New research reveals how genetic and environmental factors influence an organ that has not traditionally taken much of the blame for diabetes and cardiovascular disease.

Insulin resistance is a primary driver of both type-2 diabetes and cardiovascular disease. Numerous genetic and lifestyle factors, such as obesity and physical inactivity, can help nudge cells toward insulin resistance, which is a loss of sensitivity to the hormone that regulates the storage and use of glucose for energy. New research from Howard Hughes Medical Institute scientists reveals how these genetic and environmental factors exert their influence through an organ that has not traditionally taken much of the blame for diabetes and cardiovascular disease: the liver.

The scientists have identified genetic factors that predispose individuals to developing nonalcoholic fatty liver disease, which is a risk factor for cardiovascular disease. Their research shows the same genetic factors are linked to insulin resistance—even in individuals of normal weight.

These APOC3 variants are predisposing—so instead of getting fatty liver disease and insulin resistance when your body mass index is 30, you may get it when your BMI is only 23 or 24.

Gerald I. Shulman

Gerald I. Shulman, an HHMI investigator at Yale University School of Medicine, led the genetic analysis that linked subtle variations in the gene for apolipoprotein C3 (APOC3) to an increased risk for nonalcoholic fatty liver disease. Shulman’s team found that individuals with two particular versions of the APOC3 gene produced high levels of apolipoprotein C3, which circulates in the blood and appears to impair the body’s ability to process dietary fat. The study was published on March 25, 2010, in The New England Journal of Medicine.

Shulman’s group has been working to decipher the cellular and molecular mechanisms that contribute to insulin resistance. The National Institute of Diabetes and Digestive and Kidney Diseases estimates that more than 23 million Americans have diabetes, which is the leading cause of blindness, end-stage renal disease, and non-traumatic limb loss. Shulman says the associated health care costs are estimated to exceed $180 billion a year.

Previously, Shulman and colleagues had found that high levels of fat in the liver are associated with insulin resistance. They had also found that Asian Indian men showed markedly more insulin resistance and had three to four times the amount of liver fat than men of other ethnic backgrounds. “This got us interested in pursuing genetic causes for this,” Shulman says. His lab approached HHMI investigator Richard P. Lifton, director of the Yale Center for Human Genetics and Genomics, for help in looking for two candidate gene variants, which, based on previous research, the scientists believed might be involved.

Shulman and his colleagues focused on two sites in the promoter region of the APOC3 gene, which were known to influence the abundance of fatty acid molecules—triglycerides—in the blood, in a group of 95 healthy, normal-weight, sedentary Asian Indian men. Such a site of variation, which amounts to a change in a single letter of the genetic code, is known as a single nucleotide polymorphism (SNP).

The team found one or both of the high-triglyceride variants of the SNPs in 76 of the men in their study. None of the men who lacked these two APOC3 variants had fatty liver disease, but almost 40 percent of the men with at least one variant did. Furthermore, those individuals with fatty liver were markedly insulin resistant.

Shulman and his colleagues then looked at whether the APOC3 variants had the same correlation to fatty liver disease in 163 healthy non-Asian Indian men. In that group, nine percent of the men with the APOC3 gene variants had fatty liver disease, compared with none who lacked those variants. “We found pretty much the same thing; both of these common APO C3 gene variants predispose normal weight individuals to nonalcoholic fatty liver disease and insulin resistance across ethnic groups,” says Shulman.

Once Shulman and his colleagues had identified which individuals carried the high-risk variants of APO3C, they ran a variety of tests to see if they could figure out how the gene influenced fat buildup in the liver. They found that the relevant SNPs were associated with a 30 percent increase in fasting plasma concentrations of apolipoprotein C3. They also were linked to a 60 percent increase in fasting blood levels of triglycerides, the common chemical form of fat in the body and in food, and a 46 percent reduction in triglyceride clearance. Average plasma levels of triglycerides in the liver were higher in the men who carried one or both of the variants.

Shulman says those measurements help paint a picture of exactly how the gene variants may contribute to fatty liver: The APOC3 variants generate higher concentrations of apolipoprotein C3 in the blood. This inhibits the lipoprotein lipase enzyme, which fat cells use to break down dietary fats in the bloodstream. As these triglycerides accumulate in the blood, the liver takes them up in the form of triglyceride-laden particles called chylomicron remnants. Over time, Shulman says, this process can result in non-alcoholic fatty liver disease and hepatic insulin resistance.

Although nonalcoholic fatty liver disease is associated with obesity, Shulman’s findings show that people with the gene variants are vulnerable to the disease even if they are of normal weight. “These APOC3 variants are predisposing—so instead of getting fatty liver disease and insulin resistance when your body mass index is 30, you may get it when your BMI is only 23 or 24,” which is considered normal weight, Shulman explains. “I think the fact that we looked for the impact of these gene variants on fatty liver disease and insulin resistance in relatively young, normal-weight individuals is a critical point. I doubt we would have found these gene effects if we looked for it in overweight individuals, who typically develop both of these conditions independent of any genetic factors.”

The researchers put a subgroup of seven subjects with APOC3 variants who had fatty liver disease and insulin resistance on a calorie-restricted diet—a strategy that they have previously found to reverse nonalcoholic fatty liver disease, liver insulin resistance, and hyperglycemia in patients with type 2 diabetes - leading to a modest average weight loss. After three to six months, participants lost an average of 12 pounds, and there was a drop in the amount of fat in their livers. Also, Shulman says, “whole body insulin sensitivity improved—demonstrating that liver fat is in fact related to their insulin resistance and consistent with all of our previous work linking nonalcoholic fatty liver disease to hepatic insulin resistance and type 2 diabetes.”

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