New York Academy of Sciences
The Sum of Our Parts: Integrative Physiology
Posted July 07, 2008
Scientists have recently made enormous strides toward elucidating the multiple functions of genes in adult animals. Remarkably, this has revealed unanticipated connections between organs. This progress allows us to now ask much broader questions related to the physiology of organisms and to analyze its impact on the molecular pathogenesis of degenerative diseases.
At this meeting, presentations by top researchers summarized where the field of integrative physiology is and how we can move the field forward.
The conference, held May 14–16 2008, explored the genetic basis of the known functions of many organs, the identification of novel physiological functions for various organs, and the definition of genetic cascades leading to frequent degenerative diseases such as metabolic syndrome, heart failure and osteoporosis.
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Gerard Karsenty, MD, PhD
Gerard Karsenty is professor and chair of the Department of Genetics and Development at Columbia University Medical Center, where he studies genetic and hormonal aspects of skeleton formation and maintenance. He came to Columbia from Baylor College of Medicine in Houston, where he was professor of molecular and human genetics and served as scientific director of the Bone Disease Program of Texas. He was a postdoctoral fellow and faculty member in molecular genetics at the University of Texas M.D. Anderson Cancer Center before joining the Baylor faculty in 1998. He also performed postdoctoral studies at the National Institutes of Health and the National Cancer Institute before moving to Houston in 1987. Karsenty holds an MD-PhD degree from the University of Paris, France.
Andrew Marks, MD
Andrew Marks is chairman of the Department of Physiology & Cellular Biophysics, Clyde and Helen Wu Professor of Medicine, and director of the Center for Molecular Cardiology at Columbia University. His research focuses on the molecular mechanisms regulating contraction of normal and failing cardiac muscle, molecular triggers of cardiac arrhythmias, and coronary artery in-stent restenosis. He is former editor-in-chief of The Journal of Clinical Investigation, and was elected a member of the American Academy of Arts and Sciences and the National Academy of Sciences.
Leonard Guarente, PhD
Leonard Guarente is Novartis Professor Biology at the Massachusetts Institute of Technology, where he leads a lab that studies the molecular mechanisms that regulate aging. In research with S. cerevisiae, C. elegans, mice, and mammalian cells, he has focused primarily on the SIRT family of genes, which influence life span and the effects of caloric restriction. Guarente is a member of the American Academy of Microbiology and author of Ageless Quest: One Scientist's Search for the Genes that Prolong Youth.
Gerald I. Shulman, MD, PhD
Gerald Shulman is professor of medicine and cellular & molecular physiology at the Yale School of Medicine, and an investigator with the Howard Hughes Medical Institute. His lab is investigating the molecular mechanisms of insulin resistance in type 2 diabetes, with a particular interest in the relationship between insulin resistance and muscle glucose metabolism, the role of fatty acids in inducing insulin resistance in muscle, and the genetic factors that may predispose individuals to the disease. Through understanding these factors, Shulman hopes to identify potential therapeutic targets.
Johan Auwerx, MD, PhD
Laurie Glimcher, MD
Jonathan Graff, MD, PhD
Tamas L. Horvath, PhD
Barbara Kahn, MD
Shigeaki Kato, PhD
Richard Kitsis, MD
Leslie Leinwand, PhD
Eleftheria Maratos-Flier, MD
Ruslan Medzhitov, PhD
Bruce Spiegelman, PhD
Jil Tardiff, MD, PhD
Ira Tabas, MD
Ken Walsh, PhD
Alan Dove is a science writer and reporter for Nature Medicine, Nature Biotechnology, and Bioscience Technology. He also teaches at the NYU School of Journalism, and blogs at http://dovdox.com.
Eat a big meal, and your body will erupt into an orgy of hormonal signals: liver talks to fat, which talks back to liver, while muscles, stomach, intestines, heart, brain, and pancreas all send their own urgent communiques. These messages unify the organs in a single, critical mission: separate the food's components as quickly as possible, and sort them for either storage or elimination.
