Oxidative phosphorylation in mouse liver mitochondria during weaning

Mechanisms of Ageing and Development, 54 (1990) 121--129

121

ElsevierScientificPublishers Ireland Ltd.

OXIDATIVE P H O S P H O R Y L A T I O N IN MOUSE LIVER M I T O C H O N D R I A DURING WEANING

M. SUBRAMANIAN" and S.S. KATYARE'" Biochemistry Division, Bhabha Atomic Research Centre, Bombay 400 085 (India)

(ReceivedMay 19th, 1989) (RevisionreceivedDecember8th, 1989)

SUMMARY Oxidative energy metabolism in mouse liver mitochondria was examined during weaning using different substrates. During the post-natal development, from suckling to weanling stage, the respiration rates showed a temporary decrease. These altered levels recovered in the adults. Respiration rates with the three substrates studied namely, glutamate,/t-hydroxybutyrate and succinate showed the same pattern. However, the levels of primary dehydrogenase as well as that o f basal adenosine triphosphatase were not significantly altered during weaning. The content o f cytochromes aa 3 and b significantly decreased during this period. The results indicate that cytochrome aa 3 may be o f primary importance in the restoration of full respiratory function in mitochondria after the weaning period.

K e y words: Mitochondria; Mouse liver; Weaning; Oxidative phosphorylation; Cyto-

chrome oxidase INTRODUCTION Age is a significant factor which alters the structure and function o f cell organelles [1--3]. It also influences the mitochondrial oxidative energy metabolism in mammals [1,4--11]. Mitochondria, where tricarboxylic acid enzymes are located and fatty acid oxidation occurs, are also responsible for the oxidation o f energy rich substrates. Age-dependent alteration at molecular level is evident from a decline in the rate of inner membrane-matrix protein synthesis in the isolated rat liver mito"To whom all correspondenceshould be addressed. "'Present address: Department of Biochemistry,M.S. Universityof Baroda, Baroda 390 002, India. 0047-6374/90/$03.50

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© 1990ElsevierScientificPublishers Ireland Ltd.

122 chondria [12]. Although enormous data including various mechanisms involved in the process of aging, are available [13, 14], there is a paucity of investigations on neo and post-natal mice especially during weaning. Thus, it becomes a matter of importance to understand the biochemical aspects of energy metabolism during this period. The influence of diet on hepatic growth with age which influences the life span and dietary restriction and results in increased mean and maximum life span of the rat has been studied in detail [15]. The weaning period offers a period of growth during which animals change from the feeding habit of liquid to solid diet. It also involves a transition from predominantly lipid diet to that rich in carbohydrate [16]. Since the composition of diet has been shown to alter cellular membranes [17], it is possible that metabolic and developmental changes could also occur in the mitochondrial function during weaning period. Data on oxidative phosphorylation during foetal and early stages of post-natal development are documented [18,19]. Recent studies on rat brain development during neo- and post-natal periods have indicated that respiratory rates are low initially and increase with age. The A D P / O ratios are comparable with those of adult brain mitochondria from the first week of the post-natal period [20]. Thus, lack of data on oxidative energy metabolism in mouse liver during the weaning period warranted a detailed study to examine oxidative phosphorylation during this period. MATERIALSAND METHODS

Animals Swiss strain mice were used for these studies. The sucklings were weaned from their mothers at 21 days o f age which was designated Day 0 for weaning studies. The animals were fed on a nutritionally balanced laboratory diet containing wheat, bengal gram, fish meal, yeast powder, sesame oil and shark liver oil (62°70 carbohydrates, 17070proteins, 1.4010 lipids etc.). Isolation o f mitochondria The animals were killed by cervical dislocation and their livers quickly removed and chilled in ice. The homogenates (10070, w/v) were prepared in 0.25 M sucrose and the mitochondria were prepared as described by Katyare and Rajan [21]. The mitochondrial pellets were suspended in the isolation medium to give a concentration of 25 mg protein/ml. All operations were carried out at 0 - - 4 °C. Oxidative phosphorylation Measurements of oxidative phosphorylation were carried out as described by Katyare and Rajan [21] at 25 °C with a Clark-type oxygen electrode [22]. The assay system (1.3 ml) contained 225 mM sucrose, 10 mM potassium phosphate, 10 mM T r i s - - H C l and 5 mM MgCl 2 (pH 7.4). Glutamate (10 mM),/3-hydroxybutyrate (10 mM) and succinate (10 mM) were used as substrates. The state 3 respiration rates

