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Metabolic Research Centre, Institute for Conservation Biology, School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia; Department of Basic Medical Sciences, University of Lleida, Lleida, Spain; and Department of Biology, City College of the City University of New York, New York, New York
ABSTRACT I. INTRODUCTION II. MECHANISMS: OXIDATIVE-STRESS THEORY OF AGING A. Mitochondrial Free Radical Production B. Cellular Protection: Antioxidant Defenses C. Cellular Protection: Repair Mechanisms D. Membrane Composition and Lipid Peroxidation III. MAMMALS A. Metabolic Rate and Body Size of Mammals B. Maximum Life Span, Body Size, and Lifetime Energy Expenditure of Mammals C. Mitochondrial Free Radical Production and Antioxidant Defenses of Mammals D. Membrane Composition, Lipid Peroxidation, Body Size, and Life Span of Mammals E. Insights From Relatively Long-Living Mammals (Including Humans) IV. BIRDS A. Metabolic Rate and Body Size of Birds B. Maximum Life Span, Body Size, and Lifetime Energy Expenditure of Birds C. Insights From Bird-Mammal Comparisons D. Insights From Comparison Among Birds V. ECTOTHERMIC VERTEBRATES AND INVERTEBRATES VI. TREATMENTS TO MANIPULATE LIFE SPAN A. Calorie Restriction B. Antioxidant Defenses C. Genetic Manipulations VII. CONCLUSIONS: SO, WHAT DETERMINES A SPECIES MAXIMUM LIFE SPAN? GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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Later, Harman (127) proposed the free radical theory of aging, which provided a possible mechanistic link between metabolic rate and aging. Harman (127) proposed that normal oxygen consumption inevitably results in the production of oxygen free radicals which, in turn, damage important biological molecules (nucleic acids, proteins, and lipids, among others), resulting in the process of "aging," and eventually this accumulated damage results in death of the organism. Over the last half century, the free radical theory has developed into the oxidative-stress theory of aging which takes into account that 1) not all the damaging reactive oxygen molecules (or "species" in the chemical sense hence the abbreviation; ROS) produced by normal metabolism are free radicals, and that organisms have both 2) antioxidant defenses to minimize ROS and 3) mechanisms to repair or remove biological molecules damaged by ROS. Currently, the oxidative-stress theory is the most widely accepted mechanistic explanation of aging and life span, and there is much evidence to support it. In the past, the oxidative-stress and rate-of-living theories have been reconciled by supposing that higher levels of ROS are generated at higher levels of metabolic activity (e.g., Refs. 22, 328). However, this popular concept is flawed, because while it appears true in some situations (e.g., when broadly comparing mammals of different size), it is not true in others (e.g., between different states of mitochondrial respiration, following calorie restriction, or in bird-mammal comparisons).
Furthermore, although there is a broad correlation between body size and MLSP, there are a number of problems associated with presuming a linkage between rate-of-living and MLSP. For example, 1) voluntary exercise and its associated increase in metabolic rate does not shorten life span of rats (147) or humans (200); 2) there is no inverse relationship between life span and mass-specific metabolic rates of individuals in populations of mice (331) or fruit flies (157); 3) although caloric restriction extends life span, it does not do so by reducing metabolic rate (MR) (see later sections); 4) long-living insulin/insulin-like growth factor (IGF) mutant fruit flies do not have a decreased metabolic rate (157); 5) although birds and mammals have similar rates of living, birds are generally much longer living than similar-sized mammals; and 6) within both groups of endotherms, there are significant species differences in MLSP that cannot be explained by metabolic rate differences (see later sections). Collectively these problems show that part of the scientific jigsaw puzzle that explains aging and maximum life span is missing. In this contribution, we review the evidence that membrane fatty acid composition is this missing piece of the puzzle.
A century ago, mass-specific MR (and its physiological correlates, such as heart rate) were the obvious properties to be related to life span. There have been a variety of attempted explanations for the size-dependent differences in metabolic rate over the years, and differences in cellular composition were initially excluded (see Ref. 176). However, recently it has been shown that one aspect of cell composition, namely, membrane fatty acid composition, does vary in a systematic manner with body size in mammals (69, 164) and in birds (37, 162). It has also been shown that membrane fatty acid composition is related to MLSP (266, 271, 272, 275–277). These observations, when combined with the long-known differences in susceptibility of different fatty acids to peroxidative damage (148), were seminal in the development of the "homeoviscous-longevity" membrane theory (267) and the later "membrane-pacemaker" theory of aging (155). These modifications of the oxidative-stress theory of aging emphasize the effects of membrane fatty acid composition on lipid peroxidation (and its products) on the process of aging and how this might determine the distinctive maximum life span of a species.
In this contribution, we concentrate on the comparison of the maximal longevity (MLSP) distinctive to different species. We first discuss the mechanisms associated with aging (based on the oxidative-stress theory) and briefly describe the means by which the fatty acid composition of membranes might be important in the processes of aging. We then examine the links between body mass, metabolic rate, and maximum longevity separately for the two classes of endothermic vertebrates, mammals and birds, with an emphasis on how membrane fatty acid composition might account for variation in MLSP among species. We briefly review the limited data on this topic for ectothermic vertebrates and invertebrates. Finally, we consider how changes in membrane composition might explain how some experimental treatments can extend longevity. We restrict ourselves largely to a physiological context, paying limited attention to the genetic manipulations involved in much recent aging research.
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II. MECHANISMS: OXIDATIVE-STRESS THEORY OF AGING |
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A. Mitochondrial Free Radical Production
That oxygen can be toxic, and consequently the beginnings of the oxidative-stress theory of aging, dates back to the late 19th century. Paul Bert (25) described the toxic effects of air at high pressure, as well as oxygen alone at high concentration, and several subsequent studies showed that oxygen toxicity was particularly acute in endotherms. In ectotherms its toxicity increased with ambient temperature, which suggested a relationship with metabolic rate rather than oxygen solubility. These studies demonstrated that three organ systems (lungs, retina, and nervous system) are especially sensitive to high oxygen tensions in whole animals (195); however, a scientific explanation of the cause of these harmful reactions was lacking for many decades.
