Bioenergetics in aging

Biochimica et Biophysica ,4cta, 726 (1983) 4 1 - 8 0

41

Elsevier B i o m e d i c a l Press

BBA 86096

BIOENERGETICS

IN AGING

R I C H A R D G. H A N S F O R D

Laboratory of Molecular Aging, National Institute on Aging, Natwnal Institutes of Health, Gerontology Research Center, Baltimore City Hospitals, Baltimore, MD 21224 (U.S.A.)

(Received J u n e 23rd, 1982)

Contents 1.

Introduction ............................................................................

42

II.

Approaches and parameters measured ..........................................................

42

III.

Substrate o x i d a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. P y r u v a t e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. T r i c a r b o x y l a t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. 2 - O x o g l u t a r a t e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Succinate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. M a l a t e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. G l u t a m a t e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. A c y l c a r n i t i n e a n d fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. A s c o r b a t e ( c y t o c h r o m e c oxidase) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. C o m p o s i t i o n of the electron-transport c h a i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53 53 55 56 56 57 57 58 59 60

IV.

Phosphorylation ......................................................................... A. F r A T P a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. C r e a t i n e k i n a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

60 60 61

V.

O x i d a t i v e p h o s p h o r y l a t i o n in i n t a c t tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62

VI.

M i t o c h o n d r i a l p e r m e a b i l i t y in a g i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

64

VII.

M i t o c h o n d r i a l m e m b r a n e c o m p o s i t i o n in aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

66

VIII. M i t o c h o n d r i a l m e m b r a n e lipid p e r o x i d a t i o n in a g i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67

IX.

M o r p h o l o g y in a g i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70

X.

Bioenergetics of a g i n g insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

72

XI.

Some conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75

Acknowledgements ............................................................................

76

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

76

0 3 0 4 - 4 1 7 3 / 8 3 / 0 0 0 0 - 0 0 0 0 / $ 0 3 . 0 0 © 1983 Elsevier Science Publishers

42 I. Introduction It is a common observation that aging animals tend to be less energetic. It is therefore understandable that gerontologists have long focused attention on energy-transducing pathways in their search for the biochemical lesions which underlie the observable physiological decrements of old age. To some extent this rationale may be naive, as behavioral characteristics of the animal surely have a prime influence on movement and thus on a large part of energy demand. Nevertheless, the pathways of catabolic metabolism provide the wherewithal, and must place an upper limit on the intensity of energy transduction that can be maintained, and energy demand that can be met. This review deals with the impact of aging on these catabolic pathways, and will focus on studies of oxidative metabolism as this generates the bulk of the ATP in most mammalian tissues. Glycolytic generation of ATP would obviously be pertinent, expecially in white muscle fibers, tumor cells and erythrocytes, but is omitted for reasons of space. Equally, the use of cellular ATP by the actomysin ATPase and ion-transporting ATPases would be a very valid concern of an article on bioenergetics in aging, but is omitted because the reviewer has no special insight in that area. The proton-translocating (F1) ATPase of the mitochondrion is included as being part of the pathway of ATP generation, under physiological conditions. Much of this review will be a description of changes in mitochondrial metabolism that occur with age. Some of this is a collection of phenomena, but an attempt will be made to provide insight into mechanism at the level of enzymology or membrane transport, whenever this is accessible. Equally, age-linked changes described in work with isolated proteins or organelles will be scrutinized in the context of what is known of the physiology of the organ. Sometimes decrements found with isolated mitochondria would be quite incompatible with life, and can surely be ascribed to damage during preparation. Indeed, one theme of this article will be that the biochemist should go first to the physiology of the animal, identify age-linked changes and then try to provide a mechanism at the biochemical level, rather than make an enzymological finding, and then search for a significance.

A second theme is that membranes are central to the study of the impact of aging on bioenergetics. This arises not only because the metabolism of the mitochondrion occurs within a membrane of highly restrictive permeability properties [1,2], which has the effect of erecting barriers across the pathways of metabolism, but also because, as articulated by Mitchell [3,4], much energy transduction is impossible without membranes. Changes in membrane permeability properties appear to occur quite widely with aging and will be stressed. These may arise as a consequence of increased peroxidation of lipids, and an associated general loss of membrane fluidity [5]. However, whether the mitochondrion is the Achilles heel of the cell, promoting chaos by the peroxidation of inner membrane components and especially the components of the mitochondrial ribosomes [6,7], is by no means so sure and will be discussed. For this article, aging is taken to mean the time-dependent changes that occur after sexual maturity has been reached, and is used as a synonym for senescence. This is a matter of convenience and should not obscure the fact that growth, maturity and senescence may all be coded for at the nucleic acid level, and represent a continuum of development. Much excellent work on the development of biological oxidations in the neonate is thus ruled out, and would be worthy of a separate review. For recent reviews devoted more to theories on the mechanism of aging, the reader is referred to the articles by Miquel et al. [7], Shock [8], Sohal [9] and Zs-Nagy [10]. II. Approaches and parameters measured Perhaps the most straightforward approach to the question of whether tissues from senescent animals have a decreased capacity for oxidative phosphorylation is to isolate a mitochondrial fraction and investigate phosphorylation in vitro. This approach has been widely applied to a variety of tissues ever since the pioneering work of Weinbach and Garbus [11] showing a decreased ability of liver mitochondria from old rats to phosphorylate ADP with 3-hydroxybutyrate as substrate. In general, the results of studies of State 3 respiration have been interpreted in the light of the statement

43 of Chance and Williams [12] that State 3 respiration is limited by the activity of the respiratory chain. This has led to the relative neglect of substrate permeation and dehydrogenation as possible rate-limiting factors, and sites of age-linked changes. In fact, State 3 rates of 02 uptake may reflect either respiratory chain or substrate permeation or substrate dehydrogenation or adenine nucleotide translocase activity, depending on the substrate and the tissue of origin (Refs. 2 and 13, and this article). The loci of age-linked decrements in activity are correspondingly diverse, and this will be discussed below in relation to results with different oxidizable substrates. State 4 [12], or controlled respiratory rates, by contrast, provide evidence of the rate of dissipation of the mitochondrial proton electrochemical gradient, A~H+ (see Refs. 3 and 4), by cation cycling [14] or possibly by a dielectric breakdown of the membrane [15]. In addition, when correctly measured after the completion of a cycle of ADP phosphorylation [12], State 4 rates have a component owing to extramitochondrial ATPase, especially if the incubation medium contains Mg 2÷. The latter component can be quite large, especially in muscle preparations, and can be negated by the use of atractyloside, as an inhibitor of the adenine nucleotide translocase [16]. In most cases, State 4 rates of 02 uptake have been found to be unchanged with aging of the animal, when measured in heart [17-20], liver [21] and brain [18,21] mitochondria. Exceptions include a report by Nohl et al. [22] of a small increase in the State 4 oxidation of glutamate plus malate in heart mitochondria from senescent rats, and another indicating a small decrease, with brain mitochondria [23]. The general consensus that State 4 rates are unchanged with aging provides good evidence against any generalized deterioration of membrane structure and function, to the point that electrophoretic ion movements become elicited. Agelinked changes in membrane permeability undoubtedly do occur (see below), but evidently there is no overall 'leakiness,' as the movement of ions in a nonelectrically compensated fashion would lead to a lessening of the A/~H., and a corresponding rise in the rate of State 4 respiration [3,14,15]. There have been no direct measurements of A/~H+ or the t~/2 for proton equilibration across the

membrane of mitochondria from aging animals. Many investigators have presented respiratory control ratios as an index of intactness of mitochondrial preparations from young and old animals. In general, there is no diminution with age [17-21,24] or a diminution only to the extent that the State 3 rate of oxidation of a specific substrate is depressed [ 17,18,21]. This restates the conclusion of the paragraph above, namely, that in general State 4 rates of 02 uptake are unchanged. Measurements of P / O (usually in the form A D P / O ) ratios are a less sensitive criterion of intactness, in the sense that mitochondria showing no respiratory control (respiratory control ratio = 1) may nevertheless give an appreciable, albeit diminished, P / O ratio [25]. There are many excellent studies showing undiminished P / O ratios when mitochondria from various tissues of senescent animals are compared with their counterparts from young adults [17-21]. One startling exception is the study by Nohl et al. [22] which shows diminished P / O ratios for the oxidation of glutamate plus malate, 3-hydroxybutyrate and succinate by heart mitochondria from senescent rats. Changes in respiratory control ratio are also reported, though they are quite modest in view of the changes in P / O ratio. Nohl et al. [22] link this decreased efficiency of energy transduction to a structural change in the inner mitochondrial membrane, citing as evidence a shift in the temperature at which Arrhenius plots for several membrane-associated activities show discontinuities, and the results of spin-label studies. This will be discussed further below. It is not clear why Nohl et al. [22] find this rather pronounced decrease in the efficiency of energy coupling in heart mitochondria from senescent rats, whereas several other convincing studies, also in heart, show no such lesion [17-20,24]. It is possible to argue that there exists in vivo a fraction of damaged mitochondria which may be discarded during the making of a mitochondrial preparation. Indeed, Murfitt and Sanadi [26] have provided evidence that age-linked deterioration of function (decrease in respiratory control ratio) can clearly be shown in only one of two fractions of heart mitochondria, separated on the grounds of density. It is possible that this fraction is discarded during other preparations, conceivably as a result of using the proteolytic

TABLE I MITOCHONDRIAL SUBSTRATE OXIDATION IN SENESCENCE The significance of age-linked changes ( P < 0.05) is indicated thus (s) when claimed by the author; other decrements not so marked may also possibly be significant; n.s., not significant, P > 0.05; decrements are cited as the percentage by which the value in Tissue of origin, species, strain, sex, age

Substrate Pyruvate plus malate

Brain Rat, Fisher 344, cf. 6 and 28-32months

Rat, Charles River, M, cf. 12 and 24 months Synaptic

Nonsynaptic

Palmitoylcarnitine

2-Oxoglutarate

ADP/Ounchanged RCRdecreased State 3 decreased 34% (s)

Glutamate

ADP/Ounchanged RCRdecreased State 3 decreased 32% (s)

RCRunchanged State 3 unchanged RCRunchanged State 3 decreased 24%

Rat, Sprague-Dawley, M, cf. 12 and 24months Synaptic Nonsynaptic

Mouse, C57 BL/6J, M, cf. 9-12 and 23-26 months

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 unchanged

Heart Rat, Wistar, cf. 12-24 and 24-27 months

Rat, Fisher 344, M, cf. 8-9 and 24 months

A D P / O unchanged RCR unchanged State 2 unchanged

A D P / O unchanged RCR decreased State 3 decreased 18% (s)

Hamster, M, cf. 1, 3, 10 and 13-14 months

Hamster, M, cf. 3 and 15-16months

Rat, Wistar, M, of. 6 and 24 months

Rat, cf. 3 and 24 months

A D P / O unchanged RCR unchanged State 3 unchanged

RCR unchanged State 3 unchanged

RCRdecreased Stated 3 decreased 25% (s)

A D P / O unchanged RCR unchanged State 3 unchanged

senescence is lower than the value in the young animal. RCR, respiratory control ratio; M, male. a Respiration was stimulated by 2,4-dinitrophenol (DNP) instead of ADP. b These results are quoted by kind permission of B. Ashour. References Succinate

Glutamate plus malate

ADP/Ounchanged RCRdecreased State 3 decreased 34%

ADP/Ounchanged RCRdecreased State 3 decreased 27% (s)

3-Hydroxybutyrate

18

RCRdecreased State 3 decreased 33% (s) RCRunchanged State 3 decreased 25%

23

RCRunchanged State 3 unchanged RCRunchanged State 3 decreased 28%

81 81

21

A D P / O unchanged RCR unchanged State 3 unchanged

ADP/Ounchanged RCRunchanged State 3 unchanged

• A D P / O unchanged RCR decreased State 3 decreased 18% (s)

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 unchanged

19

A D P / O unchanged RCR decreased State 3 decreased 23% (s)

17

24

36

A D P / O unchanged RCRdecreased 15% State 3 unchanged RCR unchanged State 3 unchanged

A D P / O decreased RCR decreased State 3 decreased 30%

23

A D P / O decreased RCR decreased State 3 unchanged

28

A D P / O decreased' RCR decreased State 3 decreased 19%

22

TABLE I (continued) Tissue of origin, species, strain, sex, age

Rat, Fisher 344, cf. 6 and 28-32 months

Rat, Sprague-Dawley, M, cf. 10 and 24 months

Substrate Pyruvate plus malate

Palmitoylcarnitine

A D P / O unchanged RCR decreased State 3 decreased 49% (s)

A D P / O unchanged RCR decreased State 3 decreased 33% (s)

2-Oxoglutarate

Glutamate

A D P / O unchanged RCR decreased State 3 decreased 68% (s) A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 unchanged

Kidney Rat, Wistar, cf. 12-24 and 24-27 months

Rat, Wistar, M, cf. 6 and 24 months

State 3 decreased 307o (s)

State 3 decreased 54% (s)

State 3 decreased 35% (s)

Liver Rat, Wistar, cf. 12-14 and 24-27 months

Rat, M, cf. 4-6 and 24-26 months Hamster, M, cf. 13-14 and 20 months

Rat, BHE, cf. 1-2 and 10-17 months Rat, Wistar, cf. 1-2 and 10-17 months Mouse, C57 BL/6J, M, cf. 9-12 and 23-26 months

State 3 decreased 197o (s) A D P / O unchanged State 3 decreased 22% DNP decreased a 25% (s)

DNP decreased a 22% (s)

DNP decreased a 25% (s)

DNP unchanged a

DNP unchanged a

DNP unchanged a

Striated muscle Rat, Fisher 344, M, cf. 8-9 and 24 months

Hamster, M, cf. 1, 3, 10, 13-14 and 20 months

Hamster, M, cf. 3 and 15-16 months

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 decreased

14% Rat, Wistar, M, cf. 8 and 25 months

RCR decreased 25% (n.s.) State 3 decreased 16% (n.s.)

