Apr. 16, 2002 In experiments with fat cells, Johns Hopkins scientists have discovered direct evidence that a build-up of sugar on proteins triggers insulin resistance, a key feature of most cases of diabetes. The results underscore the importance of glycosylation – attachment of a sugar to a protein -- as a way cells control proteins' activities, the scientists report in the April 16 issue of the Proceedings of the National Academy of Sciences. The scientists found that at least two proteins involved in passing along insulin's message were unlikely to work properly when coated in extra sugar.
Type 2 diabetes, the most common form in adults, occurs when muscle, fat and other tissues stop responding to insulin's signals to mop up sugar from the blood. The resulting high blood sugar, if uncontrolled, can lead to blindness, amputation and death. Understanding sugar's precise influence on insulin's activity may help improve treatment and prevention, scientists hope.
"Cells don't respond to insulin itself. Instead, a whole cascade of events, set in motion by insulin, eventually causes cells to take in sugar," explains Gerald Hart, Ph.D., professor and director of biological chemistry in the school's Institute for Basic Biomedical Sciences. "We now have an explanation of how sugar can affect these signals, and even a hypothesis for how high blood sugar could cause tissue damage in diabetes -- by improperly modifying proteins."
Hart's lab discovered 18 years ago that sugar is used routinely inside cells to modify proteins, turning them on and off. The more commonly known protein-controller, phosphate, actually binds to some of the same building blocks of proteins as sugar does. If proteins have too many sugars on them, they can't be controlled properly by the cell and are unlikely to work correctly, suggests Hart.
"We think we've come across a major mechanistic reason for insulin resistance," says Hart. "These cells developed insulin resistance simply because their proteins, and specific proteins in fact, had more than the normal number of sugar tags."
If key proteins laden with sugar are present in patients with diabetes, the findings may provide a target for developing new strategies to deal with this growing public health threat, says Hart. While diabetes can be fairly well controlled by diet and carefully monitoring one's blood sugar levels, finding a way to remove extra sugar tags may help treat or prevent diabetes someday, the researchers suggest.
"Textbooks frequently and incorrectly show glycosylation only happening to proteins on the cell surface," says Hart. "Complex sugars are added only to proteins outside the cell, but simple sugars are used all the time in the nucleus and cytoplasm to modify proteins. It's this glycosylation that happens inside the cell, involving simple sugars, that is the key in insulin resistance."
The "simple sugar" to which he refers is O-linked beta-N-acetylglucosamine, a complex name that condenses to a difficult acronym -- O-GlcNAc -- with an ugly pronunciation -- "oh-gluck-nack." But in many ways, O-GlcNAc is a beautiful and mysterious thing, says Hart.
"O-GlcNAc is a modifier on many proteins, but if you didn't know to look for it, you'd never find it," he says. "Instruments and the usual laboratory methods have a hard time measuring it, so we developed the techniques to detect it."
O-GlcNAc is added to proteins by one enzyme and removed from proteins by another. By selectively blocking that removal, the scientists hoped to load up proteins with sugar without adding extra sugar (the way other scientists have created insulin resistance). "We wanted to see the effect of glycosylation itself, so we used a molecular sledgehammer to increase the amount of sugar bound to proteins," says Hart, whose lab proved the ability of the blocker, a molecule called PUGNAc.
Not only did the blocker increase the amount of O-GlcNAc bound to proteins, but that increase caused the cells to stop responding to insulin, say co-first authors and postdoctoral fellows Lance Wells and Keith Vosseller.
Looking for proteins in the insulin-signaling pathway that were more glycosylated than normal, Vosseller and Wells found two: beta-catenin and insulin receptor substrate-1 (IRS-1). The crucial role these proteins play in passing along insulin's messages is likely to be adversely affected by the extra sugars they carry, the researchers say.
"Our experiments show that increasing O-GlcNAc on proteins is, by itself, a cause of insulin resistance, rather than an effect or a coincidence," says Vosseller.
In the body, sugar (glucose) is changed into glucosamine, which is changed into O-GlcNAc. Other scientists have shown that giving cells or animals excessive amounts of sugar or glucosamine, along with extra insulin, leads to insulin resistance. The new findings provide an explanation for others' experience with animal and laboratory models of insulin resistance.
There has been little study of glucosamine, a commonly used dietary supplement, in people. It is suggested that people taking glucosamine consult their doctors if they are concerned about the possibility of increasing their risk of developing diabetes.
### Funding was provided by grants and National Research Service Awards from the National Institutes of Health. Professor of biological chemistry Daniel Lane, Ph.D., is also an author.
Under a licensing agreement between Covance Research Products and The Johns Hopkins University, Hart is entitled to a share of royalty received by the university on sales of the antibody used to detect O-GlcNAc on proteins. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict of interest policies.
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