After Fenton (100) discovered that Fe(II) catalyzed the oxidation of tartaric acid by hydrogen peroxide, it was proposed that hydroxyl radicals, hydrogen peroxide, and superoxide anions undergo a chain reaction that results in a net conversion of hydrogen peroxide into water (119, 120). This became known as the Haber-Weiss reaction and is now believed to be at the core of much ROS generation in cells. However, the connection between oxygen toxicity and ROS generation was not then understood (27). Using electron spin resonance, Commoner et al. (67) published the first direct evidence that free radicals were produced in living cells and found that free radical levels were higher in tissues that were more metabolically active. The free radical hypothesis of oxygen toxicity was first proposed by Gerschman et al. (109), taking into account the strong similarity of the histological alterations produced by hyperoxia and by ionizing radiation (264). Harman (127) postulated that even at normoxia, oxygen utilization causes subtle tissue damage due to production of free radicals. He integrated evidence from the electron spin resonance study, post-Hiroshima studies of radiation damage, Fenton's findings, and contemporary theories that proposed free radical mechanisms for the oxidation of organic compounds and dismutation of hydrogen peroxide by iron salts (358). Harman proposed that physiological iron and other metals would cause ROS to form in the cell via Haber-Weiss chemistry as a by-product of normal redox reactions. The ROS would damage nearby structures like mitochondrial DNA (129). He predicted that administering compounds that are easily oxidized, such as cysteine, would slow down the aging process, a suggestion recently reviewed by Dröge (83). Harman's proposal constitutes the basis of the free radical theory of aging, which is supported by a large body of scientific evidence. The discovery of the superoxide dismutase enzyme in living organisms (226) led to serious consideration of the role of free radicals in biology. If an enzyme that decomposed oxygen radicals inside the body was highly conserved during evolution, oxygen free radicals were likely important in biological systems.
A free radical is any molecule capable of independent existence that contains one or more unpaired electrons (124). ROS is a collective term that includes oxygen radicals as well as nonradical derivatives of oxygen (124). Reduction of univalent oxygen generates three main ROS: superoxide and hydroxyl radicals as well as the nonradical hydrogen peroxide. Among these, the hydroxyl radical is extremely reactive. It can be generated by the combination of superoxide radical and H2O2 in the presence of trace amounts of iron (or copper) during the Fenton-Haber-Weiss reaction. Thus H2O2, although not a free radical itself, can behave as a Trojan horse, diffusing away from sites of its production to generate the hydroxyl and other reactive radicals at other cellular locations, hereby propagating oxidative damage. In many scientific papers, use of the term ROS is restricted to what we might describe as the three primary ROS described above (especially superoxide and hydrogen peroxide); however, there are a number of other important ROS, both radicals and nonradicals (see Table 1). The roles of many of these other ROS in aging have not been fully evaluated. There is also some overlap between ROS and reactive nitrogen species (RNS) such as nitric oxide. In the case of mitochondria, nitric oxide production is much smaller than superoxide production. However, nitric oxide can still be important due to interaction with superoxide and other radicals to produce reactive species like peroxynitrite (76), which can modify many kinds of macromolecules and possibly contribute to various pathologies (65).
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The finding that the percentage of total electron flow in mitochondria directed to radical generation is not constant in different tissues, and under different conditions, suggests that ROS generation is not a constant stoichiometric by-product of mitochondrial respiration (17, 19, 113, 136), as is frequently assumed. Oxygen radical generation at the respiratory chain has been classically attributed to complex III (32). However, in agreement with early information obtained in submitochondrial particles (347), complex I is also an important ROS generator in intact functional heart and brain mitochondria from rats (20, 108, 136, 137), as well as other organs and other species (17, 184). Nowadays the concept that both complex I and complex III produce ROS is widely accepted in the scientific literature and is already part of mainstream biochemical knowledge (e.g., Ref. 253). There is, however, current debate concerning the identity of the ROS generator inside complex I. Some studies have suggested a role for flavin mononucleotide (184, 206, 380), while others favor complex I linked ubisemiquinones (191, 256). However, other studies localize the ROS generator in the electron pathway inside complex I in a place between the ferricyanide reduction site and the rotenone-binding site (108, 139, 186). This finding discards flavin and suggests that the source of ROS might be the complex I FeS clusters situated in that region (139), although a role for complex I-linked ubisemiquinones cannot be ruled out at present. Because all FeS clusters of complex I are situated in the hydrophilic matrix domain of the complex, ROS arising from them will damage targets situated in the mitochondrial matrix. In contrast, complex III ROS generation seems to be directed to the cytosolic side (342).
Apart from the degree of electron flow, other possible physiological mechanisms influencing the rate of mitochondrial ROS generation include 1) a decrease in the relative concentration of the respiratory complex(es) responsible for ROS generation; 2) the degree of electronic reduction of these generators; the more reduced, the higher their rate of ROS production (20, 113, 114, 208, 310); 3) uncoupling proteins (discussed in the next paragraph); 4) as well as chemical modification (e.g., glutathionylation) of mitochondrial ROS generators. The nonenzymatic glutathionylation of complex I increases its superoxide production, and when the mixed disulfides are reduced, superoxide production returns to basal levels (125, 351). Oxidation of the glutathione pool within intact mitochondria to glutathione disulfide also leads to glutathionylation of complex I, which correlates with increased superoxide formation. In this case, most of the superoxide is converted to hydrogen peroxide, which can then diffuse into the cytosol. This mechanism of reversible mitochondrial ROS production suggests how mitochondria might regulate redox signaling and shows that oxidation of the mitochondrial glutathione pool could contribute to the pathological changes that occur to mitochondria during oxidative stress (125, 351).
