Oakland: Food for Thought at American Aging Association Annual Meeting
The recent emphasis on caloric restriction (CR) as a means to extend lifespan didn’t stop the American Aging Association from serving up plenty of exciting presentations at their 34th annual meeting held last weekend in Oakland, California. Check back over the next few days when we’ll be posting summaries of the most appetizing fare, starting today with a summary of the 1-day pre-meeting symposium on nutrition and successful aging.
Eat, Drink, and Age Successfully
The 1-day symposium, hosted by Jim Joseph of Tufts University, Medford, Massachusetts, addressed the impact of nutrition and lifestyle on aging and neurodegenerative disease. Stephane Bastianetto, Douglas Hospital Research Centre, Verdun, Canada, got the morning off to a brisk start when he reported on experiments to identify how polyphenols derived from wine and green or black tea can act as neuroprotective agents.
The polyphenol resveratrol, found in grapes and red wine, has, of course, been linked to increased lifespan in yeast and worms (see ARF related news story), and more recently has been shown to protect against polyglutamine toxicity in roundworms (see ARF related news story). Bastianetto found that only some compounds in teas, those gallate esters of polyphenols, such as epicatechin gallate and epigallocatechin gallate (EGCG), can compete for resveratrol binding in rat brain homogenates. Non-gallate esters, such as epicatechin and epigallocatechin, did not compete with resveratrol, suggesting that polyphenols have multiple sites of action. In addition, only those compounds that did compete for resveratrol binding seem to protect against the toxic effects of amyloid-β. Bastianetto reported that EGCG protected hippocampal neurons treated with the fibrillogenic Aβ25-35 (see ARF related news story), as did epicatechin gallate, though this polyphenol was slightly less effective. Epigallocatechin or catechin itself failed to spare neurons.
How these polyphenols protect against Aβ is unclear. Resveratrol has been shown to stimulate deacetylases and may prevent apoptosis (see ARF related news story). While other polyphenols that compete for resveratrol binding might have similar modes of action, it is generally believed that these compounds are beneficial to cells and organisms because they are very good quenchers of reactive oxygen species (ROS). But when Bastianetto compared the antioxidant activity of these polyphenols, he found that EGCG and epigallocatechin are equally effective in cultured neurons, indicating that the reduction of ROS species cannot explain why EGCG alone protects against Aβ. Instead, Bastianetto suggested that this particular polyphenol might well inhibit formation of Aβ oligomers.
But the antioxidant activity of polyphenols should be not be dismissed. Polyphenolics from nuts can help reduce the risk of cardiovascular disease by up to 25 percent, Jeffrey Blumberg, Tufts University, reminded us. In fact, so good are the epidemiological data on the inverse relationship between nut consumption and coronary heart disease that the FDA came up with a “B level health claim for nuts which states: ‘scientific evidence suggests, but does not prove, that consuming 1.5 ounces per day of most nuts as part of a diet low in saturated fat and cholesterol can reduce the risk of heart disease,’” said Blumberg. Those partial to Brazil, macadamia, and cashew nuts will be disappointed to hear that those particular varieties are not included in the dietary claim, mostly because of their high saturated fat content, but walnuts, almonds, pistachios, hazelnuts, and pecans are.
So how do nuts protect against cardiovascular disease. Earlier studies showed that eating one to two ounces of almonds per day over about a month can lower total cholesterol by 6 percent, LDL cholesterol by 10 percent, and oxidized cholesterol by up to 14 percent (see Jenkins et al., 2002). It was the oxidized cholesterol finding that particularly intrigued Blumberg and colleagues.
Blumberg showed that almond skin phenolics (ASP) can increase the resistance of human LDL cholesterol to oxidation, and works in synergy with vitamin E in this regard. He also showed how a single dose of ASPs in vivo can increase the lag time to LDL oxidation, and that the polyphenols catechin, naringenin, and quercetin all increased in plasma in a time-dependent manner. Their appearance correlated with an increase in the ratio of reduced:oxidized glutathione, indicative of the antioxidant properties of the phenols.
Navindra Seeram of the UCLA Center for Human Nutrition also addressed the issue of bioavailability of polyphenols. Yes, these high-molecular-weight compounds may have a variety of effects in vitro and in vivo, but in what state do these compounds actually reach the blood or target tissues? Seeram reported some data on absorption of polyphenols from pomegranate juice. This juice is a rich source of polyphenols and has potent anti-atherosclerosis activity in ApoE-deficient mice and in in vitro models.