If a single plate of food can do all that, what are we to make of long-term processes such as aging and weight gain, or chronic diseases such as atherosclerosis and osteoporosis? Physiologists have traditionally approached these questions by studying individual components, not because they failed to appreciate the system's interconnectedness, but because their tools weren't sophisticated enough to take a broader view.
That's finally changing. "There are so many advances, both in molecular biology and genetics ... that have shown that there are many functions for many organs that were not supposed to be functions of these organs, and also many connections between organs that are not the ones that I learned ... when I was in medical school," says Gerard Karsenty, chairman of genetics and development at Columbia University and co-organizer of the New York Academy of Sciences' first meeting on the evolving field of integrative physiology.
At the meeting, held May 14–16, 2008, at the NYAS conference center, researchers from several branches of medicine and biology discussed the data behind some of those surprising new connections, revealing and abetting a convergence between fields that previously kept to themselves. The gathering featured more than a dozen main presentations, two "data blitz" sessions of brief but intense talks, and a substantial poster area. A small sample of presentations, organized around two of the meeting's most common themes, provides a glimpse of the vast territory the event covered.
The problem of plenty
Nutrition is arguably the most ancient problem in biology, so it's little surprise that energy homeostasis—how the body processes food—has been a major focus of physiological research. These days, much of that discussion centers around an irony of modern life. After four billion years under the constant threat of starvation, evolution has produced a species that has overcome the problem all too well. Humans' success at feeding ourselves has been so comprehensive, at least in the developed world, that our ancient calorie-hoarding adaptations now work against us, driving a global epidemic of obesity.
Obesity research has revealed new connections between organ systems as well as critical regulatory genes.
Unfortunately, evolution has had no time to respond, and our bodies are handling this new development poorly. Long-term obesity brings with it a suite of complications that shorten life and call for expensive interventions. "The cost to U.S. society alone exceeds $170 billion a year, and of course it's growing with this epidemic," says Yale University's Gerald Shulman, who delivered the meeting's final keynote presentation.
Eating less and exercising more can prevent or reverse obesity, but that advice is easier given than followed. Consequently, researchers are trying to develop other treatments, and to understand the full range of molecular signals controlling the condition. This work has revealed new connections between organ systems, as well as critical regulatory genes that link lifespan to energy homeostasis. "The reason that these genes might be there is that this adaptive process has selected for genes ... that can recognize food scarcity, and translate it into these effects on basic life choices, reproduction versus hunkering down and slowing the aging process," says Massachusetts Institute of Technology biologist Leonard Guarente, the meeting's other keynote speaker.
Apoptosis and taxes
Besides being biologically interesting, the link between food intake and aging also connects the obesity epidemic to the other major health transition now sweeping the globe. As starvation and many infectious diseases yield to technology, more people are living long enough to encounter medical problems that used to be rare. Collectively known as the "diseases of aging," conditions such as heart disease, cancer, dementia, and osteoporosis are reaching pandemic prevalence.
The classical view of these diseases was that they were "post-evolutionary" events. Because they generally affect people long after their prime reproductive years, the theory went, there was no selective pressure for the body to develop defenses against them. Once we've lived long enough to reproduce, biology no longer cares what happens to us, so we simply fall apart.
Aging may be a programmed process.
More recently, researchers have come to view aging as a programmed process, in which highly evolved molecular signals make deliberate—if unfortunate—choices about life and death. Atherosclerosis, or hardening of the arteries, provides a good example of this phenomenon. "Everybody sitting here has atherosclerotic lesions, and most of those will never do you any harm from now until the day you die, but some of them will," says Columbia University's Ira Tabas. The distinction between benign plaques and deadly ones is chiefly a matter of apoptosis. Tabas and his colleagues have found that in some lesions, a combination of cellular stress and a "second hit" from another signaling pathway sends macrophages into a suicide pact. The dead cells then accumulate in the plaque, creating a necrotic core that worsens the lesion. Drugs that alter or block these signals might prevent or even reverse heart disease.