Mitochondrial superoxide production is very sensitive to the proton-motive force across the inner membrane, so it can be strongly decreased by mild uncoupling. For example, one study (239) has shown that a 10-mV decrease in mitochondrial membrane potential is associated with 70% decrease in superoxide production. The decrease in ROS production by isolated mitochondria between state 4 and state 3 is likely due to the decrease in mitochondrial membrane potential between these two states. Similarly, it has been proposed that an ancestral function of uncoupling proteins (UCP) is to cause mild uncoupling and so diminish mitochondrial superoxide production, hence protecting against oxidative damage at the expense of a small loss of energy (38). For example, UCP3 (found in skeletal muscle mitochondria) is activated by increased superoxide production, and the consequent increased proton conductance will cause a mild decrease in proton-motive force and consequently a diminished superoxide production (88). The role of UCP3 activation during exercise and during the transition from state 4 to state 3 in muscle mitochondria, and its control on ROS production is one possible reason why increased voluntary exercise of mammals is not associated with a decrease in life span. It has been proposed that this negative-feedback loop will protect cells from ROS-induced damage and might represent the ancestral function of all UCPs (38, 249).
Other possible factors that modulate complex I activity are cardiolipin content (280) and S-nitrosation (45). Cardiolipin, a phospholipid of unusual structure localized almost exclusively within the inner mitochondrial membrane, is particularly rich in unsaturated fatty acids. This phospholipid plays an important role in mitochondrial bioenergetics by influencing the activity of key mitochondrial inner membrane proteins, including several anion carriers and electron transport complexes I, III, and IV (143). Mitochondrial cardiolipin molecules are possible targets of oxygen free radical attack, due to their high content of polyunsaturated fatty acids and because of their location in the inner mitochondrial membrane near the site of ROS production. In this regard, it has been recently demonstrated that mitochondrial-mediated ROS generation affects the activity of complex I (280), as well as complexes III and IV (278, 279), via peroxidation of cardiolipin following oxyradical attack to its fatty acid constituents. These findings might explain the decline in respiratory chain complexes observed in mitochondria isolated from aged animals and in pathophysiological conditions that are characterized by an increase in the basal rate of the ROS production.
In addition to cardiolipin, S-nitrosation may also serve as another regulatory system for complex I. Nitric oxide is a pleiotropic signaling molecule, with many of its effects on cell function being elicited at the level of the mitochondrion. One of the many cellular reactions of nitric oxide is S-nitrosation of protein thiols, resulting in the generation of S-nitrosothiols. There are several reasons why S-nitrosation may be an important mitochondrial regulatory mechanism. Mitochondria contain sizable thiol pools, are abundant in transition metals, and have an internal alkaline pH, all of which are known to modulate S-nitrosothiol biochemistry (102). In addition, mitochondria are highly membranous, sequester lipophilic molecules such as nitric oxide, and the formation of the putative S-nitrosating intermediate N2O3 is enhanced within membranes (42). Thus the mitochondrial respiratory chain embedded within the inner membrane would seem to be an ideal target for S-nitrosation. In this scenario, it has been described that mitochondria interact with nitric oxide at several levels, and one particularly well-characterized example is the inhibition of complex I and IV (45, 265).
Although free radicals and derived reactive molecular species can have damaging effects, at moderate concentrations they are often important signaling molecules involved in the regulation of several physiological functions (e.g., control of ventilation, erythropoietin production, immune responses, apoptosis). In such situations, ROS are typically generated by tightly regulated enzymes such as NAD(P)H oxidases (82).
B. Cellular Protection: Antioxidant Defenses
Aerobic life demands antioxidant defenses. An antioxidant is extremely difficult to define, but a broad definition is "any substance that, when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate" (124). An antioxidant reacts with and neutralizes an oxidant, or regenerates other molecules capable of reacting with the oxidant. Oxidative damage is a broad term used to cover the attack upon biological molecules by free radicals. Cellular protection against oxidative damage includes both elimination of ROS and repair of damage with antioxidants constituting the first line of this defense.
Direct ROS scavenging antioxidant enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). SOD converts the superoxide radical to oxygen and hydrogen peroxide. There are different types of SOD in different cellular compartments: Mn-SOD in the mitochondrial matrix, a Cu/Zn-SOD in the cytosol and the intermembrane space, and a different Cu/Zn-SOD in the extracellular compartment. Although SOD eliminates superoxide radicals, it is not a complete antioxidant defense because it produces H2O2. CAT and GPX are two different but kinetically complementary enzymes that can eliminate H2O2. CAT is one of the most active enzymes known and removes H2O2 by breaking it down directly to H2O and O2. However, it has a low affinity for H2O2; thus it needs high H2O2 concentrations to function effectively. Conversely, GPX enzymes (both the selenium- and non-selenium-dependent forms) are most functional at low hydrogen peroxide concentrations, since they have high affinity and low rates of catalysis. GPX(s) have the same locations as SOD, suggesting that GPX is the main enzyme that deals with H2O2 produced by Cu/Zn-SOD in the cytosol and Mn-SOD in the mitochondria (124).
As well as antioxidant enzymes, there are different kinds of endogenous nonenzymatic scavenger molecules that cooperate to limit cellular oxidative stress. After reacting with ROS, these molecules are oxidized and thus must be reduced to regain their antioxidant capacity. Their low molecular mass allows them to remove ROS at sites that larger antioxidant enzymes cannot access. Glutathione (GSH) and ascorbate are two major nonenzymatic antioxidants of the hydrophilic cellular compartments. The antioxidant activity of GSH resides in the reduced thiol group of its cysteine residue. Glutathione can react directly with ROS or can act as a cosubstrate of GPX enzymes. Oxidized glutathione (GSSG) also plays an important role in the regulation of protein function, as mentioned above for mitochondrial complex I. Glutathione reductase is the enzyme responsible for reducing GSSG back to GSH using NADPH (124).
