Obese Mice Provide Clues To A Natural System That Puts Brakes On Obesity

The finding, reported in the August 1 issue of the Journal of Clinical Investigation, marks the first time a gene has been identified that is induced, or activated, in the fat cells of obese animals. According to Rockefeller University professor Markus Stoffel, M.D., Ph.D., lead investigator of the study, the gene, called Foxa-2, inhibits young body cells from becoming mature fat-producing cells called adipocytes. In addition, when this gene is switched on in mature adipocytes, it functions as a brake to slow down further fat production and storage.

"We know a lot about the various molecular pathways that stimulate or promote fat production, and the focus has been on trying to block these pathways to fight obesity," says Stoffel. "This pathway is one of only a few that we know of that naturally work to counteract obesity.

"We have shown that Foxa-2 has two beneficial effects in mice: it counterregulates the formation of fat and it increases the activity of genes important for insulin sensitivity," Stoffel continues. "This is the ideal combination for pharmacologically treating obese or type 2 diabetic patients, or people with a risk of developing obesity."

Foxa-2 was originally discovered in the 1980s by Rockefeller scientist James E. Darnell Jr., M.D., as an activator of genes in the liver. Subsequent research by Stoffel and colleagues at Rockefeller showed that Foxa-2 also activates genes in the insulin-producing islet cells in the pancreas and it is expressed in the gut and the lung. However, previous research by scientists could not provide evidence for its expression in fat cells. The reason, says Stoffel, is "that we did not look at fat cells from obese animals."

"We were only able to find Foxa-2 in fat tissues when we extracted fat from a leptin-deficient mouse, which has a mutation in the 'ob' gene," says Stoffel, referring to studies with mice that are obese due to low levels of the hormone leptin, the protein product of the obese gene, which was identified by Rockefeller professor Jeffrey M. Friedman, M.D., Ph.D., in the mid-1990s.

Leptin is a hormone secreted by adipocytes that acts on an area of the brain called the hypothalamus. Leptin, Stoffel notes, causes mice and people to lose weight by increasing food intake and energy expenditure, while Foxa-2's effects are solely on the adipocyte at the level of the cell.

"The discovery of Foxa-2 in fat cells of obese mice was unexpected," Stoffel continues. "We were able to replicate this finding in mice that were obese due to mutations in several genes and in diet-induced obese mice."

Stoffel and colleagues found Foxa-2 expression in mature adipocytes and in pre-adipocytes, young cells that have not accumulated lipids. In addition, Foxa-2 expression was higher in visceral fat than in subcutaneous fat, which is stored under the skin. Visceral fat, found around the gut, is a form of fat that predisposes people to complications of obesity, including type 2 diabetes.

To understand Foxa-2's role in pre-adipocytes, the Stoffel and his colleagues "overexpressed," or activated above normal levels, Foxa-2 in laboratory cultures of these cells. The scientists then added the necessary hormonal and nutritional signals for these cells to turn into mature fat cells. They found that the pre-adipocytes did not accumulate lipids, and Foxa-2 did not induce genes in these cells that typically are active in mature adipocytes.

During the normal course of differentiation of pre-adipocytes into fat, the activity or expression of a gene called Pref-1 decreases. Stoffel found that Foxa-2 activates Pref-1, and that this activation inhibited the transformation of the laboratory cultured pre-adipocytes into mature adipocytes. The researchers also found overexpression of a set of genes that metabolize glucose, suggesting that Foxa-2 also directly activates these genes.

When Foxa-2 was overexpressed in mature adipocytes that normally do not express Foxa-2, Stoffel and his colleagues found increased expression of genes that break down glucose and burn fat.

"We know that adipocytes of obese animal models are insulin-resistant, so levels of glucose metabolism enzymes decrease," says Stoffel. "At the level of the mature adipocyte, Foxa-2 seems to counterregulate obesity by increasing these enzymes in the adipocyte or at least working against further decreases."

The researchers then tested the effects of removing this gene in living mice. Because both copies of Foxa-2 are necessary for survival, the researchers deleted, or "knocked out," only one copy of this gene, which effectively reduced its activity by 50 percent. When fed a high fat diet for six weeks, these knockout mice gained much more weight than genetically normal mice fed the same diet.

Analysis of fat cells from the Foxa-2 knockout mice showed that activity levels of Pref-1 and the glucose metabolism genes decreased.

"It is now apparent that when we overfeed a mouse and it becomes obese, Foxa-2 is induced, which then activates a set of genes that work against obesity," says Stoffel. "Obviously that's not sufficient to prevent obesity, but it is sufficient to slow it down. Without this force, the mice accumulate more fat.

"It's an ineffective system, since otherwise people wouldn't get obese, but it is clear that if you take the system away, there is an increase in obesity."

The next step for the researchers is to identify stimuli that induce Foxa-2 in people.

"We know that Foxa-2 is expressed in human adipocytes, and if we knew the stimuli that are responsible for inducing Foxa-2 we could potentially induce Foxa-2 activity in people at risk for developing obesity and possibly counteract the development of obesity as well as increase insulin sensitivity," says Stoffel. Both obesity and insulin resistance are risk factors for type 2 diabetes.

In their research with mice, Stoffel and his colleagues showed that growth hormone can induce Foxa-2 in cultured cells that normally don't express the gene.

Stoffel cautions that growth hormone is not the answer for treating obesity in people.

"We do not believe that growth hormone is responsible for the induction of Foxa-2 in obesity because we know that obese patients actually have a relative deficiency of growth hormone," Stoffel says. "And, we can't give growth hormone to patients because there are too many negative side effects."

Stoffel's co-authors are Christian Wolfrum, David Q. Shih, Satoru Kuwajima at Rockefeller, and Andrew Norris and C. Ronald Kahn at the Joslin Diabetes Center.

This research was supported in part by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, Medical Scientists Training Program, Human Frontier Science Program and German Research Council.