Modification of mitochondrial respiration by aging and dietary restriction

Mechanisms o f Ageing and Development, 12 (1980) 375-392

375

© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

MODIFICATION OF MITOCHONDRIAL RESPIRATION BY AGING AND DIETARY RESTRICTION

RICHARD H. WEINDRUCH, MARSHALL K. CHEUNG, M. ANTHONY VERITY and ROY L. WALFORD Department of Pathology, School o f Medicine, University of California, Los Angeles, CA 90024 (U.S.A.)

(Received September 5, 1979; in revised form December 23, 1979) SUMMARY Effects of aging and of dietary restriction on mitochondrial recovery and respiratory capacities have been assessed in mice. Old mice ( 2 3 - 2 6 months) did not differ from adult mice ( 9 - 1 2 months) in amounts of protein recovered in mitochondrial fractions of liver, brain and spleen, but did show a decline in specific activity of cytochrome c oxidase (cyt. c ox.) in liver and spleen. Age effects on in vitro respiration by mitochondria occurred in liver and spleen. In liver, only one substrate (fl-hydroxybutyrate) of four tested was respired at a different rate by old than by young mitochondria. Depression of state 3 respiration and 2,4-dinitrophenol (DNP)-uncoupled rates was observed for this substrate; however, this effect depended on expressing respiration on the basis of mitochondrial protein and was less overt if data were expressed per unit of cyt. c ox. activity. Old spleen mitochondria exhibited a grosser defect, showing a 40% decrease in the respiratory control index (RCI) for (succinate + rotenone)- supported respiration (the only substrate tested) due to a possible increase in state 4 rates. Effects of dietary restriction were assessed in liver and brain of 3-7-month-old mice underfed since weaning. Dietary restriction reduced recovery of total liver mitochondrial protein and liver cyt. c ox. specific activity. Liver mitochondria from restricted mice generally showed increased state 3 rates with no differences from controls in state 4 rates for respiration supported by glutamate or pyruvate + malate, resulting in an increased RCI for these substrates. DNP-uncoupled rates were also raised by dietary restriction. Unlike effects observed in old versus young mice, these differences obtained whether the data were expressed on the basis of mitochondrial protein or on cyt. c o x . activity. Electron microscopy of liver mitochondrial preparations revealed more non-mitochondrial contaminants in old mice and larger mitochondria in dietarily restricted mice. These findings are compatible with reports of age-dependent losses of liver mitochondria and suggest that dietary restriction may retard this loss.

INTRODUCTION Age-dependent mitochondrial changes occur in certain tissues of mammals. The number of mitochondria in mouse hepatocytes decreases between 8 and 44 months as

376 determined by electron microscopy [1]. Similar decreases have been reported for liver of rat [2] and human [3]. Between 12 and 24 months of age, clear decreases in both total mitochondrial DNA and protein have been reported for rat liver [4]. Rat heart cytochrome content and cytochrome oxidase activity also decrease with age [5]. In addition to actual losses of mitochondria, age-dependent alterations in respiratory activity have been repeatedly observed in isolated mitochondria. Oxidative phosphorylation supported by ~-hydroxybutyrate is reduced 40% in mitochondria from 24-month-old rat liver compared to preparations from 3-4-month-old rats [6]. After 20 months of age, rat heart mitochondria show reduced ADP-stimulated (state 3) respiration supported by/~-hydroxybutyrate, palmitylcarnitine, glutamate + malate, and glutamate + pyruvate [7]. Furthermore, state 3 rates of O2 uptake due to fatty acid oxidation are reduced in rat heart mitochondria between 6 and 24 months of age [8]. In contrast, no changes in ~-hydroxybutyrate-supported respiration were observed in rat liver, heart, or kidney mitochondria between the ages of 12-14 months and 2 4 - 2 7 months [9]. Defective respiration of glutamate + malate by a dense subpopulation of mitochondria isolated from old rat heart has recently been reported and was interpreted as being indicative of increased mitochondrial degeneration in old animals [10]. It is well known that dietary restriction uniquely prolongs maximal survivorship and slows actuarial rates of aging in rodents [ 11 ]. The mechanism of these effects remains obscure. It is possible that the lifespan-prolonging effect of dietary restriction relates in part to cellular metabolism dependent upon maintenance of levels of high-energy phosphorylated intermediates. The combined restriction of dietary protein and energy increases both oxygen consumption per unit body weight and maximal survivorship in mice [12]. Dietary restriction increases the specific activity of hepatic succinoxidase in both rats [13] and mice [14]. Other studies assessing effects of life-prolonging dietary restriction on mitochondria-associated processes have, to our knowledge, not been carried out. We herein describe effects of dietary restriction on several respiratory parameters using mitochondria isolated from liver and brain of dietarily restricted mice. In addition, respiration of mitochondria isolated from liver, brain, and spleen of normally fed young and old mice was compared.