Another thiol-related redox-active substance is the protein thioredoxin (238). Thioredoxin has a redox-active disulfide/dithiol at the active site that regulates transcription factors (such as nuclear factor 
B and AP-1). It is induced by various oxidative stresses and is translocated to the nucleus. Thioredoxin is cytoprotective against oxidative stress by scavenging ROS in cooperation with peroxiredoxin/thioredoxin-dependent peroxidase (238).
Apart from glutathione, ascorbate (vitamin C) is the next most abundant reduced nonenzymatic antioxidant inside cells. It is endogenously synthesized in most vertebrates (although not in human beings, fruit bats, or guinea pigs), and it is maintained at levels as high as 1 mM in tissues. After reacting with ROS, the oxidized form of ascorbate must be reduced by NADPH-, GSH-, or NADH-dependent reductases to regain antioxidant capacity (14, 124).
Tocopherols and carotenoids are the main radical scavenger antioxidants that act in lipophilic environments of cells. The major scavenger inside membranes is D-
-tocopherol (vitamin E). Most membranes are thought to contain approximately 1 tocopherol molecule per 1,000 lipid molecules (369). Vitamin E acts on lipid peroxyl groups inside membrane bilayers, reducing them to hydroperoxides, and thus inhibiting the propagation of the peroxidative chain reaction. It breaks the chain reaction of lipid peroxidation but is itself converted to a radical during the process. Vitamin E also reduces lipid alcoxyl radicals to lipid alcohols. Oxidized vitamin E can be recycled back to its reduced form by ascorbate or ubiquinone (coenzyme Q). Carotenoids quench singlet oxygen and interact with other ROS at physiological tissue oxygen partial pressures. Ubiquinol, the reduced form of coenzyme Q, is an important antioxidant. It is a hydroquinone that is synthesized and present in all cellular membranes (15), and its antioxidant activity is exhibited through scavenging of lipids radicals or reduction of vitamin E radical. Regeneration of coenzyme Q is performed by reductases that use NADPH or NADH as cofactors. Methionine residues in proteins (see sect. IIC) can also be considered as scavengers of oxidants.
C. Cellular Protection: Repair Mechanisms
Despite the plethora of antioxidant defense systems, oxidative damage still occurs in vivo. The next level of cellular protection against oxidative stress is the repair/removal of oxidatively damaged macromolecules. As with antioxidant defenses, space limitations restrict our coverage here. The reader is directed elsewhere (e.g., Ref. 124) for more detailed presentation.
To avoid the gradual accumulation of oxidative DNA base lesions and thus maintain genomic stability, DNA damage is repaired by a number of mechanisms. In general, oxidized DNA bases are removed by base excision mechanisms, and although these correcting mechanisms show high fidelity, DNA lesions still accumulate with age. Such accumulation is most dramatic in mitochondrial DNA (312), and less is known about mitochondrial than nuclear DNA repair (31). DNA polymerase enzymes have proofreading and error-correcting facilities, and when this capacity of mitochondrial DNA polymerase was removed in mice, there was a three- to fivefold increase in mitochondrial mutations and a corresponding decreased life span (185, 357).
Damaged proteins can either be repaired or removed. For example, methionine residues of proteins are among the amino acids most susceptible to oxidation by ROS, and the sensitivity of proteins to oxidative stress increases as a function of their number of methionine residues (337). Oxidation of methionine residues generates methionine sulfoxide in proteins, which deprives them of their function as methyl donors, and may lead to loss of their biological activity. Methionine sulfoxide can be repaired by the enzyme methionine sulfoxide reductase, which uses the reducing power of thioredoxin (248). In this context, it is very illustrative that knocking out methionine sulfoxide reductase A lowers MLSP and increases protein carbonyls and sensitivity to hyperoxia in mice (248). The opposite manipulation, overexpression of methionine sulfoxide reductase A, increases life span and delays the aging process in Drosophila (304). The oxidized form of thioredoxin, produced during the reduction of methionine sulfoxide, can be converted back to reduced thioredoxin by the enzyme thioredoxin reductase. In agreement with the methionine oxidation hypothesis of aging, it has been reported that overexpression of thioredoxin reductase increases longevity in mice (238). However, protein repair mechanisms are generally limited, and most oxidized proteins are removed by proteolysis either via lysosomal degradation or via the proteasome. The proteasome complex recognizes amino acid residues that are exposed following the oxidative rearrangement of secondary and tertiary protein structures (117).
Protection of membranes is achieved by a more complex system that involves three mechanisms: lipid repair, lipid replacement, and scavenging of lipoperoxidation-derived end products. The action of chain-breaking antioxidants can result in the production of lipid hydroperoxides. Some of these are metabolized by GPX antioxidant enzymes, which act on H2O2 and also on free fatty acid hydroperoxides, reducing them to fatty acid alcohols. However, the peroxidized fatty acids must be first released from the membrane lipids. A phospholipid hydroperoxide glutathione peroxidase (PHGPX) has also been described in some mammalian tissues and is capable of acting on peroxidized fatty acid chains still esterified to membrane lipids and is thus a very important antioxidant defense for membranes in situ (167). An important mechanism for removing peroxidized lipids is membrane lipid remodeling. While the enzyme PHGPX is important in removing peroxidized acyl chains from phospholipids, other enzymes are also involved in the continual deacylation/reacylation of phospholipids, and this turnover of membrane acyl chains is very rapid. Phospholipase A2 is an important enzyme for removing acyl chains from phospholipids while acyltransferase and transacylase enzymes are responsible for reacylation of phospholipids (98). When isolated rat liver cells are subjected to oxidative stress, lipid peroxidation is increased; however, there is no change in either the fatty acid composition of or in the rate of acyl turnover of membrane phospholipids. There is, however, a decrease in the polyunsaturated fatty acid (PUFA) content of cellular triacylglycerols. It is suggested that rapid constitutive recycling of membrane phospholipids rather than selective in situ repair is responsible for eliminating peroxidized phospholipids, with triacylglycerols providing a dynamic pool of undamaged PUFA for phospholipid resynthesis (110). Finally, lipid peroxidation-derived end products resulting from membrane lipid oxidative damage can be detoxified by glutathione S-transferases (GST; Ref. 348).