RCR decreased (29% n.s.) State 3 unchanged

RCR unchanged State 3 unchanged

References Succinate

Glutamate plus malate

3-Hydroxybutyrate

A D P / O unchanged RCR unchanged State 3 unchanged

ADP/Ounchanged RCRdecreased State 3 decreased 54% (s)

ADP/Ounchanged RCRdecreased State 3 decreased 59% (s)

A D P / O unchanged RCR unchanged State 3 unchanged

18

20

A D P / O unchanged RCR unchanged State 3 unchanged State 3 decreased 40% (s)

19

60

A D P / O unchanged RCR unchanged State 3 unchanged State 3 unchanged

19

63

A D P / O unchanged State 3 unchanged

A D P / O unchanged State 3 decreased 7%

24

62

62 A D P / O unchanged RCR unchanged State 3 unchanged

ADP/Ounchanged RCRdecreased State 3 decreased 12% (s)

21

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR decreased State 3 decreased 18% (s)

17

A D P / O unchanged RCR unchanged State 3 unchanged

A D P / O unchanged RCR unchanged State 3 decreased 20%, cf. 1 and 14 months

24

A D P / O unchanged RCR unchanged State 3 decreased 17%

36

RCR decreased 25% (n.s.) State 3 decreased 15% (n.s.)

unpublished work (this laboratory) b

TABLE II IMPACT OF SENESCENCE ON ENZYMES OF CATABOLISM Homo, measurements in whole-tissue homogenates; Mito, measurements in mitochondrial preparations. M, male; F, female, a The % change was approximated by the reviewer from graphical presentations in the original paper. Tissue of origin, species, strain, sex, age

Enzyme activity Pyruvate dehydrogenase complex (EC 1.2.4.1 +2.3.1.12 + 1.6.4.3)

Citrate synthase (EC 4.1.3.7)

NAD-isocitrate dehydrogenase (EC 1.1.1.41)

unchanged Homo

decreased 42% Homo

2-Oxoglutarate dehydrogenase complex (EC 1.2.4.2)

Glutamate dehydrogenase (EC 1.4.1.3)

Brain Rat, Sprague-Dawley, M, cf. 7 and 29 months Rat, CD, M, cf. 3 and 24 months Rat, Sprague, Dawley, M, cf. 14 and 32 months

decreased 69% a Mito decreased a 78% Mito

cf. 4-5 and 32 months Rat, Wistar, M, cf. 8 and 20 months

decreased 57% Mito

Rat, Sprague-Dawley, M, cf. 3 and 24 months

Rat, Sprague-Dawley, M, cf. 3 and 24 months

decreased 21% (nonsynaptic). decreased 43% (synaptic) Mito unchanged (synaptic and nonsynaptic) Mito

Mouse, C5 BL/6J, M, cf. 9-12 and 23-26 months

Rat, Wistar, MF, cf. 6 and 20 months Rat, CFN, MF, cf. 3 and 24 months Rat, Sprague-Dawley, cf. 3 and 12 months

Heart Rat, Wistar, M, cf. 12-14 and 24-27 months Rat, Sprague-Dawley, M, cf. 6 and 24 months Rat, Sprague-Dawley, M, cf. 5 and 26-29 months

unchanged Mito

decreased 50% Mito a

increased 15% Homo a unchanged Homo decreased 46-55% Mito

References Succinate dehydrogenase (EC 1.3.99.1)

3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)

Cytochrome oxidase (EC 1.9.3.1)

decreased 16% H o m o

94

47

decreased a 35% Mito decreased a 51% Mito

decreased a 30% H o m o decreased a 47% H o m o

93 93

48

81

23

unchanged Mito Homo

21

49

263

264

decreased 16% Mito unchanged Mito

64

decreased 28% H o m o 27% Mito decreased 17% Mito

19

94

TABLE II (continued) Tissue of origin, species, strain, sex, age

Rat, Wistar, M, cf. 6 and 24 months

Enzyme activity Pyruvate dehydrogenase complex (EC 1.2.4.1 +2.3.1.12 + 1.6.4.3)

Citrate synthase (EC 4.1.3.7)

NAD-isocitrate dehydrogenase (EC 1.1.1.41)

2-Oxoglutarate dehydrogenase complex (EC 1.2.4.2)

unchanged Mito

decreased 18% Homo

decreased 16% Homo

decreased 19% Homo

increased 25% Mito a

decreased 20% Mito a

Rat, Wistar, MF, cf. l0 and 21 months

Glutamate dehydrogenase (EC 1.4.1.3)

Liver Rat, Wistar, M, cf, 12-14 and 24-27 months Rat, Sprague-Dawley, M, cf. 6 and 24 months Mouse, C57 and C3H, MF, cf. 6 and 30 months

Rat, CFN, F, cf. 3 and 24 months

increased 94% Homo a

Rat, Sprague-Dawley, M, cf. 7 and 29 months Rat, Sprague-Dawley, M, cf. 112 and 495 g

unchanged Homo

Rat, Wistar, M, cf. 8 and 20 months

decreased 39% Mito

Mouse, C57 BL/6J, M, cf. 9-12 and 23-26 months

Rat, Wistar, MF, cf. 6 and 21 months Kidney Rat, Wistar, M, cf. 12-14 and 24-27 months Rat, Sprague-Dawley, M, cf. 6 and 24 months Rat, Sprague-Dawley, M, cf. 7 and 29 months Rat, Wistar, M, cf. 6 and 24 months

Striated muscle Rat, Wistar, MF, cf. 5 and 15-30 months White muscle Red muscle

unchanged Mito

unchanged Mito

References Succinate dehydrogenase (EC 1.3.99.1)

3-Hydroxyacyl-CoA dehydrogenase (EC !.1.1.35)

Cytochrome oxidase (EC 1.9.3.1)

41

decreased decreased 33% Mito 27% Homo

50

increased 170% Mito a

unchanged Mito

19

64

increased 36% Mito decreased 70% (M) a 72% (F) a Homo

33

263

unchanged Homo

94

42

48

decreased 19% Mito 20% Homo

265

increased 140% Mito a

unchanged Mito

19

64

decreased 31% Mito unchanged Homo decreased 177o Mito

decreased 35% Mito decreased 53% Mito

21

94

unpublished work (this laboratory)

34

52 TABLE II (continued) Tissue of origin, species, strain, sex, age

Enzyme activity Pyruvate dehydrogenase complex (EC 1.2.4.1 +2.3.1.12 + 1.6.4.3)

Citrate synthase (EC 4.1.3.7)

NAD-isocitrate 2-Oxoglutarate Glutamate dehydrogenase dehydrogenase dehydrogenase (EC 1.1.1.41) complex (EC 1.4.1.3) (EC 1.2.4.2)

Hamster, M, cf. 3 and 13-14 months Rat, Wistar, M, cf. 3 and 28-36 months Soleu~ Diaphragm Extensor digitorum longus Human, 20-65 years Vastus lateralis

decreased 30% Homo unchanged Homo unchanged Homo unchanged Homo

Human, cf. 13-48 and 65-78 years Vastus iateralis Rat, Wistar, M, cf. 6 and 24 months Soleus Diaphragm ;~

unchanged

decreased 36% Homo decreased 25% Homo

enzyme, Nagarse. However, the reviewer feels strongly that measurements of the efficiency of oxidative phosphorylation in intact organs or tissues place a stringent limit on how large such a damaged fraction of mitochondria could be, and this is discussed below. As an alternative to measuring flux through a metabolic pathway directly, it is also possible to infer What the maximum flux might be, t h r o u g h measurement of nonequilibrium enzymes, in vitro, under Vmax conditions. Such an approach has been advocated by Crabtree and Newsholme [27]. It is perhaps particularly appropriate to the study of aging in that the potential activity of a tissue can be determined rather easily and thus in a large groul~ of animals, and in that age-linked decrements are normally reflected in changes in Vmax (see, ~e.g., Refs. 28 and 29). It demands, however, the identification of nonequilibrium reactions in a

decreased 33% Homo decreased 36% Homo

decreased 56% Homo decreased 19% Homo

pathway, and this has by no means been understood in the aging field. Thus, for the tricarboxylate cycle, the NAD'isocitrate dehydrogenase (EC 1.1.1.41) and 2-oxoglutarate dehydrogenase (EC 1.2.4.2) reactions are nonequilibrium, as is perhaps the citrate synthase (EC 4.1.3.7) reaction [30,31]. Succinate dehydrogenase (EC 1.3.99.1), the mitochondrial enzyme most widely studied as a function of age [32-35], by contrast catalyses a near-equilibrium reaction (see Ref. 13), though the overall succinoxidase reaction is nonequilibrium, as a consequence of the lack of equilibrium at the cytochrome c oxidase step. Thus, an age-linked decrement in the activity of succinate dehydrogenase, as measured by the reduction of artificial electron acceptors [32-35], will probably not translate into a proportional decrease in flux through the tricarboxylate cycle, whilst such a decrement in 2-oxoglutarate dehydrogenase activity will do so.

53

References Succinate dehydrogenase (EC 1.3.99.1)

3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)

Cytochrome oxidase (EC 1.9.3.1)

24

decreased 16% Homo

210

increased 45% Homo a

increased Homo

266

increased 260% decreased 52% Homo decreased 19% Homo The following sections present the results of aging studies with a variety of mitochondrial substrates, with some thoughts on the rate-limiting reactions involved. Rates of 0 2 uptake are presented in Table I, and the activities of the appropriate nonequilibrium enzymes are presented in Table II. III. Substrate oxidation

IliA. Pyruvate There are several convincing papers showing no change in State 3 0 2 uptake with senescence in mitochondria from rat and hamster heart [17,20, 24,28,36] and hamster skeletal muscle [24]. By contrast, Chiu and Richardson [18] in an equally convincing study show a 49% decrement with age in heart muscle preparations. The reason for the discrepancy is not clear, but it may reflect the

129

decreased 54% Homo decreased 45% Homo

29

greater age of the animals used by Chiu and Richarckson (28-32 months for the senescent group, compared to 24 months used by Chen et al. [17] in studies with the same rat, the Fisher F344). If so, the decline in the ability to oxidize pyruvate occurs later in the life of the animal than the decline reported by others in fatty acid [17,28] and glutamate plus malate [17] oxidations. It is not clear what rate limits pyruvate oxidation in muscle mitochondria. Table III presents State 3 rates of 0 2 uptake with pyruvate and glutamate plus malate as substrates, separately and in combination. It is seen that with rat heart mitochondria there is a modest degree of summation, suggesting that pyruvate oxidation alone is not limited by phosphorylation or the respiratory chain, but presumably by pyruvate dehydrogenase or pyruvate permeation on the p y r u v a t e / H + symport system [37,38]. On the other hand, some contribution to

54

TABLE III MAXIMAL RATES OF MITOCHONDRIAL 02 UPTAKE WITH COMBINATIONS OF N A D - L I N K E D SUBSTRATES IN SENESCENCE Rates are given as mean State 3 rate of 02 uptake+S.E. (number of preparations), a The rate of oxidation of this substrate combination is Significantly ( P < 0.05) greater than that of pyruvate plus malate, b The rate of oxidation of this substrate combination is significantly ( P < 0.05) greater than that of palmitoylcarnitine (heart) or glutamate plus malate (striated muscle) alone, n.s., not significant, in the appropriate comparison. Results for heart and liver mitochondria are unpublished results of F. Castro and R. Hansford; results for striated muscle mitochondria are unpublished results of B. Ashour and R. Hansford. Substrate combination

Rate of 02 uptake (ngatom O / m i n per mg protein) Heart

A. 25°C (1) 5 mM L-glutamate+5 mM L-malate (2)

(3)

(4)

(5)

2.5 mM pyruvate+ 1 mM L-malate 20/,tM palmitoyl-L-carnitine + 20 #M serum albumin+ 1 mM malate 5 mM L-glutamate+5 mM L-malate + 2.5 mM pyruvate

(2)

(3)

(4)

(5)

2.5 mM pyruvate+ 1 mM L-malate 20 p,M pamitoyl-L-carnitine + 20 #M serum albumin+ 1 mM L-malate 5 mM L-glutamate+5 mM L-malate + 2.5 mM pyruvate

5 mM L-glutamate + 5 mM L-malate + 20/xM pamitoyl°L-carnitine + 20 # M serum albumin

Striated muscle

6 months

24 months

6 months

24 months

6 months

24 months

258±6 (5)

264± 13 (5)

90+20 (3)

73+3 (3)

447+20 (4)

382±26 (4)

391±15 (5)

369±16 (5)

48+10 (3)

40±1 (3)

445±15 (6)

372±20 (5)

357±15 (5)

313±13 (5)

87±19 (3)

69±3 (3)

313+12 (6)

338±25 (8)

449+17 a (5)

435±8 a (5)

84±23 (3) n.s.

73±4 (3) n.s.

506±34 (3) n.s.

476±38 (3) n.s.

408± 14 b (5)

92±17 (3) n.s.

75±5 (3) n.s.

538+40 b (3)

512±36 b (3)

638± 15 (5)

619+25 (5)

182±44 (3)

126+9 (3)

759 + 22 (5)

749 ± 38 (5)

80 ± 17 (3)

72 ± 2 (3)

687±21 (5)

637±25 (5)

135±34 (3)

95± 10 (3)

5 mM L-glutamate+5 mM L-malate + 20 # M palmitoyl-L-car453±20 b nit±he + 20 # M serum albumin (5)

B. 37°C (1) 5 mM L-glutamate+5 mM L-malate

Liver

808±20

763±10

181±39

155+7

(5)

(5)

(3)

(3)

n.s.

n.s.

n.s.

n.s.

