Posts

Robert J. Freishtat working in the lab

Detecting early signs of type 2 diabetes through microRNA

Robert J. Freishtat working in the lab

Obesity is a major risk factor for insulin resistance and type 2 diabetes. Now researchers understand the pathogenesis better among teens with mid-level obesity, thanks to clues released from circulating adipocyte-derived exosomes.

Researchers know that exosomes, tiny nanoparticles released from fat cells, travel through the bloodstream and body, regulating a variety of processes, from growth and development to metabolism. The exosomes are important in lean, healthy individuals in maintaining homeostasis, but when fat gets ‘sick’ – the most common reason for this is too much weight gain – it can change its phenotype, becoming inflammatory, and disrupts how our organs function, from how our skeletal muscle and liver metabolize sugar to how our blood vessels process cholesterol.

Robert J. Freishtat, M.D., M.P.H., the chief of emergency medicine at Children’s National Health System and a professor of precision medicine and genomics at the George Washington University School of Medicine and Health Sciences, and Sheela N. Magge M.D., M.S.C.E., who is now the director of pediatric endocrinology and an associate professor of medicine at the Johns Hopkins School of Medicine, were curious about what this process looked like in teens who fell in the mid-range of obesity.

Obesity is a major risk factor for insulin resistance and type 2 diabetes, but Dr. Freishtat and Dr. Magge wanted to know: Why do some teens with obesity develop type 2 diabetes over others? Why are some teens in this mid-range of obesity metabolically healthy while others have metabolic syndrome? Can fat in obese people become sick and drive disease?

To test this, Dr. Freishtat and Dr. Magge worked with 55 obese adolescents, ages 12 to 17, as part of a study at Children’s National. The participants – 32 obese normoglycemic youth and 23 obese hyperglycemic youth – were similar in age, sex, race, pubertal stage, body mass index and overall fat mass. The distinguishing factor: The hyperglycemic study participants, the teens with elevated blood sugar, differed in where they stored fat. They had extra visceral fat (or adipose tissue) storage, the type of fat that surrounds the liver, pancreas and intestines, a known risk factor for type 2 diabetes.

Dr. Magge and Dr. Freishtat predicted that circulating exosomes from the teens with elevated blood sugar are enriched for microRNAs targeting carbohydrate metabolism.

They used three tests to examine study participants’ metabolism, body composition and circulating exosomes. The first test, an oral glucose tolerance test, measures how efficiently the body metabolizes sugar; the second test is the whole body DXA, or dual-energy x-ray absorptiometry, which analyzes body composition, including lean tissue, fat mass and bone mineral density; and the third test, the serum adipocyte-derived exosomal microRNA assays, is an analysis of circulating fat signals in the bloodstream.

They found that teens with elevated blood sugar and increased visceral fat had different circulating adipocyte-derived exosomes. These study participants’ exosomes were enriched for 14 microRNAs, targeting 1,304 mRNAs and corresponding to 179 canonical pathways – many of which are directly associated with carbohydrate metabolism and visceral fat.

Dr. Magge will present this research, entitled “Changes in Adipocyte-Derived Exosomal MicroRNAs May Play a Role in the Progression from Obese Normoglycemia to Hyperglycemia/Diabetes,” as an oral abstract at the American Diabetes Association’s 79th Scientific Sessions on Saturday, June 8.

Dr. Freishtat envisions having this information will be especially helpful for a patient in a mid-range of obesity. Exosomes primarily consist of small non-coding RNAs. In the current study, the altered RNAs affect P13K/AKT and STAT3 signaling, vital pathways for metabolic and immune function.

“Instead of waiting until someone has the biochemical changes associated with type 2 diabetes, such as hyperglycemia, hyperlipidemia and insulin resistance, we’re hoping physicians will use this information to work with patients earlier,” says Dr. Freishtat. “Through earlier detection, clinicians can intervene when fat shows sign of illness, as opposed to when the overt disease has occurred. This could be intervening with diet and lifestyle for an obese individual or intervening with medication earlier. The goal is to work with children and teens when their system is more plastic and responds better to intervention.”