In mice, punicalagin, one of the major pomegranate polyphenols, has been found to cross the gut into the blood even though it is a relatively large molecule (mol wt 1,084). But what about in humans? Are we getting equivalent doses when we imbibe? Seeram found that, in fact, we are not. Instead, elagic acid, a product of punicalagin hydrolysis, turns up in the plasma and urine of human volunteers given the juice after being starved of polyphenols for four days. The finding suggests that we should take care not to rely too heavily on mouse bioavailability data when considering human nutrition. In fact, bioavailability turns out to be a very complex affair, something you might want to consider next time you peruse or plan a menu. Steven Schwartz from Ohio State University showed how carotene uptake can be profoundly influenced by the other food on your plate.
Carotenes come in two flavors, hydrocarbon carotenes like lycopene, and their oxygenated analogs, the xanthophylls. There are over 700 natural carotenes identified to date and they include vitamin A and provitamin A, and other essential compounds such as lutein and zeaxanthin, which prevent the photo-oxidation that causes macular degeneration. Epidemiological studies show that carotene may help fight against a wide variety of cancers including prostate cancer.
But carotenes are hydrophobic and their absorption may depend on emulsification or other processing in the intestine. Lycopene, for example, found in tomatoes as predominantly the all trans variety, turns up in mammalian tissues as 65-80 percent cis lycopene (cis and trans refer to the relative positions of carbon chains about a carbon-carbon double bond, which is rotationally constrained). Even in cooked tomatoes, lycopene is all trans, so clearly there are some chemical rearrangements going on in vivo, either during or after absorption from the gut. And yet, cooking your tomatoes might turn out to be not such a bad idea. Schwartz found that lycopene is more readily absorbed from tomato sauce or tomato soup than from V8 tomato juice, indicating that cooking or food processing helps to make the carotene more biologically available.
And for those who like tomatoes raw, consider using full fat dressing on your next salad. Schwartz showed how different dietary components, particularly the presence of lipids, can dramatically impact lycopene uptake. He showed how full fat dressing gave maximal uptake of lycopene from raw tomatoes. Low fat dressing did help, but with non-fat dressing, the amount of lycopene absorbed was virtually nil. The findings may help explain why the Mediterranean diet, though drizzled as it is with olive oil, is still good for you. And for those of you who insist on that non-fat dressing, try spicing up your salad with avocado. Schwartz found that the pear-shaped veggie, weighing in at 17 percent lipid, is not so green when it comes to helping the absorption of lycopene from tomatoes, working almost as well as the full fat dressing. Guacamole and salsa, anyone?
Dietary balance also served as the theme for Rui Hai Liu, from Cornell University, New York. Liu emphasized how important it is to consider the whole package of micronutrients rather than focusing on one or two flavors of the month. “Why has the single antioxidant approach to therapy never proven to be effective?” he asked. In a recent study that tested the potential of β-carotene to prevent cancer, for example, those taking the supplement actually did worse than did those on placebo. Is this because the dose wasn’t optimal, or is it because we are better sticking to whole fruits and vegetables? he asked (see also recent ARF related news story on vitamin E and Alzheimer disease and ARF related news story on the potential benefit of vitamin E in reducing the risk of Parkinson disease).
Here’s some evidence for the whole food approach. Liu reported how there are about 5.7 mg of vitamin C in 100 g of your average apple. But the same amount of apple contains the equivalent of 1,500 mg of vitamin C when the total antioxidant power of the fruit is measured. In fact, Liu presented data showing how, in a dose-dependent manner, apple extract can prevent the growth of breast cancer cells in mice. Apple extract also reduces the total number of tumors and the average tumor size.
Other fruit extract has similar properties. Grape extract, he showed, can prevent expression of proliferating cell nuclear antigen (PCNA), a tumor cell marker, in mice, while cranberry or apple extract can induce arrest of mitosis in the G1 phase of cell division in MCF7 breast cancer cells. There are over 8,000 known phytochemicals that we can access in our diet, and Liu suggested that some, if not many, of these may act synergistically. The EC50s for the two polyphenols catechin and chlorogenic acid, for example, are halved if the compounds are given together.