MATERIALS AND METHODS Animals C57BL/6J male mice were used to study age effects. Two age groups were compared: young mice (9-12 months, except for the spleen study in which 4-month-old mice were used) and old mice ( 2 3 - 2 6 months). Old mice were autopsied at sacrifice and only disease-free animals used for these experiments. Female (C3H.S WSn × C57B10.RIII(71NS)/Sn)FI mice (hereafter abbreviated to C3B10FI) were used to study effects of dietary restriction. Two diet groups were compared: normally fed mice ( 3 - 7 months, = 4.5 months) and dietarily restricted mice ( 3 - 6 months, )? = 4.7 months). This hybrid combination was selected for its potential as a longqived strain based on parental longe-

377 vities [15]. C57BL/6J mice were caged in groups of 4 - 6 whereas C3B10F~ mice were housed individually so that food consumption could be accurately monitored. Diets

C3B10F~ female mice were weaned at 2 1 - 2 4 days o f age and randomly assigned to a cohort to be either normally fed or dietarily restricted. Two semi-synthetic diets were designed and prepared (twice monthly) to yield two very different levels of caloric intake while providing all mice similar weekly amounts o f protein (casein), salts, and vitamins. Only the weekly intake of sucrose, cornstarch, corn oil, and fiber were markedly less for restricted mice. The composition of these diets and feeding strategies are shown in Table I. Diet 1 (20% casein) was fed in nearly a d l i b i t u m amounts to produce "normally" fed mice. Animals fed diet 2 (35% casein) consumed about one-half the number of calories as mice fed diet 1. To avoid introducing variations due to fasting intervals, all experiments using diet mice were done on Saturday mornings (24 hours after restricted mice were given one-half of their weekly food allotment). C57BL/6J mice were not dietarily manipulated and were allowed free access to Purina Lab Chow. Reagents

Ultrapure sucrose was purchased from Schwarz/Mann (Orangeburg, N.J.). 2,4Dinitrophenol (DNP) was obtained from Calbiochem (Los Angeles, Calif.). Trizma, cytochrome e (horse heart, type III), rotenone, succinic acid, pyruvic acid, malic acid, glutamic acid, /3-hydroxybutyrate, disodium ADP, bovine serum albumin (BSA), and potassium EGTA (ethyleneglycol-bis-(2-aminoethyl)-tetraacetate) were purchased from Sigma Chemical Company (St. Louis, Mo.). Unless otherwise stated, all other reagents used were o f AR grade. TABLE I COMPOSITION OF DIETS (g/kg DIET) Constituents

Diet 1 a

Diet 2 b

Casein, vitamin-free test Cornstarch Sucrose Corn off (Mazola) Nonnulxitive fiber Salt mixture, USP XIV Zinc oxide Vitamin mixture (ICN Pharmaceuticals) Brewers' yeast (ICN Pharmaceuticals)

200.0 260.8 260.8 135.0 56.4 60.0 0.05 23.0 4.0

350.0 157.6 157.6 135.0 40.0 110.1 0.1 42.2 7.4

aDiet 1 : 20% casein diet with normal levels of salts and vitamins. Fed as seven 3.0 g feedings per week (one daily feeding on Monday through Thursday, three feedings on Friday) providing about 85 Cal/ week. All food always consumed. (Please note that Cal is the abbreviation for the nutrionists calorie and 1 Cal = 1 kcal.) bDiet 2: 35% casein diet richer in salts and vitamins than diet 1. Fed as four 2.5 g feedings per week (one daffy feeding on Monday and Wednesday, two feedings on Friday) providing about 40 Cal/week.

378 Preparation o f mitochondria Liver ( 1 - 2 g) and whole brain (approximately 0.45 g) were rapidly removed from each mouse and immediately placed in 5 ml of ice cold homogenization medium containing 0.35 M sucrose, 10 mM Tris.HC1 (pH 7.4) and 0.5 mM EGTA as previously described [16]. A 0.1 ml volume of 5% BSA was added prior to homogenization in an ElvehjemPotter glass homogenizer loosely fitted with a Teflon pestle using ten up and down strokes (350 rpm) over a 30-second period. After centrifugation of the homogenate at 5100 rpm for 1 minute (2064 g, Rmax) using a Beckman 50 angle rotor in a Spinco Model L centrifuge, the supernatant ($1) was removed and saved. The remaining pellet was resuspended in 4 ml of fresh homogenization medium and recentrifuged at 2064 g, Rmax- The $1 supernatants were combined, centrifuged at 15 000 rpm (17 838 g, Rmax) for 10 minutes, and the supernatant ($2) discarded. The resulting pellet (P2) was washed three times by resuspension in 5 ml aliquots of a medium containing 0.35 M sucrose and 10 mM Tris. HC1 (pH 7.4) and centrifuged at 12 000 rpm (11 430 g, Rmax) for 4 minutes. After the final wash, the mitochondrial pellet was resuspended in the Tris-HC1 buffered sucrose medium to 0.5-1.5 ml and was used for the determination of protein, respiration, and cytochrome c oxidase activity. Aliquots were also taken and processed for electron microscopy as described below. For the preparation of spleen mitochondria, spleens from 5 - 7 mice were pooled, the mitochondria harvested and washed as described above. Protein determinations Protein was determined by the method of Lowry et al. [17] using crystalline BSA as a standard. Cytochrome c oxidase determinations Mitochondrial cytochrome c oxidase (cyt. cox.) was determined by a modification of the method of Cooperstein and Lazarow [18] as previously described [19]. Enzyme activity was calculated from first-order kinetics assuming an extinction coefficient for cytochrome c of 0.96 X 107. One unit of activity is defined as 1/zrnole of cytochrome c oxidized per minute. Measurement o f respiratory rates, control, and ADP/O ratios Respiratory rates of mitochondria were recorded polarographically in a closed system using an oxygen electrode assembly (Yellow Springs, Colo.) and the respiratory control index (RCI) and ADP/O ratio were calculated as previously described [20]. The respiration medium contained 100 mM KCI, 5 mM KH2PO4, 20 mM Tris.HC1 (pH 7.4) and 1 mM potassium EGTA. A typical assay consisted of 2.5 ml of medium, 0.1 ml of 5% BSA, 0.1 ml of substrate, and 50-200 gl of the mitochondrial preparations ( 1 - 3 mg of protein). Respiration rates were expressed as #g atoms of oxygen utilized per minute per 100 mg mitochondrial protein or per 100 units of cyt. c o x . activity. RCI was determined by the addition of 0.3-0.6/~moles of ADP to the system and is defined as the ratio of the respiration rate in the presence of ADP to that after utilization (state 3/state 4) [21]. The ADP/O ratio was calculated from the total amount of oxygen utilized during state 3