Though integrative physiology is still in its infancy, researchers are optimistic about its potential. For inspiration, Kitsis cites James Clerk Maxwell's famous equations, which unified electricity and magnetism a century ago. "I would like to think that sometime when I'm still around, we will have some connections between [physiological pathways] that explain mechanistically how these things interface with each other," says Kitsis. It's certainly food for thought.
Alan Dove Alan Dove is a science writer and reporter for Nature Medicine, Nature Biotechnology, and Bioscience Technology.
- Contrary to conventional wisdom, fat calories and carbohydrate calories behave differently in the body.
- The growth factor FGF-21, secreted by liver and muscle, is a critical regulator of lipid metabolism.
- Type 2 muscle fibers secrete "myokines," which stimulate the liver to burn fat.
- Sirtuin proteins connect calorie intake, lifespan, metabolism, and many of the diseases of aging at the molecular level.
The other energy crisis
The obesity epidemic sweeping the industrialized world is easily one of the most written about, worried about, and thought about problems in public health. Despite all of that ink, angst, and intellect, though, the causes and solutions are, on one level, embarassingly simple: people are eating more calories than they burn, storing the excess as fat.
Paradoxically, part of the problem may be that they're trying to eat healthy diets. Beginning in the late 1970s, the U.S. Department of Agriculture, in collaboration with public health experts, began a campaign against high-fat foods. Fatty food is calorically denser than carbohydrates, and fats are major drivers of cardiovascular disease pathogenesis. At the same time, the Department needed to find a way to dispose of a rising glut of cheap American-grown corn, which is easily processed into carbohydrate-rich foods. The final result was the well-known and much-criticized Food Pyramid, which recommends eating more carbohydrates and fewer fats.
Since 1992, when the Pyramid was first published, Americans have increasingly widened their own bases. "The U.S. population got fatter and fatter and fatter eating low fat, high carbohydrate diets," says Eleftheria Maratos-Flier, an endocrinologist at the Beth Israel Deaconess Medical Center who presented her work on the molecular basis of this problem.
Nor is the problem strictly aesthetic. In the final keynote presentation, Gerald Shulman summarized his group's extensive work on obese patients and their medical complications, which include such devastating conditions as diabetes and heart disease. None of this is easy to manage in the clinic. "The cost to U.S. society alone exceeds $170 billion a year, and of course it's growing with this epidemic," says Shulman. Within 20 years, at least 300 million people worldwide will be affected by obesity and its sequelae.
The skinny on fat
Fortunately for researchers, there are excellent mouse models available for studying obesity. One of these, the diet-induced obesity model, doesn't even require genetic engineering; simply feeding normal mice a diet of chow that is, essentially, junk food causes the animals to plump up like couch potatoes.
To examine the role of fat calories in obesity, Maratos-Flier and her colleagues placed mice on four different diets: unlimited quantities of conventional mouse chow, a caloric restriction regimen with limited quantities of conventional chow, a 22% fat diet known to induce obesity, or an 88% fat "ketogenic" diet. Consistent with previous results, the controls on unlimited conventional chow maintained normal weight, the calorie-restricted animals got leaner, and those fed a 22% fat diet became obese.
The surprise was the ketogenic diet. Despite eating mostly fat, and consuming the same number of calories as the mice on the obesity-inducing diet, the animals on the ketogenic diet actually lost some weight. Closer analysis revealed that the ketogenic diet caused the mice to increase their energy expenditure as well as their caloric intake, overturning the conventional wisdom that all food calories are equivalent. "While 'a calorie's a calorie' is true if you put the stuff in a bomb calorimeter, if you actually feed it to an animal, the physiological changes actually trump the thermodynamic effects," says Maratos-Flier.