837± 34 b (5)

789±22 b (5)

175±43 (3)

132±8 (3)

n.s.

n.s.

55 rate limitation by the respiratory chain cannot be excluded, as the higher rate in the presence of both substrates may reflect a higher steady-state level of reduced cytochrome c, and hence of flux through the limiting cytochrome c oxidase [39]; i.e., the nonequilibrium cytochrome oxidase does not operate under Vma x conditions. In liver, there is some evidence for a decline in pyruvate oxidation in hamsters over the age range 13-20 months [24]. On the other hand, results from aged mice unequivocally show that pyruvate oxidation is unimpaired up to 23-26 months of age [21]. In liver mitochondria, the State 3 rate of pyruvate oxidation is much lower than that of other NAD-linked substrates, and so rate limitation can be ascribed with some confidence to pyruvate permeation or pyruvate dehydrogenase. Pyruvate transport is inactive relative to the transport of most other mitochondrial substrates, and is thus a plausible site of rate limitation [2]. It has been implicated as contributing to rate limitation of gluconeogenesis in isolated hepatocytes, in experiments involving the inhibitor a-cyano-4-hydroxycinnamate [40]. There is some conflict over the impact of aging on pyruvate oxidation by mitochondria from brain. Chiu and Richardson [18] report a substantial decline for the senescent Fisher rat, whereas Weindruch et al. [21] find no such decline in senescent mice. A partically interesting study is that of Deshmukh et al. [23] which compares properties of synaptosomal and nonsynaptosomal mitochondria from rat brain. They find a decreased ability to oxidize pyruvate in both cases, but an undiminished activity of pyruvate dehydrogenase, when this is measured as the total, i.e., after dephosphorylation of the inactive enzyme. The authors reach the conclusion that pyruvate permeability, rather than pyruvate dehydrogenase activity, may limit flux in this system. If so, the age-linked decrement must reside at the level of the mitochondrial transport process. Interestingly, the unchanged pyruvate dehydrogenase activity rules out a trivial explanation of the diminished flux, namely, an increased contamination of the mitochondrial preparation with inactive proteih in old age. In fairness, it should be pointed out that some of these changes may be developmental, as the largest decline in pyruvate oxidation by syn-

aptic mitochondria occurs between 3 and 12 months of age. In the nonsynaptic population there is a further decrease in activity from 12 to 24 months, though the statistical significance of this is unclear. It is noted that there is no evidence for a decline in pyruvate dehydrogenase activity with senescence in any tissue studied. In addition to the invariant activity noted by Deshmukh et al. [23] in brain, Hansford [41] also reported no change in heart mitochondria from old rats, and Hoffmann et al. [42] found no change in whole liver pyruvate dehydrogenase content. The latter findings did include dramatic decreases in enzyme activity in epididymal adipose tissue, but this was probably a response to obesity rather than senescence in this rat (Sprague-Dawley), especially since the older animals were only slightly more than 1 year old. Pyruvate dehydrogenase catalyses a clearly nonequilibrium reaction, committing pyruvate carbon to complete oxidation or lipogenesis, and is thus a central control point. In this light, the lack of change reported in these aging studies [23,41,42] becomes noteworthy.

IIIB. Tricarboxylates The reviewer is not aware of any studies of the effect of aging on the mitochondrial oxidation of added tricarboxylates. Such oxidation is definitely limited by permeability in muscle mitochondria (see Ref. 43 for rates of substrate oxidation), and maybe in liver, depending on the availability of malate [44]. In brown adipose tissue [45] and in kidney [60] there is evidence that the dehydrogenase is limiting, at least at low substrate concentrations, on the basis of an activation of State 3 02 uptake by low levels of Ca 2÷, a proven activator of the purified NAD-isocitrate dehydrogenase [46]. The reaction catalysed by NAD-isocitrate dehydrogenase is nonequilibrium, and this enzyme has a major role in the regulation of flux through the tricarboxylate cycle (for a review, see Ref. 13). Enzyme activity has been shown to decline slightly in rat heart muscle homogenates in old age [41], and to a greater extent in homogenates of diaphragm and the soleus [29]. The degree of loss of activity is the same for citrate synthase, NAD-isocitrate dehydrogenase and 2-oxogiutarate dehydro-

56 genase in heart [41 ], raising the possibility that the mitochondrial content is simply 18% less, on a wet weight basis, perhaps as a consequence of an increased content of connective tissue. In brain, NAD-isocitrate dehydrogenase declines substantially and significantly with age, whether measured in homogenates [47] or isolated mitochondria [48,49]. In liver, a large and significant decline was also found in isolated mitochondria [48]. Other data have been reported [50], but the absolute activities are too low to be able to support known metabolic fluxes. This is probably a consequence of the omission of ADP, required to activate and stabilize the enzyme [51], during the extraction and assay [50]. Thus, the response of NAD-isocitrate dehydrogenase to aging is quite tissue specific. The results obtained with brain and liver mitochondria would suggest a substantial decrease in flux through this reaction in old age. On the other hand, the unchanged rate of pyruvate oxidation in isolated heart [17,20,28] and insect flight muscle [52] mitochondria argues for an undiminished flux through NAD-isocitrate dehydrogenase in mitochondria from those tissues, in view of the inability of tricarboxylates to escape from those mitochondria [53-55]. 111C. 2-Oxoglutarate 2-Oxoglutarate dehydrogenase controls the flux through the second segment of the tricarboxylate cycle (i.e., from 2-oxoglutarate to malate [56,57]) and a measurement of the activity of this enzyme can be advocated as the best single way of characterizing the maximal potential of a tissue to carry Out the tricarboxylate cycle. Surprisingly, it is very seldom measured in aging work. In one report, this activity is shown to decline slightly in homogenates of heart muscle and diaphragm, and to a much greater extent in the soleus [29]. The soleus was chosen in this study as being a red, highly oxidative muscle. It may not be correct to generalize these results to other striated muscle, as the hind limbs may suffer particularly from atrophy in the artificial situation of the caged laboratory rat [58]. 2-Oxoglutarate oxidation by isolated mitochondria may be limited in State 3 by the dehydrogenase. Certainly this is true of muscle (heart and

striated) mitochondria oxidizing low concentrations (0.5 or 1 mM) of 2-oxoglutarate, for under these conditions 02 uptake is stimulated by micromolar concentrations of free Ca 2÷ [59, 60]. Ca 2+ has been shown to activate purified 2-oxoglutarate dehydrogenase, decreasing the K m for 2-oxoglutarate [61], and not to affect 2-oxoglutarate transport into heart mitochondria [60]. The stimulation of State 3 02 uptake holds also for adipose tissue [45] and brain [60] mitochondria, but not for liver or kidney [60]. 2-Oxoglutarate permeability can be inferred to be rate limiting in liver mitochondria, as rates are lower than those with other NAD-linked substrates [43]. There are two studies of 2-oxoglutarate oxidation by muscle mitochondria, as a function of aging, and they reveal an unchanged activity in heart [17] and in striated muscle (Table I and unpublished data from this laboratory). In the case of heart, this could be squared with the enzyme data cited above for homogenates if indeed the mitochondrial content is diminished by about 18% in old age on a wet weight basis. In liver, an age-linked decrement in uncoupler-stimulated 2-oxoglutarate oxidation was identified in mitochondria from BHE rats, a strain associated with a short life span and maturity-onset lipemia and glycemia [62]. However, no such decrement was seen in State 3, and the result is hard to interpret in view of the nonidentical experimental conditions used for the two age groups [62]. IIID. Succinate A review of the literature suggests that State 3 rates of succinate oxidation are undiminished with age in rhitochondria from most tissues. This is true of heart [17,18,20,24,36], striated muscle [17,24], brain [18] and liver [21,24,631 mitochondria. There are a few dissenting opinions, with Nohl et al. [22] reporting a small decrease in 02 uptake with heart mitochondria, and Chiu and Richardson [18] a small decrement with brain mitochondria. The latter is evident only when 6-month values (not 3-4-month values) are compared with 28-32month values, and is of unstated statistical significance. There is also an appreciable decrement in preparations of kidney mitochondria, obtained in a study by the author involving male Wistar rats (Table I). One has to be cautious in interpreting these results, as these rats have grossly sclerotic

57 kidneys in old age. On the other hand, one should not dismiss the finding out of hand, as decrements in oxidative activity are substrate specific (Table I) and quite small in the case of cytochrome oxidase (17%). By contrast, several laboratories have reported age-linked changes in succinate dehydrogenase activity, when this is measured by the reduction of added electron acceptors. Results include decreases measured with liver [33], kidney [321, prostate [35] and striated muscle [24] homogenates and with kidney mitochondria [64], as well as rather substantial increases in enzyme activity with aging, when this is measured in heart [50] or liver [64] mitochondria. These results may not be incompatible with the generally unchanged State 3 rates of mitochondrial 02 uptake (see above), as succinate dehydrogenase is probably not rate limiting for this overall activity. Instead, rate limitation may be exerted at the level of adenine nucleotide permeation, in liver mitochondria, or succinate permeation, as has been suggested by Palmieri et al. [65]. If this is so, the bulk of the available evidence would suggest that these permeability processes are unchanged with age, except possibly in the kidney. IIIE. Malate

Malate oxidation forms part of the malateaspartate shuttle by which most mammalian tissues oxidize glycolytically derived NADH [66,67], and generally occurs in isolated mitochondria only when the product oxaloacetate is removed, by citrate formation or by transamination. An exception would be a tissue (e.g., a tumor) with an active mitochondrial NAD-malic enzyme (EC 1.1.1.39) which forms pyruvate from malate [68-70]. In vitro malate oxidation is generally linked to transamination with glutamate, and Table I contains data obtained with mitochondria from animals of different ages. It is seen that there is a conflict in the results obtained from heart muscle, with several reliable studies indicating unchanged oxidation of glutamate plus malate with aging [24,28], and an equal number demonstrating a decreased activity [17,18]. The more important" aspects of the incubation conditions (substrate concentration, Pi concentration, pH) were similar, and so the discrepancy may relate to the making

of the organelle preparation. In striated muscle preparations there is also some evidence for a loss in activity of glutamate plus malate oxidation with aging (Table I). The oxidation of glutamate plus malate is not quite active enough to give a maximal rate of electron transport in heart (Table III) and kidney mitochondria (not shown). In view of the very high measured activities of malate dehydrogenase and aspartate aminotransferase, flux is probably limited by 2-oxoglutarate-malate or glutamate-aspartate exchange. For the reasons given by LaNoue and Schoolwerth [2], the latter is the more likely candidate. With liver mitochondria the rate of 02 uptake equals that achieved in the additional presence of pyruvate or palmitoylcarnitine, probably because adenine nucleotide translocation is limiting: uncoupling agents relieve this limitation, and the flux through transamination is then probably restricted by the activity of glutamate-aspartate exchange, which is severely depressed by uncoupling agents [71 ]. IIIF. Glutamate

Glutamate oxidation proceeds quite largely through transamination [72-75], with the exact partition of flux between glutamate dehydrogenase and aspartate aminotransferase depending on the tissue content of glutamate dehydrogenase (high in liver), the availability of ADP as an activator of glutamate dehydrogenase [76,77] and the activity of the glutamate/hydroxyl antiport system, which is mandatory for the oxidative pathway. The latter tends to be sluggish. Thus, the relative importance of the two pathways varies with the tissue, and the locus of any age-linked decrements in the overall oxidation varies accordingly. The oxidation of glutamate by the transamination pathway involves 2-oxoglutarate dehydogenase in an obligatory fashion, whereas the oxidation of glutamate plus malate does not, as the 2-oxoglutarate can leave the mitochondrion on the 2-oxoglutarate/malate antiporter. However, it may be somewhat arbitrary to make a distinction between the oxidation of glutamate and of glutamate plus malate (Table I), as the latter could involve flux through 2-oxoglutarate dehydrogenase too, depending on the activity of 2-oxoglutarate-malate exchange. Studies in this laboratory have implicated 2-oxoglutarate

58 dehydrogenase as being rate limiting in the overall oxidation of glutamate by rat heart and brain mitochondria, in that they have shown a stimulation of State 3 02 uptake by micromolar concentrations of Ca 2÷ (please see above). There was no such stimulation in liver, kidney or striated muscle mitochondria: in kidney and striated muscle, the system may be limited at the level of glutamate-aspartate exchange. This is suggested on the grounds of low measured transport activity in mitochondria from tissues in which these measurements have been made [78], and on the grounds that the carrier does not seem active enough in studies of intact cells to allow the distribution of glutamate and aspartate to reach equilibrium with the membrane potential [79,80]. Glutamate oxidation was found to be substantially decreased with age in rat heart [18] and kidney [60] mitochondria (Table I). In the kidney study, aging was also found to give less active oxidation of glutamine (30% decrement) and of glutamate plus glutamine (40% decrement). Glutamine oxidation involves glutamate dehydrogenase, as no extramitochondrial glutamate is available to countertransport with aspartate generated within the mitochondrion: glutamine oxidation may be limited in rate by glutamine permeation or, more probably, by glutaminase or glutamate dehydrogenase (see Ref. 2). Thus, a decrement with aging is likely to have a different mechanism from a decrement obtained with glutamate. In aging brain, Chiu and Richardson [18] report decreased oxidative activity with glutamate. Deshmukh and Patel [81], in a very interesting paper, link an impaired ability to oxidize glutamate plus malate to less active mitochondrial substrate transport. In the light of the above discussion, one might be more specific and suggest a less active glutamate-aspartate exchange. In brain mitochondria, glutamate plus malate oxidation seems to involve oxidation rather than outward transport of 2-oxoglutarate, from 14CO2 release results [81], i.e., it resembles the oxidation of glutamate alone. The age-linked decrement occurs mainly between 3 and 12 months of age in this study, and thus may reflect partly maturation rather than senescence: in the p o p u l a t i o n of n o n s y n a p t o s o m a l mitochondria, however, a further decrease in 02 uptake occurs from 12 to 24 months of age [81].