379 respiration induced by the addition of a known amount of ADP. RCI and ADP/O ratios are reported only for data expressed on the basis of mitochondrial protein as these ratios are independent of protein and cyt. c o x . normalization. Fully uncoupled rates of respiration were determined by the addition of DNP to a final concentration of 0.1 mM.

Electron microscopy An aliquot of the mitochondrial preparations was centrifuged at 30 000 rpm in a Misco microcentrifuge for 10 minutes. The supernatant was removed and the pellet fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer as previously described [16]. Statistics Data are expressed as .~(S.E.). Each experiment compared adult vs. old or normally fed vs. dietarily restricted mice. Such paired data were tested for statistical significance using a paired t-test. A p value < 0.05 was considered to be of statistical significance.

RESULTS

Effects o f age on mitochondrial recovery from liver, brain and spleen C57BL/6J mice of ages 9 - 1 2 months (average body weight = 32.1 + 0.7 g) and 2 3 - 2 6 months (37.3 + 0.9 g) were compared. Mitochondria were harvested from whole liver and whole brain, and from pools of 5 - 7 spleens as described above. Liver wet weights for young mice (1.62 -+ 0.08 g, n = 12) were less (p < 0.01) than those of old mice (1.88 + 0.05 g, n = 12). Brain wet weights for young animals (0.43 -+ 0.01 g, n = 8) did not differ from those of the old (0.45 + 0.01 g,n = 8). There was no significant age effect on total recovery of mitochondrial protein from liver, brain, or spleen (Table II). Further, the mitochondrial protein recovered per gram wet weight of either liver or brain was not influenced by age. However, Table III reveals a significant decline in the specific activity of cyt. c o x . (a known enzyme marker for inner mitochondrial membrane) in liver and a marginal decline in spleen mitochondria but not in brain mitochondria from old animals. The recovery of cyt. c o x . activity per gram wet weight of brain also did not change with age, contrasting with a significant age-related decrease (approximately 20%) in the recovery of activity per gram wet weight of liver (Table III). Effects o f age on liver mitochondrial respiration Liver mitochondrial respiration rates were determined for mice of both age groups using glutamate, malate + pyruvate, succinate + rotenone, and/3-hydroxybutyrate as substrates (Table IV). Data are expressed in terms of both mitochondrial protein (Table IV, A) and cyt. c o x . activity (Table IV, B). Data normalized to mitochondrial protein showed no age effects on respiration supported by either glutamate, malate + pyruvate, or succinate + rotenone. In contrast, old mice exhibited lower rates of state 3 and DNPuncoupled respiration when/3-hydroxybutyrate was used as substrate. This depression of

380 TABLE II EFFECTS OF AGE ON MITOCHONDRIAL RECOVERY FROM LIVER, BRAIN AND SPLEEN Values represent .g(S.E.).

Young

Old

p

Liver (n = 12) Total mitochondrial protein (mg) Mitochondrial protein per wet wt. (mg/g)

27.68(1.58) 17.10(0.97)

28.71(1.89) 15.46(0.82)

NS NS

Brain (n = 8) Total mitochondrial protein (mg) Mitochondrial protein per wet wt. (mg/g)

11.47(0.82) 26.88(1.76)

11.43(0.72) 25.50(1.55)

NS NS

0.41(0.06)

0.55(0.14)

NS

Spleen (n = 3 experiments, 5 - 7 spleens per experiment) Average mitochondrial protein per spleen (rag)

TABLE III EFFECTS OF AGE ON CYTOCHROME c OXIDASE ACTIVITIES OF LIVER, BRAIN AND SPLEEN MITOCHONDRIA Values represent .~(S.E.). Activity equals/~moles cytochrome c oxidized per rain.

Young

Old

p

Liver (n = 12) Activity per mg mitochondrial protein Activity per g liver wet wt.

1.72(0.14) 29.63 (3.15)

1.57(0.13) 23.60(1.79)

<0.05 <0.02

Brain (n = 8) Activity per mg mitoehondrial protein Actiity per g brain .wet wt.

1.41 (0.20) 36.54(3.30)

1.39(0.21) 34.28(4.37)

NS NS

Spleen (n = 3 experiments, 5 - 7 spleens per experiment) Activity per mg mitochondrial protein 2.27(0.21)

1.67(0.16)

NS (<0.1)

state 3 respiration coinciding with a lack o f age effect on state 4 respiration produced a significant decrease (13%, p < 0.01) in the RCI o f liver mitochondria from old mice. When respiration was expressed in terms o f unit cyt. c o x . activity, age-related increases in state 4, state 3, and DNP-uncoupled rates in the presence o f glutamate, malate + pyruvate, and succinate + rotenone were seen. However, statistically significant differences were found only for the glutamate-supported parameters and for state 4 respiration o f malate + pyruvate. No significant differences occurred when/~-hydroxybutyrate-supported respiration was normalized to cyt. c ox. activity. Nevertheless, a numerically larger state 4 plus a concomitant decrease in state 3 respiration produced the observed decrease in the RCI o f old mouse liver mitochondria oxidizing this substrate.