Hoping to understand why the ketogenic diet caused the mice to burn more calories, the researchers used the popular Affymetrix gene chip technique, probing all of the genes expressed in mouse liver to see which ones were more or less active in the fat-fed mice compared to the chow-fed mice. The results were mostly as expected. "What you see is a dramatic down-regulation of all the enzymes involved in fatty acid synthesis in the liver. What's increased is enzymes that are involved in ketogenesis and enzymes that are involved in breakdown of fatty acids," says Maratos-Flier. Besides the enzymatic changes, the ketogenic diet also boosts expression of the cellular signaling molecule FGF-21.
The investigators then created a small interfering RNA (siRNA) that suppresses FGF-21 expression, and gave it to the mice on the ketogenic diet, with dramatic results: the animals' livers grew huge, turned white, and filled up with triglycerides, and their circulating triglyceride levels also skyrocketed. Looking more closely at liver gene expression, the team found that FGF-21 acts directly and acutely on this organ to balance the effects of the ketogenic diet.
The results suggest that FGF-21 sits at a critical junction in fat metabolism, which could make it a useful target for treating obesity. "We think that obesity is likely to be an FGF-21-resistant state, and it can be overcome pharmacologically," says Maratos-Flier.
Myo Mouse lets himself go
Maratos-Flier's data fit nicely with the work Ken Walsh and his colleagues at the Boston University School of Medicine have been doing for the past several years. The team is generally interested in figuring out how diabetes and obesity influence cardiovascular disease. Initially, the most obvious place to look for those links was in fat cells.
While they're best known for storing energy and destroying sexy physiques, adipocytes also serve an important role as an endocrine organ, secreting numerous hormones that regulate the behavior of other tissues. In general, adipose-derived hormones, or adipokines, cause a lot of trouble in obesity. For example, many adipokines are proinflammatory, keeping the immune system on its toes in healthy individuals. "These normal adipocytes, which are normally making low levels of proinflammatory cytokines ... protect the body from stresses," says Walsh. In the obese, the expanded adipocytes secrete a surplus of the compounds, shifting the immune system into self-destructive overdrive.
Adiponectin, though, follows a different pattern. Fat cells' secretion of this adipokine varies inversely with the amount of body fat, so it's more abundant in normal-weight subjects than in obese ones. Among its many effects, adiponectin is insulin-sensitizing, protects against cardiac damage, and inhibits the formation of atherosclerotic plaques. Excess body fat, then, turns inflammatory signals up too high while dialing protective signals down too low, shifting the hormonal balance toward disaster.
What was missing was the other side of the balance: some tissue must be secreting hormones that keep the adipokines in check, or the slightest increase in body fat would cause the system to run amok. Walsh took an educated guess that perhaps skeletal muscle offsets the fat signals.
Pursuing that idea, the researchers developed the "Myo mouse," which carries an inducible transgene for the Akt signaling protein, driven by a muscle-specific promoter. Akt promotes growth, and weightlifters show increases in the molecule in their muscles after working out. Satisfyingly, inducing Akt in the transgenic mice causes them to bulk up, adding about 5% to their muscle mass by growing more type 2, or "fast twitch" fibers. This is the same type of fiber weightlifters develop, and the type that normally declines with age.
Myokines from muscles counteract the negative effects of fat.
If the Myo mice get a standard high-fat diet before their transgene is activated, they become obese, just like normal mice, but turning on the transgene then reverses the obesity. This happens even though the mice don't increase their activity—the animals seem to induce increased activity in their livers, which then burn off excess fat. "It is the mouse that we all want to be," says Walsh, adding that "they're basically total couch potatoes, they eat terrible food ... they get less exercise than a normal mouse, and yet they're metabolically normal and they've built a bunch of beach muscle."
Using gene profiling chips, the researchers have now found at least a dozen "myokines" that help drive this fat-burning metabolic shift. One of these molecules is FGF-21. "Muscle makes as much FGF-21 as liver from a fasted animal, and you have to figure there's so much muscle in the body, it's probably a pretty important source," says Walsh. Drug developers at Eli Lilly are now working on developing FGF-21-targeting compounds to bring similar benefits to humans.