Fight Fire with Antioxidants
So how might dietary nutrients be of help to the aged and aging? Simin Meydani from Tufts University reported on the effects of antioxidants in aged macrophages. These cells are involved in inflammatory responses, which of course have been linked to a variety of diseases including cardiovascular, autoimmune, and neurodegenerative diseases. Meydani showed how macrophages from older animals produced more prostaglandin E2 (PGE2) when exposed to the inflammatory toxin bacterial lipopolysaccharide (LPS) than do those cells isolated from younger animals. As PGE2 stimulates the release of proinflammatory cytokines, this may explain, in part, why inflammatory responses are more pronounced in the aged.
What leads to the increase in PGE2? Meydani’s work has shown that the increased production of the prostaglandin in the LPS-treated older macrophages is due to increased activity of cyclooxygenase (COX). This enzyme comes in two isoforms, COX-1 and COX-2. Inhibitors of the latter have been studied for their potential to slow the progression of AD, but those trials have been stopped due to safety concerns (see ARF related news story). COX-2, rather than COX-1, also appears to be the culprit in the increased PGE2 in older macrophages as Meydani showed that the activity of this enzyme is significantly and dramatically increased when these cells are treated with LPS.
There are several modulators that can influence production of COX-2, including glutathione and glucocorticoids, which decrease its expression, and cytokines such as interleukin-1 (IL-1), IL-6, and the sphingolipid ceramide, which all increase expression of the oxygenase. It was ceramide and glutathione that Meydani and colleagues found to modulate COX-2 in older macrophages. Ceramide and glutathione are, in fact, related, because glutathione inhibits the activity of sphingomyelinase, which is necessary for ceramide synthesis.
Meydani and colleagues found that old animals produce much higher levels of ceramide than do young during the initial response to LPS. Work in her lab has also shown that ceramide can lead to an increase in NF-κB, and that the NF-κB inhibitor I-κB is less abundant in older macrophages.
So how might dietary nutrients affect these signaling mechanisms? At first, Meydani looked at the influence of antioxidants because oxidizers such as peroxides have been implicated in the mechanism of action of the COX enzymes. She found that macrophages isolated from old mice that had been fed a diet high in vitamin E for 30 days had significantly lowered production of PGE2 in response to LPS than did cells isolated from mice fed normal lab chow. In contrast, vitamin E made no difference to production of PGE2 in macrophages from young mice.
But surprisingly, Meydani found that the COX-2 activity in the older, vitamin-supplemented macrophages was unchanged from controls. But she did find that vitamin E quenched the activity of peroxynitrite, a nitric oxide derivative that drives cyclooxygenase catalysis. This theory fits with another observation from the Meydani lab, namely, that levels of nitric oxide synthase are elevated in old macrophages (see Wu and Meydani, 2004).
The effect of vitamin E on macrophages might be clinically relevant as it has been shown to improve T cell-mediated function. Meydani also reported that when supplemented in the diet, it can reduce the incidence of upper respiratory infection in elderly nursing home residents.
Other interventions, including green tea and oat extracts, can also reduce PGE2 levels, reported Meydani. In fact, caloric restriction in humans has been shown to reduce PGE2 in blood mononuclear cells. PGE2 may be a very useful marker of aging, Meydani suggested.
Brain Food or Pie in the Sky?
Does brain food really exist? There are plenty of models of cognitive function that can be used to address this issue, not to mention studies on human volunteers. Michael Forster, North Texas Health Science Center, Forth Worth, reported on water maze experiments designed to test the effect of dietary supplements on cognition in the mouse.
Forster and colleagues tested vitamin E and coenzyme Q10 (CoQ) to determine if their antioxidant prowess could lead to smarter laboratory mice. When given as lifelong supplements, neither compound had much effect on survival (mice on the highest does of Q10, 370 mg/Kg body weight/day, actually tended to have a lower lifespan), and had no effect on performance in the classic Morris water maze test, where the animals must search for and climb to safety on a hidden platform in a water tank.
Similarly, vitamin E given late in life (18 months) failed to halt the normal cognitive decline in the laboratory mice, but while CoQ had no effect in the standard water maze test, when Forster and colleagues tried a slight variation on the theme, that is, moving the platform to a different location, they found that, compared to mice on normal chow, the CoQ-treated mice spent a significantly longer time searching the section of the tank where the platform used to be. This suggests that CoQ, when given late in life, does help to improve learning and working memory.