IIIG. Acylcarnitine and fatty acids Fatty acid oxidation provides a prime source of energy in some tissues, e.g., heart. When studied with isolated heart mitochondria, there is little doubt that fatty acid and acylcarnitine oxidation is less active in old age [17,28]. There is no one single locus of this effect, but rather an orchestrated decline in the activity of several enzymes involved in fatty acid oxidation. Common to the oxidation of the acylcarnitine species is a decrease in the activity of carnitine-acylcarnitine exchange across the inner mitochondrial membrane [28]. This transport is mediated by a mersalyl-sensitive carrier protein described by Pande and Parvin [82- 84] and Ramsay and Tubbs [85]. Investigations in this laboratory showed that the first-order rate constant for exchange was unchanged with age, but that the pool of intramitochondrial carnitine available for exchange was diminished [28]. This is not a trivial consequence o f the isolation of the mitochondria from old animals, as estimates of the total carnitine content of whole heart also show a 40% decline with old age [29,86]. Whether the decreased transport rate of the acylcarnitine species into the mitochondria is the mechanism of the decreased rates of 02 uptake is not entirely clear, and is discussed in the original paper [28]. At least for acetylcarnitine oxidation, transport activity is in bare excess over the flux through the pathway, judged from titration with the inhibitor mersalyl. A limitation by transport in the case of palmitoylcarnitine is less likely, as the transport rate is almost as high as that achieved with acetylcarnitine, and the complete oxidation of each palmitoyl moiety gives at least 10-times as much 02 uptake. Nevertheless, in vitro transport experiments are carried out under artificial conditions, and net transport in vivo will be slowed by competition with other more abundant acylcarnitine species, and by futile exchanges. Hence, it would be premature to rule that this decrement in transport does not affect palmitoyl group oxidation. The 3-hydroxyacyl-CoA dehydrogenase activity (EC 1.1.1.35), often taken as an index of the capacity of the tissue for t-oxidation (see, e.g., Ref. 87), is also diminished by approx. 30% in both mitochondria and heart homogenates from old rats [28,29,41]. This may not be part of the mechanism of the overall decrease in the rate of

59 palmitoylcarnitine oxidation [17,28], however, as it is not clear that this is a nonequilibrium step. By contrast, it is quite clear that a 40% diminution in the activity of acyl-CoA synthetase (EC 6.2.1.2 and 6.2.1.3) is the likely cause of the decreased activity of octanoate oxidation seen with mitochondria from the aging heart [28]. The oxidation of octanoate does not involve acylcarnitine formation or translocation, and thus age-linked inactivation of those systems is not the cause in this instance. Palmitoylcarnitine oxidation has also been found to be markedly decreased in old age when studied in kidney mitochondria (Table I). Acetylcarnitine oxidation shows a comparable decrease (60% decrement) and thus the two acylcarnitine species show a specific decrement over and above the decrease in activity seen with the other respiratory substrates (Table I). Rates of acylcarnitine translocation are clearly pertinent, but have not been measured. No such decrement has been found with striated muscle mitochondria either in earlier studies in which oxaloacetate availability may have been a problem owing to the omission of malate [17], or in a recent study in which malate was added and in which absolute rates of substrate oxidation were higher [60] (see Table I). These findings may be important, in view of the large mass of the striated muscle, and the effect that active fatty acid oxidation by this tissue will have in sparing glucose for, e.g., the brain, during the stress of starvation. It is tempting to speculate on whether the significant loss of capacity to oxidize fatty acids that occurs in heart and kidney (at least) mitochondria may relate to thyroid status. It has been shown that heart homogenates from thyroxine-treated guinea pigs oxidize palmitate more actively, and have a raised content of carnitine and a raised activity of carnitine palmitoyltransferase (EC 2.3.1.2), when compared to controls [88]. At the same time, it has been reported that circulating levels of T 3 and T 4 are decreased in senescence [89,90], though significant changes only occur when 2-3-month-old, and not 10-12-month-old rats, are compared to the 30-32-month-old senescent group [90]. Hypothyroidism is also reported to lead to a loss of other mitochondrial components, including citrate synthase (discussed above as a function of

age) and cytochrome c content (to be discussed below), To this extent, it is not a complete explanation of the rather more restricted decrements in fatty acid oxidation seen with mitochondria from the aging heart [28], but it should certainly be borne in mind. Endurance training leads to an increased activity of fatty acid oxidation in skeletal muscle and to a preferential use of fatty acid over carbohydrate during exercise of moderate severity (reviewed in Ref. 91). It would be interesting to known whether a treadmill-running regimen would prevent agelinked decrements in fatty acid oxidation from occurring. The original finding of Weinbach and Garbus [ 11 ] that fl-hydroxybutyrate oxidation is less active in old age has been amply confirmed, both for liver [21,24] and for heart [17,18,22]. In liver, the decrement is quite specific [21], as it is seen in preparations showing unimpaired activity with other substrates. IIIH. Ascorbate (cytochrome c oxidase) Cytochrome oxidase constitutes the terminal . complex, or electron-transferring leg of a redox loop [3], of the mitochondrial respiratory chain, and is often used as a marker enzyme for the inner mitochondrial membrane. Its activity is commonly assayed by measuring 02 uptake in the presence of ascorbate to donate reducing equivalents to cytochrome c [92], and is found to be depressed with senescence in both homogenates and isolated mitochondria from brain [93], heart [94] and liver [21]. Activity is also markedly decreased in homogenates of striated muscle, whether from the soleus or from the diaphragm [29]. Abu-Erreish and Sanadi [94] have compared the decreased specific activities of heart mitochondrial preparations with those of homogenates, and concluded that the mitochondrial content of the myocardium is the same at 5 and 26 months of age of the rat, though it is somewhat elevated at an intermediate age (14 months). They infer moreover that the composition of the inner mitochondrial membrane, of which cytochrome oxidase forms a part, must be changed with age to embrace the change in specific activity found for mitochondrial preparations. This may be so, but really needs confirmation from the demonstration of a differ-

60 ent change in the content of another inner membrane component. Otherwise, one may invoke the explanation used by Weindruch et al. [21] to explain rather similar findings in liver, namely, that there was an increased degree of contamination of the organelle preparation from the senescent animals. The composition, as opposed to the activity, of the respiratory chain is dealt with below.

IIII. Composition of the electron-transport chain To complete this survey of oxidoreduction processes as a function of age, this paragraph summarizes findings on the content of the carriers of the respiratory chain, and specifically the cytochromes. Abu-Erreish and Sanadi [94] found lowered cytochrome content in heart homogenates in senescence, when 15- and 26-month-old rats were compared. Values were 0.56 vs. 0.74, 0.26 vs. 0.47 and 0.39 vs. 0.48 n m o l / m g mitochondrial protein, for cytochromes b, c plus c~, and a plus a3, respectively, with the value in senescence presented first. They interpreted the constant proportion between the cytochromes as indicating that mitochondria from the old animals contained fewer respiratory assemblies, but that the composition of the assemblies themselves was invariant. A different conclusion emerged from a study of liver mitochondria in human, where there was a marked increase in cytochrome c plus c~, with no change in cytochromes a plus a 3 (cytochrome oxidase) above age 55 years [95]. There is no reason to doubt either study, and it is noted that there are major differences between human and rat respiratory chains, for instance, in the cytochrome c plus c J a a 3 ratio and in the specific activity of cytochrome oxidase, in heart [96]. The increased cytochrome c plus c~ content of human liver mitochondria in old age could, it seems, be an adaptation to the sort of decrements in substrate dehydrogenation discussed above and a means of maintaining an adequate concentration of reduced cytochrome c (and hence cytochrome a) for the terminal nonequilibrium, oxidase step.

IV. Phosphorylation IVA. FI-A TPase The overall process of oxidative phosphorylation was featured in the sections above and in Table I, being quantitated in terms of State 3 rate

of 0 2 uptake and P / O ratio. This section merely presents measurements of activity of the enzymes involved in phosphoryl group transfer. The FI-ATPase is probably better termed an ATP synthetase and, under physiological conditions, catalyses the transfer of the phosphoryl group of Pi to ADP, with the formation of ATP. This reaction is accompanied by the vectorial transfer of protons and is thus sensitive to the proton electrochemical gradient across the mitochondrial membrane [3]. The reaction only reverses, to give an ATPase, when the proton electrochemical gradient is collapsed, either by proton ionophore uncoupling agents [3] or by dissolution of the membrane by detergents. This has not been widely realized in aging research. Thus, Nohl et al. [22] reported a decline in ATP hydrolysis catalyzed by heart mitochondria when 23month-old rats were compared with 3-month-old controls. However, in another paper [5], detergent was used to solubilize the mitochondria and substantially higher absolute rates of ATP hydrolysis were obtained, together with the disappearance of the age-linked decrement. The author [5] interpreted these findings to mean that the effect of age was not in the F~-ATPase protein per se, but lay at the level of lipid-protein interactions, a conclusion strengthened by a shift in the discontinuity in the Arrhenius plot of whole-mitochondrial ATPase activity [22]. This concept may be very fruitful, and is discussed in Section IX. However, the detergent also clearly had the effect of removing latency barriers to the measurement of maximal enzyme activity. The reviewer suggests that the experiments could be done using uncoupling agenttreated submitochondrial particles: these would preserve lipid-protein interactions, yet allow ATPase action unrestrained by substrate or proton permeability. Mitochondrial ATPase has also been measured with respect to age in homogenates of heart, diaphragm and hind limb muscle of the rat [97]. Apparently, uncoupling agents were not added and thus the measurement would pertain more to the degree of mitochondrial breakage than to the true total activity. It seems that it should be possible to measure FI-ATPase in homogenates by using EDTA to inactivate extramitochondrial ATPases, and adding an uncoupling agent. If necessary,

61 specificity could further be determined by the degree of inhibition by oligomycin. Certainly, we lack knowledge at the moment of any age-linked changes in the activity of this enzyme. 1VB. Creatine kinase ATP synthesized in the mitochondrion is transferred to the cytosol by the adenine nucleotide translocase (for a review, see Ref. 16), which will be discussed in Section VI. There is kinetic evidence of some sort of functional coupling with the mitochondrial creatine kinase (EC 2.7.3.2) in muscle tissue, such that creatine acts as the preferred phosphoryl group acceptor [99,100]. The reverse reaction then occurs at the myofibrils, catalyzed by a different isozyme of creatine kinase [101], such that ATP is reformed in the immediate vicinity of the actomyosin ATPase. There has been considerable interest in the impact of aging on these relations, with somewhat contradictory resuits. Thus, Ermini [102] reported unchanged creatine kinase activities in homogenates of both red and white striated muscle from rat, whereas Chesky et al. [103] reported a decline in activity in partially purified preparations from rat heart. The latter decrement, variously cited as being 25 and 75% [103], seems to occur mainly between 2 and 11 months, an earlier portion of the life span than that mainly emphasized in this article, but one over which a decrease in isotonic velocity and extent of shortening of heart muscle preparations has been shown [104]. Results of a recent study by the same group [105] are interpreted as showing that a chronic exercise regimen reduces the agelinked decrement in myocardial creatine kinase activity: certainly the exercised rats show a higher specific activity for this enzyme at all ages studied. In human skeletal muscle a convincing study has shown a decrease in creatine kinase activity from 24-47 to 64-84 years of age [106]. This change was quite large ( - 5 3 % ) on the basis of DNA content but smaller on the basis of soluble protein, owing to the decreased water content of the aging muscle. Interestingly, this study provided no evidence for immunologically cross-reacting but catalytically inactive creatine kinase molecules, a prediction of the 'random-error' school of aging research [107,108]. An element missing in these studies has been the conscious discrimination of aging

effects on the two isoenzymes of creatine kinase, though the preparation methods used [103] may in fact have inactivated the mitochondrial enzyme. One study in which such a distinction was made has appeared recently [109] and reports no agelinked difference in activity for either enzyme, in preparations from rat cardiac muscle (6-8 vs. 26-28 months). At the same time, there has been considerable interest in the role of creatine kinase in reestablishing levels of creatine phosphate, after the cessation of a bout of vigorous exercise. The earlier work in this field was interpreted as showing that creatine phosphate levels were lower at rest in skeletal muscles from older animals and fell to lower values during exercise, relative to young controls [110,111]. However, this work was marred by the slowness of freezing of the muscles, which led to improbably low values for the A T P / A D P ratio. This criticism has been reinforced recently by the measurement of these parameters in resting human striated muscle, in a study which shows a statistically significant but very small decrease in the creatine phosphate/total creatine ratio in old age (0.58 vs. 0.61) and an unchanged A T P / A D P ratio [112]. Recently, the work of Ermini [110,111, 113] has been extended by McNamara et al. [114], who used rapid in situ freezing to quench metabolism, and who studied the reestablishement of creatine phosphate levels in heart and brain, as well as striated muscle, after a period of stimulation. They found that results were quite tissue specific, with a marked failure to reestablish creatine phosphate concentrations in striated muscle within a 3 min recovery period, a partial failure in brain, and complete recovery in heart, when aged animals were compared with young controls. Moreover, the age range over which the changes occurred was not the same for brain and for striated muscle [114]. The results underline the authors' conclusion that it is unwise to generalize about age-dependent changes from results obtained with one tissue, and without including animals representative of middle age. These results are certainly relevant to a discussion of energy metabolism in old age, but probably not relevant to a discussion of creatine kinase. Thus, it seems likely that the lag in the reestablishment of creatine phosphate levels to the resting

62 values reflects more an impairment in ATP generation than a decrease in creatine kinase activity. This would be consistent with the report that a high-glucose diet allows more rapid reestablishment of creatine phosphate levels in old rats [115], which points a finger towards blood glucose levels, glucose transport or glucose phosphorylation as being the locus of the age-linked change.

