The Gladstone Connection

Facing Up to Fat Obesity and overweight are epidemic in America. Nearly two-thirds of Americans are carrying too many extra pounds, including about 15% of children, a prevalence that has doubled since the 1970s. And it’s not just Americans—much of the rest of the developed world is also rapidly getting fatter. From one standpoint, obesity represents a success story: the body has stored large quantities of potential energy for the future. Unfortunately, the excess stored fat can cause problems, including tissue and organ failure. At least one of five obese people develops type 2 (adult-onset) diabetes, and many more develop abnormalities of sugar metabolism called impaired glucose tolerance, a pre-diabetic state. Just as obesity is a major risk factor for diabetes, diabetes and its precursors are major risk factors for cardiovascular disease (e.g., heart attacks, strokes), kidney failure, nerve dysfunction, and blindness. Thus, obesity is a huge public health epidemic.		Obesity trends among U.S. adults. During the past 20 years, obesity rates have risen sharply in the United States. Obesity is defined as a body mass index (BMI) of 30 or more. For a 5’ 4” tall woman, that represents about 30 extra pounds. BMI is calculated as weight (in kilograms) divided by height squared (in centimeters). Overweight is defined as a BMI of 25 to 29.9. Source: CDC (Behavioral Risk Factor Surveillance System)
The laboratory of Robert Farese, Jr., in the Gladstone Institute of Cardiovascular Disease has long studied how fats are synthesized and has recently turned its attention to understanding the basic biology of triglyceride synthesis. Their work has elucidated the roles of key enzymes involved in triglyceride synthesis and has identified drug targets for pharmaceutical companies, who are racing to develop safe and effective treatments for obesity and diabetes.

Triglyceride Synthesis and DGAT Enzymes
Triglycerides and disease. When energy input exceeds energy output, the excess energy is stored primarily in the form of triglycerides. Excessive accumulation of triglycerides in the adipose tissue results in obesity. In the liver and skeletal muscle, fat accumulation is associated with insulin resistance and diabetes.		The majority of the fat in our bodies (more than 95%) is in the form of triglycerides. Triglycerides are found in nearly all cells but are concentrated in tissues, such as adipose tissue, liver, muscles, and mammary glands, where they serve to store energy. Tissues cannot directly take up triglycerides; instead, these lipids must be made from precursor molecules (glycerol and fatty acids) within the cells that store them. Triglycerides are also synthesized in large amounts in the small intestine, where fats are absorbed. Like many biochemical processes, triglyceride synthesis involves a series of steps. The final step—
the joining of a fatty acid derivative to diacylglycerol—is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. Because other molecules in the triglyceride synthesis pathway can be utilized for other biochemical reactions, the DGAT step is the only committed step in triglyceride synthesis.

Sowing the Seeds of Discovery
The Farese group was the first to identify a gene that encodes a DGAT enzyme. They examined related genes that participate in cholesterol metabolism and discovered one gene that encoded a DGAT, which is now known as DGAT1. By studying DGAT1’s function in mice, the team determined that a second gene must exist, and with collaborators at Calgene, they discovered the gene for DGAT2. The DGAT2 gene is part of a larger gene family that is still being characterized. The identification of DGAT genes has provided molecular tools for studying the basic biology of triglyceride synthesis in cells and energy metabolism in mammalian organisms.		Obesity resistance. Mice lacking DGAT1 (Dgat–/– mice) are resistant to diet-induced obesity. Mice of the three genotypes were fed a high-fat diet and weighed over time. N = 6–8 mice per genotype.

Harvesting the Rewards of Research

Having discovered the DGAT genes, the Farese laboratory was then able to inactivate them in mice. These powerful gene-knockout models have provided significant insights into triglyceride synthesis.

Loss of the DGAT2 gene is incompatible with life, at least in mice. Mice without DGAT2 have a near-complete absence of triglycerides and lack specific lipids that help coat the skin and prevent dehydration. As a result, these mice die shortly after birth. Thus, DGAT2 appears to be an essential “house-keeping” enzyme. Without DGAT2, cells and the organism cannot keep triglyceride supplies in order, and the consequences are severe.

In contrast, mice lacking DGAT1 are viable and relatively healthy. However, they have a lower triglyceride content than normal mice and about half as much adipose tissue. Moreover, they are resistant to developing obesity and diabetes. When fed a Western-style diet rich in saturated fats and cholesterol, they remain slim, while their littermates with DGAT1 become obese. The mechanism for the protection against obesity in mice lacking DGAT1 is not reduced food intake. In fact, they eat as much as or more than their littermates with normal DGAT1 expression. Instead, the absence of DGAT1 somehow increases energy expenditure (calorie burning) by up to 15% per day. As a result, excess triglycerides do not accumulate, and the mice don’t develop obesity or diabetes.

Given these findings, several pharmaceutical companies have identified DGAT1 inhibition as a prime strategy for preventing or treating obesity and diabetes. Enzymes are attractive drug targets, and many enzyme inhibitors are used to treat diseases. The cholesterol-lowering statin drugs, for example, inhibit the enzyme HMG-CoA reductase. Within a few years, the first DGAT1 inhibitors will be tested for efficacy and side effects in experimental animals and possibly in humans.

From Obesity to Acne, Hair Loss, and Breast Cancer

Work on the DGAT1 knockout mice has yielded some unexpected twists that ultimately may have relevance to medicine. For example, female mice that lack DGAT1 do not make milk after giving birth. This lactation defect is due in part to the absence of DGAT1 in the fatty tissue that surrounds the developing mammary glandular cells—without DGAT1, these cells do not develop normally. This raises the question of whether DGAT1 deficiency in the mammary tissue might prevent the development of breast cancer. Work is in progress to address this question.

Mice without DGAT1 also have intriguing changes in their skin and fur. DGAT1 deficiency causes atrophy of the sebaceous glands, which are associated with hair follicles and secrete oils that coat the hair and skin. Thus, DGAT1 inhibition may be a potential treatment for acne. In addition, DGAT1-deficient mice have abnormalities of hair growth. These mice, particularly post-pubertal male mice, lose fur on their backs. Recent work in the Farese laboratory has begun to elucidate mechanisms for the hair loss and even to identify treatments that cause the hair to grow back.

Delving Into Unanswered Questions

Much remains to be learned about DGAT1. How does the lack of DGAT1 trigger an increase in energy expenditure? How does the absence of DGAT1 cause an increased sensitivity to insulin (which prevents the development of diabetes)? Even if DGAT1 inhibitors do not work out as a viable drug therapy for obesity, an understanding of the underlying mechanisms triggered by DGAT1 deficiency may provide new targets for pharmaceutical-based therapies.

One possible new lead relates to the discovery that adipose tissue lacking DGAT1 appears to produce hormonal factors that promote resistance to obesity and diabetes. The Farese laboratory showed that transplanting very small pieces of fat from mice that lack DGAT1 into normal mice could confer partial protection from obesity and diabetes caused by high-fat feeding. This finding strongly suggests

that DGAT1-deficient fat makes more of one or more factors that prevent fat accumulation. Current work is focused on identifying these factors, which themselves may turn out to be useful therapeutic agents.

Conclusions

It is often impossible to predict where breakthroughs will come in biomedical science. Just a few years ago, the Farese laboratory was firmly focused on cholesterol metabolism and knew little about the processes underlying obesity. Now, with the support of Gladstone and numerous funding agencies, this laboratory is at the forefront of the field where lipid metabolism intersects with diseases such as obesity and diabetes. Continued work on the basic mechanisms of triglyceride synthesis holds promise for enhancing our understanding of these metabolic diseases.