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Research matters

News from the Harvard Medical School research community.

Research matters
Research matters
SIRT3 and SIRT4 may now be potential drug targets for diseases associated with aging. (Noah Seelam/AFP/Getty Images)
Research matters
Glucose-sensing neurons may play a role in Type 2 diabetes.
Research matters
Fatty liver: low-glycemic index foods, like most vegetables, raise blood sugar slowly. (Lakruwan Wanniarachchi/AFP/Getty Images)

News from the Harvard Medical School research community.

Pathology

Researchers find connection between caloric restriction and longevity

For nearly 70 years, scientists have known that caloric restriction prolongs life. In everything from yeast to primates, a significant decrease in calories can extend lifespan by as much as one-third. But getting under the hood of the molecular machinery that drives this longevity has remained elusive.

Mitochondria are vital for sustaining the health and longevity of a cell.

As reported in the September 21 issue of the journal Cell, researchers from Harvard Medical School, in collaboration with scientists from Cornell Medical School and the National Institutes of Health, have discovered two genes in mammalian cells that act as gatekeepers for cellular longevity. When cells experience certain kinds of stress, such as caloric restriction, these genes rev up and help protect cells from diseases of aging.

"We've reason to believe now that these two genes may be potential drug targets for diseases associated with aging," says David Sinclair, associate professor of pathology at Harvard Medical School and senior author on the paper.

The new genes that Sinclair's group have discovered, in collaboration with Anthony Sauve of Cornell Medical School and Rafael de Cabo of NIH, are called SIRT3 and SIRT4. They are members of a larger class of genes called sirtuins. (Another gene belonging to this family, SIRT1, was shown last year to also have a powerful impact on longevity when stimulated by the red-wine molecule resveratrol.)

In this paper, the newly discovered role of SIRT3 and SIRT4 drives home something scientists have suspected for a long time: mitochondria are vital for sustaining the health and longevity of a cell.

Mitochondria, a kind of cellular organ that lives in the cytoplasm, are often considered to be the cell's battery packs. When mitochondria stability starts to wane, energy is drained out of the cell, and its days are numbered. In this paper, Sinclair and his collaborators discovered that SIRT3 and SIRT4 play a vital role in a longevity network that maintains the vitality of mitochondria and keeps cells healthy when they would otherwise die.

When cells undergo caloric restriction, signals sent in through the membrane activate a gene called NAMPT. As levels of NAMPT ramp up, a small molecule called NAD begins to amass in the mitochondria. This, in turn, causes the activity of enzymes created by the SIRT3 and SIRT4 genes-enzymes that live in the mitochondria-to increase as well. As a result, the mitochondria grow stronger, energy-output increases, and the cell's aging process slows down significantly. (Interestingly, this same process is also activated by exercise.)

"We're not sure yet what particular mechanism is activated by these increased levels of NAD, and as a result SIRT3 and SIRT4," says Sinclair, "but we do see that normal cell-suicide programs are noticeably attenuated. This is the first time ever that SIRT3 and SIRT4 have been linked to cell survival."

In fact, the mitochondria appear to be so essential to the cell's life that when all other energy sources inside the cell-including the nucleus-are wiped out, yet the mitochondria are kept intact and functional, the cell remains alive.

"Mitochondria are the guardians of cell survival," says Sinclair. "If we can keep boosting levels of NAD in the mitochondria, which in turn stimulates buckets more of SIRT3 and SIRT4, then for a period of time the cell really needs nothing else."

Sinclair and his colleagues have coined a phrase for this observation: the Mitochondrial Oasis Hypothesis.

SIRT3 and SIRT4 may now also be potential drug targets for diseases associated with aging. For example, in recent years scientists have become increasingly aware of the importance of mitochondrial function in treating diseases such as cancer, diabetes, and neurodegeneration.

"Theoretically, we can envision a small molecule that can increase levels of NAD, or SIRT3 and SIRT4 directly, in the mitochondria," says Sinclair. "Such a molecule could be used for many age-related diseases."
According to Suave of Cornell, "This study also highlights how advanced technological methods can help resolve fundamental biological questions in ways that were hard to achieve as recently as a few years ago."

Cardiology

A step toward tissue-engineered heart structures for children

Infants and children receiving artificial heart-valve replacements face several repeat operations as they grow, since the replacements become too small and must be traded for bigger ones. Researchers at Children's Hospital Boston have now developed a solution: living, growing valves created in the lab from a patient's own cells.