Effects o f age on brain mitochondrial respiration No age effects were apparent in brain mitochondrial respiration in the presence o f glutamate, malate + pyruvate, or succinate + rotenone (Table V) expressed either on the basis o f mitochondrial protein (Table V, A) or unit cyt. c ox. activity (Table V, B).

381 TABLE IV EFFECTS OF AGE ON LIVER MITOCHONDRIAL RESPIRATION

State 4

State 3

A. Respiration per mitochondrial proteina Glutamate (n = 11) Young 2•18(0•18) Old 2.20(0.15) Malate + pyruvate (n = 8) Young 1.88(0•23) Old 1•89(0•23) Succinate + rotenone (n = 8) Young 5•91(0•65) Old 5•66(0•61) ~-Hydroxybutyrate (n = 10) Young 2.75(0.28) Old 2•82(0.28)

2,4.DNP

10.04(0•65) 10.33(0.64) 4.32(0•60) 4.16(0.45)

RC1

8•32(0•91) 4.68(0.15) 8.69(0•87) 4•73(0.11) 4•08(0.55) 3.92(0.48)

ADP/O

3.05(0•10) 3.09(0.13)

2•28(0.12) 3•10(0.14) 2•24(0•14) 3•25(0.16)

17•14(1•54) 15.27(1.64) 2•97(0•13) 2•31(0•13) 16.51(1.48) 14•71(1•50) 2•98(0•17) 2•19(0•08) 8.90(0.63) e 7.81(0•48)

8.88(0.70) e 3.39(0.21) d 2.97(0•16) 7.81(0.62) 2.94(0.23) 2.92(0.12)

B. Respiration per unit of cytochrome c oxidaseb Glutamate Young 1.35(0.12) e 6.24(0.50) d 5.02(0.48) e Old 1.50(0.14) 7•03(0•63) 5•70(0.45) Malate + pyruvate Young 1.32(0.14) e 3•01(0•36) 2•87(0.39) Old 1•46(0.19) 3•17(0.34) 2.95(0.29) Succinate + rotenone Young 4.17(0•49) 12.17(1.17) 10•74(1•09) Old 4•49(0•77) 13.04(1.82) 11•63(1.76) #-Hydroxybutyrate Young 1.52(0.14) 5•10(0.54) 5•62(0•89) Old 1.70(0.15) 4•87(0•48) 5•33(0•69) aValues represent X(S.E.) expressed as ~g atom O per min per 100 mg protein. Respiratory control index (RCI) Is defined as the ratio of the resptratlon m the presence of ADP to that after utilization (state 3/state 4) [21]• Substrate concentrations are: 10 mM glutamate, 4 mM malate + 5 mM pyruvate, 10 mM succinate + 0.5 gg/ml rotenone, and 10 mM #-hydroxybutyrate. bData from A normalized to each animal's cytochrome c oxidase activity and expressed as #g atom O per min per unit of activity. One unit of activity equals 1 gmole of cytochrome c oxidized per minute x 100. esignificant age effect with p < 0.05. dsignificant age effect with p < 0.01. •

.

•

~

.

•

.

Effects o f age on spleen mitochondrial respiration Results of three experiments each using mitochondria from pools of 5 - 7 spleens from young and old mice are shown in Table VI. Succinate + rotenone was the only substrate tested. Spleen mitochondria were less well coupled than those isolated from liver and brain. A significant decrease in RCI of approximately 40% was observed in mitochondria isolated from old mouse spleens, produced by an apparent decreased state 3 respiration. If expressed on the basis of cyt. c o x . , old spleen mitochondria displayed a significant increase in state 4 only (data not shown). The DNP-uncoupled rates were not influenced by age, suggesting an unaltered rate of maximum electron transport in old spleen mitochondria.

382 TABLE V EFFECTS OF AGE ON BRAIN MITOCHONDRIAL RESPIRATION State 4 A. Respiration per mitochondrial protein a Glutamate Young 1.24(0.13) Old 1.22(0.13) Malate + pyruvate Young 1.35(0.16) Old 1.40(0.17) Succinate + rotenone Young 3.38(0.39) Old 3.36(0.41)

State 3

2,4-DNP

RCI

ADP/O

2.91(0.31) 3.01(0.35)

4.12(0.61) 3.97(0.56)

2.37(0.08) 2.43(0.08)

2.97(0.14) 3.03(0.09)

4.17(0.55) 4.14(0.53)

5.16(0.72) 5.52(0.80)

3.08(0.17) 2.97(0.11)

3.31(0.13) 3.19(0.20)

7.29(0.83) 7.05(0.73)

7.61(0.80) 7.61(0.98)

2.18(0.09) 2.13(0.06)

2.12(0.08) 2.20(0.09)

B. Respiration per unit of cytochrome c oxidaseb Glutamate Young 0.98(0.16) 2.25(0.30) Old 1.00(0.18) 2.44(0.44) Malate + pyruvate Young 1.09(0.20) 3.28(0.61) Old 1.12(0.19) 3.25(0.50) Succinate + rotenone Young 2.71(0.53) 5.83(1.12) Old 2.84(0.68) 5.78(1.10)

3.17(0.54) 3.16(0.57) 3.92(0.58) 4.18(0.54) 5.67(0.79) 5.61(0.99)

avalues represent X(S.E.) for n = 8 pairs (young vs. old) for each substrate tested and are expressed as ~g atom O per min per 100 mg of protein. See Table IV for substrate concentrations. Data from A normalized to each animal's cytochrome c oxidase activity and expressed as ug atom O per min per unit of activity. One unit of activity equals 1 tamole of cytochrome c oxidized per minute x 100.