123 (initiated by addition of 150 nmol of ADP in 15--20/~l volume) and the state 4 respiration rates (after the depletion of added ADP) were recorded. Calculations of ADP /O ratios and respiratory control ratio (RCR) were as described by Katyare et al. [23], RCR being defined as the ratio of state 3 and state 4 respiration rates. Determination o f cytochrome contents Mitochondria were solubilized in phosphate-buffered isolation medium with suitable aliquots of Triton X-100 and deoxycholate and the difference spectra of dithionate reduced minus ferricyanide-oxidised cytochromes were recorded [23]. Cytochrome contents were calculated by using the wavelength pairs and using millimolar extinction coefficients (1 cm light path) of 24, 23.4 and 18.7 for cytochromes aa3, cytochrome b and cytochrome c + c~, respectively [24--26]. Enzyme assays Activities of ATPase, glutamate dehydrogenase,/3-hydroxybutyrate dehydrogenase and succinate dehydrogenase were estimated using standard methods [27--30]. All chemicals used were of analytical reagent grade. Protein estimation was carried out according to Lowry et al. [31]. Student's t-test was used to determine statistical significance. RESULTS Both the body weight and the liver weight of mice showed a gradual increase during the weaning period (Table I). The protein content of hepatic homogenate and mitochondria decreased on Day 4 of weaning and then gradually recovered to reach adult values. The body weight remained significantly lower than the adult level on Day 12 of weaning. Data on oxidative energy metabolism in mouse liver mitochondria during weaning are given in Table II. Weaning significantly altered state 3 and state 4 respiration rates. The state 3 respiration with glutamate as substrate showed an 18070decrease as compared with the respiration observed in suckling mice. However, state 4 respiration increased by 10807o resulting in the lowering of the respiratory control ratio (RCR). State 3 respiration with/3-hydroxybutyrate as substrate, on the other hand, was increased in the weaning mice compared to the suckling (14070) but the state 4 respiration increased by 282070, again resulting in the lowering of RCR. State 3 respiration rate with succinate as substrate increased by 22070 and state 4 respiration by 26007o during weaning, resulting in the decrease of RCR. A D P / O ratios with respect to cell substrate used remained unaltered in all groups investigated viz. suckling, weanling and adult. Data on changes in the RCR in mouse liver mitochondria as a function of weaning period are given in Fig. 1. The patterns observed showed that there was a sudden decrease in the RCR between 0 and 4 days of weaning with three substrates studied.

11.97 ± 0.27(20) 0.56 ± 0.02 (20)

22.97 _ 1.05 (20)

207.73 ± 5.02(20)

0

Days after weaning

13.66 ± 0.72 ±

0.57(14) 0.03 (14)

16.23 ±

2.30 (6)

192.81 ± 17.00(6)

4 14.56 ± 0.37(23) 0.67 ± 0.02(23)

21.75 ± 2.20 (9)

210.31 ± 9.16 (8)

8 0.50(14) 0.05 (14)

22.03 ±

3.29 (4)

226.21 _+ 20.04 (4)

18.23 ± 1.03 ±

12

25.64 ± 1.09 (10)

216.33 _+ 2.76(10)

24.25 ± 1.21 (10) 1.10 _+ 0.06(10)

Adu/t

Suckling Weanling Adult Suckling Weanling Adult Suckling Weanling Adult

Glutamate

30.46 24.95 47.32 23.45 26.70 30.94 48.87 59.72 82.81

± 1.39 (20)' _ 1.96 (12) _ 2.20 (14) c _+ 1.73 (18) ± 1.98(14) ± 2.58 (12) ± 2.88 (18) a _ 4.35 (14) ± 4.83 (12) b

+ ADP

Rate o f oxidation (nmol O / m i n p e r mg prot.)

