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From left: Marc Montminy, Reuben Shaw, and Ron Evans

Diabetes: Looking under the hood

Salk scientists are teaming up to unravel the complex metabolism behind diabetes


Think of the most futuristic, intricate hybrid car possible, one that effortlessly and efficiently balances battery and fuel use to whisk you to your destination. It won't be one fraction as complicated as human metabolism—the production and use of energy that provides our motion, our life.

Now think of all the things under the hood that can go wrong in the fantastically involved electronics that match fuel use with need, and you get an idea of what could be happening when diabetes, a disease of energy utilization, develops.

Salk researchers are trying to understand the human "hybrid car" of metabolism and what happens when this biological system breaks down. The problem is attracting a growing number of scientists worldwide, given the increasing burden that diabetes and other metabolic dysfunctions have on human health and society.

According to the latest figures from the American Diabetes Foundation, nearly 25.8 million Americans have type 2 diabetes, and an estimated 79 million people are at risk of developing the condition. It is the sixth leading cause of death, and treatment of the disease costs the country $116 billion annually. If current trends continue, the U.S. Centers for Disease Control and Prevention estimate that one in three Americans will have diabetes by 2050, making it by far the most dominant and costly disease to manage.

Despite a growing focus on diabetes research in centers around the world, progress in understanding and treating the disease is hard won. But at the Salk Institute, a unique collaboration among three molecular biologists—Ronald Evans, Marc Montminy and Reuben Shaw—is decoding the labyrinth of genetic switches that control human metabolism. With a generous grant from the Leona M. and Harry B. Helmsley Charitable Trust, these investigators established the Salk Center for Nutritional Genomics in 2009, helping to meld their strengths into a powerful and cohesive unit.

The research in the three labs in the Center for Nutritional Genomics employs a molecular approach to nutrition and its impact on the role of metabolism in diabetes, obesity, cancer, exercise physiology and lifespan, thereby increasing the understanding of how nutrients affect health. It includes a metabolic core facility and an interdisciplinary fellows program. Their work has already uncovered key discoveries regarding genetic switches that potentially point to new ways of controlling the body's production of glucose, the simple sugar that is the source of energy in human cells and the central player in diabetes.

"If you control those switches, you can control the production of glucose, which is really at the heart of the problem of type 2 diabetes," says Marc Montminy, professor and head of the Clayton Foundation Laboratories for Peptide Biology.

Ronald Evans, professor in the Gene Expression Laboratory and a Howard Hughes Medical Institute investigator, adds that understanding diabetes requires more than just looking at what a person eats. "The use of food to power our bodies is an old story," he says. "The new story is that the foods we eat interact with genes that control nutrient metabolism. To tackle the enormous problem of diabetes and metabolic disease, we have to understand how the genome controls the flow of energy in the body."

Food fuel in the day, fat batteries at night

To understand the exquisite metabolic machine that is a human, it helps to think of that hybrid car.

During the day, humans burn "gas," the high-octane glucose derived from the food we eat. This is the fuel that supplies the muscles, the brain and all other parts of the body expending energy. At night, when we sleep, we revert to our "batteries"—our stored fat—as a source of very dependable but slowly released energy.

This shift from burning one kind of fuel to burning the other is controlled by pancreatic islet cells, which can be viewed as the "central transmission." Islet cells control the release of insulin from the pancreas during the day. The role of insulin is to allow high-octane glucose, produced from the food we eat, to be taken up into muscle. Insulin also tells the liver to stop making glucose, with the net effect of lowering glucose levels in the blood.

At night, the body shifts to glucagons, hormones that are also produced in the pancreatic islets. That changes the nutrient mix from one based on glucose to one based on fat. (There is an exception: during the night, the liver needs to provide enough glucose to keep the brain and red blood cells going.)

Glucagon, which is also released during a fasting state, such as during starvation, is thus the opposite of insulin; it tells the liver to convert stored fat into glucose, which is released into the blood. Glucagon and insulin are part of a feedback system designed to keep blood glucose at a stable level.