As this research evolves, Dr. Freishtat continues to look at the intergenerational effects of circulating adipocyte-derived exosomes. Through ongoing NIH-funded research in India, he finds these exosomes, similar in size to lipoproteins, can travel across the placenta, affecting development of the fetus in utero.

“What we’re finding in our initial work is that these exosomes, or ‘sick’ fat, cross the placenta and affect fetal development,” Dr. Freishtat says. “Some of the things that we’re seeing are a change in body composition of the fetus to a more adipose phenotype. Some of our work in cell cultures shows changes in stem cell function and differentiation, but what’s even more interesting to us is that if the fetus is a female sex that means her ovaries are developing while she’s in utero, which means a mother’s adipocyte-derived exosomes could theoretically be affecting her grandchild’s phenotype – influencing the health of three generations.”

While this research is underway, Dr. Freishtat is working with JPOD @ Boston, co-located with the Cambridge Innovation Center in Cambridge, Massachusetts, to develop a test to provide analyses of adipocyte-derived exosomal microRNAs.

“It’s important for families to know that these studies are designed to help researchers and doctors better understand the development of disease in its earliest stages, but there’s no need for patients to wait for the completion of our studies,” says Dr. Freishtat. “Reaching and maintaining a healthy body weight and exercising are important things teens and families can do today to reduce their risk for obesity and diabetes.”

Human Rhinovirus

Finding the root cause of bronchiolitis symptoms

Human Rhinovirus

A new study shows that steroids might work for rhinovirus but not for respiratory syncytial virus.

Every winter, doctors’ offices and hospital emergency rooms fill with children who have bronchiolitis, an inflammation of the small airways in the lung. It’s responsible for about 130,000 admissions each year. Sometimes these young patients have symptoms reminiscent of a bad cold with a fever, cough and runny nose. Other times, bronchiolitis causes breathing troubles so severe that these children end up in the intensive care unit.

“The reality is that we don’t have anything to treat these patients aside from supportive care, such as intravenous fluids or respiratory support,” says Robert J. Freishtat, M.D., M.P.H., chief of emergency medicine at Children’s National Health System. “That’s really unacceptable because some kids get very, very sick.”

Several years ago, Dr. Freishtat says a clinical trial tested using steroids as a potential treatment for bronchiolitis. The thinking was that these drugs might reduce the inflammation that’s a hallmark of this condition. However, he says, the results weren’t a slam-dunk for steroids: The drugs didn’t seem to improve outcomes any better than a placebo.

But the trial had a critical flaw, he explains. Rather than having one homogenous cause, bronchiolitis is an umbrella term for a set of symptoms that can be caused by a number of different viruses. The most common ones are respiratory syncytial virus (RSV) and rhinovirus, the latter itself being an assortment of more than 100 different but related viruses. By treating bronchiolitis as a single disease, Dr. Freishtat says researchers might be ignoring the subtleties of each virus that influence whether a particular medication is useful.

“By treating all bronchiolitis patients with a single agent, we could be comparing apples with oranges,” he says. “The treatment may be completely different depending on the underlying cause.”

To test this idea, Dr. Freishtat and colleagues examined nasal secretions from 32 infants who had been hospitalized with bronchiolitis from 2011 to 2014 at 17 medical centers across the country that participate in a consortium called the 35th Multicenter Airway Research Collaboration. In half of these patients, lab tests confirmed that their bronchiolitis was caused by RSV; in the other half, the cause was rhinovirus.

From these nasal secretions, the researchers extracted nucleic acids called microRNAs. These molecules regulate the effects of different genes through a variety of different mechanisms, usually resulting in the effects of target genes being silenced. A single microRNA typically targets multiple genes by affecting messenger RNA, a molecule that’s key for producing proteins.

Comparing results between patients with RSV or rhinovirus, the researchers found 386 microRNAs that differed in concentration. Using bioinformatic software, they traced these microRNAs to thousands of messenger RNAs, looking for any interesting clues to important mechanisms of illness that might vary between the two viruses.

Their findings eventually turned up important differences between the two viruses in the NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway, a protein cascade that’s intimately involved in the inflammatory response and is a target for many types of steroids. Rhinovirus appears to upregulate the expression of many members of this protein family, driving cells to make more of them, and downregulate inhibitors of this cascade. On the other hand, RSV didn’t seem to have much of an effect on this critical pathway.