3.57 7.44 3.46 2.35 8.98 2.82 5.04 18.15 4.27

_+ 0.64 (20) c _+ 0.84 (12) _+ 0.89 (14) ~ _ 0.57 (18) ~ _+ 1.03 (14) _+ 0.81 (12) c _ 1.14(18) b _+ 4.35 (14) ± 1.46(12) b

- ADP 8.98 3.49 13.68 9.98 3.17 10.97 9.70 3.34 19.39

RCR

2.40 2.90 2.21 2.92 2.82 2.46 1.56 1.88 1.56

ADP/O ratio

Values are mean ± S . E . of number of animals as mentioned in the parentheses. ap < 0.05; b p < 0.01 and ~P < 0.001, as compared to the corresponding weanlings. Sucklings were 20 days old and weanlings 29 days old, when they were killed

Succinate

fl-hydroxybutyrate

Animals

Substrate

CHANGES IN THE 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 MOUSE-LIVER M I T O C H O N D R I A USING DIFFERENT SUBSTRATES D U R I N G WEANING

TABLE II

Values are mean + S.E. of number of animals as mentioned in the parentheses. • Expressed as g/animal. a Expressed as m g / g liver.

Bodywt: Liver wt." Protein content: Hepatic homogenate b Hepatic mitochondria b

Parameters

CHANGES IN BODY W E I G H T , LIVER W E I G H T A N D RELATED PARAMETERS IN MICE D U R I N G W E A N I N G

TABLE I

125

~) 2O I

z

0¢J >. no i.< hi 14J

10

I

K

4

l

I

8 12 DAYS AFTER W E A N I N G

ii I

Adult

Fig. 1. Changes in respiratory control ratio (RCR) as a function of weaning period. -A-A- represents succinate, -O-O- represents glutamate and -D-D- represents/3-hydroxybutyrate as substrate.

The RCR levels in the weanlings were lower than adult values even on Day 12 post weaning. With a view to understanding the biochemical basis of the impairment in the respiratory potential as measured by RCR, we extended our investigations to measure the levels of primary dehydrogenases and examine functional alterations in the mitochondrial electron chain components. From the data presented in Fig. 2 it can be noted that succinate and glutamate dehydrogenases did not show any significant change during weaning. However, /3-hydroxybutyrate dehydrogenase significantly 0.5

z

0"4

Ld I'-

m

o

a

0.3 E "

0.2

o

0.1

iiiiiii

SDH

c

GDH

13-OH BDH

Fig. 2. Changes in the dehydrogenase levels in mouse liver mitochondria during weaning. Enzyme activities are represented as A, A / m i n per mg protein and values are mean +_ S.E. of 7--20 animals, ap < 0.05, bp < 0.01 and cp < 0.001, as compared to the corresponding weanlings. D, suckling; ra, weaning and B, adult.