In a special issue of Circulation published September 11, they described making pulmonary valves through tissue engineering. These valves, which provide one-way blood flow from the heart's right ventricle into the pulmonary artery, are often malformed in congenital heart disease, putting an extra burden on the heart.

"The heart valve is a complex organ," says Virna Sales, MD, a researcher in Children's Department of Cardiac Surgery and the study's first author. "It must open and close synchronously, withstand pressure, and be pliable and elastic. We are one of the few labs in the US that's attempting to make heart valves through tissue engineering. We hope these could just be implanted in a child just once, instead of the many heart operations most children have to go through as they get older."

The researchers, led by Sales and senior investigator John Mayer, MD, in Children's Department of Cardiac Surgery, first isolated endothelial progenitor cells (precursors of the cells that line blood vessel walls) from the blood of laboratory animals. They then ‘seeded' the cells onto tiny, valve-shaped biodegradable moulds and pre-coated with proteins found in the natural ‘matrix' that surrounds and supports cells.

Experimenting with different matrix proteins and growth factors, they were able to make pulmonary valve leaflets that had the right mechanical properties - sturdy yet pliable. Tests showed the original cells had differentiated to form both endothelial cells and smooth-muscle-like cells and added to the surrounding matrix to hold them together.

With grants from the American Heart Association and the Cambridge, Mass.-based Centre for Integration of Medicine and Innovative Technology (CIMIT), Sales is now refining the lab-grown valves by exposing them to mechanical stress in a bioreactor. She is also using a ‘cardiac jelly' - a cushiony material rich in matrix components and growth factors - to encourage cells to differentiate and form a heart valve on their own, with only minimal reliance on an artificial scaffold. "I would like to mimic what really happens in the embryo - what Mother Nature does," she says. The next step would be to implant the living valves into animals.

Sales and surgical research fellow Bret Mettler, MD, have already used tiny tissue-engineered patches in sheep to rebuild a portion of the pulmonary artery - an area that often needs augmentation in patients with congenital heart disease. Eventually, Sales hopes to use tissue-engineering techniques to create ‘living stents' for adults with atherosclerosis.

Diabetes

A role for glucose-sensing neurons in Type 2 diabetes

In cases of Type 2 diabetes, the body's cells fail to appropriately regulate blood glucose levels. Research has suggested that this results from two simultaneous problems: the improper functioning of pancreatic beta cells and the impairment of insulin's actions on target tissues, including the liver, fat and muscles.

But now, research led by scientists at Beth Israel Deaconess Medical Centre (BIDMC) and Oregon Health & Science University has identified a third abnormality that could play an important role in the development of obesity-induced Type 2 diabetes. Reported in the journal Nature, the study describes a previously unrecognised role for glucose-sensing neurons in the onset of the disease - in other words, an important component of Type 2 diabetes may indeed be ‘in your head'.

"For many years, we've known that subpopulations of neurons in the brain become ‘excited' by glucose," explains Bradford Lowell, MD, PhD, an investigator in the Division of Endocrinology, Diabetes and Metabolism at BIDMC and Professor of Medicine at Harvard Medical School (HMS). "But we haven't understood exactly how or why this is significant. "With this study, we show that these neurons sense increases in glucose and then initiate responses aimed at returning blood-glucose levels to normal. This is the first demonstration that glucose-sensing by neurons plays an important role in responding to rising blood glucose levels." This finding, adds Lowell, who served as the study's co-senior author together with Michael Cowley, PhD, of the Division of Neuroscience, Oregon Health & Science University, could potentially lead to novel treatments for Type 2 diabetes.

Knowing that the pro-opiomelanocortin (POMC) neurons regulate body weight in both mice and humans, co-lead authors Laura Parton, PhD, Chian Ping Ye, PhD, Roberto Coppari, PhD, and Pablo Enriori, PhD, decided to study the electrical properties of these cells in an animal model.

"New advances in genetic techniques have allowed us to express green fluorescent proteins [GFP] specifically in one cell type," explains Parton, a member of the Lowell laboratory at BIDMC and Postdoctoral Research Fellow at HMS. "The advantage of expressing a fluorescent marker specifically in one type of neuron is the ability to identify and distinguish these cells from the many hundreds of other cell types that are present in the brain."
As predicted, the electrophysiology experiments demonstrated that POMC neurons became electrically excited by a rise in glucose, similar to what would occur after eating a meal. The authors then went on to disrupt glucose-sensing abilities specifically in the POMC neurons - and confirmed that these neurons play a critically important role in regulating blood-glucose levels in mice. And, as is the case in pancreatic beta cells, the glucose-sensing ability of POMC neurons was shown to be defective in the mice with obesity-induced Type 2 diabetes.