V. Oxidative phosphorylation in intact tissues The strength of the studies using isolated mitochondria and enzymes, which have been described above, is that the metabolism of specific substrates can be monitored as a function of age and that maximal activities or metabolic fluxes can be measured. The weakness of these studies is that cell fractions may be damaged during isolation, or even improved by the loss of aberrant organelles [116], and that it is impossible to reconstruct fully in the cuvette the environment facing the organelle or enzyme in the cell. By contrast, whole tissue or organ preparations have the strength that they preserve the intracellular milieu, but the weakness that the access of substrate or 02 may be a problem. Further, the metabolic state of most of the whole tissues used in aging research has been quite ill defined. Thus, the energy metabolism of an incubated but unstimulated muscle will probably relate quite largely to damage incurred during preparation, as this will affect the movement of ions across the plasma membrane, membrane potential and consequently ATP usage by the ( N a + + K÷)-ATPase and the Cae÷-ATPase of the plasma membrane and sarcoplasmic reticulum. Thus, age differences using such preparations are difficult to interpret. In addition, metabolism may be artificially restrained by substrate or 02 availability, as pointed out for the rat soleus preparation by McCarter et al. [58]. When very thin muscles are used and damage to the plasma membranes can be avoided [58] the 02 uptake and substrate fluxes measured are nevertheless resting values, in the absence of electrical stimulation, and are not very useful as indices of the ability of aged animals to transduce energy in response to stress, e.g., severe exercise. Some or all of these criticisms are thought to apply to most of the earlier aging studies, involving the measurement of substrate

oxidation o r 0 2 uptake by slices or sections from arterial tissue [117], elastic cartilage [118], heart muscle [119], kidney [119], liver [119], skin [120] and striated muscle [121]. Nevertheless, the more physiological measurements are necessary to place the biochemical findings in context, and to restrain the more enthusiastic speculations [113]. One system which avoids most of the problems mentioned above is the isolated perfused heart. Not only is substrate and 02 supply generally adequate, though 02 may become limiting at high work loads especially in the aged [122], but known work loads can be enforced. In one such study in which senescence was studied [86], it was found that hearts from senescent rats responded adequately to an increased work load, elicited by an increased left atrial filling pressure and increased aortic resistance, when paced electrically at 240 beats per min. Rates of 02 uptake and palmitate oxidation were modestly, but significantly, lower in the hearts from the senescent animals, when expressed on the basis of heart weight. These differences were obscured when data were expressed per heart, owing to the hypertrophy of the aging heart [86]. Notably, the efficiency of oxidative phosphorylation was seen to be essentially unimpaired with age, when this was approximated by the quotient 'peak systolic pressure x heart r a t e / O 2 consumption' [861. This finding seems to the reviewer to limit the credibility of studies with isolated cardiac mitochondria which show large decreases in P / O ratio [22,26]. Equally, the successful maintenance of high A T P / A D P ratios and creatine phosphate content by the aging heart when challenged by a high work load (30 min perfusion at 175 mmHg aortic peak systolic pressure) is evidence for the integrity of oxidative phosphorylation in aging heart [86] and flatly contradicts the results of earlier, less controlled, experiments showing large decreases in these parameters [123]. The study by Abu-Erreish et al. [86] also places limits on the extent to which fatty acid oxidation is impaired with age in the intact heart, an impairment inferred to occur from studies with isolated mitochondria [17,28]. The evidence appears in an unchanged ratio of palmitate oxidation to pressure development in this study [86]. This suggest an equally effective competition by palmitate with

63 glucose in the hearts of each age group. It is possible, however, that maximal work loads in this study still do not reach those achieved in vivo (see Ref. 124), and that the decrements in fatty acid oxidation seen at the mitochondrial level, which are decrements in Vmax activity [17,28], will appear only when the system is fully stressed. In a very recent study by McCarter et al. [58], the 02 uptake of an intact striated muscle preparation (the lateral omohyoideus muscle) was found to change very little with age. There was a significant decrease at 18 months of age, but very little change when the whole adult life span was investigated (cf. 6 and 27 months for the control rats and cf. 6 and 36 months for rats showing an extended life span owing to dietary deprivation). At the same time there was an appearance with aging of a few Type 2A (oxidative) fibers in a muscle otherwise entirely composed of Type 2B (glycolytic) fibers. The 02 uptake measured was a resting value and thus the physiological implications are for basal metabolic rate in age rather than for the potential to face the stress of acute exercise. In nervous tissues there is the possibility of fully expressing the potential activity of catabolic metabolism by depolarizing the cells, in response to a high-K + medium, and this approach has been followed recently by Gibson and Peterson [125]. Somewhat surprisingly, they found only very modest age-linked decrements in the oxidation of [U14C]glucose and [2-14C]pyruvate - and none for [1-14C]pyruvate - when the brain slices were incubated in a high-K + medium, though age-linked decrements were substantial both for [u-tac]glucose and [1-14C]pyruvate oxidation when the tissue was not depolarized. In the latter finding, this work [125] reproduced the results of Patel [126] and Parmacek et al. [127], who found decrements with both glucose and 3-hydroxybutyrate in old age, in nondepolarized tissue slices. Thus, in this instance [125], maximal velocities of enzymes involved in catabolic metabolism seem little changed in old age, and one might speculate instead that membrane leakiness to Na ÷ and K ÷ might be changed, resulting in a different level of activity of the ( N a + + K+)-ATPase in the steady state (my speculation). Finally, a promising approach to the question

of the integrity and functional capacity of mitochondria in situ in tissues from senescent animals was recently developed by Brouwer et al. [128]. They studies the degree of respiratory enhancement by the uncoupling agent dinitrophenol, added to isolated hepatocytes respiring in a controlled state, as enforced by oligomycin or atractyloside. The in situ respiratory control ratio so obtained was not diminished when the cells were isolated from 36-month-old rats, though in absolute units there was a slight decrease in the dinitrophenol-stimulated rate (cf. 36 and 12 months). It could be claimed that the cell fractionation technique might preferentially select the 'fitter' cells, though the authors maintain that this was not so in this case [128]. Certainly, attempts to prepare cardiac myocytes from 24-month-old rats failed miserably in the reviewer's laboratory, though they were prepared successfully from 6month-old animals. Nevertheless, the approach seems valuable and could be applied to intact organs, e.g., perfused liver, to circumvent fractionation problems. In conclusion, from the small number of studies available in which oxidative phosphorylation has been strongly and reproducibly stimulated in intact tissues, the decline in activity with old age seems quite limited. This is also true of efficiency, or in situ respiratory control ratio [128] or P / O ratio [86]. This is not to deny importance to the large decrements in activity seen in enzymes catalysing certain nonequilibrium reactions, especially in striated muscle, and detailed in the text above and Table II. These surely must limit metabolic fluxes during intense physical work; however, such a stress does not seem to have been successfully applied to whole tissues, or organs, in the laboratory. In the intact animal, other factors come into play, and indeed decreased 02 transport to peripheral tissues has been invoked (see Ref. 129) as a cause of the decreased maximal 02 uptake seen in man in old age [130]. This would clearly be the conclusion from a recent elegant study by Sylvia and Rosenthal [131] who applied noninvasive optical techniques to the study of the steady-state reduction of cytochrome a a 3 in the brain of living rats. Their conclusion was that aging had no impact on the percentage reduction of cytochrome a a 3 , but that this was acutely sensi-

64 tive to lung pathology. This implicated the provision of 02 to the brain tissue as a limiting factor, a conclusion which would have to be strengthened by the very high degree of steady-state reduction (30%) obtained in normal resting brain in their studies, in distinction to the minimal reduction seen with isolated mitochondria [12].

VI. Mitochondriai permeability in aging Recent work has identified rather sizeable (30-40%) age-linked decrements in the activity of several transport systems present in heart mitochondria. These include acylcarnitine-carnitine translocation [28], adenine nucleotide translocation [132] and the transport of Ca 2+ [133]. These decreases are noteworthy in that they do not reflect a general deterioration of mitochondrial membrane structure, nor a greater contamination of mitochondrial preparations with inactive protein, as the preparations from the old animals show undiminished rates of substrate oxidation, and respiratory control ratios, with some substrates [17,28,132]. The mechanisms underlying these permeability changes may not, however, be identical, and this is examined below. In the case of carnitine-acylcarnitine translocation (as discussed above), the first-order rate constant was found to be unchanged with age, and differences in activity were attributed to a decreased intramitochondrial pool of carnitine and acylcarnitine species, in old age [28]. Decreased rates of acylcarnitine translocation may rate limit acylcarnitine oxidation in heart mitochondria, especially for acetylcarnitine [28]. In the case of adenine nucleotide translocation, elegant work by Nohl and Kr~imer [132] using quench-flow apparatus to study early time points in ATPi,-ADPou t exchange has established a 40% decrease in the amount of exchange at each time point, as a consequence of senescence. The availability of the tight-binding inhibitor [3H]carboxyatractyloside allowed Nohl and Kr~imer [ 132] further to determine that there was no change in the number of binding sites in the mitochondria from the senescent rats and to attribute the diminished rates of transport to a diminished turnover number for the carrier. This was thought to be due to changes in membrane composition, notably a loss

of negatively charged phospholipid species, which were shown to be required for translocase activity in a reconstituted system [134], and a decreased degree of fatty acid unsaturation, which will be discussed below. At the same time Nohl and Kr/imer [132] showed that the sum of mitochondrial ATP plus ADP (the translocated species) was diminished by 25% in senescence, but implied that this would not change the measured rate of translocation. It seems to the reviewer that the lowered intramitochondrial pool size of adenine nucleotide would indeed be expected to contribute to the decreased velocity of translocation, as this has been shown to be substantially first order with respect to the sum of ATP + ADP (see Ref. 16). It further seems that there is some analogy with the age-linked changes in the carnitine-acylcarnitine translocase system mentioned above. Interestingly, adenine nucleotide translocation has been shown recently to be very sluggish in mitochondria isolated from the neonate, and to be enhanced rapidly after birth, with the buildup of the intramitochondrial adenine nucleotide pool [135]. A dependence of State 3 rates of oxidation on the pool size could only be shown when this was much lower than that achieved in later development [135]. Equally, whether the decrease in transport activity noted by Nohl and Kr/imer in senescence in any way limits oxidative phosphorylation in heart mitochondria is not established, as State 3 rates of oxidation of glutamate plus malate plus pyruvafe, a substrate combination that gives a maximal rate of oxidative phosphorylation, are not significantly lowered in old age (Table III). In the case of mitochondrial Ca 2+ transport, age-linked decrements are apparent in the activity of both the uptake pathway, which involves an electrogenic uniport [136-138], and in the egress, which consists of an electroneutral Ca2+-2Na ÷ exchange, in heart [139,140]. The two transport processes are further characterised by differential sensitivity to inhibition by elements of the lanthanide series [141] and by the inhibition, solely, of the uniporter by the compound ruthenium red [139,142,143]. The results of some studies of the age dependence of these transport systems, described fully in Ref. 133, are presented in Table IV. It is seen that there is an approx. 30% decrease

65 TABLE IV MITOCHONDRIALCa2+ TRANSPORTIN SENESCENCE Ca2÷ uptake and release by rat heart mitochondria were monitored with the metallochromic indicator arsenazo III and a dual-wavelength spectrophotometer,at 25°C. For further details, the reader is referred to Ref. 133. n.s., not significant. Rate of uptake or release (ngion/min per mg protein)

(A) Ca2+ uptake [Ca2+] (/~M) 10 30 (b) Ca2+ release Km for Ca2+ release (nmol Ca2+/mg protein) V,,ax for Ca2+ release (nmol Ca2+/min per mg protein)

in the rate of Ca 2+ uptake in senescence. This is equally true at three different concentrations (two are presented) giving different degrees of saturation of the carrier, and is presumed to reflect a decrease in VmaX. It has not been possible to measure Vmax directly in these experments, as the V vs. S relationship is sigmoidal [144,145], and Ca 2+-uptake velocities at high Ca 2+ concentrations may be limited by the activity of the respiratory chain rather than by that of the uniporter [146]. Generation of the A~p which drives Ca 2÷ uptake was presumably not compromised in the mitochondria from the senescent animal, in the sense that the respiratory control shown with the respiratory substrate used (glutamate plus malate) was undiminished. Ca 2+ release was studied with very small mitochondrial Ca 2+ loads, which are inferred to be within the physiological range, both from direct measurements using electron probe analysis (Ref. 147, albeit in another muscle), and from the fact that pyruvate and 2-oxoglutarate dehydrogenases only show Ca2+-dependent modulation of activity over the range 0 - 2 nmol C a : + / m g mitochondrial protein in heart [148,149]. This work allowed the determination of an So 5 value for Ca 2+ egress, atad this is unchanged in senescence (Table IV). An apparent activity coefficient of approx. 10 -3 determined for intramitochondrial Ca 2+ [149] allows the conversion of these values of So.5 from contents per mg protein, to concentrations of free intramitochondrial Ca 2+. The appropriate values

Difference (%) (P)