126 increased (50%) in the weaning period followed by a decrease to suckling levels in the adult mice. Analysis o f the intramitochondrial cytochromes content (Table III) showed that weaning significantly lowered the levels of cytochromes aa 3 and b (59 and 50%, respectively). Although there was an increase in the levels o f these cytochromes in the adult, the contents o f these two hemoproteins were lower than those of sucklings. Unlike these two cytochromes, the cytochrome c + c~ did not show any marked change during weaning. The ratios o f these three cytochromes indicated that the changes observed during three stages of development were different in the cytochromes studied. The cytochromes b and aa~ segments of the respiratory chain appeared to be affected to a greater extent by a change o f diet during the weaning period. Further, we examined the mitochondrial ATPase activity in suckling, weanling and adult mice. I n s i t u ATPase functions as A T P synthase [32]. The results are depicted in Fig. 3. In sucklings, hepatic mitochondrial ATPase activity at basal level -Mg 2" and -Dinitrophenol (DNP) was found to be 2.35 /~mol/h per mg protein. Addition o f Mg 2" or D N P stimulated by the ATPase activity and the increase observed was 2.2- and 3.1-fold, respectively. Maximal stimulation was observed when both were added together. In weanlings the basal activity was comparable with that of sucklings. ATPase activity induced by D N P showed a significant increase (49%) whereas that induced by Mg 2÷ showed a significant decline (25%). Compared with weanlings, the basal ATPase activity in the adults decreased by 32%. Though the basal ATPase activity as well as that induced by D N P were low in adults, ATPase induced by Mg 2÷and D N P together, was more in these animals as compared to weanlings. DISCUSSION The weaning period is a transition stage in which animals change their food habit from predominantly lipid-containing liquid diet to carbohydrate-rich solid diet. Our TABLE IlI CHANGES IN THE CYTOCHROMECONTENT OF MOUSE LIVER MITOCHONDRIADURING WEANING Cytochrome

Suckling

Weanling

Adult

aa3

0.17 ± 0.003 (10)c 0.28 ± 0.006(10)c 0.26 ± 0.008(10) 1.00 : 1.65 : 1.53

0.07 ± 0.010(5) 0.14 ± 0.025(5) 0.26 ± 0.028(5) 1.00 : 2.00 : 3.71

0.12 ± 0.003(14)c 0.23 ± 0.009(12)c 0.25 ± 0.016(12) 1.00 : 1.91 : 2.08

b c + c~ Ratio ofaa 3: b : c + c~

Cytochromecontents were expressed as nmol/mg protein. Values are mean ± S.E. of number of animals as mentioned in the parentheses. cp < 0.001, as comparedto the correspondingweanlings.

127

15-a

~O <~10--

cii

13- ®

-6 5 E

Additions Mg 2+

(-)

(+)

(-)

(+)

DNP

(-)

(-)

(+)

(+)

Fig. 3. Changes in ATPase activity in mouse liver mitochondria during weaning. ATPase activity is given as P~ liberated/h per mg protein. D, suckling; [], weaning and [], adult. Values are mean -+ S.E. of 4 - 16 animals. "P < 0.05, cp < 0.001, as compared to the corresponding weanlings.

study showed that during this transition period between suckling and adult stage, there was a significant change in the composition and function of hepatic mitochondria. The analysis of different parameters of oxidative phosphorylation showed a slight impairment in the respiration function during weaning. But the energy metabolism recovered to normal levels in the adult. Our results showed that though the ADP/O ratios remain unaltered, the RCR values declined during weaning. RCR is a more sensitive indicator of mitochondrial malfunction than the ADP/O ratio [8]. The altered ratio, however, returned to normal values in adults. In our studies, we used succinate, glutamate and/3-hydroxybutyrate as substrates. The former two support maximum rates of respiration and consequently are most likely to reveal age-related changes in the electron transport or phosphorylation [8]. We tried to correlate the observed changes in oxidative phosphorylation with compositional changes in energy transducing systems including ATPase. The structural integrity of mitochondrial function in terms of ATPase activity also was evaluated. The structural alterations on the mitochondrial membrane can be inferred from changes in the Mg~*-ATPase [33]. Compared with sucklings, weanlings showed significantly lesser amount of Mg2÷-ATPase. It is, therefore, evident from our results that mitochondria from weanlings show an increased basal ATPase activity. /