To see if these effects translated into cells making more inflammatory molecules in this pathway, the researchers searched for various members of this protein cascade in the nasal secretions. They found an increase in two, known as RelA and NFkB2.

Based on these findings, published online Jan. 17, 2018, in Pediatric Research, steroids might work for rhinovirus but not for RSV, notes Dr. Freishtat the study’s senior author.

“We’re pretty close to saying that you’d need to conduct a clinical trial with respect to the virus, rather than the symptoms, to measure any effect from a given drug,” he says.

Future clinical trials might test the arsenal of currently available medicines to see if any has an effect on bronchiolitis caused by either of these two viruses. Further research into the mechanisms of each type of illness also might turn up new targets that researchers could develop new medicines to hit.

“Instead of determining the disease based on symptoms,” he says, “we can eventually treat the root cause.”

Study co-authors include Kohei Hasegawa, study lead author, and Carlos A. Camargo Jr., Massachusetts General Hospital; Marcos Pérez-Losada, The George Washington University School of Medicine and Health Sciences; Claire E. Hoptay, Samuel Epstein and Stephen J. Teach, M.D., M.P.H., Children’s National; Jonathan M. Mansbach, Boston Children’s Hospital; and Pedro A. Piedra, Baylor College of Medicine.

Fat Cells

Cellular signals may increase atherosclerosis risk

Fat Cells

Fat cells from obese patients have the ability to send signals that can accelerate biological processes leading to atherosclerosis.

Obesity has been linked to a variety of adverse health conditions, including Type 2 diabetes, cancer, heart attack and stroke – conditions that may begin as early as childhood in patients whose obesity also begins early. While this much is known, it has been unclear how extra fat mass might lead to these chronic health conditions.

New research from Children’s National Health System scientists might help answer this question. In findings presented at the 2017 annual meeting of the Pediatric Academic Societies, the research team shows that exosomes – nanosized chemical messages that cells send to each other to regulate protein production – isolated from very obese teenage patients behave very differently from those derived from lean patients and could be key players in heightening the risk of developing atherosclerosis. This hardening of the arteries can, in turn, increase the risk of heart disease and stroke in adulthood.

A research team led by Robert J. Freishtat, M.D., M.P.H., chief of emergency medicine at Children’s National, is exploring possible links between extra belly fat and obesity-related diseases, such as atherosclerosis, a buildup of plaque in arteries that can harden and restrict blood flow. More precise knowledge of the mechanisms by which obesity ratchets up heart risks holds the promise of helping the next generation of kids avoid experiencing chronic disease.

The working theory is that exosomes derived from belly fat from obese patients have the distinct ability to accelerate biological processes leading to atherosclerosis.

The research team isolated exosomes from five obese teenagers and compared them to five sex-matched lean adolescents. It turns out that exosomes derived from fat pick up their marching orders from microRNA content likely to target cholesterol efflux genes, which help reduce cholesterol buildup in cells.

The research team looked at differences in cholesterol efflux gene expression in THP-1 macrophages. Uptake of low-density lipoprotein cholesterol, “bad” cholesterol, was 92 percent higher than in those exposed to exosomes from obese patients compared with their lean counterparts. Exposure to obese exosomes also reduced cholesterol efflux.

“Atherogenic properties of fat-cell derived exosomes from obese patients differ markedly from the non-atherogenic profile of exosomes from lean patients. It is especially concerning that we see biological clues of heightened risk in teenagers, and the finding underscores how the seeds for atherosclerosis can be planted very early in life,” Dr. Freishtat says.

The presentation is the latest finding from a research team that, over years of work, is unraveling the mechanisms of cellular signaling by fat cells.  By closely examining very obese children – who have the most severe cardiometabolic disease – the team identified strong molecular signals of disease risk that they can search for in leaner patients who may be at risk for disease years from now.

“We know that morbidly obese patients have cardiovascular issues,” explains Dr. Freishtat. “An unanswered question is for patients with no clinical symptoms who are a little overweight. Can we look at them and say whether they are at risk for developing atherosclerosis, insulin resistance or Type 2 diabetes five or 10 years down the line? That’s the whole rationale for doing this work.”