William Milgram, University of Toronto, Scarborough, Canada, reported similarly encouraging data from studies on cognitive performance in beagle dogs.
Dogs develop natural amyloid plaques with very similar pathology to that seen in humans. Magnetic resonance imaging of dog brain shows increased ventricular volume as the animals age, much like in humans, and the changes seem to correlate with cognitive decline.
Milgram and colleagues reported results from two trials of dietary antioxidant therapy, one in young and one in old dogs. The antioxidants included vitamin E and C, lipoic acid, L-carnitine and other controlled ingredients. Measurement of cognitive function consisted of testing the animal’s ability to discern one odd object out of a set of three. Correct choices were rewarded with food.
The results showed that two years on the antioxidant diet had little effect on the young animals. The older animals, however, had much better performance in the discrimination task. Those dogs that had also been treated with a special behavioral enrichment program also did better, while dogs that received both the antioxidant and the special program did best of all. Postmortem analysis showed that dogs that did better in the cognitive tests also had reduced plaque load in the entorhinal, parietal, and occipital cortices, but not in the prefrontal cortex.
Finishing up the day’s symposium, Greg Cole, VA Medical Center, UCLA, reported on a theme much covered on Alzforum, the turmeric spice curcumin (see ARF related news story). Cole has previously shown that the spice works at multiple levels. In transgenic mice expressing human Aβ precursor protein (AβPP), curcumin reduces the number of plaques. In vitro it appears to activate microglia surrounding plaques, and it can also disassemble Aβ fibrils, and reduce oxidative damage. Because the spice is widely used, has been through many toxicological studies, and is readily available, it could be of enormous benefit in slowing the progression of AD. So logically, the next step is a pilot clinical trial, which Cole reported is now ongoing at UCLA. The plan is to test 40 or so volunteers for a variety of parameters including bioavailability, oxidative damage, cognitive function, brain imaging, and cerebrospinal fluid markers.
In the pre-conference symposium, Donald Ingram, National Institute on Aging, and Larry Reagan, University of South Carolina, gave presentations on caloric restriction in primates and insulin resistance in aging, respectively, which we shall report in our coverage of the main conference.—Tom Fagan.
- How Does Calorie Restriction Extend Life?
- Huntington Disease: Three Ways to Tackle Triplet Disorder
- Aβ Induces Neuronal Death via the Fas Apoptosis Pathway
- Who Says Chivalry is Dead?—Sir2 Fights Against Aging in Mammals
- Early Intervention Trial Bears Little Fruit, but Sows Hope
- Search and Rescue in Parkin Fly Mutants—DA Neurons <i>Are</i> Lost, but Saved by GST
- Safety Concerns Halt ADAPT Trial
- Curry Ingredient Spices Things Up by Blocking Aβ Aggregation
- Jenkins DJ, Kendall CW, Marchie A, Parker TL, Connelly PW, Qian W, Haight JS, Faulkner D, Vidgen E, Lapsley KG, Spiller GA. Dose response of almonds on coronary heart disease risk factors: blood lipids, oxidized low-density lipoproteins, lipoprotein(a), homocysteine, and pulmonary nitric oxide: a randomized, controlled, crossover trial. Circulation. 2002 Sep 10;106(11):1327-32. PubMed.
- Wu D, Meydani SN. Mechanism of age-associated up-regulation in macrophage PGE2 synthesis. Brain Behav Immun. 2004 Nov;18(6):487-94. PubMed.
Oakland: Caloric Restriction Set for Primate Time?
There is now overwhelming evidence in the scientific literature that caloric restriction (CR) increases longevity in a variety of organisms including yeast, roundworms, fruit flies, and rodents (see ARF related news story). But does CR have a similar benefit for primates, including humans? Despite the lack of hard evidence, there are some individuals who have gone with their gut—literally—and reduced their caloric intake by up to 30 percent in the hope of postponing the inevitable. So will the sacrifice be worth it?
The jury is still out, but the relationship between caloric restriction and human health was one of the major themes in Oakland last weekend at the American Aging Association annual meeting, organized by association president Andrzej Bartke, University of Southern Illinois, and colleagues.