The lean gene
Another promising target for obesity treatments originally surfaced in experiments seemingly far removed from mammalian biology. Since the early 1990s, Leonard Guarente and his colleagues have studied the general phenomenon of biological aging, at first using the yeast Saccharomyces cerevisiae as a relatively simple model. Genetic studies on this fungus highlighted the SIR2 gene as an aging regulator. That was an interesting basic research finding, but it seemed unlikely to have any broader implications.
Surprisingly, though, the team then found that the orthologous gene in the round worm Caenorhabditis elegans serves precisely the same function, regulating lifespan in response to the abundance of food. The two organisms are separated by hundreds of millions of years of evolution, so a mechanism that's so highly conserved between the two is probably a fundamental requirement for life. "This is a rather dramatic conclusion that was greatly expanded from the notion that we were simply studying a baroque system in budding yeast," says Guarente, who gave the meeting's first keynote presentation.
Since then, work in several labs has confirmed that SIR2-like genes are also present in mammals. The gene product is a histone deacetylase that requires the metabolic molecule NAD for activity, which means that the sirtuins, as the gene family is now known, link metabolism to gene regulation and lifespan. "The reason that these genes might be there is that this adaptive process has selected for genes like SIR2 that can recognize food scarcity, and translate it into these effects on basic life choices, reproduction versus hunkering down and slowing the aging process," says Guarente.
A pill that slows the aging process while burning off excess calories would obviously be a blockbuster, but a dilute version of it may already be on the market—in the grocery store's produce section. Resveratrol, a compound found in the skins of grapes, cranberries, and a few other fruits, activates SIRT1, the human version of SIR2. Mice fed the compound, albeit at levels impossible to attain from eating fruit alone, are leaner, fitter, and live longer than controls.
Johan Auwerx, director of the Institut Clinique de la Souris at the University of Strasbourg, has studied the effects of resveratrol extensively. So far, he and his colleagues have determined that the compound increases energy expenditure, decreases the concentration of lipids in brown fat cells, and boosts the number of mitochondria in muscle cells.
Before gorging on grapes, though, potential resveratrol dieters should note a key limitation of the compound: it does not completely prevent weight gain in the diet-induced obesity mouse model, which most closely represents the human obesity problem. However, compounds that hit some of SIRT1's targets more specifically may be effective in treating the condition. Auwerx and his colleagues found that one such compound, designated SRT1720, completely prevents the mice from becoming overweight. "We've never seen that. With resveratrol, recall that there was moderate weight gain, but here there was no weight gain at all," says Auwerx.
Fighting the resistance
Excessive weight gain is the defining feature of obesity, but the problems that accompany the condition only begin with fat. Indeed, researchers and public health experts are increasingly concerned about metabolic syndrome, a suite of conditions that often follow obesity. The syndrome's definition includes diagnostic markers such as insulin resistance, elevated blood pressure, and a generally pro-inflammatory immune state, all conspiring to increase the patient's risk of heart disease and type 2 diabetes.
In studying the underlying causes of insulin resistance, Beth Israel Deaconess Medical Center's Barbara Kahn and her colleagues Timothy E. Graham and Qin Yang discovered another molecule that's elevated in insulin-resistant patients: retinol binding protein 4, or RBP4. Levels of this protein correlate very closely with metabolic syndrome—its level rises before the onset of other symptoms, and treatments as diverse as exercise and gastric banding surgery cause it to decline. "It seems like this could be a useful marker ... to indicate which people need to get into medical care," says Kahn.
RBP4 could be a good marker for metabolic syndrome.
Diagnostic markers are good, but Kahn and her colleagues also wanted to understand the mechanism behind the molecule, especially whether RBP4 is helping cause metabolic syndrome, or simply indicating it. To answer that question, the investigators had to narrow the problem down. Normally, RBP4 delivers vitamin A to cells, which metabolize it to retinoic acid, a highly active signaling molecule that regulates the expression of more than 300 genes.
