhibit this type of protein," said Hotamisligil. "Many people were skeptical that it could be done at all."

The result was the drug known as BMS309403, described in the Nature article as a "rationally designed, potent, and selective inhibitor of aP2" that blocks the protein's ability to bind with fatty acids -- the function that leads to inflammatory and metabolic havoc when a high-fat or high-cholesterol diet is consumed. The drug appears to have no effect on lean animals eating a normal diet, the scientists said, suggesting that blocking aP2 may not be harmful in people -- though this remains to be determined.

The report describes tests of the inhibitor on cell systems in culture and in live mice. In one experiment, mice genetically engineered to be highly susceptible to atherosclerosis were fed high-fat diets and assigned to receive the drug or an inert substance. One set of mice was put on a high-fat "western" diet at five weeks of age; half received the inhibitor drug. A second group of animals ate the western diet for eight weeks, at which time they developed severe atherosclerosis. Then they were started on the drug to determine whether the drug could halt or reverse the disease process.

In both experiments, the drug treatment reduced the size of fatty plaques in the animals' aortas by more than 50 percent compared to control mice.

To study the impact of the aP2 inhibitor on diabetes, the scientists used a genetic animal model of obesity and insulin resistance, as well as normal mice fed a diabetes-inducing diet.

In both animal models, the mice that received the inhibitor drug were found to have lower blood sugar and triglycerides -- a component of "bad" cholesterol -- and significantly improved insulin sensitivity compared to control mice. Tests also showed that the drug reduced the activity of gene pathway called JNK that triggers the inflammatory and insulin-resistance responses to fatty diets.

As an "added bonus," commented Hotamisligil, the drug also exerted a marked protective effect against another metabolic disorder, fatty liver disease.

While the genetic variant studies in humans indicate that a drug like the one used in the current experiments is likely to work in humans, Hotamisligil said that this remains to be proven. The next step is to perform more detailed toxicity tests in animals, and then move toward human testing.

The important lesson from the new study, the researchers wrote in their report, is the demonstration that aP2 can be blocked by a small-molecule, oral compound. In turn, they add, the results suggest that targeting aP2 "can lead to a new class of powerful therapeutic agents to prevent and treat metabolic diseases such as type 2 diabetes and atherosclerosis."

Said Hotamisligil, "This work turned out to be a perfect example of out-of-the box thinking, inter-institutional and inter-disciplinary science, to bridge discovery with application and fruitful industry-academia collaboration."