In the pre-conference meeting that focused on the impact of nutrition and lifestyle on aging and neurodegenerative disease, Donald Ingram from the National Institute on Aging (NIA), Baltimore, Maryland, reviewed some of his data on the effects of caloric restriction in non-human primates. First, the bad news. The study, initiated by the NIA to address the dearth of data on primates, has failed to detect any statistically significant extension in lifespan in rhesus monkeys, some of which have been on a CR diet since 1987. However, because the average lifespan of a rhesus monkey is about 25 years (maximum lifespan is about 40 years) and the study is only 18 years old, it may be too early to tell if the CR regimen is having any real effect on longevity.
Instead, the silver lining suggests that if the monkeys on the reduced calorie diet are not living longer, they are at least living healthier. For example, the CR animals have much better insulin sensitivity and lower plasma insulin levels—two parameters that have been correlated to longer life in humans. And in many standard tests, such as those that might be encountered in a regular clinical setting, the younger monkeys on the low calorie diet did better. They had lower blood pressure and triglycerides and increased HDL cholesterol, changes that are indicative of healthy living. They also seemed to be protected from some hormonal changes. The gradual increase in plasma follicle stimulating hormone that normally occurs during aging was attenuated in monkeys on the CR diet, as was the decline in plasma dehydroepiandrosterone sulfate (DHEAS). But it was perhaps appearance-wise where the monkeys differed most. Those fed a normal diet looked heavier, greyer, and somewhat tired and worn out compared to their CR-diet counterparts.
On the first day of the meeting proper, data presented by Richard Weindruch, VA Hospital in Madison, Wisconsin, seemed to support Ingram’s findings. Weindruch has been studying the effects of a CR diet on rhesus monkeys since 1989 and has found that levels of fat, C-reactive protein, oxidative damage, and osteoarthritis of the spine are all reduced in animals on reduced calories. Further signs of healthier aging include increases in insulin sensitivity and adiponectin, a cytokine that accentuates insulin signaling. The study is ongoing, slated to be completed in 2020.
But what about humans? Not too long ago, the prospect of carrying out a human study on caloric restriction and aging seemed like nothing more than idle, after-dinner chatter. The general consensus then was that finding sufficient numbers of volunteers who would agree to such a drastic cut in calories (about 30 percent) would be hard enough. But finding volunteers who would stay on the diet would be the real sticking point. Well, enter the CRONies.
John Holloszy, Washington University School of Medicine, reported how with the help of the Calorie Restriction Society he was able to recruit dedicated volunteers who were already practicing caloric restriction with optimal nutrition (CRON). Data collected from this group suggest that those who have been on the CRON regimen for between 3 and 14 years have much healthier physiological and biochemical profiles than do those on regular diets, or even those on regular diets who get plenty of exercise.
The exercise group, for example, had an average body weight of about 70 Kg; that’s about 11 Kg lower than the average sedentary volunteer. But the CRONies weighed in at another 11 Kg lighter, or 59 Kg (that’s about 130 pounds), evidence that they are serious about their CR regimen. The CRONies also had much lower trunk fat (2.4 percent) compared to those in the exercise group (7.5 percent).
The CRONies had lower average LDL cholesterol (91 mg/DL) than did the exercise or control groups (95 and 122 mg/DL, respectively), lower triglycerides (57 mg/DL vs. 65 and 162 mg/DL), and higher HDLs. Blood pressure was also markedly different in the CRON, exercise, and control groups (103/62, mmHg vs. 126/73, and 132/83 mmHg, respectively), as were plasma insulin (1.6, 1.8, and 8.2 μU/mL), glucose, leptin, and other factors that are indicative of less than optimal health. These included C-reactive protein (0.23, 0.73, and 1.27 mg/L) and tumor necrosis factor α (0.8, 1.4, 1.4 pg/mL).
The only downside to the CRON regimen appears to be cold intolerance. These volunteers always complain of being cold, Holloszy said. This may be related to their lower levels of T3 thyroxine, which regulates metabolism. In terms of diastolic function, the CRONies are about 10 years younger than the control group, suggested Holloszy. They also have decreased numbers of white blood cells and other lymphocytes—parameters that are also reduced in animals on CR diets.
Holloszy also reported data from a 1-year intervention study that suggest the benefits of caloric restriction come quickly. In this study, improvements in lipid biology and blood pressure were all seen within a year.
