Mitochondria and Aging
Tory Hagen, Ph.D.
"All would live long, but none would be old." --Benjamin Franklin
Most people enthusiastically welcome knowledge of new ways to slow or reverse aging because aging affects us all. While much attention has been focused on cosmetic remedies, the underlying mechanisms that lead to aging are still not well understood and will have to be better defined before any meaningful ways of making us age more gracefully can be achieved. Despite our meager understanding, it is becoming increasingly clear that oxidants (also called "reactive oxygen species") and the accumulation of oxidative damage are important factors in the overall decline that we experience as we age.
My research is focused on understanding the role that mitochondria play in aging. We are keenly interested in the mitochondria because these organelles are the chief producers of both energy and oxidants inside the cell. Oxidants released from the mitochondria can damage important biomolecules, such as DNA, lipids, and proteins (see Frei's article in the Fall/Winter 1997 Newsletter). If DNA damage were not repaired or repaired improperly, then it could be converted into mutations and passed on to daughter cells. Over the lifespan of an individual, DNA mutations would be expected to accumulate and eventually lead to the cellular changes evident in aging or to age-related diseases like cancer. Moreover, mitochondria may themselves be important targets for aging because of their vital importance to overall cellular metabolism.
As Professor Don Reed discussed in a previous newsletter article (Spring/Summer 1997), mitochondria are cellular organelles that convert unusable forms of energy into a usable chemical form known as adenosine triphosphate (ATP), so that vital cellular chemical reactions throughout the body can occur. They do this by oxidizing (burning) fuels, such as lipids from the diet, and transporting free electrons liberated from these oxidation reactions through a series of proteins called the "electron transport chain." An electrical potential develops across the inner mitochondrial membrane as a result of this movement of electrons. Energy liberated from these oxidation reactions is then used as the driving force for ATP synthesis. It is clear that factors and conditions that cause mitochondrial dysfunction could severely affect overall cellular metabolism and, ultimately, our energy levels and survival.
There is tantalizing evidence that mitochondrial decay may be a primary factor in the aging process. As already noted, mitochondria are the chief source of endogenous oxidants, including hydrogen peroxide and the superoxide and hydroxyl radicals. This high flux of oxidants would not only be expected to damage the cell overall, but also certainly would damage the mitochondria in which the oxidants are produced. Despite an impressive array of antioxidant defenses, such as vitamin C, glutathione and vitamin E, the level of oxidative damage to mitochondria is enormous. We have shown that oxidative damage to mitochondrial DNA (the only organelle with its own DNA outside the nucleus) is 8 to 10-fold higher than the levels found in nuclear DNA and accumulates even higher levels of damage with age. This high level of damage, if not repaired properly, could be converted into mutations and may be the cause of the age-related 17-fold higher accumulation of DNA mutations in the mitochondria compared to the cell nucleus. As the vicious cycle continues, the increased level of mutations would adversely affect mitochondrial electron transport and further increase the rate of oxidant production. Oxidative damage may also adversely affect the inner mitochondrial membrane, which is a specialized structure that serves as the site of ATP production. With age, the fatty acid constituents comprising the phospholipids of the inner mitochondrial membrane become altered in composition, making them even more prone to oxidative damage. These changes could also affectmembrane fluidity, which, in turn, may change the shape of proteins embedded in the lipid bilayer of the membrane. Alterations to membrane fluidity as well as the oxidation of critical functional groups on proteins could seriously impair the ability of mitochondria to meet cellular energy demands.
Cellular and mitochondrial antioxidant status also becomes limited. This loss may reflect increased utilization because of the heightened cellular oxidative stress in aged tissue or the decline in gastrointestinal absorption of vitamin C or cysteine, an amino acid necessary for glutathione synthesis. Loss of antioxidants, coupled with increased oxidant production, may lead to heightened susceptibility of mitochondria to oxidative damage. Carnitine, an amino acid synthesized in a series of vitamin C-dependent reactions from the dietary amino acids, lysine and methionine, and also found in meat and dairy products, is necessary for the transport of fatty acids into the mitochondria. It also becomes limited, which may deprive mitochondria of its most important fuel source and cause ATP levels to decline.
Ironically, it appears that mitochondria cause their own decay and may ultimately adversely affect survival of the whole organism. This decay may result from constant oxidative damage to mitochondria, leading to accumulation of mitochondrial DNA mutations, damage to constituents of the inner mitochondrial membrane and loss of critical micronutrients supplied by the cell. While reversing the effects of mitochondrial mutations directly through genetic engineering is currently unfeasible, it may be possible to supply mitochondria with micronutrients through dietary supplementation that may reverse the age-associated decline in their levels or enhance the endogenous antioxidant defenses and therefore maintain mitochondrial function.
Most studies examining the role of mitochondria in aging have used mitochondria isolated from a particular tissue to determine age-related changes to the organelle. However, mitochondria become very fragile and susceptible to rupture with age, and purified mitochondria from aged tissue may not truly represent those found in vivo. Moreover, once mitochondria have been removed from the cell, it is impossible to determine what influence age-related changes in their function may have on the cell. Clearly, a means to assess mitochondrial function within the cell will provide new insights in their role in the aging process.