128

Also changes in the lipid composition of the diet alter biochemical composition of rat hepatic endoplasmic reticulum membranes [17]. Therefore, it is most likely that mitochondrial membranes of weaning mice are subjected to a similar change on account of exposure to a new diet. It is an accepted fact that cytochrome a a 3 (oxidase) plays a crucial role in mitochondrial oxidative energy metabolism [34]. Our results showed that this enzyme followed decline of RCR during weaning and its restoration in the adult stage. Results also indicated that the mitochondrial cytochrome a a 3 content in the suckling was more than that of adults. In conclusion, our studies showed that weaning was associated with a temporary decrease in the oxidative energy metabolism of the mouse hepatic mitochondria. This altered level recovered in the adult stage. Our results indicate that cytochrome a a 3 may be of primary importance in the restoration of respiratory function. ACKNOWLEDGEMENT

The authors wish to thank Dr. D.S. Pradhan, Associate Director, Biochemical Group, for advice and encouragement and Drs. K.G. Raghavan, U. Tarachand and T.P.A. Devasagayam, Biochemistry Division, for helpful suggestions. REFERENCES 1 2

3 4 5 6 7 8 9 10 11 12 13

B.L. Strehler, Time, Cells andAging (2nd edn.), Academic Press, New York, 1977, pp. 243--247. T.P.A. Devasagayam, C.K. Pushpendran and J. Eapen, Changes in enzymes of hepatic rough and smooth microsomes during post-natal development and aging of rats. Mech. Ageing Dev., 21 (1983) 365--375. B.M. Stanulis-Praeger, Cellular senescence revisited: a review. Mech. Ageing Dev., 38 (1987) 1--48. J. Miquel, A.C. Economos, J.E. Fleming and J.E. Johnson, Jr., Mitochondrial role in cell aging. Exp. Gerontol., 15 (1980) 575--591. J.E. Fleming, J. Miquel, S.F. Cottrell, L.S. Yengoyan and A.C. Economos, Is cell aging caused by respiration dependent injury to the mitochondrial genome? Gerontology, 28 (1982) 44--53. Z.A. Medveder, Age related polyploidization of hepatocytes: The cause and possible role. A minireview. Exp. GerontoL, 21 (1986) 277--282. H. Ikeda, H. Tauchi and T. Sato, Fine structural analysis of lipofusion in various tissues of rats of different ages. Mech. Ageing Dev., 33 (1985) 77--93. A.A. Horton and J.A. Spencer, Decline in respiratory control ratio of rat liver mitochondria in old age. Mech. Ageing Dev., 17 (1981) 253--259. D.L. Knook, Organ ageing in relation to cellular ageing. In A. Viidik (ed.), Lectures on Gerontology, VoI.I; on Biology o f Ageing, Pt. A, Academic Press, London, 1982, pp. 213--251. M.T. Clandinin and S.M. Innis, Does mitochondrial ATP synthesis decline as a function of change in the membrane environment with ageing. Mech. Ageing Dev., 22 (1983) 205--208. D. Harman, Free radical theory of aging: Consequences of mitochondriai aging. Age, 6 (1983) 86-94. D.L. Marcus, N.G. lbrahim and M.L. Freedman, Age related decline in the biosynthesis of mitochondrial inner membrane proteins. Exp. GerontoL, 17(1982) 333--341. B.L. Strehler, Genetic instability as the primary cause of human aging. Exp. Gerontol., 21 (1986) 283--319.