"What is apparently happening," says Parton, "is that an increase in the activity of the mitochondrial uncoupling protein 2 (UCP2), is behind the loss of glucose-sensing ability in the POMC neurons. Increased activity of UCP2 is known to cause loss of glucose-sensing and defective insulin secretion by pancreatic beta cells and this study now shows that a similar phenomenon also occurs in neurons."

"These new findings add to our understanding of Type 2 diabetes at a critically important time," adds Lowell. "The incidence of the disease has risen to epidemic proportions, and obesity is a big risk factor for the disease. The discovery that defects in glucose-sensing by the brain may also be contributing to Type 2 diabetes could help lead to new therapeutic strategies for this widespread problem."

Dermatology

Specialised recipient cells determine where pigment is deposited in epidermis and hair

The pigment melanin, which is responsible for skin and hair colour in mammals, is produced in specialised cells called melanocytes and then distributed to other cells. But not every cell in the complex layers of skin becomes pigmented. The question of how melanin is delivered to appropriate locations may have been answered by a study from researchers at the Massachusetts General Hospital (MGH) Cutaneous Biology Research Centre (CBRC).

"Pigment recipient cells essentially tell melanocytes where to deposit melanin, and the pattern of those recipients determines pigment patterns," says Janice Brissette, PhD, who led the study. "Recipient cells act like the outlines in a child's colouring book; as recipient cells develop, they form a ‘picture' that is initially colourless but is then ‘coloured in' by the melanocytes." The report appeared in the Sept. 7 issue of Cell.

In humans, melanin is deposited in both the skin and the hair; but in some other mammals such as mice, melanin is primarily deposited in the coat, leaving the skin beneath the coat unpigmented. Melanocytes deposit melanin via cellular extensions called dendrites that reach out to other cells in the epidermis (the outer layer of skin) or the hair follicles. But the mechanism determining whether melanin is delivered to a particular cell has been unknown.

The MGH-CBRC researchers theorised that a mouse gene known as Foxn1 might play a role. Lack of Foxn1 is responsible for so-called ‘nude mice,' which have hair that is so brittle it breaks off, resulting in virtually total hairlessnes, and other defects of the skin. A similar phenomenon exists in humans with inactivation of the corresponding gene.

When the researchers developed a strain of transgenic mice in which Foxn1 is misexpressed in cells that do not usually contain melanin, they found those normally colourless areas became pigmented. Examining the skin of the transgenic mice revealed that melanocytes were contacting and delivering melanin to the cells in which Foxn1 was abnormally activated. No pigment was observed in the corresponding tissues of normal mice. Examination of human skin samples showed that the human version of Foxn1 was also expressed in cells known to be pigment recipients. Further experiments revealed that Foxn1 signals melanocytes through a protein called Fgf2, levels of which rise as Foxn1 expression increases.

"Foxn1 makes epithelial cells into pigment recipients, which attract melanocytes and stimulate pigment transfer, engineering their own pigmentation," says Brissette, an associate professor of Dermatology at Harvard Medical School. She and her colleagues note that the Foxn1/Fgf2 pathway probably has additional functions in the skin and that it is probably not the only pathway responsible for the targeting of pigment.

"We know that Foxn1 and Fgf2 act in concert with other factors and function within a larger network of genes. Our next step will be to identify other genes that can confer the pigment recipient phenotype or control the targeting of pigment," Brissette adds. Her research may eventually be relevant to disorders such as vitiligo - in which pigment disappears from patches of skin - age spots, the greying of hair and even the deadly melanocyte-based skin cancer melanoma.

The co-first authors of the Cell report are Lorin Weiner, PhD, and Rong Han, PhD, both of the MGH-CBRC. Additional co-authors are Jian Li, PhD, Kiyotaka Hasegawa, MS, and David Lee, MGH-CBRC; Bianca Scicchitano, PhD, now at La Sapienza/University of Rome; and Maddalena Grossi, PhD, University of Lausanne, Switzerland. The study was supported by the National Institutes of Health and by the CBRC through the support of Shiseido Co. Ltd.