The team found that fenretinide, a synthetic retinoid drug that causes RBP4 to be excreted through the kidneys, reduces insulin resistance in obese mice. Interestingly, the compound does not cause the animals to lose weight. "It's not because the mice are leaner ... so it's truly an insulin sensitizing agent," says Kahn.
In other studies, the team has elucidated several steps of a signaling pathway RBP4 regulates in the liver, and determined that in normal-weight humans, the protein's level may be regulated by physiological states. Taken together, the results suggest a causative role for RBP4 in insulin resistance, making it a promising drug target as well as a biologically interesting molecule.
Even as researchers continue to add to the list of promising drug targets, though, developing drugs that hit those targets could take years. In the meantime, patients may have to turn to an old, effective, but stubbornly unpopular remedy for obesity: eating less and exercising more.
- Sirtuin proteins connect calorie intake, lifespan, metabolism, and many of the diseases of aging at the molecular level.
- Inappropriate apoptosis drives the formation of deadly atherosclerotic plaques.
- Estrogen may signal osteoclasts to eliminate themselves, preventing osteoporosis.
Something to look forward to
From cholera to measles to Yersinia, the gradual improvement of both infrastructure and medicine—at least in the developed world—have virtually eliminated most of the classical plagues. Instead, modern humans increasingly live long enough to have problems their ancestors seldom would have encountered: cancer, heart disease, osteoporosis, and dementia.
For most of the 20th century, scientists thought of these "diseases of aging" as a product of simple decay. Over time, the thinking went, the body simply accumulates so many insults that something is bound to fail. Things fall apart; the center cannot hold.
A handful of molecular signals could underlie multiple diseases.
That view seems to fit well with evolutionary theory, at least. Diseases of aging occur long after the prime reproductive years, so there would be little selective pressure to evolve defenses against them. More recently, though, data from systems as diverse as budding yeast and mice have revealed a more complicated picture.
When their food intake is restricted, organisms tend to live longer, indicating that they have some biological control over their own aging. Evolutionarily, it makes sense to reproduce fast and die young, making room for the offspring, as long as those offspring are likely to survive. When times are lean, though, living longer to wait for better conditions is probably a better strategy.
In the emerging field of integrative physiology, researchers are trying to understand how this basic choice works, and especially how it influences the diseases of aging. The work has revealed surprising connections between organ systems, and uncovered the tantalizing possibility that a handful of molecular signals could underlie multiple diseases.
Help for the hard-hearted
One of those signals is the histone deacetylase SIRT1, the human version of a gene originally discovered in yeast by Leonard Guarente and his colleagues. By linking overall metabolic activity to gene regulation, SIRT1 indirectly induces or inhibits a vast range of physiological activities, and it appears to be the key controller of the lifespan extension seen in calorie restriction experiments.
Since discovering SIRT1, Guarente's team has focused on tracing its many activities downstream to different diseases of aging. Looking at a mouse that lacks the SIRT1 gene, for example, the researchers found unusually low levels of HDL, or "good cholesterol" in the bloodstream, but high cholesterol in tissues. That suggested a role for SIRT1 in cardiovascular disease, a condition driven partly by the inexorable increase in cholesterol with age. The mouse phenotype also provided a testable hypothesis. "Maybe the process of reverse cholesterol transport ... from cells out of the body through HDL is defective in the SIRT1 knockout," says Guarente, who delivered one of the meeting's keynote presentations.
The investigators soon found that the primary transporter in this process, a protein called ABCA1, is sufficiently reduced in the mutant mice to account for the cholesterol accumulation. Additional experiments revealed that SIRT1 normally deacetylates a regulatory protein called LXR, which then regulates ABCA1 levels to control cholesterol transport. Drugs targeting this pathway might eventually be useful for treating or preventing cardiovascular disease.