129

14 15

16 17

18 19

20 21 22 23

24 25 26 27 28

29 30 31 32

33 34

P.A. Ceruti, Modern Biological Theories o f Aging, H.R. Warner, R.N. Butler, R.L. Sprott and E.L. Schneider (eds.), Raven Press, New York, 1987, pp. 199--209. B.J. Merry, A.M. Holehan, S.E.M. Lewis and D.F. Goldspink, The effects of ageing and chronic dietary restriction on in vivo hepatic protein synthesis in rat. Mech. Ageing Dev., 39 (1987) 189-200. B.R. Sonawane, P.M. Coates, S.J. Yaffe and O. Koldovsky, Influence of prenatal nutrition on hepatic drug metabolism in the adult rat. Dev. PharmacoL Ther., 6 (1983) 323--332. C.T. Hammer and E.D. Wills, The role of lipid components of the diet in the regulation of the fatty acid composition of the rat liver endoplasmic reticulum and lipid peroxidation. Biochem. J., 174 (1978) 585--593. J.K. Pollak, The maturation of the inner membrane of foetal rat liver mitochondria. Biochem. J., 150 (1975) 477--488. J.K. Pollak, Mitochondrial heterogeneity in foetal and suckling rat liver, differential leucine incorporation into the proteins of two mitochondrial populations. Biochem. Biophys. Res. Commun., 69 (1976) 823--829. R. Rajan, Studies on hormonal regulation o f brain development and drug membrane interaction, Ph.D Thesis, Univ. of Bombay 0989). S.S. Katyare and R. Rajan, Enhanced oxidative phosphorylation in rat liver mitochondria following prolonged in vivo treatment with imipramine. Br. J. Pharmacol., 95 (1988) 914--922. B. Chance and G.R. Williams, Respiratory enzymes in oxidative phosphorylation. Kinetics of oxygen utilization. J. BioL Chem., 217 (1955) 383--393. S.S. Katyare, M.V.J. Joshi, P. Fatterpaker and A. Sreenivasan, Effect of thyroid deficiency on oxidative phosphorylation in rat liver, kidney and brain mitochondria. Arch. Biochem. Biophys., 182 (1977) 155--163. B.F. Van Gelder, On cytochrome c oxidase. 1. The extinction coefficients of cytochrome a and cytochrome a 3. Biochem. Biophys. Acta, 118 (1966) 36--45. W.S. Zangg and J.S. Rieske, The quantitative estimation of cytochrome b in submitochondrial particles from beef heart. Biochem. Biophys. Res. Commun., 9 (1962) 213--217. E. Margoliash, N. Frohwirt and E. Wiener, A study of the cytochrome c haemochromogen. Biochem. J., 71 (1959) 559--570. J.G. Satav and S.S. Katyare, Effect of experimental thyrotoxicosis on oxidative phosphorylation in rat liver, kidney and brain mitochondria. Molec. Cell. Endocrinol., 28 (1982) 173--189. F. Leighton, B. Poole, H. Beaufy, P. Baudhuim, J.M. Coffer, S. Flower and C. De Duve, The large-scale separation of peroxisomes, mitochondria and lysosomes from livers of rats injected with triton X-100. Improved isolation procedures, analysis and biochemical properties of fraction. J. Cell Biol., 3 7 (1968) 482--513. M. Miyahara, K. Utsumi and D.W. Deamer, Selective interaction of D-/~-hydroxybutyrate dehydrogenase with intracellular membranes. Biochim. Biophys. A cta, 641 (1981 ) 222--231. A.J. Caplan and J.W. Greenwalt, The effect of osmotic lysis on the oxidative phosphorylation and compartmentation. J. Cell Biol., 36 (1968) 15--31. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with folin phenol method. J. BioL Chem., 193 (1951) 265--275. H.S. Penefsky, M.F. Pullman, A. Datta and E. Racker, Partial resolution of the enzymes catalyzing oxidative phosphorylation. II. Participation of a soluble adenosine triphosphatase in oxidative phosphorylation. J. BioL Chem., 235 (1960) 3330--3336. S.S. Katyare and J.G. Satav, Impaired mitochondrial oxidative energy metabolism following paracetamol-induced hepatotoxicity in the rat. Br. J. PharmacoL, 96 (1989) 5 i--58. R.A. Capaldi, F. Malatesta and V.M. Darley-Usmar, Structure of cytochrome c oxidase. Biochim. Biophys. Acta., 726(1983) 135--148.