Eric Ravussin’s solution to volunteers straying from their dietary regimen is to accommodate them at the Pennington Biomedical Research Center in Baton Rouge, Louisiana. The center is set up with a “metabolic kitchen” which ensures the diets are of good quality and calorically accurate.
Ravussin reported results of an intervention study designed to assess the benefit of calorie restriction, or a combination of calorie restriction and exercise, on 48 healthy but overweight male and female volunteers who spent the first 3 months and last 2 weeks of a 6-month study at the center.
The volunteers were split into four groups. In one, calories were reduced to 25 percent of that required to keep the subjects' weight stable. The second group was put on mild caloric restriction (12.5 percent of that required to maintain weight), plus enough physical activity to soak up another 12.5 percent of their calories. In a third, a very low-calorie diet (LCD) was designed so that 15 percent of body weight would be rapidly lost, but then the volunteers would be allowed to consume enough calories to maintain that weight. A fourth group, with no intervention, served as controls.
Not unexpectedly, except for the control group, all of these volunteers lost weight. Those in the intervention groups also had lower plasma insulin than did controls (33, 24, and 10 percent lower for the CR, CR plus exercise, and LCD groups, respectively). All the groups demonstrated a 25-30 percent improvement in insulin sensitivity. Though magnetic resonance imaging revealed that there was no difference in levels of muscle fat among the four groups, those on intervention had up to 50 percent lower fat in the liver.
Ravussin and colleagues are also set up to measure cellular parameters that might shed some light on what changes occur during calorie restriction. They found, for example, that spontaneous oxidation of DNA was lower in the three intervention groups compared to controls, though they detected no difference in protein carbonyls, which herald protein oxidation. Ravussin and colleagues also have ongoing microarray analysis of protein expression in muscle and adipose tissue, and they are examining the effects of CR on mitochondrial biogenesis. They found, for example, that levels of the mitochondrial protein PGC1α are increased in the CR and LCD group, but not in the CR plus exercise group. They also found that levels of SIRT1, a protein that has been implicated in longevity in yeast, and mammalian cells (see ARF related news story), are elevated almost twofold in the CR volunteers.
Ravussin reported that volunteers in all the intervention groups had a lower 24-hour energy expenditure than would be expected, even correcting for body weight. This suggests that there has been some metabolic adaptation to the caloric restriction. The basis for this is currently being investigated.
But what about humans who live to ripe old ages? Is there any correlation between longevity as we know it now, and caloric intake? There just might be. Bradley Willcox from the Pacific Health Research Institute, Honolulu, Hawaii, showed some data from the world’s longest-running population-based study on centenarians, the Okinawan Centenarian Study.
The islands of Okinawa are home to the highest concentration of centenarians in the world. The people there also have the longest disability-free life expectancy in the world, reported Willcox. So why is that?
Willcox suggested that there may well be some genetic influence because siblings of Okinawan centenarians generally live longer, too. But he also emphasized the role of diet. Historically, Okinawa has been the most undernourished prefecture in Japan, and data from the Okinawan Centenarian Study supports the theory that reduced caloric intake has contributed to their longevity. Okinawans have had the lowest average body weight of all Japanese, for example, and levels of DHEA, which inversely correlates with caloric intake in animal studies, are much higher in male and female Okinawans than in age-matched Americans, Willcox reported.
However, things are changing in Okinawa. Unfortunately, Okinawans now rank as some of the heaviest Japanese, so unbeknownst to many people from that prefecture, they may be taking part in one of the largest studies to date on caloric intake and longevity. Should Okinawa also lose its standing in the centenarian tables, one might conclude that it is because they are spending more time at the dinner table. Only time will tell.—Tom Fagan.
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Oakland: Are Mitochondria the Key to Longevity?
The benefit of caloric restriction (CR) on health and longevity was one of the major themes at the 34th annual meeting of the American Aging Association, held June 3-6 in Oakland, California. CR can increase lifespan in yeast, fruit flies, and mammals by up to 20 percent or more, and it can also lead to healthier living (see Oakland report on CR in primates). So how do fewer calories translate into health benefits?