D. Membrane Composition and Lipid Peroxidation
Oxygen radicals can attack many cellular molecules. In addition to protein and DNA modification (reviewed in Ref. 312), damage to membrane lipids is also very relevant for life span determination. The susceptibility of membrane lipids to oxidative damage is related to two traits. The first is that oxygen and many radical species are several times more soluble in lipid membrane bilayers than in the aqueous solution (246). The second property is related to the fact that not all fatty acid chains are equally susceptible to damage. It is this second property that is the key to the link between membrane composition and oxidative damage to membranes. The carbon atoms that are most susceptible to radical attack are the single-bonded carbons between the double-bonded carbons of the acyl chains (124). The hydrogen atoms attached to these carbons are called bis-allylic hydrogens. This means that saturated and monounsaturated fatty acyl chains (SFA and MUFA, respectively) are essentially resistant to peroxidation while PUFA are damaged. Furthermore, the greater the degree of polyunsaturation of PUFA, the more prone it is to peroxidative damage. Holman (148) empirically determined (by measurement of oxygen consumption) the relative susceptibilities of the different acyl chains (see Fig. 1). Docosahexaenoic acid (DHA), the highly polyunsaturated omega-3 PUFA with six double bonds, is extremely susceptible to peroxidative attack and is eight times more prone to peroxidation than linoleic acid (LA), which has only two double bonds. DHA is 320 times more susceptible to peroxidation than the monounsaturated oleic acid (OA) (148). With the combination of the relative susceptibilities of different fatty acids with the fatty acid composition of membrane lipids, it is possible to calculate a peroxidation index (a measure of the susceptibility to peroxidation) for any particular membrane.1 The peroxidation index of a membrane is not the same as its unsaturation index (sometimes also called its "double bond index"), which is a measure of the density of double bonds in the membrane. For example, a membrane bilayer consisting solely of MUFA will have an unsaturation index of 100 and a peroxidation index of 2.5, while a membrane bilayer consisting of 95% SFA and 5% DHA will have an unsaturation index of 30 and a peroxidation index of 40. This means that although the 5% DHA-containing membrane has only 30% the density of double bonds of the monounsaturated bilayer, it is 16 times more susceptible to peroxidative damage.
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The hydroperoxides and endoperoxides, generated by lipid peroxidation, can undergo fragmentation to produce a broad range of reactive intermediates, such as alkanals, alkenals, hydroxyalkenals, glyoxal, and malondialdehyde (MDA; Ref. 95) (see Fig. 2). These carbonyl compounds (collectively described as "propagators" in Fig. 2) have unique properties contrasted with free radicals. For instance, compared with ROS or RNS, reactive aldehydes have a much longer half-life (i.e., minutes instead of the microseconds-nanoseconds characteristic of most free radicals). Furthermore, the noncharged structure of aldehydes allows them to migrate with relative ease through hydrophobic membranes and hydrophilic cytosolic media, thereby extending the migration distance far from the production site. On the basis of these features alone, these carbonyl compounds can be more destructive than free radicals and may have far-reaching damaging effects on target sites both within and outside membranes. These carbonyl compounds, and possibly their peroxide precursors, react with nucleophilic groups in proteins, resulting in their modification. The modification of amino acids in proteins by products of lipid peroxidation results in the chemical, nonenzymatic formation of a variety of adducts and cross-links collectively named advanced lipoxidation end products (ALEs; Ref. 353). These can be useful indicators of lipoxidative stress in vivo (Fig. 2).
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The amino group of aminophospholipids can also react with carbonyl compounds and initiate some of the reactions occurring in proteins, thus expanding the negative biological effects of such nonenzymatic modification (291). In this context, biological processes involving aminophospholipids can potentially be affected by this process. Among these, the following may be highlighted: asymmetrical distribution of aminophospholipids in different membranes, translocation between and lateral diffusion in the membrane, membrane physical properties; biosynthesis and turnover of membrane phospholipids, and activity of membrane-bound proteins that require aminophospholipids for their function (291).
Thus lipid peroxidation should not be perceived solely in a "damage to lipids" scenario, but should also be considered as a significant endogenous source of damage to other cellular macromolecules, such as proteins and DNA (including mutations). In this way, variation in membrane fatty acid composition, by influencing lipid peroxidation, can have significant effects on oxidative damage to many and varied cellular macromolecules. For example, peroxidized cardiolipin in the mitochondrial membrane can inactivate cytochrome oxidase by mechanisms both similar to hydrogen peroxide and also mechanisms unique to organic hydroperoxides (251). Such an effect could be very detrimental if it results in a greater degree of reduction of other respiratory chain intermediates and consequently increases production of superoxide. Figure 3 depicts the mitochondrial processes producing ROS, the effects of ROS on lipid peroxidation, and some of the consequent lipoxidative damage to mitochondrial DNA and proteins.
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As with many biochemical processes that can be measured in vitro, it is very difficult to assess the rate of lipid peroxidation in vivo, and consequently to be able to compare rates of lipid peroxidation either between species or between treatments. One method is to measure the exhalation rate of hydrocarbons produced from lipid peroxidation. Ethane is one of the end products of n-3 PUFA peroxidation, while pentane is produced from peroxidation of n-6 PUFA. Both are volatile and excreted from the body via respiration, and the rate of their exhalation has been used to assess the in vivo rate of lipid peroxidation (178).