6 months

24 months

24.0 +0.8(9) 94.5 + 4 . 9 ( 5 )

16.4 + 1.5(9) 68.6+5.1(9)

-32 ( < 0.001) -27 ( < 0.001)

6.05 +0.46(5) 9.95 + 0.69(5)

6.64+ 1.33(5) 7.28 _+0.42(5)

+ 10 (n.s.) - 27 ( < 0.01)

are about 6 #M. In contrast to the unchanged So.5 value, the Vmax of release is diminished by approx. 30% in old age (Table IV). This work on Ca 2+ transport [133] could be improved by the measurement of binding sites, which should at least be possible for the uniporter by using ruthenium red. This would allow the discrimination between a changed number of carriers and a changed turnover number, as the mechanism for the reduction in Vmax. Analysis of the results obtained with these four transport systems provides no evidence for altered carrier molecules, as seen in changes of Km, but rather consistently shows changes in Vmax. One may envisage these changes as being caused by: (a) nonsaturation of the carrier with the substrate which is countertransported, in which case the change in Vmax is only apparent; (b) an altered carrier protein; or (c) altered carrier protein-lipid interactions. Mechanism a seems to account for the decrement in carnitine-acylcarnitine translocation [28], and at least a part of the decrement in adenine nucleotide translocation [132]. Mechanism b has not been identified in age-linked changes in mitochondria, whereas mechanism c has been strongly supported by Nohl [5] and Nohl and Kr~imer [132]. They emphasize that age-linked changes in heart mitochondria are seen only in activities catalysed by proteins which are closely associated with lipid, and that when these interactions are destroyed with detergent the age-linked changes disappear [5,22]. Examples are ATPase,

66

succinate dehydrogenase and succinate oxidase, and fl-hydroxybutyrate dehydrogenase [22]. In each case there are not only decreased specific activities with aging, but also shifts in the discontinuities in Arrhenius plots, in each case in the direction of higher temperature in old age, which are consistent with a less fluid membrane in old age. This was shown more directly by ESR studies of vesicles formed from phospholipid extracted from mitochondria of young and old animals, in the presence of the spin label 2-[3-carboxypropyl]-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl. These resulted in a break at 22.5°C for the young and at 27.1°C for the senescent, in Arrhenius-type plots [22]. Thus, a fluidity change in the bulk phase of the lipids could lead to a lessening in activity in old age of the transport systems discussed above. Moreover, changes in the lipid composition of more localized domains could affect all of the membrane-associated processes mentioned, several of which have been shown to have specific lipid requirements (see, e.g., Ref. 134 for adenine nucleotide translocation; Refs. 150 and 151 for 3-hydroxybutyrate dehydrogenase]. This view is coherent and attractive in that it postulates a unified mechanism for the spectrum of age-linked changes seen in membrane-associated activities of mitochondria. Moreover, compositional changes in the lipid of mitochondrial membranes clearly do occur in aging, and more evidence will be presented below. However, it is probably premature to regard the importance of age-linked changes in the lipid microenvironment of these various enzymes as having been established [5,22], as permeation of substrates between bulk phases is also involved in the measurement of these activities. Thus, detergent will remove latency caused by permeability barriers, as well as perturb microenvironments. This has been mentioned in connection with F~-ATPase, above, where permeability (probably to protons) was clearly limiting, in the studies of Nohl et al. [22]. Further, the oxidation of glutamate plus malate, claimed by Nohl et al. [22] to be immune to the effects of detergent as well as aging, though involving a dehydrogenase (malate dehydrogenase) which is not m e m b r a n e bound, is probably rate limited in intact mitochondria by permeation of adenine nucleotides (liver) or substrate (glutamate/aspartate, in heart: see

discussion above). Thus, one cannot fully support the thesis [5] that all membrane-associated activities are affected by age-linked changes in membrane composition. An equally convincing explanation of the changes in activity that do occur [5,22] is that there are age-dependent effects on substrate permeation between the bulk phases of the mitochondrial matrix and the cytosol. This of course is the inference from the direct studies of permeability discussed above [28,132], as well as being in accord with the conclusion of Patel et al., based on the lack of correspondence of age-linked changes in brain mitochondrial pyruvate [23] and glutamate [81] oxidation with the changes seen in the appropriate isolated enzymes. It would seem that studies with submitochondrial particles would be informative, when the enzyme activity survives sonication, as this system preserves local proteinlipid interaction while removing permeability barriers, in general. In the context of permeability, there are also several studies, originating with the paper of Weinbach and Garbus [11], which show an increase with age in unspecific permeation of the mitochondrial membrane. This may appear as a loss of coupling [24,152] or of macromolecules [153] in response to prolonged storage [24] or a brief exposure to a hypotonic medium [ 152,153]. It does not seem likely that mitochondria from old animals are leakier in situ, rather that the membrane is more. fragile and susceptible to damage during isolation. Thus, these studies provide indirect evidence for a change in mitochondrial membrane composition with aging.

VII. Mitochondrial membrane composition in aging Some evidence for a decreased fluidity of the mitochondrial membrane in old age [5,22] has been discussed above. Direct measurements of the lipid composition of this membrane perhaps provide a mechanism for this change, though there is considerable variation between tissues. Thus, Nohl [5] found on aggregate a decrease in the ratio unsaturated/saturated fatty acids with old age, when studying extracts of the inner membrane of rat heart mitochondria. An increase with aging in linoleic acid content was in opposition to this general trend. By contrast, Grinna [154] found a

67 slight age-related increase in the average degree of unsaturation of phospholipid fatty acids from both liver and kidney mitochondria. In liver there was a significant decrease in linoleic acid (18:2) but an increase in docosahexaenoic acid (22 : 6) with age. In kidney phospholipid, there was an increase in oleic acid (18 : 1). At the same time, there was a change in the type of phospholipid present in mitochondria from both tissues, with a decline in cardiolipin and phosphatidylethanolamine and an increase in phosphatidylcholine [154]. Apropos of membrane fluidity, the changes in fatty acid species in liver and kidney mitochondria clearly would not engender a decreased membrane fluidity with age in those tissues. However, Grinna [154] claims an increase in cholesterol content of both kidney and liver mitochondria and this would make the membrane more rigid. In view of the high cholesterol content of microsomal membranes [154], extensive purification of the mitochondrial fraction would seem imperative for this type of study, and the use of mitochondrial inner membrane vesicles [155] would also be an improvement, as mitochondrial energy transduction primarily involves the inner membrane. The reproduction of the finding of Grinna [154] in such a more purified system would be very significant. Finally, membrane fluidity is affected by protein-lipid interactions, and it is a change in these that is thought to give rise to fluidity changes in the membrane of erythrocytes during aging [156]. However, there is no evidence for a change in mitochondrial lipid/protein ratio with aging [157]. Thus, the generality of the age-related decrease in mitochondrial membrane fluidity shown by Nohl [5,22] for heart remains to be established.

VIII. Mitochondrial membrane lipid peroxidation in aging One possible mechanism for a decrease in membrane fluidity with age is an increased peroxidation of membrane lipid, and the evidence that this happens in old age is quite convincing, and will be discussed in this section. A minority of the flux of electrons down the respiratory chain gives rise to the generation of superoxide radicals ( 0 2 ) via the single-electron reduction of 02 (Refs. 158 and 159; and see Ref.

160 for a review). The main site of superoxide generation appears to be ubiquinone [161], though cytochrome b-566 has also been implicated [162]. In addition, N A D H dehydrogenase was recognized very recently as a source of superoxide radicals, albeit a more minor one [163]. By virtue of the presence of an Mn-requiring superoxide dismutase (see Ref. 164 for a review) within the mitochondrial matrix [165], 02- radicals are converted to H 202. Mitochondria are protected against this by the presence of catalase and of glutathione peroxidase, as outlined in Fig. 1. It has been assumed that catalase is quantitatively more important in the removal of H202 in prokaryotes and glutathione peroxidase in eukaryotes, but recent work with rat heart mitochondria has established that catalase is the more significant in that system [166]. A fraction of the H202 apparently reacts with superoxide radicals via the Haber-Weiss [167] reaction (see Fig. 1) generating the highly reactive hydroxyl radical, OH" The evidence for this is mainly indirect, involving the requirement for both H202 and 02- for the induction of lipid peroxidation, and the scavenging effect of ethanol and benzoate (see Ref. 168). However, there is also a recent report claiming the detection of OH" radicals in a heart mitochondrial system, in an ESR study using spin-trap reagents [162]. The OH" radical will attack a wide range of organic molecules generating other radicals, including lipid, lipid peroxy, purine and pyrimidine radicals. Lipid peroxy radicals formed in the mitochondrial membrane will in turn react energetically and nonspecifically; their reaction with polyunsaturated fatty acids will generate lipid hydroperoxide, which spontaneously breaks down to give malondialdehyde, ethane and pentane among other products. These relations are illustrated in Fig. 1 and have been reviewed by Chance et al. [160], Fridovich [164,168], Leibovitz and Siegel [169], Logani and Davies [170], Miquel et al. [7] and Tappel [171]. Malondialdehyde reacts with the ~-amino group of lysine, causing the cross-linking of proteins, and with other primary amino groups on phospholipids and nucleic acids. These products are Schiff bases, have a characteristic fluorescence, and appear to comprise the so-called 'age-pigments.' A major constituent has been identified as peroxidized phosphatidylethanolamine [ 172]. The case

68

(A) PRODUCTIONOF SUPEROXIDERADICALr HYDROGENPEROXIDE AND HYDROXYLRADICAL [ ELECTRONTRANSPORTCHAIN] 02

.~

~

e-

02-

SUPEROXlDE RADICAL

02-'~./ ~

"

,,0

(2REACTIONI~Oz

z

OH-+OH HYDROXYLRADICAL

IB) RADICAL-MEDIATED LIRDPEROXIDATION R-CH=CH-CHz-R ~ - - kO0

H" •

02

R-CH=CH-CH-R

~--

~ R-CH=CH-CH-R

ALIYI.ICPOLY- UNSATURATED FATTYACIDRADICAL

LH ~ 1

I

rcno^llJl~t

LIPtOPEROXY RADICAL

L'~

2GSH

OH

0 O

I

~ DECOMPOSITION MALONDtALDEHYDE,ETHANE, PENTANE,ETC

(C) CROSS-LINKINGREACTIONSMEDIATEDBY MALONDIALDEHYDE eg

0 0i, PROTEiN-NH z -I- H-C-CHz-CH4- HzN-PROTEIN a b

1

PROTEIN-N= CH-CH=CH- NH-POOTEIN a b FLUORESCENTCROSS-LINKEDPRO~CT

Fig. 1. Radical generation and the role of radicals in causing lipid peroxidation and the ultimate cross-linking of proteins, and other macromolecules containing primary amino groups. GSH, reduced glutathione; GSSG, oxidized glutathione; LOO', lipid peroxy radical; L; lipid radical; LH, polyunsaturated lipid.

for a relationship between lipid peroxidation and aging has been put eloquently by Harman [6,173] and by Tappel [174], who have drawn attention

not only to the metabolic havoc that could be wrought by the cross-linking of membrane enzyme proteins but also to the likelihood of this happening in the mitochondrion, with both polyunsaturated fatty acid and heme iron present to potentiate lipid peroxidation. In the context of membrane fluidity, which is a major interest of this article, these processes act via two mechanisms [175]. The peroxidative attack on unsaturated fatty acids lowers their content in the membrane directly, and the cross-linking of both phospholipid and protein molecules also introduces an increased rigidity. A direct demonstration of loss of fluidity with nonenzymic peroxidation has recently been made by Dobretsov et al. [176], using three different fluorescent probe systems. The following section is designed to bring up to date what is known about the impact of aging on these processes. Aging effects both the rate of production of superoxide by the mitochondrial respiratory chain and the rate of removal of superoxide by superoxide dismutase, and of hydrogen peroxide by the action of catalase and glutathione peroxidase. Thus, Nohl and Hegner [165] showed that coupled heart mitochondria from old rats produce O2radicals more actively than those from young controis. This is the more significant in that no electron-transfer inhibitors were used, in distinction to much of the previous work (e.g., see Ref. 177). The same age difference was found for submitochondrial particles, this time incubated in the presence of antimycin [ 165]. Mitochondrial superoxide dismutase was found to be unchanged with age in heart mitochondria [178], but the capacity for the removal of H202 was found to be increased. Thus, Nohl et al. [ 178] report increases in the activity of both catalase and the selenium-dependent glutathione peroxidase in rat heart mitochondria, in old age. The authors interpret these as adaptive responses to the raised content of peroxidized lipids which they identified in senescence [165], and implicate catalase as the major mechanism for the removal of H202, and glutathione peroxidase as catalysing the reduction of lipid peroxides [166,178]. The activity of glutathione peroxidase is of course contingent upon the provision of reduced glutathione by glutathione reductase, but the latter does not seem to be

69 limiting in the rat heart mitochondrion in old age, judged by the high, and unchanged, steady-state level of reduced glutathione [178]. There is evidence that despite an increased activity of the enzymes which protect mitochondria from the attack of free radicals, peroxidative damage does accumulate in old age [165]. Similarly, a fluorescence study of intact rat liver mitochondrial membranes showed an increase with age in the content of a pigment with a fluorescence spectrum resembling aminoiminopropene [180]. Suggestively, the same spectrum was generated when membranes from young animals were treated with malondialdehyde [181]. In another study, membrane extracts of heart mitochondria from senescent rats were shown spectroscopically to have increased contents of peroxidized lipid [22]. Spectrally identical or similar compounds were measured when mitochondria from young animals were incubated with xanthine and xanthine oxidase, as a source of superoxide radicals [22]. The analogy is attractive, though quantitative aspects of the comparison are not good, with more peroxidized product necessary in the in vitro system to give rise to the same decline in functional properties of the heart mitochondria [22]. It is also entirely proper to point out at this stage that the results of Nohl et al. [22] showing a functional decline in heart mitochondria of old animals, the presumed correlate of the raised content of lipid peroxide, represent a minority opinion (see text above and Table I). Antioxidants also have the capability of limiting peroxidative reactions (see Ref. 169 for a review) and dietary antioxidants have been investigated for possible effects on longevity, with varied results [182-185]. The question of whether such a dietary regimen can prevent age-dependent changes in membrane structure and function is still open. Grinna [186] found no evidence that dietary a-tocopherol prevented either age-dependent changes in structure (the degree of unsaturation of microsomal fatty acids; the fluorescence of anilinonaphthalenesulfonate in mitochondrial membranes) or function (succinate-cytochrome c reductase and 3-hydroxybutyrate dehydrogenase activity). However, the conclusions are clouded by the fact that it was not possible to generate a true vitamin E deficiency state in the older animals.