Because of these concerns, we elected to isolate intact cells from a particular organ and then develop ways to monitor mitochondrial function within those cells. To accomplish this, we used rat liver cells (hepatocytes) as our model because of the ease with which abundant cells can be isolated. As an assay for mitochondrial function, we measured the ability of mitochondria to generate the electrical potential energy (see above) that is used as the driving force for ATP synthesis. This was achieved by incubating hepatocytes isolated from young and old rats with a fluorescent dye that accumulates specifically in the mitochondria based on their generation of energy or membrane potential. The dye has two characteristics that make it an optimal compound to examine mitochondrial function within cells: it is not toxic to the cell, and it does not alter mitochondrial function. Thus, we can quickly determine whether aging impairs the ability of mitochondria to meet cellular energy demands based on the amount of dye that binds to mitochondria.
Results from our studies showed that three distinct populations of hepatocytes in rats develop with age. The largest population of cells (accounting for 67% of the total) from old rats had mitochondria with a significantly lower membrane potential, which could impair their ability to meet cellular energy demands. The smaller subpopulations of cells from old rats had mitochondria that were either moderately impaired or retained the same functional characteristics as seen in cells from young rats. This type of age-related heterogeneity has not been reported before and provides a valuable perspective on the consequences of mitochondrial dysfunction. We then successfully separated these cell subpopulations from each other so that we could better characterize the underlying causes that may have led to appearance of these functionally different populations with age.
Most of the cells containing mitochondria with the lowest functional ability were the least metabolically active. Their mitochondria were more inefficient and released significantly higher amounts of oxidants, which resulted in significantly higher levels of oxidative damage to the mitochondria and to the cell as a whole. Because of mitochondrial decay, the majority of the liver cells were experiencing a higher level of oxidative stress with age, which almost certainly produces deleterious effects. The other cell subpopulations from old rats also showed varying degrees of the same age-related alterations. The majority of cells also had substantially lower levels of cardiolipin, a key molecule necessary for proper mitochondrial function. The levels of another critical molecule, carnitine, were also markedly lower with age, which could profoundly affect the transport of fatty acids into the mitochondria for fuel (see above). Moreover, the antioxidant status of these cells was compromised; hepatocytes from old rats had significantly lower vitamin C and glutathione levels than cells from young rats.
Because carnitine levels decline significantly with age, we supplemented the diet of old rats with a derivative of carnitine called acetyl-L-carnitine (ALCAR) as a means to replenish this substance within cells. ALCAR was used instead of carnitine because it is taken up slightly better from the diet. However, both compounds are readily absorbed from the gastrointestinal tract, and few, if any, side effects have been reported from their use as dietary supplements. One month of ALCAR supplementation not only substantially improved hepatic carnitine levels but also significantly increased overall cellular metabolism, mitochondrial energy production, and cardiolipin values. However, ALCAR supplementation also slightly increased the rate of oxidant production, indicating that the efficiency of mitochondrial electron transport had not improved. Because ALCAR supplementation might result in increased oxidative stress, we also sought to establish dietary feeding regimens of antioxidants that could reverse their age-associated decline and reduce the risk of oxidative injury.
We found that dietary supplementation for two weeks with R-lipoic (alpha-lipoic) acid, a potent naturally occurring antioxidant that is abundant in organ meats and green leafy vegetables, increased hepatic vitamin C and glutathione levels significantly. Moreover, cellular oxidant levels markedly declined, even when ALCAR was co-administered to the animals. Dietary supplementation with ALCAR, particularly in combination with R-lipoic acid, improves mitochondrial function in rats without a significant increase in oxidative stress. Overall, these results show that mitochondria within cells exhibit age-related decay that could severely affect cellular function and that efficient mitochondrial function in aged tissue may be restored by providing key metabolites, such as carnitine and lipoic acid, in the diet.
In human clinical studies to test the effect of large daily doses of ALCAR on the progression of Alzheimer's disease (published in Neurology in 1996) or the effect of intravenously administered carnitine in healthy subjects (published in the European Journal of Clinical Pharmacology in 1988), no side effects were observed. The supplemental use of lipoic acid also seems to be well tolerated--daily doses of several hundred milligrams for three or four months have been given to diabetic patients (published in Diabetes Care in 1997) in Germany with no signs of toxicity or side effects. Indeed, lipoic acid has been used in Germany for over forty years to treat diabetes.
These results provide compelling evidence that implicates mitochondrial dysfunction in aging and also suggest that this process can be slowed or even reversed by dietary means. In my laboratory at the Linus Pauling Institute, we plan to delve more thoroughly into the underlying causes of mitochondrial decay and how they affect cardiac and neurological function.
Last updated May, 1998.
Honoring a Scientific Giant with Research Toward Longer, Better Lives