Unsaturated fatty acids are also responsible for the "fluid" nature of functioning biological membranes. It has been shown that it is the introduction of the first double bond to an acyl chain that is primarily responsible for its fluidity at normal physiological temperatures, and introduction of more and more double bonds to the acyl chain has relatively little additional effect on membrane fluidity (39). Thus substitution of fatty acids with four or six double bonds with those having only two (or sometimes three) double bonds will strongly decrease the susceptibility to lipid peroxidation while maintaining membrane fluidity. This phenomenon may be helpful in longevous animals, and in view of membrane acclimation to low temperature in ectotherms, has been called homeoviscous longevity adaptation (267).
The importance of membrane fatty acid composition for understanding aging (described above) and its addition to the standard scenario describing the oxidative-stress theory of aging is illustrated in Figure 4. It emphasizes that membrane fatty acid composition influences 1) metabolic rate (via effects on the physical properties of membrane bilayers) and 2) the degree of oxidative stress and damage to cellular molecules (via the peroxidative susceptibility of fatty acyl chains).
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III. MAMMALS |
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A. Metabolic Rate and Body Size of Mammals
Body size is an important species characteristic that influences almost every aspect of organismal biology (49). As species increase in size, they obviously require more energy, but metabolic rate does not increase in direct proportion to increases in body mass. This is largely because of the constraints of geometry. For every doubling in size, the surface area of an object increases by only 59% and not by 100% (i.e., surface area is related to mass0.67). The basal metabolic rate (BMR) of mammals similarly scales allometrically with body mass, and depending on the particular study, BMR has been reported to only increase by 59–69% with every doubling of body mass (i.e., it is proportional to mass0.67–0.76; Refs. 176, 371). The constraints imposed by such surface limitations are manifest in the following calculation. If a mouse increased in size to that of a horse and its BMR increased in direct proportion to the increase in body mass, the horse-sized mouse would need a surface temperature of 
100°C to rid itself of the heat produced by its BMR (134). Obviously if mammals, ranging in size from shrews to elephants, are to maintain essentially the same body temperature, then surface constraints (for heat loss, nutrient uptake, waste excretion, etc.) mean BMR cannot be directly proportional to body mass (see Ref. 160 for a more detailed discussion; the reader is also referred to the other papers in May 2005 issue of Journal of Experimental Biology "Scaling functions to body size: theories and facts" for a detailed coverage of the scaling of physiological functions to body size). When BMR data are expressed as mass-specific metabolic rate, the metabolic rate of mammals is related to the –0.24 to the –0.33 power of body mass, depending on the particular compilation of mammalian BMR values. In other words, small mammal species have higher mass-specific BMRs than do larger mammals, such that for every doubling in body mass, there is a 15–20% decrease in mass-specific BMR. The compilation of mammalian mass-specific BMR values presented here shows it to be proportional to mass–0.31 (see Fig. 5A; the data for birds in Figs. 5 and 6 will be discussed in sect. IV, and we will only consider the mammalian data in this section).
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70% of the BMR of humans is contributed by internal organs that constitute only 7% of body mass (317). The variation in BMR among species is thus due to variation in both tissue size and tissue metabolic rate (159). Although the relative importance of different cellular processes that constitute tissue metabolic rate varies between tissue types, an interesting finding over the last couple of decades has been that membrane-associated activities constitute a large component of cellular metabolic activity during rest (302). For example, it has been estimated that the maintenance of two transmembrane ion gradients (namely, the Na+ gradient across the plasma membrane and the H+ gradient across the mitochondrial inner membrane) together are responsible for about half of the energy turnover associated with the BMR of mammals (302). Another interesting finding has been that although cellular metabolic rate decreases with increasing body mass in mammals, the relative contribution of various processes to total metabolic activity does not vary. For example, in a comparison of the respiration rate of isolated hepatocytes from eight mammalian species, ranging in size from mice to horses, although hepatocyte respiration rate decreased by 13% for every doubling of species' body mass (i.e., proportional to mass–0.20), the relative contribution of mitochondrial ATP production, mitochondrial proton leak, and nonmitochondrial processes did not change with body mass (288). When BMR of mammals varies with body mass, all subcellular processes that constitute metabolic activity seem to vary in unison (see Ref. 159). B. Maximum Life Span, Body Size, and Lifetime Energy Expenditure of Mammals
Although death may not always be due to the decline in physiological function and/or homeostatic imbalance that occur during aging, MLSP is nevertheless an important species characteristic. Reported MLSPs vary more than 40,000-fold across the animal kingdom, and within mammals by at least 2 orders of magnitude. Generally, MLSP of mammals increases with body size, such that the smallest mammals (shrews) live 1 yr while the reported maximum life span for elephants is 80 yr (50). Thus maximum life span is positively related to body mass in mammals, such that for every doubling of body mass in mammals, there is on average a 16% increase in MLSP (i.e., MLSP is proportional to mass0.22, see Fig. 5B and Ref. 330). As has long been known, both BMR and MLSP are correlated with body mass in mammals, but the MLSP of mammals is not as strongly correlated with body mass as is mammalian BMR. For example, whereas body mass variation can statistically explain 64% of BMR variation, it only explains 35% of the variation in MLSP of mammals (see Fig. 5, A and B).
The core of the rate-of-living theory ascribes species differences in longevity to differential rates of energy expenditure, such that a short maximum life span will be associated with a high mass-specific metabolic rate. When the MLSP data for the 267 mammal species in Figure 5B is plotted against their mass-specific BMR data in Figure 5A, it can be seen that the MLSP of mammals is indeed negatively correlated with BMR (Fig. 6). Although the relationship is statistically significant, it is obvious that there is a huge amount of variation in the relationship. For example, BMR can statistically explain only 26% of the variation in MLSP of mammals, which is less than the predictive power of body size for mammalian MLSP (35%, see Fig. 5B) and also less than the predictive of body size for the BMR of mammals (64%, see Fig. 5A). The variation obvious in Figure 6 is a clear demonstration that the rate-of-living generalization is only a rough predictor of how long a mammal species can maximally live. Its inability to precisely describe the maximum longevity of a mammal suggests other factors are involved in the determination of maximum life span.