Certainly there is evidence for decreased lipid peroxidation in response to dietary antioxidants, as shown by a lesser accumulation of age-pigment [187], and, more recently, by a lower rate of production of ethane and pentane when vitamin Edeficient diets were supplemented with vitamin E [188-190]. These hydrocarbons are products of peroxidation of o~3 and ~6 polyunsaturated fatty acids, and are not further metabolized. Presumably, membrane lipid is a major source of the polyunsaturated fatty acid. However, a protection of membrane structure and function by these agents during aging has still to be shown. In addition to the peroxide generation by the respiratory chain described above, an enzymic NADPH-dependent lipid peroxidation has also been described in preparations of liver mitochondria [191]. It is essentially similar to the microsomal system [192] but seemed not to be present in microsomes contaminating the mitochondrial preparation [191]. In view of its reactivity with exogenous nicotinamide nucleotide, one might surmise that this is an outer membrane function. Of relevance to this review is the finding the N A D P H dependent lipid peroxidative activity was enhanced in liver mitochondrial preparations from senescent rats [193]. Finally, reference must be made to the view that mitochondria are the Achilles heel of the cell [6,7], actively carrying out lipid peroxidation by virtue of their content of polyunsaturated fatty acid and heme iron and high rate of 02 utilization. On this view, damage to the mitochondria leads to impairment of ATP production and loss of cellular homeostasis [6,7]. This theory has been refined recently [7] to take note of the fact that peroxide generation takes place in the inner membrane and is thus so situated as to cause damage to the mitochondrial DNA; to this one might add, presumably, damage to the proteins involved in replication, transcription and translation. This then highlights the polypeptides synthesized by the mitochondria as being loci of age-linked damage, and here this theory seems to fall down. Thus, of the few polypeptides synthesized in mitochondrial ribosomes three are subunits of cytochrome oxidase (see Ref. 194 for a review) and four are subunits of the oligomycin-sensitive ATPase [194]. However, the previous sections of this article have shown

70 that substrate oxidation is not invariably depressed in aging, even in fixed postmitotic tissues like the heart, and the activity of cytochrome oxidase is depressed modestly [94], if at all. Equally, the coupling of respiration to phosphorylation is unimpaired [17-19,24] and respiration is sensitive to inhibition by oligomycin [60,128]. Thus, there is no reason to invoke an especially severe impairment of protein synthesis by the mitochondrion, in mammals. The decreased synthesis of mitochondrial protein shown recently in the aging heart in response to the stress of a high work load [20] is very interesting in its own right but does not bear directly on this question, as leucine incorporation into mitochondrial protein in the intact heart reflects the cooperation of both cytosolic and mitochondrial protein-synthesizing machinery, with the major quantitative contribution being cytoplasmic [194]. Thus, there is little evidence that deranged synthesis of mitochondria leads to cellular aging and death, in mammals. This is in keeping with the relatively rapid turnover of these organelles, even in fixed post-mitotic tissues: the half-lives of mitochondrial protein and lipid are of the order of a few days [195]. In the plant kingdom there is much better evidence linking membrane lipid peroxidation to a decline in mitochondrial function. Thus, Rana and Munkres [196] showed that the growth of an inositol auxotroph of Neurospora crassa on media with limitingly low inositol led to greatly increased mitochondrial membrane lipid peroxidation and a decline in N A D H oxidase and cytochrome oxidase activities. That the lipid peroxidation was the cause of the respiratory decline was inferred from a protective effect of radical scavengers and hydrocortisone, which stabilizes the membrane. This study forms part of a body of excellent work by Munkres and co-workers, to which references are given in Ref. 175. It is not discussed further here only for fear that aging in plants may not be a good model for aging in animals.

IX. Morphology in aging There has been a great deal of work dealing with the number and structure of mitochondria in aging tissue, as determined with the electron microscope. Morphological studies can complement

biochemical measurements of catabolic enzymes of the sort presented above, as mitochondrial structure is the visible manifestation of the potential for function. Thus, the area of the inner (cristael) membrane may reasonably be correlated with the capacity for oxidative phosphorylation, and this has been done [197]. However, electron microscope studies also allow the differentiation between different cell types present in a tissue [198] and between cells of the same type present in different environments, e.g., periportal and centrilobular regions of liver [198]. They may also identify populations of aberrant mitochondria which are lost during tissue fractionation [116]. Thus, these studies may provide insights not available from homogenisation and biochemical assay methods. Finally, histochemistry allows the localisation of specific enzyme activities to different loci in the cell, albeit in a semiquantitative fashion. All of these techniques have been applied to aging tissues and some results are given below. There is no clear consensus on the impact of senescence on mitochondrial number and contribution to the mass of the cell (Table V). However, one could support the view that in liver the number of mitochondria decreases with aging, but that the individual size increases [199-202]. In heart, there is a flat contradiction (Table V). Possibly some of these differences are the differences between species. When the area of the inner mitochondrial (cristael) membrane was measured, this was found to fall when expressed on the basis of cell volume, in both liver and heart [197]. However, the mechanism was different, in that in liver there was a change in mitochondrial structure, with a reduction in inner membrane area as well as a reduction in the number of mitochondria in the cell, whereas in heart only a reduction in mitochondrial number was found [197]. In both tissues, the net reduction in area in old age was 35%, which might be expected to translate into a decrease in respiratory activity, though it is noted that the composition of the inner mitochondrial membrane is not immutable, but may change with age [94,203]. One can compare this morphological result with the results of a recent biochemical study by Stocco and Hutson [204], in which the mitochondrial content of liver was estimated by DNA and protein analysis following fractionation

71 TABLE V SOME MORPHOLOGICAL RESULTS PERTAINING TO M I T O C H O N D R I A IN SENESCENCE 1' denotes an increase, $ a decrease and ~ no change in the parameter measured, n.s., not statistically significant ( P > 0.05). References are given in parentheses. Change in parameter measured

Tissue Heart

Number of mitochondria/tissue area

1"(267) 1'(268) ],(197) J, (205)

Liver

Striated muscle

1"(269) ---}(270) J,(197) ],(199)

---}(129)

(200) J, (201) n.s J, (202) J, (205) Size of mitochondria

---}(197) (205) (268)

T(i99) 1"(200) 1"(201) 1'(202) ---, (197) (205) (270) $ (269)

J,(129)

Mitochondrial fraction of cellular volume

~ (267) ~ (268) $(205)

$ (202) n.s. -~ (269) $(197) $ (205)

4 (129)

Surface density of ---, (197) cristael membrane/unit mitochondrial volume

$(197)

by rate-zonal centrifugation. This technique allowed the recovery of 85% of the cytochrome oxidase o f the original homogenate and thus tends to rule out the loss during fractionation of any other than a minor fraction of mitochondria of altered density. This study indicated a fall in the number of liver mitochondria with senescence, on the basis of mitochondrial DNA. This then accords with the electron microscope data of Tate and Herbener [ 197]. Apart from a quantitation of mitochondrial mass, there has been major interest in the aging field in the presence of mitochondria of aberrant structure in senescent animals. Thus, Wilson and Franks [116] reported large mitochondria with a

light, foamy, vacuolated matrix and attenuated cristae in the liver of old mice. These could be seen side by side with mitochondria of more normal appearance. Interestingly, the enlarged, altered mitochondria were not recovered in mitochondrial preparations from these livers [116]. Similarly, an earfier study by Tauchi and Sato [199] of human liver mitochondria had revealed the presence of giant mitochondria in old age, together with mitochondrial inclusions comprising osmiophilic granules, myelin-like figures, and fibrillar or crystalline structures. These findings can be questioned, however, on the grounds that other more recent studies reported no such aberrant structures in liver mitochondria in old age [200]. Furthermore, the tissue fractionation by rate-zonal centrifugation mentioned above [204] revealed a very similar size distribution for both young and old, when either cytochrome oxidase or lipoamide dehydrogenase was measured. In heart, most reports do not mention the presence of mitochondria of an altered appearance [197,205,206]. There is the possibility that the result of Wilson and Franks [116] reflected a fixation artifact, though this reviewer does not have the competence to assert this. Certainly, fixation conditions are critical for the preservation of mitochondrial structure (see, e.g., Ref. 206), and mitochondria from senescent tissues may be perturbed more easily by osmotic shock [11,153]. In tissues other than heart and liver it seems clear, however, that mitochondria of altered structure occur in aging. Thus, in the parathyroid gland of old dogs arrays of strangely cup-shaped mitochondria, some with cristae running longitudinally rather than transversally, were seen [207]. Further, in nervous tissue altered mitochondria have been seen in old age, both in the dorsal column nuclei of aging mice [208] and in spinal ganglion neurons of aging rats [209]. In the latter study, the mitochondrial matrix was more electron opaque (cf. Ref. 116) and the mitochondria contained strange inclusions, comprising filaments, electron-dense globules and glycogen-like particles. The application of histochemical methods to mitochondrial enzyme activities has revealed no change with age in the distribution of succinate dehydrogenase along the nephron of the kidney

72 [32] or within lobules of the liver [198]. The latter study did reveal an interesting dependence of distribution within the lobule on the degree of oxygenation of the tissue, but this was not altered in aging. However, an elegant study by Bass et al. [210] of the aging of striated muscle did reveal changes in the distribution of succinate dehydrogenase and actomyosin ATPase activity. Thus, the mosaic pattern of succinate dehydrogenase staining tends to be lost with age in the extensor digitorum longus, with the larger (low activity) fibers showing increased activity. The mosaic pattern of ATPase staining characteristic of the extensor digitorum longus and soleus of the young animal is also muted with aging. These results attest to the 'de-differentiation' of these muscles with aging, and are uniquely revealed by the histochemical approach.

X. Bioenergetics of aging insects Insects offer many advantages for the study of aging. With the exception of their reproductive organs their tissues are postmitotic [21 l], and thus more likely to show the accumulation of the effects of environmental insult and of the byproducts of oxidative metabolism. Further, the flight muscles of insects are among the most actively respiring tissues known [212,213], which should potentiate the accumulation of peroxidized age products, if these are an inevitable correlate of high oxidative fluxes (see Ref. 7). Finally, insects offer the advantage that their life span is generally measured in days, so that they age more quickly than the investigator. For these reasons there is a great deal of work on this subject, much of which is reviewed in a recent and comprehensive article by Miquel et al. [7]. In general, this article takes the view that peroxidative damage occurs with aging, with the mitochondrion being the primary target. The consequent decline in ATP production leads to a decline in the ability to maintain cellular homeostasis, and this is seen in a decreased ability to sustain high levels of motor activity, i.e., to fly [7]. The present reviewer feels that although elements of the evidence needed to sustain this hypothesis are available, some of the data are over-interpreted. The present article will present a different perspective.