To answer that question, many people have looked to the mitochondria. These organelles are not only the major power plants in most eukaryotic organisms, consuming most of the calories to keep up with demand for life-sustaining adenosine triphosphate (ATP), but they are also primary polluters, pumping out toxic reactive oxygen species (ROS), which have been considered a causative factor in a variety of age-related disorders, including Alzheimer and Parkinson diseases (see ARF related news story). Mitochondria are also the major instigators of apoptosis, a suicidal spiral in which many damaged cells find themselves caught up.
So what impact does aging have on mitochondrial health, and how might caloric restriction change that? Michal Jazwinski, from the Louisiana State University Health Sciences Center in New Orleans, addressed the first part of the question. Using yeast as a model system, he has studied the relationship between longevity and fitness of mitochondria.
Though it may appear at first blush that yeast can grow ad infinitum, they do age. There is a limit to the number of times a yeast cell can bud or divide to yield a daughter cell, for example. This limit is commonly called the replicative lifespan. However, like humans, not all yeast live to the ripest of old age. Some small yeast cells, or “petites,” always seem to live longer than their larger relatives.
It has been known since the 1950s that these petites are missing some of their mitochondrial DNA, thus linking these organelles to longevity. Jazwinski described how dysfunctional mitochondria set off what is known as the retrograde response, a complicated signal transduction pathway regulating the expression of many genes, which has been traced largely by work from Ron Butow’s lab at the University of Texas Southwestern Medical Center in Dallas. A protein called Rtg2, which triggers the transcription of many genes, is primarily involved in initiating the response. Jazwinski and colleagues found that activation of the retrograde response increases replicative lifespan in normal yeast. The response also seems to increase reproductive fitness—as yeast get older they take longer and longer to produce daughter cells, but if the retrograde response is triggered, this increase in regeneration time is postponed, again suggesting that the retrograde response protects yeast from the ravages of aging.
So might there be something akin to the retrograde response that occurs during normal aging? Jazwinski’s group found that there is an increase in mitochondrial mass as yeasts age, but that the increase in electric potential across the mitochondrial membranes is fivefold less, suggesting that, on average, mitochondrial membrane potential is fivefold lower than in young yeast cells. Concomitantly, there is a proportionate induction of the retrograde response. Life extension is more pronounced the greater the activation of the retrograde response. The results suggest that the mitochondrial membrane potential may be related to longevity—drops in mitochondrial membrane potential have, in fact, been linked to neurodegeneration (see ARF related news story). The extent of the retrograde response also increases with age and is also higher in petite cells, which suggests, said Jazwinski, that the retrograde response is not simply a reaction to the absence of some mitochondrial DNA, but is actually some kind of compensatory mechanism for mitochondrial dysfunction (which may or may not be a result of genomic changes). The nature of the signal that triggers this response is as yet unclear, he said.
Petite cells, though they live longer than regular yeast cells, are also packed with extrachromosomal ribosomal DNA circles (ERC). These circles are normally deleterious to yeast, but petite cells seem to accumulate them at over twice the normal rate. Jazwinski showed that these circles are also related to the retrograde response because Rtg2, the response trigger, can also suppress the formation of ERCs. However, it appears that Rtg2 is not that good at multitasking, so when the retrograde response is set to full steam ahead, as when the mitochondria are not functioning normally, ERCs accumulate faster, Jazwinski reported. These recent findings from his lab, recently reviewed in the journal Gene (see Jazwinski, 2005), link mitochondrial dysfunction, compensatory catabolic mechanisms such as the glyoxylate pathway, genomic instability, and chromatin-dependent gene activation.
Similar mechanisms might be at work in humans. Jazwinski suggested that the transcription factor myc might be a human equivalent of yeast Rtg2 because myc expression is induced in several human cell lines that have naturally occurring losses of mitochondrial DNA (see Miceli and Jazwinski, 2005). Also of interest is the fact that in yeast, Rtg2 complexes with the SAGA/SLIK multisubunit histone acetyltransferases. This links the life-lengthening retrograde response with histone acetylation, and perhaps more interestingly, with polyglutamine diseases such as spinocerebellar ataxia—Sgf73, a homolog of human ataxin-7, is a component of yeast SAGA/SLIK. In fact, it has just been shown that though a polyglutamine expanded ataxin-7 can lead to normal assembly of SAGA, the complex is devoid of acetyltransferase activity and fails to acetylate nucleosomes (see McMahon et al., 2005).