"We are really looking at how the whole body constantly reprograms its metabolism in response to changes in nutrition, hormones, environment or day and night cycles," says Reuben Shaw, an assistant professor in the Molecular and Cell Biology Laboratory and a Howard Hughes Medical Institute early career scientist.

Multiple organs constantly talk to each other during this metabolic dance, but in diabetes, communication within this complex system breaks down in two different ways. One is the inability of muscle and fat tissues to recognize the insulin signal that is coming primarily during the day. The other is the inability of the islet cells in the pancreas to produce enough insulin to meet the demands. As a result, the liver increases its glucose production, resulting in high levels of "sugar" in the blood.

"It's kind of remarkable that we don't all develop diabetes, given that these very sensitive systems are naturally affected by age, as well as genetics, not to mention body weight," Montminy says. "The Salk provides an ideal environment in which to study this problem. Because we have no barriers between different laboratories, we are in an excellent position to piece together the very complex puzzle of human metabolism."

Looking under the hood at the metabolism's wiring system

diabetes

Confocal microscope image of an insulin producing cell. Cell nuclei are labeled in blue, AktPH protein in green and the cell membrane in red. Upon stimulation with insulin, the AktPH protein translocates to the outer cell membrane, illustrated as a yellow overlap.

Image courtesy of Sam Van de Velde, Montminy lab and taken in the Waitt Advanced Biophotonics Center Core Facility.

In their quest to decode metabolism, the Salk researchers focus on different tissues: the liver (Shaw and Montminy), muscle and gut (Evans and Shaw) and pancreas (Montminy and Evans). Much of their work has centered on the genetic "switches" that regulate metabolic control.

Montminy's lab has for years focused on the central switches that control glucose production in the liver and others that control glucose sensing and insulin production in the pancreas.

Among his key findings are how a protein (CREB) controls glucose production; that two genetic "fasting" switches (CRTC2 and FOXO1), are needed to turn on glucose-making genes during fasting; and how a glucagon hormone (GLP-1) turns on a series of switches inside the pancreas that increases production of insulin.

Evans's lab studies many different aspects of metabolism, including the way muscles use glucose and how that process can be reprogrammed. One of his seminal discoveries was a "superfamily" of at least 50 different cell receptors that regulates the absorption, storage and burning of fat.Some of these receptors also control metabolism of sugar, salt and the amount of calcium stored in bones. He has also found a new hormone that appears to trigger the formation and expansion of fat cells.

It was Shaw's arrival at Salk in 2006 that sparked a focused collaboration between the three different labs, resulting in a transformative discovery in 2011—the precise sequences of switches that turn on the liver's production of glucose when blood sugar levels drop. The hope is that this finding may result in an exquisitely sensitive drug that lowers glucose in diabetes, which would reduce the need for replacement insulin in patients.

The glue linking the labs

Shaw came to Salk from Harvard Medical School, where, as a postdoctoral researcher, he found how a pathway involved in cellular hunger links diabetes and cancer. This circuit tells cells to slow down and stop dividing when food, in the form of glucose, is scarce. The finding helped explain why people with type 2 diabetes have an elevated risk of certain forms of cancer and also provided insights into how popular diabetes drugs work.

diabetes

In response to glucagon, HDACs quickly move into the nucleus, where they help turn on genes needed for the production of glucose in the liver.

Image courtesy of Maria Mihaylova, Salk Institute for Biological Studies.

The day he realized that an enzyme (LKB1), which was important in cancer, acted on a key metabolism protein (AMPK), Shaw says he knew the discovery was going to set the course of his career.

"AMPK was well studied, and it was known to be a central regulator of glucose metabolism," he says. "But no one knew it had any role in cancer."