The critical issue is what exosomes are up to. Dr. Freishtat says in lean people, they’re active and are very important in maintaining stable metabolism and homeostatic processes.

“When a person becomes obese, however, exosomes evolve,” he says. “They no longer support insulin signaling, which is helpful, and drive processes in the reverse direction, repressing insulin signaling – which can be harmful,” he adds.

Ultimately, the research team aims to revolutionize how chronic diseases like Type 2 diabetes are diagnosed. For far too long, clinicians have relied on symptoms like high glucose levels and excess urination to diagnose diabetes.

“By the time you have symptoms, it’s too late,” says Dr. Freishtat. “In many cases, damage has been done by relentless exposure to high sugar levels. The biological processes that underlie the Type 2 diabetes process began five, 10, 15 years earlier. If we can detect it earlier, before symptoms arise, intervention is going to have a more significant impact on improving and extending patients’ lives.”

Robert J. Freishtat

A game changer for detecting complications from obesity

Robert J. Freishtat

The work that Children’s National Health System physician-scientist Robert J. Freishtat, M.D., M.P.H., and colleagues are doing could soon be a game changer when it comes to early intervention and prevention of obesity-related illnesses.

They already knew there’s a direct relationship between the amount of visceral adipose, or belly fat, a person has and development of some of the most common and life-threatening complications of obesity, including cardiovascular disease and the insulin resistance that leads to diabetes. What remained unclear, until recently, were the precise mechanisms for how the increase in belly fat triggers the onset of additional disease.

Dr. Freishtat, senior author of “Adipocyte-Derived Exosomal miRNAs: A Novel Mechanism for Obesity-Related Disease,” published by Pediatric Research, studies the adipocytes, or fat cells, of visceral adipose in both lean and obese patients to understand exactly how these fat cells can and do wreak havoc — not just locally but throughout the body. Cells leverage exosomes to communicate among themselves, but in overweight patients those cellular communications can go awry.

“As the body’s visceral fat grows, somewhere on the path to obesity the fat cells change and begin to release different exosomes than lean adipose cells do. These new messages disrupt some important processes that eventually prevent the body from effectively dealing with sugar and cholesterol,” says Dr. Freishtat, chief of Emergency Medicine at Children’s National, and associate professor of Pediatrics, Emergency Medicine, and Integrative Systems Biology at the George Washington University.

Dr. Freishtat describes exosomes as “biological tweets”— short messages shed by all cells that allow for intercellular communication and alter gene expression. In the case of the adipocytes that exist in large quantities of visceral fat, these “tweets” actually cause the downregulation of proteins impacting two key signaling pathways — TGF-β and Wnt/β-catenin — associated with controlling chronic inflammation and fibrotic disease throughout the body. These signaling changes make morbidly obese patients more vulnerable to systemwide issues, such as cholesterol accumulation and changes to how the liver processes fat.

Details of the study

The study authors surgically collected fat tissue from lean and obese female patients aged 11 to 19 and used modified bead-based flow cytometry to separate, identify, and compare the exosomal RNA shed by the fat cells in both lean and obese samples. To confirm the unique impact of the obese adipose exosomes on gene expression, the research team then exposed lung cells in vivo to the exosomes shed by both lean and obese adipose. They measured the impact of exposure and uptake on a single receptor type — activin receptor type-2B — known to have a major influence on the TGF-β pathway. The exosomes from obese adipose caused the receptor to slow down, leading to significant changes in the function of the TGF-β pathway.

The team continues to explore how the exosomes shed from excess amounts of visceral adipose spread throughout the body and how the function of organs such as the liver, the heart, and the brain are impacted by the migrating fat cells.

A Look into the future

Successfully identifying and isolating these exosomes also has opened the door to developing a test to detect them, an idea that may permit even earlier intervention to delay or prevent the onset of obesity-related illnesses.

“It is entirely plausible, and is on its way to happening very soon, that someone could walk into their physician’s office for a routine physical and, via a urine test, find out that they are on the road to some dangerous additional side effects of significant weight gain,” says Dr. Freishtat. “That type of early detection could really be a game changer for the millions of Americans who are on track to developing heart, liver, and other diseases resulting from morbid obesity.”