Starting with the physiology, there is little evidence that old flies cannot fly as intensely as young. The original classic work on the subject by Williams et al. [214] has been misinterpreted as showing that fruitflies (Drosophila funebris) exhibit a decreased wing-beat frequency in old age, whereas in fact Williams et al. [214] showed a decreased total number of wing beats, a consequence of a shorter flight time, but one still measured in tens of minutes. The wing-beat frequency was unchanged, indicating an undiminished rate of energy transduction, given an unchanged wing area [215]. Equally, expeiments with aging blowflies showed an undiminished wing-beat frequency [215] and rate of O 2 uptake [216]. The latter finding suggests that flux through glycolysis, glycerol phosphate oxidation and the tricarboxylate cycle is undiminished with age, in view of the marginal changes in steady-state concentrations of the relevant intermediates that occur on flight [52,217]: the latter finding further demonstrates that the efficiency of oxidative phosphorylation, the overwhelming contributor to ATP production in this tissue (see Ref. 218 for a review), is also undiminished with age. Thus, physiology provides scant reason to expect major changes with aging in rates of substrate oxidation, or efficiency of oxidative phosphorylation, in insect flight muscle mitochondria, despite near-universal claims to the contrary [219-222]. The work of Williams et al. [214] might instead highlight the control of sugar transport and glycogen synthesis and degradation, as it is a shortage of stored glycogen that limits the flight of the senescent Drosophila. In line with the results of experiments on the physiology of flight, Hansford [52] showed that senescent blowflies were capable of sustaining undiminished flight muscle A T P / A D P ratios, when compared with young adults. This finding became the more persuasive as a result of an unwitting experiment carried out by the animal caretaker, namely the maintenance of the flies in the absence of a source of protein. Under these conditions, the senescent flies were still able to maintain flight, and undiminished A T P / A D P ratios, although there was a clear change in the pattern of glycolytic intermediates, including a much diminished flight muscle concentration of pyruvate [52]. The latter put a stress on the tricarboxylate cycle, to

73

the extent that the fall in citrate content upon flight which is near universal in insect flight muscle [55,223,224] was no longer seen. Nevertheless, A T P / A D P ratios were maintained. When fed a more complete diet, the senescent blowflies showed an essentially unchanged pattern of glycolytic and tricarboxylate cycle intermediates during flight, when compared to the young adult [52]. As criticisms of this work one might cite the lack of measurement of wing-beat frequency, though only female flies with intact wings were used and the asynchronous, and resonating, nature of the flight muscle and exoskeleton probably maintains an unchanged frequency (see Ref. 215). Perhaps more seriously one can maintain that only a uniquely flighty subpopulation survives beyond the median life expectancy, and this is a problem that is intrinsic to aging research. Despite these indications of unchanged physiological performance, several studies have shown grossly impaired oxidative phosphorylation in work with flight muscle mitochondria isolated from old flies. The earlier studies were marred by absolute values of 02 uptake, respiratory control ratio and P / O ratio that were too low to be really credible [225,226]. A criterion of credibility here is a respiratory control ratio at least approaching the 60-fold increase in 02 uptake seen when a fly takes to flight [212]. A contribution to the low rate of pyruvate oxidation in these studies may have been oxaloacetate insufficiency, in that the exogenous malate used to 'spark' pyruvate oxidation does not penetrate the membrane of fly flight muscle mitochondria [55,227]. Work by Bulos et al. [222,228], however, also showed an age-linked decrement in State 3 rates of pyruvate oxidation by blowfly mitochondria in the presence of proline, which acts as a source of intramitochondrial tricarboxylate cycle intermediates. This was in contradiction to work in this laboratory which showed no age-linked decrement in rates of pyruvate oxidation, sparked by the presence of ATP and H C O 3 [52]. This had previously been shown to potentiate maximal rates of pyruvate oxidation in dipteran flight muscle mitochondria [229]. This issue was clarified, if not solved, with the demonstration by Wohlrab [230] that the same flight muscle mitochondria showing an age-linked decrement in the rate of oxidation of pyruvate plus

proline showed undiminished, and absolutely higher, rates of oxidation of pyruvate in the presence of ATP and HCO 3. It is possible that ATP maintains structure and assists in the slow energy-linked accumulation of potassium phosphate that occurs before competent respiration is established [231,232], as well as allowing the slow carboxylation of pyruvate [55]. In any event, as the mitochondria are exposed to ATP and HCO 3 in vivo, it seems unlikely that there is a physiological decrement in pyruvate oxidation in old age. In keeping with this view, the activities of the pyruvate dehydrogenase complex, citrate synthase and NAD-isocitrate dehydrogenase were all found to be undiminished with age when measured in extracts of blowfly flight muscle mitochondria [52]. Absolute activities of these rate-limiting enzymes are compatible with the known flux through pyruvate oxidation. Equally, there are undiminished flavin-linked glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and malate dehydrogenase activities in mitochondria from senescent houseflies [233]. The first result is particularly important, as this enzyme activity controls ADP-stimulated rates of oxidation of glycerol 3phosphate, which is produced in amounts equimolar with pyruvate by insect glycolysis (see Ref. 218 for a review). This is certainly true at physiological concentrations of glycerol 3-phosphate [52,217], as under these conditions State 3 glycerol 3-phosphate oxidation is markedly activated by micromolar concentrations of Ca 2+, which activates the dehydrogenase [234]. Finally, more evidence that the enzymic complement of these mitochondria is not affected by senescence is the unchanged content of cytochromes b, cytochromes c + c t and cytochromes a + a 3 [228,230]. Arginine kinase (EC 2.7.3.3) subserves the same function in insect muscle that creatine kinase, discussed above, serves in the mammal. There is evidence that this activity passes through a maximum in early adulthood in the fly, with the subsequent decline being intrinsic to the development of the fly, and not a function of 'wear and tear' [235,236]. The evidence for this latter statement is a parallel decline in a mutant fly with vestigial wings [237]. A similar age dependence of the cytosolic NAD-linked glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is seen in fly (Drosophila and

74

Phormia) flight muscle [238], with the same sort of

domestica). This would be expected to lower the

evidence for the importance of an intrinsic developmental program. The reaction catalysed by NAD-glycerol-3-phosphate dehydrogenase seems to be near-equilibrium [239] and thus it is not clear that this age-dependent decline in activity would have an appreciable effect on flux through the glycerol 3-phosphate shuttle [239,240]. Equally, the decreased arginine kinase activity seems not to affect flight muscle power transduction, as there is no drop in wing-beat frequency to match the decline in enzyme activity, in the blowfly Phormia [215,235]. It should be stated that this interpretation is the converse of that of the original author [215,235], who has maintained that the decline in arginine kinase and glycerol-3-phosphate dehydrogenase activity is part of a programmed decline in enzyme activity and physiological function. However, evidence for the decline in function is lacking [214,215]. For excellent reviews on the sometimes conflicting results of morphological investigations of aging in insects, the reader is referred to the articles by Baker [215] and Sohal [241]. The more recent work tends to negate earlier conclusions of widespread degeneration of flight muscle mitochondria in old age [242,243]. Thus, Rockstein et al. [244] report no deterioration in mitochondrial structure in male houseflies at 19 days, an advanced age for that sex of housefly. Similarly, Webb and Tribe [245] found little evidence of degeneration in old Drosophila, Calliphora (blowfly) or Musca (housefly), at an appropriately advanced age for each species. Interestingly, the omission of a protein source from the diet of Calliphora resulted in some distorted mitochondria, with misaligned cristae. It is recalled that the same diet of sugar and water alone also induced biochemical changes in aging blowflies [52]. The same fly fed a complete diet does not show these structural alterations. The presence of myelin-like whorls f o r m e d of i n n e r mitochondrial membrane which was originally described in senescent blowflies by Sacktor and Shimada [246] has been widely confirmed. However, recent work has found them to be quantitatively a very minor component [245,247]. Turturro and Shafiq [247,248] have recently described a reduction in the area of cristael membranes in the flight muscle of senescent houseflies (Musca

potential for oxidative phosphorylation and could be confirmed, presumably, by measurements of cytochrome oxidase or Ft-ATPase activity in homogenates. The flies used were very old (68 day) females and no information is available on their capacity for work performance. Interestingly, a recent freeze-fracture study by the same authors revealed a lesser number of clustered particles on the outer face of the inner mitochondrial membrane, and an increase in single 90-120 A particles, in old age [248]. Although the function of this protein is not known, the apparent change in aggegation properties may reflect some of the compositional changes in the membrane reported for other tissues in old age, and described earlier in this article. Finally, there has been considerable interest in the accumulation of lipofuscin-like products in insect tissues with aging, since their original description in Drosophila [249,250]. One might expect these pigments to accumulate most in flight muscle, in view of their formation as a byproduct of oxidative metabolism [158-165] and the postmitotic nature of the tissue [211], but this is not so. In fact, the fly head contains the largest amount of extractable fluorescent age product, and some of the abdominal organs contain the most as visualized by fluorescence microscopy. [251]. The fluorescent pigment content of houseflies increases with age, and more rapidly in a population which loses its flight ability, and then its life, prematurely [252]. There is moreover some evidence that the more active flies accumulate pigment more rapidly, and age more rapidly [252]. Thus, pigment accumulation is an index of physiological aging. Interpretation of these studies is complicated by the measurement of pigment in the whole insect. However, studies with honey bees show an increase in thoracic pigment, albeit a slight one, with age [253] and this at least suggests an increase in the flight muscle. In the sense that the bees were collected in the wild, no discrimination between the effects of exercise and the passage of time can be made. In terms of mechanisms serving to protect against peroxidations in insect flight muscles, vitamin E content and the activity of glutathione peroxidase have been reported to be minimal [253]. The mitochondrial superoxide dismutase has been

75

shown to be present but to decrease with aging in

Drosophila [254]. Thus, fluorescent pigments, the presumed product of lipid peroxidation and Schiff-base reactions (see Section VIII) accumulate with age in insect tissues and seem to mark 'physiological age' rather than 'chronological age'. However, their relative paucity in the most highly oxidative tissue, the flight muscle, is curious, and there is no reason to assume that they have any adverse effect on function. This follows from the unimpaired physiological performance of at least some fly species in old age [215,216] and is also consistent with recent work in mammals in which the cellular performance of nervous tissue was measured directly and was found to be unaffected by the accumulation of fluorescent age-pigments [255]. XI. Some conclusions

Credence can no longer be given to the results of experiments showing grossly impaired oxidative phosphorylation in suspensions of mitochondria from senescent animals [22,225,226]. Instead, it seems that mitochondria are substantially undamaged both biochemically (see Table I) and morphologically [197,205,206,245,247] but show some substrate-specific changes (generally decreases) in activity (Table I). These can be tentatively attributed either to the dehydrogenase involved [17,28] or to the carrier catalyzing transport across the mitochondrial membrane [23,28,81]. Changes in mitochondrial permeability have recently been established as occurring with senescence in rat heart mitochondria [28,132,133], and one can assume that more instances will come to light, as more transport systems are surveyed. This is because there is evidence that rather substantial changes in mitochondrial membrane composition may occur in old age, involving changed phospholipid/cholesterol ratios, changes in acidic phospholipid content and changes in the average degree of unsaturation of the phospholipid fatty acids [5,154]. These have the result of increasing the viscosity of the membrane in old age, at least in heart mitochondria [5,22], and this may be expected to inhibit transport processes (see, e.g., Refs. 256 and 257). In addition to this general effect, the change in membrane lipid composition

may have more specific effects on membrane integral proteins, including transport proteins, as shown by a requirement for specific phospholipid and fatty acid types of the successful reconstitution in artificial systems of several membrane-associated activities (see, e.g., Refs. 134, 258 and 259). It is felt that further transport studies in mitochondria from senescent animals would be very worthwhile. One mechanism that could contribute to a decreased content of polyunsaturated fatty acid in the mitochondrial membrane is an increased lipid peroxidation in old age [22,165,180]. This may be caused by an increased rate of peroxide generation by the respiratory chain [165] or decreased scavenging by antioxidants. There is currently no evidence for a decline in mitochondrial catalase or glutathione peroxidase activity in old age [178], these providing the other defense against radicalinduced damage (Fig. 1). Schiff-base formation between the products of lipid peroxidation and primary amines, e.g., of proteins, gives rise to age-pigment or lipofuscin, which accumulates in cells and thus bears witness to peroxidative damage. The brain and heart muscle of the mammal are particularly rich sources, as are certain insect tissues, though not the flight muscle (see Refs. 260 and 261 for reviews). The view that aging is caused by peroxidative damage at the mitochondrial level followed by an inability of the mitochondria to maintain cellular ATP levels [6,7] is, I think, harder to sustain. One reason is that when stressed appropriately in situ the mitochondria of senescent animals have not failed to maintain cellular ATP (vide supra). Another is that mitochondria are replaced with a half-life of the order of days, and which is unchanged in old age in a variety of mammalian tissues [195]. Errors could accumulate, it is true, if the mitochondrial protein-synthesizing machinery were damaged by peroxidative reactions [7] but that seems not to happen as mitochondria from senescent tissues have essentially unchanged activities of cytochrome oxidase and F~-ATPase, activities which are dependent on mitochondrial protein synthesis. Most of what has been presented in this article derives from experiments with isolated mitochondria. That reflects the bias of the literature which

76

has been surveyed. However, it is very clear that most of the problems which have arisen need a concerted approach at different levels of organization. Thus, as described in detail above, the measurement of the whole-homogenate activity of an enzyme catalysing a nonequilibrium reaction may be a very valid, as well as simple, way of assessing the maximum potential flux through a metabolic pathway. Some such results are presented in Table II. But the study of enzymes or mitochondria should be tempered by the humility which comes from physiology. Thus, the in vitro perfused aging heart carries out energy transduction with an efficiency [86] that questions some of the age-linked decrements shown in mitochondrial work [22]. Equally, the aging fly flies with an undiminished wing-beat frequency and 02 uptake [215,216], questioning some of the mitochondrial work [225,226]. Further than that, different factors may limit metabolism in the whole animal from those which limit in mitochondrial, cell or organ studies. Thus, in fatty acid oxidation by the heart, the transport of f a t t y acid across the plasma membrane of the cardiac myocyte may be rate limiting in the animal [262], whereas in experments with isolated mitochondria palmitoylcarnitine translocation into the mitochondrion or acyl-CoA dehydrogenation may be limiting [28]. Similarly, when brain metabolism is studied in the intact rat, the diffusion of 02 to the mitochondria clearly becomes a factor from the reduced status of the cytochrome a a 3 [131], whereas in in vitro experiments O z is almost always saturating. In addition, sensitivity to the different nature of tissues is essential, in investigating bioenergetics and aging. Classically, gerontologists talk of fixed postmitotic tissues, like nerve and heart, in comparison to more rapidly dividing tissues like liver, or spleen, to be more extreme. This distinction may not be so vital in the study of mitochondria, for the reason that they are constantly turning over, with similar half-lives in these different tissues [195]. However, the metabolic pattern of tissues varies widely and they do not all age at the same rate. For instance, there is clear evidence that some brain enzyme activities begin to decline earlier in the life span than do those of heart [23,81,41].

Thus, it is felt that the rather subtle changes in catabolic metabolism that do seem to occur in old age, e.g., a lesser importance of fatty acid oxidation in heart muscle and kidney in old age (Refs. 17 and 28 and Table I), need to be investigated at a variety of different levels of organisation, rather than relying on one experimental preparation.

Acknowledgements I should like to thank Frances Castro and Badr Ashour for permission to use their unpublished data, and Dana Jarvis for her unstinting help with the typing.

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