Montminy and Evans, too, were well aware of AMPK. The protein acts as a hunger sensor and thus is an important regulator of glucose in muscle, liver and the pancreas. When food (glucose) gets scarce, AMPK pushes the cell into a low-energy state, and Shaw found that LKB1 was the molecule that flipped on the AMPK switch. Shaw showed that LBK1 is often mutated in cancer, meaning that cancer cells can continue to grow even in a low-energy state. He then found that the most popular diabetes drug in the world, metformin, works by controlling blood glucose levels via LKB1.

But it was unknown exactly how AMPK regulates metabolism— or, in other words, no one knew the precise sequences of switches that controlled the system. Shaw became familiar with Evans's work in the metabolism of muscle and how the tissue that stores glucose is reprogrammed during exercise. He also studied Montminy's research into central switches that control glucose production in the liver.

"And it turns out that the thing in common between their different kinds of switches, the thing that controls Marc's switches and separately but directly controls Ron's switches, is actually the LKB/AMPK pathway that I had been studying," Shaw says. "That was not known, and it was unexpected. It totally makes sense now that we have worked out much of the details over the past five years."

Shaw says the pathway's role is to normalize the production and use of glucose and lipids in the cell, which impacts both cell growth and hormones like insulin that are released from the pancreas based on the level of glucose present in the blood. "If you eat too much sugar, it's basically trying to deal with that problem," he says. "It's saying, 'No, no, no, you took in way too much sugar, so we have to deal with it—get rid of it this way, store this part of it over there, and then shut off the liver. You don't need to make your own glucose, because you just ate five hamburgers, so stop making glucose and tell your muscles to take up more glucose because you have too much in your blood.'"

AMPK was the glue that linked the three laboratories, and in 2011, the researchers discovered that a group of enzymes (histone deacetylases, or HDACs) are the molecules that activate the production of glucose in the liver when glucose levels run low or after prolonged periods of fasting or during the night.

diabetes

Confocal microscope image of a pancreatic islet. Cell nuclei are labeled in blue, insulin-producing beta cells in red and glucagon-producing alpha cells in green.

Image courtesy of Sam Van de Velde, Montminy lab and taken in the Waitt Advanced Biophotonics Center Core Facility.

The study, published May 13, 2011, in Cell, put all the pieces together. Spearheaded by a Ph.D. student in Shaw's lab named Maria Mihaylova, but with critical assistance from researchers in both the Montminy and Evans labs, they showed that in liver cells, these HDACs normally stay outside the cell's nucleus. Strikingly, in response to fasting signals (from glucagon), they move into the nucleus, where they activate glucose synthesis from scratch inside the liver.

They further discovered that AMPK turns off the HDAC enzymes, by transferring them outside the cell nucleus, where they can no longer function.

Evans, who studies the circadian basis of metabolism (use of glucose during the day and fat at night), notes that AMPK controls that clock mechanism. It revs up production of glucose as you wake up, as a Science paper from his lab showed in 2009, with assistance from Shaw's lab. Evans's lab also found that a small molecule that activates AMPK can promote endurance in inactive mice by reprogramming metabolism genes in their skeletal muscle—the so-called "exercise pill."

The new HDAC findings offer another connection with cancer research. Oncologists are currently testing HDAC inhibitors as anticancer therapies. The exact mechanism by which these agents block cancer is unknown, but two different HDAC inhibitors have already been approved for treating a form of lymphoma.

Shaw is excited about testing how effective an inhibitor targeting the specific subclass of HDAC they found might be in shutting off glucose production in the liver. "These drugs all go to the liver first, so that is a big advantage because that is where we want them to function," he says. "And if an HDAC inhibitor with the correct specificity could cut off glucose production at the source in the liver, diabetic patients would not need as much insulin as they do now because there would already be much less sugar in the blood."

The researchers recently talked about their research with hundreds of interested listeners—including many from San Diego and the surrounding region—at a Salk Exchange event on diabetes, held at Salk last September (see page 10). "We were able to air these new findings and have a discussion with the audience," Shaw says. "It was fantastic because the level of interest in the community is so high. Everyone knows what is at stake, and they want to know what can be done, both personally and for society."