The initial version of the rate-of-living theory posited that body mass-related variation in mass-specific BMR and MLSP would mathematically cancel each other, such that different-sized mammals would have a relatively constant mass-specific lifetime energy expenditure (LEE). In other words, it suggested that because the exponents (as a function of body mass) for MLSP and mass-specific BMR have opposite signs and are similar in value, when these two variables are multiplied together, their product (LEE) would show little dependence on body mass. Regardless of the size of the species of mammal, a gram of any animal would be expected to expend approximately the same amount of energy before it dies at MLSP.
When lifetime resting energy expenditure (LEE) is calculated for the mammal data illustrated in Figure 5, A and B, and plotted against body mass (see Fig. 5C), there is a significant negative slope such that, for every doubling of body mass, LEE of mammals decreases by 6% (i.e., it is proportional to mass–0.09). A similar recent compilation of LEE for 240 mammal species similarly showed that there was a small (but statistically significant) negative allometric relationship of LEE with body mass (330). Lifetime resting energy expenditure is thus not constant (as predicted by the rate-of-living theory) but declines with increasing body mass in mammals. Such average trends, however, mask a huge amount of variation. Statistically, body mass only explains 12% of the variation in LEE between mammals, and the average decline with body mass described above (6% for every doubling of mass) is very small compared with the 81-fold range of values across all mammal species.
Of course, mammal species do not spend all their lives resting in a thermoneutral environment, and thus BMR is not a good measure of total lifetime energy metabolism. Physical activity level can account for as little as 20% of the total energy expenditure in sedentary individuals to as much as 50% in active individuals (104). As well, the energetic cost of maintaining body temperature varies with environmental conditions, body size, and insulation. Similarly, foods eaten may variably influence postprandial diet-induced thermogenesis. Field metabolic rate (FMR), which incorporates all of these influences on energy turnover, may be a better measure of lifetime energy requirements and thus test of the rate-of-living theory. FMR has been measured in a wide range of different-sized mammal species by use of the doubly labeled water technique, which can measure the rate of CO2 production in free-living individuals. A recent compilation of such values for 79 mammal species shows that FMR of mammals is proportional to body mass0.73 (252), while another mammalian data set suggests it is proportional to mass0.62 (330). These studies suggest that for every doubling of body size in mammals, there is on average a 17–23% decrease in mass-specific FMR. When lifetime mass-specific FMR was calculated for 49 mammal species (all weighing <4 kg), there was a statistically highly significant negative relationship with species body mass, which is also contrary to the rate-of-living prediction of a relatively constant lifetime energy turnover per gram of animal (330).
A key premise of the combination of the rate-of-living and oxidative-stress theories is that the more energy an organism expends, the more oxidative damage it will accrue, and the quicker it will die. Although it does not fully explain the differences between mammal species, might it be an explanation within the lifetime of an individual mammal, or between individuals within a species? If this explanation was completely adequate, couch potatoes would lead long lives, while those who follow modern trends for promoting good health by exercising regularly would die sooner. In both humans and mice, although voluntary exercise increases metabolic rate, it does not reduce life span (147, 200). Recent studies have revealed that this theory is fraught with exceptions and problems. Regardless of whether data are expressed per gram body mass, per whole animal or per lifetime, a different pattern emerges when intraspecific variation is examined relative to interspecific mammalian comparisons. Intraspecific studies on dogs (333), mice (234, 332), and humans (301) reveal a positive association between maximum life span and mass-specific metabolic rate and a negative relationship between life span and body size. For example, large dog breeds have lower mass-specific metabolic rates than smaller dogs and also show a reduction in maximum life span. However, confounding any definitive conclusions that may be drawn from this finding, other studies focusing within a particular dog breed (107, 283) revealed no clear relationship between body size and life span. The mass-specific metabolic rate of three dog breeds that differ in maximum longevity (from 8.5 to 14 yr) varied in the opposite direction to that predicted by the rate-of-living hypothesis, namely, the smallest and longest-living breed had the highest rate of living (333).
Several intraspecific studies using mice and rats (40, 146, 202, 332, 333) have not observed an inverse relationship between mass-specific metabolic rate and MLSP. Indeed, some of these studies show the opposite of rate-of-living predictions, namely, that mice with high mass-specific metabolic rates tend to live longer than those individuals with low metabolic rates. One study reported that the individual mice with highest BMR had higher levels of mitochondrial proton leak and higher activation of both UCP-3 and adenine nucleotide translocase (333). Uncoupling of mitochondrial activity to promote thermogenesis may also contribute to the combined effects of higher metabolic rate and longevity in smaller dog breeds. Such uncoupled mitochondrial metabolism has been suggested to result in lower generation of ROS but greater generation of heat (36). Clearly, the simplistic concept that metabolic rate and ROS production are directly correlated in a direct linear manner does not appear to be true. Thus insight into the relationship between metabolism and longevity requires a better understanding of mitochondrial function and efficiency.
C. Mitochondrial Free Radical Production and Antioxidant Defenses of Mammals
The decrease in mass-specific BMR with increasing body size in mammals is also associated with a decrease in tissue density of mitochondrial inner membranes (92), the site of mitochondrial ROS production. The in vitro production rates of both superoxide (327) and hydrogen peroxide (326) by isolated liver mitochondria have been measured in mammals ranging in size from mice to cattle. Allometric analysis of these two data sets shows there is a 10% decrease in superoxide production (it is proportional to mass–0.15) and a 18% decrease in hydrogen peroxide production (i.e., proportionality to mass–0.29) per milligram of mitochondrial protein with every doubling of body mass of the mammal species. It is of interest that a separate study of liver mitochondria from mammals ranging from mice to horses (289) reported that the amount of mitochondrial membrane per milligram mitochondrial protein decreases by 7% for every doubling of body mass (i.e., proportionality to mass–0.10). Thus the link between maximum life span and mass-specific metabolic rate in mammals of different body size is associated with appropriate changes in production rates of primary ROS (i.e., superoxide and hydrogen peroxide) by isolated mitochondria, at least in the liver.