While cholesterol is a major cause of cardiovascular disease, most patients are more concerned about the condition's effects, the most infamous of which is a myocardial infarction, or heart attack. When this occurs, the loss of blood flow, or ischemia, around a section of cardiac tissue causes the cells to die; if enough cells die, the patient goes with them. Besides killing ischemic tissue, though, a myocardial infarction also prompts adjacent cells with normal blood flow to initiate apoptosis, or programmed cell death, creating a zone of destruction much larger than the ischemia alone.
For Richard Kitsis, a leading apoptosis researcher who presented his results at the meeting, this collateral damage—or any other tissue pathology, for that matter—is no accident. "I don't believe there is accidental cell death," he says, adding that "If an anvil fell out of the ceiling and hit my head and killed me, I would say the cells in the top of my head that died by getting hit with the anvil will have actually participated in their own death by initiating a program.". Even if it is deliberate, though, excessive cell death in cardiac tissue is clearly a bad biological choice.
To figure out what causes cells to pick this unfortunate option, Kitsis and his colleagues have focused on an apoptosis regulator called ARC, which is induced in breast cancer and inhibited in mouse models of myocardial infarction. Mice lacking the gene for ARC are substantially less sensitive to breast tumors, but suffer much greater cardiac damage if they're given heart attacks. Mildly overexpressing ARC in cardiac tissue before inducing a myocardial infarction prevents much of the tissue damage. "There's a causative effect between the ARC going away and the [cell] death occurring. If you could keep the ARC around, maybe you could make infarcts smaller just using a natural product that the body makes," says Kitsis.
Because of its yin/yang role in infarctions and cancer, though, simply giving a patient a constant dose of an ARC-stimulating drug would probably be a bad idea—saving heart tissue might come at the expense of encouraging tumor cells. Instead, Kitsis envisions developing compounds that a patient with cardiovascular disease would take only at the first signs of a heart attack, limiting the size of the resulting infarct even before reaching the emergency room.
The second shooter
Not all atherosclerosis ends with a call to 9-1-1, though, as the disconcerting introduction of Ira Tabas's presentation made clear. "Everybody sitting here has atherosclerotic lesions, and most of those will never do you any harm from now until the day you die, but some of them will," says Tabas. He adds that "if you take a look to the left of you and the right of you, one of these people will die of this disease."
For atherosclerosis, the difference between harmless and deadly lies in the core of the plaque. Benign plaques have unremarkable centers, but the cores of bad plaques are full of necrotic tissue and dead macrophages. The macrophage graveyard appears to be a product of excessive apoptosis, and also inadequate phagocytosis, the mechanism that normally clears away dead cells. But what causes these two processes to go haywire in some plaques and not others?
Using cultured cells and mouse models, Tabas and his colleagues have found that stress on the endoplasmic reticulum (ER), the cellular organelle responsible for secreting proteins, is a major contributor to the problem. When the ER gets overloaded, it releases calcium into the cytosol, triggering multiple intracellular signaling pathways that try to alleviate the stress. Intracellular calcium release is a common occurrence in the cell, though, and cells don't spontaneously commit suicide. The key, says Tabas, is signal strength. "When it is participating in a death pathway, it is incredibly amplified, prolonged, et cetera, and that's what differentiates the death pathway from normal physiology," he says.
But even a roaring ER stress signal isn't enough to kill the cell by itself. In addition, the macrophages must suffer a "second hit," which tips the balance from adaptation and survival toward resignation and death. One of the most important second hits is a signal from Toll-like receptors, which have evolved as part of the innate immune response to recognize specific, ancient infectious diseases. In cultured cells, the researchers found that this signal is highly synergistic with ER stress—either signal alone causes modest cell death, but the two together stimulate a massive die-off.
Only some atherosclerotic plaques promote apoptosis.
Several studies have now confirmed that ER stress occurs routinely in bad atherosclerotic plaques, and these lesions also host a long list of plausible second hit signals. Genetically engineered mice with alterations in either signaling pathway are much more resistant to heart disease than wild-type controls.
The results certainly help explain how bad atherosclerotic plaques form, and even suggest a few good targets for drugs to reverse this pathology, but they also raise a question: why would macrophages, which form the backbone of the immune system, be so prone to killing themselves?