Effects o f dieta~ restriction on body growth, liver weight, and brain weight Post-weaning weight gain in dietarily restricted mice was markedly inhibited (Fig. 1). The 40 Ca.l/week re[~'nen produced animals with average b o d y weights at 200 days of about 14 g compared with about 32 g for controls consuming 85 Cal/week. Despite the severity of the restriction, the underfed mice appeared healthy and extremely active. During the first 3 months of life, some deaths did occur among the restricted animals. Deaths of restricted mice older than 3 months have only very infrequently been observed. Wet weights of liver and brain were reduced in restricted mice (Table VII). Liver of control mice weighed 1.22 +- 0.06 g (n = 13) as compared to 1.10 -+ 0.05 g (n = 13) for restricted mice. This is a significant difference (/9 < 0.05). The liver index [(g liver)/ (g b o d y weight) X 100] of restricted mice exceeded that of controls (6.9 +--0.2 vs. 4.5 -+ 0.2, p < 0.01). Brains of controls weighed 0.45 +- 0.01 g (n = 5) vs. 0.39 +- 0.01 g (n = 5) for restricted mice (p < 0.05). The brain index o f restricted mice is larger than that of controls (2.6 -+ 0.1 vs. 1.7 -+ 0 . 1 , p < 0.01).

383

TABLE VI EFFECTS OF AGE ON SPLEEN MITOCHONDRIAL RESPIRATION

Experiment No. State 4

1 2 3

Young

Old

5.35 9.53 6.94

8.06 9.24 10.43

7.27(1.22) a State 3

1 2 3

2,4-DNP

1 2 3

RCI

1 2 3

p

9.24(0.68)

12.54 14.20 12.84

10.92 9.90 11.72

13.19(0.51)

10.85(0.53)

9.70 14.38 12.40

11.64 10.31 11.59

12.16(1.36)

11.18(0.44)

2.34 1.49 1.85

1.36 1.07 1.12

1.89(0.25)

1.18(0.09)

NS

NS

NS

<0.05

avalues represent ,~(S.E.) of 3 experiments (5 - 7 spleens pooled per experiment) expressed as #g atom O per min per 100 mg of protein. The substrate in all tests was 10 mM succinate + 0.5 #g/ml rotenone.

55-

30-

NORMAL

25-

--& ~- 2 0 -

15o m

=¢'-'.---....=,,,'~Y

RESTRICTED

I0-

5-

20 3 0 4 0 5 0

100 150 AGE ( DAYS )

200

Fig. 1. Body weights of C3B10F~ mice. Values represent X(S.E.) of 5 0 - 6 0 mice per group.

384 TABLE VII EFFECTS OF DIETARY RESTRICTION ON MITOCHONDRIAL RECOVERY FROM LIVER AND BRAIN Values represent ,~(S.E.).

Normal

Restricted

p

Liver (n = 13) Wet weight Total mitochondrial protein (mg) Mitochondrial protein per wet wt. (mg/g)

1.22(0.06) 33.35(1.45) 27.25(1.09)

1.10(0.05) 26.60(1.29) 23.43(1.28)

<0.05 <0.01 <0.01

Brain (n = 5) Wet weight (g) Total mitochondrial protein (mg) Mitochondrial protein per wet wt. (mg/g)

0.45(0.01) 11.02(0.44) 24.70(0.65)

0.39(0.01) 10.41 (0.48) 26.96(0.91)

<0.05 NS NS

Effects o f dietary restn'ction on mitochondrial recovery from liver and brain The recovery o f total liver mitochondrial protein from restricted mice was significantly less (20%) than from normally fed mice (Table VII). There was a similar decline (14%) when recovery was expressed on the basis o f liver wet weight. Differences in brain mitochondrial protein recovery were not observed. The recovery o f cyt. c o x . activity from liver mitochondria is shown in Table VIII. Specific activity expressed on the basis o f mitochondrial protein was marginally reduced b y dietary restriction. However, activity recovered per gram wet weight of liver was reduced 26% in restricted mice.

Effects o f dietary restriction on liver mitochondrial respiration Liver mitochondrial respiratory rates were determined for restricted and control mice using glutamate, malate + pyruvate, succinate + rotenone, or fl-hydroxybutyrate as substrates. Data were expressed in terms o f b o t h mitochondrial protein (Table IX, A) and cyt. c o x . activity (Table IX, B). Both forms o f data expression yielded similar trends. Mitochondria from restricted mice generally showed increased state 3 rates with no differences in state 4 rates for glutamate or malate + pyruvate used as substrates. This resulted in an increase in the RCI suggesting better coupling o f oxidative phosphorylation to elecTABLE VIII EFFECTS OF DIETARY RESTRICTION ON CYTOCHROME c OXIDASE ACTIVITIES OF LIVER MITOCHONDRIA Values represent X(S.E.) for n = 7. Activity equals #moles of cytochrome c oxidized per min.

Activity per mg of mitoehondrial protein Activity per g of liver wet wt.