Judd Aiken, University of Wisconsin, Madison, also addressed the impact of mitochondrial fitness on normal aging—this time in mammalian skeletal muscle tissue. Muscles, by their very nature, consume vast amounts of energy and are packed with mitochondria. But some muscle fibers, such as in the soleus which flexes the foot, and the vastus lateralis which extends the leg, undergo a gradual atrophy as animals age. Could this be related to dysfunctional mitochondrial?
To test this theory, Aiken and colleagues looked for changes in activity of mitochondrial electron transport chain (ETC) components in and around sites of sarcopenia, the age-related muscle wasting that affects many elderly. The researchers found that atrophy correlates with loss of cytochrome c oxidase (COX) and hyperactive succinate dehydrogenase (SDH).
During aging, the cross-sectional area of the muscle fiber shrinks, but this shrinkage is not uniform. Aiken showed how the COX/SDH phenotype was normal in fibers that are of normal size, but is abnormal in areas where the fiber has thinned out most. Intriguingly, in muscles that do not undergo age-related atrophy, for example, the adductor longus, which helps squeeze your thighs together, there appears to be no age-related change in COX or SDH activity, suggesting that some muscles are protected against electron transport changes that might bring on sarcopenia.
Could spontaneous mitochondrial mutagenesis explain muscle atrophy? Aging has been shown to have a profound effect on the quality of mitochondrial DNA (see, for example, ARF related news story on the effect of aging on mitochondrial promoters and how mitochondrial fitness may impact nuclear DNA). Using laser capture microdissection, Aiken and colleagues have isolated individual fibers from human vastus lateralis and have found that they harbor abnormal ETC genes. Point and deletion mutations accrue in this muscle, but can be different from fiber to fiber, he said.
One question that does need to be addressed is the true level of mitochondrial mutations in any given fiber. Remember, each muscle cell is packed with mitochondria, which may have both qualitative and quantitative differences in DNA content. Aiken and colleagues are still working on this, but have found that near sarcopenia-related fiber breaks, up to 90 percent of mitochondrial DNA can be mutated in some form.
Might a slowdown in age-related mitochondrial changes explain how caloric restriction increases longevity? Brian Merry, from the Institute of Human Ageing at the University of Liverpool, England, addressed this possibility.
One theory linking caloric restriction to mitochondria suggests that decreased levels of reactive oxygen species might explain the increase in lifespan elicited by eating fewer calories. But the evidence supporting this is weak, said Merry. Experiments show, for example, that the metabolic rate in CR animals is no different from controls once they have become accustomed to their lower calorie diet (though see Sohal et al., 1994). And Merry and colleagues (see Lambert et al., 2004) showed that state IV respiration is normal in mitochondria isolated from the various organs of CR animals, including brain, liver, and heart (state IV respiration, measured in the absence of ADP, is when production of ROS by the ETC is at its highest). So what explains such findings that superoxide and hydrogen peroxide are lower in mitochondria isolated from the heart and liver, respectively, of CR animals?
To investigate this question, Merry and colleagues focused on the proton leak across the mitochondrial membrane. They found that for a given inner mitochondrial membrane potential, the proton leak across the membrane was higher in organelles isolated from CR animals than from controls. This was unexpected, suggested Merry, because CR has been shown to decrease ROS, which are generated by leaks in the electron transport chain. And, in fact, Merry acknowledged that other researchers, including Jon Ramsey and Richard Weindruch at the University of California, Davis, just showed the opposite, that leak across the membrane is lower in CR-derived mitochondria (see Hagopian et al., 2005).
But though the CR mitochondria have no change in stage IV metabolic rate, Merry did find that the resting membrane potential is actually lower across these mitochondria membranes than across those from control mitochondria. It is this lower membrane potential that explains the reduced production of ROS in CR, suggested Merry, because it is the extent to which the ETC components are reduced, rather than the flow through the chain, that determines the rate of formation of ROS, he contends.
Whether this is the correct explanation for reduced ROS in CR remains to be seen. However, Merry did have one other interesting observation. He showed that insulin, which is dramatically reduced in the plasma of CR animals (see related Oakland news), lowers leakage across the membrane, but at the same time increases the membrane potential. So if the overall reduction rather than flux through the ETC turns out to be the key to ROS production, then CR, insulin, and ROS may be inextricably linked.—Tom Fagan.
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