Massachusetts General Hospital, established in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of nearly $500 million and major research centres in AIDS, cardiovascular research, cancer, computational and integrative biology, cutaneous biology, human genetics, medical imaging, neurodegenerative disorders, regenerative medicine, systems biology, transplantation biology and photomedicine. MGH and Brigham and Women's Hospital are founding members of Partners HealthCare HealthCare System, a Boston-based integrated health care delivery system.

Nutrition

Quick-burning carbs may cause fatty liver

Diets rich in rapidly-digested carbohydrates not only expand waistlines, but may also cause fatty liver, a condition that can lead to liver failure and death, finds a new study in mice. If confirmed in humans, the findings suggest that fatty liver disease - on the upsurge among Americans as a by-product of the obesity epidemic - may be preventable and possibly treatable through dietary changes. The study appeared in the September issue of the journal Obesity.
The researchers, led by David Ludwig, MD, PhD, director of the Optimal Weight for Life program at Children's Hospital Boston, fed mice either a high- or a low-glycemic index diet. High-glycemic index foods, including white bread, white rice, most prepared breakfast cereals and concentrated sugar, raise blood sugar quickly. Low-glycemic index foods, like most vegetables, fruits, beans and unprocessed grains, raise blood sugar slowly.

On the high-glycemic index diet, mice ate a type of cornstarch that is digested quickly whereas on the low-glycemic index diet, mice ate a type of cornstarch that is digested slowly. The diets had equal amounts of total calories, fat, protein, and carbohydrate, and the mice were otherwise treated identically.

After six months, the mice weighed the same. However, mice on the low-glycemic index diet were lean, with normal amounts of fat in throughout their bodies. Mice on the high-glycemic index diet had twice the normal amount of fat in their bodies, blood and livers.

When sugar melts out of high-glycemic index food, Ludwig explains, it drives up production of insulin, which tells the body to make and store fat. Nowhere is this message felt more strongly than in the liver, because the pancreas, which makes insulin, dumps the hormone directly into the liver, where concentrations can be many times higher than in the rest of the body. Fat buildup in the liver, or fatty liver, is usually symptomless, but it increases the risk for liver inflammation, which can progress to hepatitis and, in some cases, liver failure.

Fatty liver is becoming more common in Americans, especially in children, says Ludwig. Many cases in adults can be explained by alcoholism, but not the paediatric cases. Where just one case of fatty liver was reported in children in 1980, now between 1 in 4 and 1 in 2 overweight American children are estimated to have the condition. As these millions of children age, some will progress to full-blown liver disease. "This is a silent but dangerous epidemic," says Ludwig. "Just as Type 2 diabetes exploded into our consciousness in the 1990s, so we think fatty liver will in the coming decade."

A previous study found that Italians who ate higher-glycemic index diets had fattier livers, but the study wasn't tightly controlled. The new study makes clear that the type of carbohydrate can cause fatty liver in animals, independent of other elements of diet or lifestyle. "Our experiment creates a very strong argument that a high-glycemic index diet causes, and a low-glycemic index diet prevents, fatty liver in humans," says Ludwig. Ludwig and colleagues now hope to confirm this in a just-launched clinical trial - and to show that a low-glycemic index diet can reverse fatty liver in overweight children. The children, aged 8 to 17, will be randomised to either the low-glycemic diet or a low-fat diet.

Low-fat diets are currently the standard treatment, Ludwig says, but many children with fatty liver don't respond to them. "We think it is a misconception that the fat you're eating goes into the liver," he says.

Ludwig, author of Ending the Food Fight: Guide Your Child to a Healthy Weight in a Fast Food/Fake Food World, hypothesises that obesity, sedentary lifestyles and increased consumption of refined carbohydrates are ‘synergistically' fuelling a fatty liver epidemic in children. Ironically, low-fat diets have only made matters worse, replacing fat with sugar or starchy foods that actually increase fat deposition in the body.

"Two low-fat Twinkies, billed as a health food, contain the same amount of sugar as an oral glucose tolerance test - a test used to determine how much sugar someone can digest," Ludwig says. He notes that the French delicacy pate de fois gras - the fatty liver of a duck or goose - is produced by over-feeding the animals with high-glycemic index grains.

Research Matters brings together selected research being conducted at Harvard Medical School's 18 affiliated institutions. For more information, visit the Harvard Medical School website at www.hms.harvard.edu.

This article is provided courtesy of Harvard Medical International. © 2007 President and Fellows of Harvard College.

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