Normal

Restricted

p

2.06(0.14) 51.24(2.56)

1.83(0.12) 37.80(3.98)

NS (<0.1) <0.05

385 TABLE IX EFFECTS OF DIETARY RESTRICTION ON LIVER MITOCHONDRIALRESPIRATION State 4

A. Respiration per mitochondrial proteina Glutamate (n = 10) Normal 2.44(0.39) Restricted 2.22(0.25) Malate + pyruvate (n = 14) Normal 1.96(0.12) Restricted 1.93(0.13) Succinate + rotenone (n = 10) Normal 6.83(0.90) Restricted 6.36(0.72) #-Hydroxybutyrate (n = 6) Normal 3.19(0.42) Restricted 3.19(0.39)

State 3

11.92(1.34) 13.03(1.25)

2,4-DNP

9.11(1.38) 8.27(1.36)

RC!

ADP/O

5.16(0.31) d 6.01(0.25)

3.39(0.13) 3.46(0.17)

4.72(0.33) d 5.30(0.41) d 2.45(0.08) d 6.18(0.50) 6.53(0.60) 3.29(0.13) 18.46(1.96) 16.34(1.93) 18.54(1.93) 17.03(2.01)

2.79(0.08) 2.96(0.08)

11.32(0.79)d 10.30(0.61) d 3.73(0.32) d 8.96(0.50) 7.30(0.47) 2.96(0.28)

3.11(0.10) d 3.46(0.15) 2.20(0.06) 2.13(0.05) 3.08(0.12) 3.36(0.13)

B. Respiration per unit of cytochrome c oxidaseb Glutamate (n = 7) Normal 1.44(0.18) 7.33(0.91) 5.52(0.73) Restricted 1.47(0.15) 8.67(0.68) 5.52(0.70) Malate + pyruvate (n = 9) Normal 1.13(0.03) 2.82(0.17) d 3.10(0.18) e Restricted 1.26(0.08) 4.09(0.32) 4.32(0.32) Succinate + rotenone (n = 7) Normal 4.18(0.41) 11.58(1.19) 10.05(1.14) Restricted 4.21(0.32) 12.16(1.00) 11.12(1.01) /3-Hydroxybutyrate (n = 6) Normal 1.49(0.17) 5.40(0.43) 4.90(0.36) e Restricted 1.76(0.19) 5.04(0.37) 4.09(0.30) avalues represent ,~(S.E.) and are expressed as #g atom O per 100 mg of protein. See Table IV for substrate concentrations. bRespirafion data normalized to each animal's cytochrome c oxidase activity and expressed as/~g atom O per rain per unit of activity. One unit of activity equals 1 ttmole of cytochrome c oxidized per minute × 100. esignificant diet effect with p < 0.05. dSignificant diet effect with p < 0.01.

tron transport in dietarily restricted mitochondria. However, DNP-uncoupled rates were also raised for malate + pyruvate (but not for glutamate) suggesting an increased potential rate of electron transport in the preparations from restricted animals. No differences between diet groups in any of the respiratory parameters were observed for respiration supported by succinate + rotenone. In contrast to these findings, the RCI, state 3, and DNP-uncoupled rate of respiration supported by/%hydroxybutyrate were greater in mitochondria from normally fed mice. For all substrates tested, ADP/O ratios were not influenced by diet with the exception of a possible increase in the ADP/O for respiration of malate + pyruvate by mitochondria from restricted mice.

386 Effects o f dietary restriction on brain mitochondrial respiration No dietary effects were apparent regarding brain mitochondrial respiration in the presence of glutamate, malate + pyruvate or succinate + rotenone (Table X). Effects o f age or dietary restriction on the morphology o f isolated liver mitoehondria Mitochondria isolated and resuspended in a slightly hypertonic (0.35 M sucrose) medium were processed for electron microscopy as described in Materials and Methods. Representative micrographs of mitochondria isolated from livers of aging and diet animals are shown in Fig. 2. Mitochondria in all preparations retained a condensed configuration. Qualitative analysis suggested that mitochondrial preparations from young mice (Fig. 2A, B, and C) contained less nonmitochondrial contaminants than those from old mice (Fig. 2D). The contaminants included structures resembling uricosomes, lysosomes, and degenerating mitochondria. Preparations from dietarily restricted mice appear to be the least contaminated and exhibited the largest mitochondria.

DISCUSSION The present experiments demonstrate tissue-specific effects on mitochondrial recovery and function caused by either aging or dietary restriction. Aging (as studied by comparing 9-12-month-old mice to 23-26-month-old mice) did not influence the amount of protein recovered in mitochondrial fractions of liver, brain and spleen but did affect a decline in specific activity of cyt. c ox. in liver and spleen. Age effects on in vitro mitochondrial respiration occurred in liver and spleen but not in brain. In liver, only one substrate (~-hydroxybutyrate) of four tested was respired at a different rate by old than by young mitochondria. The former showed depression of state 3 and DNP-uncoupled rates. This effect depended on expressing respiration on the basis of mitochondrial protein and was less clear when data were expressed per unit of cyt. c o x . activity. Electron microscopy of liver mitochondrial preparations revealed more nonmitochondrial contaminants TABLE X EFFECTS OF DIETARYRESTRICTIONON BRAIN MITOCHONDRIALRESPIRATION Substrate Glutamate Normal Restricted Malate + pyruvate Normal Restricted Succinate + rotenone Normal Restricted

State 4

State 3

2,4-DNP

RCI

ADP/O

1.10(0.04) 1.10(0.06)

2.46(0.10) 2.39(0.22)

3.73(0.53) 3.29(0.47)

2.24(0.12) 2.18(0.12)

2.72(0.20) 2.87(0.18)

1.21(0.07) 1.20(0.12)

3.33(0.19) 3.52(0.24)

4.58(0.64) 4.24(0.40)

2.76(0.08) 2.93(0.16)

3.42(0.13) 3.36(0.14)

2.82(0.12) 2.69(0.15)

6.54(0.37) 6.55(0.30)

6.76(0.41) 6.95(0.43)

2.32(0.08) 2.44(0.06)

1.99(0.05) 2.09(0.11)

Values represent .~(S.E.) for n = 5 pairs (normal vs. restricted) for each substrate tested and are expressed as ~g atom O per rain per 100 mg of protein. See Table IV for substrate concentrations.