In a word, tuberculosis. This microbe, and a few other intracellular pathogens, can evade the normal immune response by hiding inside macrophages. If the infected macrophages undergo apoptosis, though, they will be endocytosed by roving phagocytic cells, which will then digest the infectious agent and present its antigens on their surfaces, blowing its cover. "One of the major characteristics of virulent TB is that it keeps macrophages alive," says Tabas. Avirulent strains of the mycobacterium are susceptible to macrophage apoptosis. Apparently, an adaptation that helped our ancestors survive infection now primes us for heart disease.
The leg bone's connected to the ... estrogen?
Besides heart disease, everyone who lives long enough can anticipate worrying about osteoporosis. The master regulator SIRT1 crops up in this condition, too, regulating osteoblastogenesis and osteoclastogenesis. Disrupting the balance between these two processes causes a range of bone diseases.
Osteoporosis, the most common skeletal disease in developed countries, generally involves either a surplus of osteoclast cells, which break down old bone tissue, or a deficit of osteoblasts, which build up the bone. Both problems occur in mice that lack the gene for SIRT1. Knocking out the gene only in the osteoclast cell lineage also causes osteoporosis, and produces mostrous, multinucleated osteoclasts. "This to me strongly suggests that SIRT1 is functioning as a restraint, a negative regulator of osteoclast differentiation, such that when you take it away, differentiation goes up markedly," says Guarente, whose lab did this work.
Estrogen and androgens also help regulate bone buildup and loss, which is why osteoporosis hits postmenopausal women particularly hard. As women's estrogen levels decline, their osteoclasts become more abundant, breaking down more bone than the osteoblasts generate. But what connects estrogen to the osteoclasts at the molecular level?
Shigeaki Kato of the University of Tokyo is studying that question, and at the meeting he presented results from an impressive series of mouse genetics studies on it. After a conventional deletion of the estrogen receptor revealed tantalizing but incomplete results, Kato and his colleagues realized that they needed another mouse strain. "We need a knockout in an androgen receptor and estrogen receptor to compare the sex hormone action," says Kato.
Deleting the androgen receptor is tricky, though. Males without this protein have testicular feminization: female anatomy, but with testes in place of ovaries. That makes the mice sterile, and therefore impossible to maintain as a strain. Instead, the researchers generated conditional knockouts using the popular Cre/Lox system, which allowed them to breed a line of parent animals normally, inducing the gene deletion in a new litter at will.
In males, androgens may be responsible for maintaining bone mass.
Inducing the androgen receptor deletion causes testicular feminization, as expected, and also makes the males become obese later in life. The males also develop osteoporosis. Females without the receptor, however, do not develop either problem. "In the normal condition in a male ... maybe androgen is important to maintain and attenuate bone mass loss, but in a female maybe estrogens are important," says Kato.
The researchers then developed several additional mouse lines, including ones that lacked either androgen or estrogen receptors only in osteoclasts. Taken together, the results help clarify the connections between hormones and bone density. "We think that female estrogen is a kind of a signal to ask mature osteoclasts to retire from their job ... suggesting that maybe estrogens induce the apoptosis and the shortening [of] the lifespan of mature osteoclasts," says Kato.
Viewed as a set, these presentations and others at the meeting reinforced the new idea that aging and its associated diseases are part of a regulated process. We may not know how to turn back the clock yet, but at least we're starting to understand some of its gears.
Can drugs targeting FGF-21 reverse obesity in humans?
What are the most important triggers for necrosis in atherosclerotic plaques?
Will resveratrol-mimicking compounds produce the same life-extending benefits as calorie restriction?
What drives the loss of type 2 muscle fibers with age?
Do very low resveratrol doses, such as those obtained from drinking red wine or eating certain fruits, also extend life?
Can muscle-produced myokines counteract the signals of fat-produced adipokines?
Does the androgen/estrogen balance fully explain why women are more susceptible to osteoporosis than men?