However, these studies only included mammal species that followed the rate-of-living theory so the results obtained could also be interpreted as a correlate of that phenomenon. That is, small mammals with short MLSP show high mitochondrial ROS production because their rates of mitochondrial oxygen consumption are higher. Many biochemical reactions apart from oxygen consumption occur at an accelerated rate when the metabolic rate is high, and some of them, unrelated to ROS production, could also, in principle, be responsible for the accelerated aging rate of the short-lived mammals.
It may be that it is enhanced antioxidant defenses that are responsible for the longer MLSP of larger mammals. Thus it was initially surprising for investigators to find that the level of endogenous antioxidant defenses (both enzymatic and scavenger antioxidant systems) of long-living mammal species are lower than those of short-living mammals (2, 17, 285, 312). As can be seen from Table 2, for a number of antioxidant defense systems in a number of tissues, there is a significant negative relationship (or no significant correlation) between the endogenous level of the antioxidant system and MLSP of mammals. This suggests that enhanced antioxidant defenses of mammals are not the reason for the extended longevity of some mammal species but rather are more likely a reflection of the degree of oxidative stress experienced by the tissues of such long-living mammals. These results are compatible with both the notion that endogenous antioxidant defenses are already at optimal levels in animals and that their supplementation, while possibly improving general health of a population (and thus increasing mean longevity), generally has no significant influence on MLSP (see Tables 3 and 4).
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Initially, attempts to understand the basis of the body-size trends in mammalian BMR discounted differences in cell composition between species as an explanation (e.g., Ref. 176). However, nearly 30 years ago, Gudbjarnason et al. (118) published a little-noticed relationship between the DHA content of cardiac phospholipids and resting heart rate (a direct correlate of mass-specific BMR) of mammal species ranging from mice to whales. To our knowledge, this is the first record of a relationship between the fatty acid composition of cellular membranes and metabolic intensity. Later, it was demonstrated that this relationship was not restricted to heart membranes, but was also manifest in the fatty acid composition of the membranes of other important tissues of mammals (69, 164). For every doubling of body mass of mammal species there is a 12–24% decrease in the relative DHA content of cellular membranes of mammals (164), which is not dissimilar to the 19% decrease in their mass-specific BMR. The cellular membranes of small mammal species are more polyunsaturated than those of large mammal species, and in mammals, this body size-related variation in membrane DHA content is the dominant influence on the body size-related variation in membrane composition. A substantial number of membrane-associated processes have also been shown to vary with body size of mammals and have been related to this difference in membrane composition. Indeed, it has been proposed that it is the difference in membrane fatty acid composition that is causal to the differences in BMR, and this proposal has been called the membrane-pacemaker theory of metabolism (refer to Refs. 156, 158–160 for detailed explanation). This theory notes that the cellular membranes of animals with high rates of mass-specific metabolism have high degrees of polyunsaturation and proposes that such high membrane polyunsaturation results in physical properties of membrane bilayers that speed up the activity of membrane-associated proteins and consequently the metabolic rate of cells, tissues, and the whole animal. While this theory is supported by a large body of correlational evidence, there is also important experimental evidence (e.g., from "species cross-over" studies) that substantiates the theory (93, 341, 374).
At the same time that the fatty acid composition of membranes from a variety of mammalian tissues was related to variation in BMR (69, 158, 159), Pamplona and colleagues (266, 271, 272, 275–277) also measured the membrane fatty acid composition of a number of mammalian tissues and related it to MLSP of the particular species. Thus the novel and unexpected finding that the composition of membrane bilayers varies systematically in mammals provided a link between metabolic rate and maximum life span of mammalian species. In agreement with this, it was demonstrated that in long-lived mammals, the low degree of membrane fatty acid unsaturation was accompanied by a low sensitivity to in vivo and in vitro lipid peroxidation (269, 272, 275–277) and a low steady-state level of lipoxidation-derived adducts in both tissue and mitochondrial proteins of skeletal muscle, heart, liver, and brain (267, 268, 274, 277, 292, 307). These findings were consistent with the negative correlation between MLSP and the sensitivity to lipid autoxidation of mammalian kidney and brain homogenates that had been described some years earlier (72).
If the fatty acid composition of membrane phospholipids is known, it is possible to calculate a peroxidation index (PI; see sect. IID), which is a number that expresses the calculated susceptibility of the particular membrane to lipid peroxidative damage. When this is done for both liver mitochondrial phospholipids and skeletal muscle phospholipids from a range of different-sized mammal species, it can be seen that there is a strong inverse relationship between PI and MLSP of mammals (see Fig. 7). Interestingly, the relationship between liver mitochondrial phospholipid PI and MLSP has a similar slope to that between skeletal muscle PI and MLSP. The liver mitochondrial membrane PI of mammals is proportional to their MLSP–0.40, which means that a 24% decrease in their peroxidative susceptibility is associated with every doubling of maximum life span. For skeletal muscle membranes, the corresponding value is that a 19% decrease in peroxidative susceptibility is associated with every doubling of MLSP in mammals (i.e., muscle PI is proportional to MLSP–0.30).
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We know almost nothing of the ways in which the regulatory mechanisms controlling membrane fatty acid composition differ between species. With the assumption of the dietary availability of PUFAs, the control of membrane fatty acid composition will reside in the synthetic pathways for fatty acyl chains, the pathways of de novo synthesis of phospholipids, and also in the rapid membrane-remodeling pathways that involve enzymatic deacylation/reacylation cycles. Because PUFA cannot be synthesized de novo by higher animals, they are essential components of t