387

A

B

C

D

Fig. 2. Effect of age or dietary restriction on the morphology of isolated liver mitochondrial fractions. Magnification is ×17000. A. Normally fed C3B10FI; B. dietarily restricted C3B10F~; C. young C57BL/6J; D. old C57BL/6J.

388 in samples from old mice. Compared to liver, old spleen mitochondria exhibited a grosser defect: the RCI for (succinate + rotenone)-supported respiration was 40% lower than controls due principally to higher state 4 rates. Post-weaning dietary restriction influenced recovery and in vitro respiration of liver but not of brain mitochondria. Recovery of total liver mitochondrial protein and liver cyt. c o x . activity was lower in restricted mice. Liver mitochondria from restricted mice generally showed increased state 3 rates with no differences from controls in state 4 rates for respiration supported by glutamate or malate + pyruvate, thus causing an increased RCI. DNP-uncoupled rates were also raised for most of these substrates by dietary restriction. These diet influences on respiration obtain whether data are expressed on the basis of mitochondrial protein or per unit of cyt. c ox. activity. Electron microscopy revealed that liver mitochondrial preparations from dietarily restricted mice exhibited larger mitochondria with less nonmitochondrial contamination than controls. Although the recovery of protein in the mitochondrial fraction of liver was not reduced in old mice, the actual yield or numbers of mitochondria may well be lower. The greater contamination of preparations from old mice seen in the electron micrographs very likely falsely inflates what is estimated as "mitochondrial" protein. Also, the reduction in specific activity o f c y t , c o x . in preparations from old mouse liver argues for fewer mitochondria recovered, a notion supported by previous electron microscopic studies [ 1 - 4 ] . In contrast, Wilson et al. [22] observed no differences between 6-month-old and 30-month-old C57BL mice in liver mitochondria cyt. c o x . activity. Herbener's finding [ 1] that in C57BL/6J mice the greatest loss of liver mitochondria occurs between 30 and 4 3 - 4 4 months of age suggests that the mice studied in the present experiments and by Wilson et al. [22] may have been in an early stage of losing liver mitochondria. Based on the present results, in vitro respiratory capacities of brain and liver mitochondria are not overtly affected by aging. No age effects were observed on brain mitochondrial respiration, a finding in accord with results of respiration by brain slices from young and old hamsters [23]. Liver mitochondria from old mice showed lower state 3 rates of /3-hydroxybutyrate-supported respiration when data were expressed per mitochondrial protein. This agrees with observations in rat liver by Weinbach and Garbus [6] but contrasts to those of Gold et al. [9] who did not detect any changes with age. Decreases in DNP-uncoupled rates were also noted in the present study suggesting a defect in electron transport. Alternatively, Chen et al. [7] suggest that the lower state 3 13hydroxybutyrate-supported respiration by heart mitochondria from old rats may be due to changes in a specific dehydrogenase for this substrate (one that is bound to the inner mitochondrial membrane [24] ) or a reduced availability of NAD(H) with no alterations in electron transport perse. Normalization of/3-hydroxybutyrate data to cyt. c o x . activity lessens these effects, indicating that reduced numbers of normal mitochondria capable of oxidizing this substrate may exist in old liver. Interestingly, old liver mitochondria showed higher state 4, state 3, and DNP-uncoupled rates of NAD-linked glutamate respiration when results were normalized to cyt. c o x . activity, which would rule out a general deficit of NAD in old liver mitochondria. In general, (succinate + rotenone)- and (pyruvate + malate)-supported respiration by liver mitochondria were not altered by aging.

389 Spleen mitochondria preparations from old mice differed from those of young mice in their utilization of succinate + rotenone. The low yield of mitochondria from splenocytes necessitated the pooling of 5 - 7 spleens per experiment, which only allowed determination of respiration supported by this one substrate. Due to the large number of mice required, only three experiments could be done in aging mice, and none in dietarily restricted mice. Lower state 3 rates by old mitochondria were observed in all three experiments and higher state 4 rates in two of three tests when data were expressed on the basis of mitochondrial protein. This resulted in a lower RCI for old spleen mitochondria. Normalizing respiration to cyt. c o x . activities produced higher state 4 rates for preparations from old spleens with no age effect on state 3. DNP-uncoupled rates were not affected, suggesting no age effect on maximal electron transport rates. The apparently uncoupled state of spleen mitochondria from old mice may therefore be more related to an inability of these mitochondria to transport ADP and/or to a primary defect in oxidative phosphorylation. Taken together, the present findings point toward age-related effects on mitochondrial respiration being most severe in the spleen, of lesser severity in the liver and nondetectable in the brain. This situation may relate to different rates of cell and mitochondrial turnover in these tissues. It is well known that mitotic activity in spleen > liver > brain. Similarly, turnover rates of mitochondria, although not altered by aging [ 2 5 - 2 7 ] , do differ from tissue to tissue. Menzies and Gold [26] report half-lives of liver mitochondria of 9.3 days versus 24.4 days in brain. Although data were not presented, it was stated that spleen mitochondria show at least three components with half-lives ranging from 1 to 30 days. It is possible that more rapid mitochondrial turnover rates in certain tissues may be associated with respiration defects in late life. The extent to which mitochondrial defects in spleen cells from old mice may contribute to immunologic abnormalities of senescence is unclear but a report of improved humoral immune function in old mice treated in vivo with coenzyme Q [28] supports this notion. A decreased mitochondrial generation of ATP might in part account for the very low levels of cAMP and other aberrations of cyclic nucleotide metabolism observed in unstimulated spleen cells with age [29]. Dietary restriction caused a major inhibition of body weight gain and a slight reduction of liver and brain weights. Dietary restriction of rats reduced the number of hepatocytes per liver [30], an effect probably occurring also in our underfed mice. The liver and brain weight indices of restricted mice exceeded those of controls, indicating that the relative decrease in weights of these organs was not as great as the decrease in body weight. This contrasts to what occurred for spleen and thymus indices, which were reduced in mice by regimens of dietary restriction less severe than those herein employed [31 ]. Thus, tissues of the immune system appeared more affected by underfeeding than were brain and liver, as judged by organ weight indices. Dietary restriction reduced mitochondrial recovery in liver but not in brain. The mitochondrial pellet was smaller and a darker brown. Since electron micrographs showed less contamination in liver mitochondrial preparations from restricted than control mice, the decline in recoverable mitochondrial protein in the former may not be indicative of a

390 decline in yield of actual mitochondria. Nevertheless, restricted mice show a marginal decrease (p < 0.1) in cyt. c o x . activity per mg mitochondrial protein along with less cyt. c o x . activity per liver wet weight, thereby supporting the possibility of reduced numbers of mitochondria in livers of restricted mice. This possibility is further supported by the fact that livers from restricted mice weigh less than those of controls and probably contain fewer hepatocytes [30]. Quantitative electron microscopy of tissue sections from normal and restricted animals would clarify this issue. It is likely that these influences of dietary restriction on mitochondrial yield are partially mediated by thyroid hormones since dietarily restricted rats show decreased plasma levels of TSH, T4 and T3 [32], and thyroidectomy increases the half-life of rat liver mitochondria by 45 % [33]. Even in the face of apparently normal morphological parameters and cyt. c ox. content, respiration by isolated mitochondria from liver was greatly affected by dietary restriction whereas brain mitochondria were not influenced thereby. Mitochondria from restricted livers showed higher state 3 rates with no differences in state 4 rates for respiration supported by glutamate or malate + pymvate expressed either on the basis of mitochondrial protein or cyt. c o x . activity. The state 4 and state 3 effects resulted in an increased RCI which initially suggests better coupling of oxidative phosphorylation to electron transport. DNP-uncoupled rates were also generally higher for malate + pyruvatesupported respiration. These data may signify that dietary restriction caused increased efficiency of both electron transport and oxidative phosphorylation. An effect in the opposite direction (decreased state 3, DNP-uncoupled rates and lowered RCI) occurred in /3-hydroxybutyrate respiration. This might be due to the reduction of total fats fed to restricted mice causing lower rates of transport and induction of fl-hydroxybutyrate dehydrogenase. How may these findings relate to the ability of post-weaning initiated dietary restriction to increase maximum lifespan and slow actuarial rates of aging? Sacher's analysis [11 ] of Ross' data [30] for lifespan effects in dietarily restricted rats shows that the lifetime caloric intake per gram of rat is nearly constant (about 100 cal/g) for five diet regimens which varied in daily energy intake over about a four-fold range. Lifespan among these groups was linearly related to caloric intake with a slope of about -3.9 days per cal per day. Sacher concludes that the very small variance in lifetime energy intake per gram of tissue occurring in rats differing widely in levels of daily food intake and lifespan reflects a fundamental aspect of the longevity potential of rat cells. This apparent limitation in energy metabolism may relate to losses of or abnormalities in mitochondria occurring late in life. It may be relevant to note that heart mitochondria produce free oxygen radicals (O~) and that mitochondria from 23-month-old rats produce O~: radicals at a 25% faster rate than those from 3-month-old rats [34]. Evidence exists, furthermore, that with age superoxide dismutase, which functions to remove excess O~, declines in liver, brain, and heart [35, 36]. Accompanying the age-dependent increase in O~- generation in the heart is a concomitant increase in peroxidized lipids in the inner mitochondrial membrane [34]. Perhaps the better coupling of mitochondria isolated from dietarily restricted mice results in the reduction of free radical generation in the intact animal and partially prevents subsequent mitochondrial damage. Dietary restriction might thereby

391 p o s t p o n e the onset o f age-related losses o f m i t o c h o n d r i a by slowing m i t o c h o n d r i a l damage and t u r n o v e r t h r o u g h o u t life. F u r t h e r studies are needed to test these possibilities.

ACKNOWLEDGEMENTS We t h a n k Monica Wong for doing the electron m i c r o s c o p y and J a m e s Kristie for preparing the m o u s e diets. This study was supported by U S P H S research grants A G - 0 0 4 2 4 and H D - 0 5 6 1 5 . R . H . W . was s u p p o r t e d b y National Research Service p o s t d o c t o r a l fellowship C A 9 0 3 0 f r o m the National Cancer Institute.

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