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Mitochondrial oxidative phosphorylation and intracellular glutathione compartmentation during rat liver regeneration
Mitochondrial Oxidative Phosphorylation and Intracellular Glutathione Compartmentation During Rat Liver Regeneration GIANLUIGI VENDEMIALE, 1 FERRUCCIO GUERRIERI, 2 IGNAZIO GRATTAGLIANO, 1 DOMENICO DIDONNA, 1 LEONILDE MUOLO, 2 AND E M A N U E L E ALTOMARE ~
The rate of mitochondrial oxidative phosphorylation and the cytosolic and mitochondrial total and oxidized glutathione concentrations were studied in regenerating rat livers after partial (70%) hepatectomy. The rate of mitochondrial oxidative phosphorylation progressively decreased during the early prereplicative phase of liver regeneration. This was accompanied by a progressive decrease in mitochondrial, but not cytosolic, glutathione concentration. Twenty.four hours after partial hepatectomy, both the rate of adenosine triphosphate (ATP) synthesis and the amount of mitochondrial glutathione were depressed by 50% with respect to controls (sham. operated animals). During the second replicative phase, both the oxidative phosphorylation rate and mitochon. drial glutathione concentration were recovered; however, the kinetics of the recovery were different, being the total amount of mitochondrial glutathione completely restored 48 hours after partial hepatectomy, whereas 72 hours were needed for the recovery of oxidative phosphorylation. The decrease in the rate of oxidative phosphorylation, during the early phase of liver regeneration, appeared to be secondary to the decreased content of the catalytic subunit fl-F, of the ATP synthase complex, which in turn was shown to be linearly related to the decrease of intramitochondrial glutathione. These observations suggest that the two phenomena may be due to the previously reported increased free radical production during the early phase of liver regeneration. The depression of mitochondrial glutathione after partial hepatectomy may play a contributory role in structural and functional alterations of mitochondria occurring in the first retrodifferential phase of liver
regeneration. (HEPATOLOGY1995;21:1450-1454.) The liver is a useful organ for the study of growth regulation in mammals. Under different experimental conditions, such as regeneration or neoplasia, the hepatocytes of adult animals can easily enter the replicative Abbreviations: GSH, total glutathione; GSSG, oxidized glutathione; ATP, adenosine triphosphate; PH, partial hepatectomy. From the 1Institute of Clinica Medica I, and the 2 Institute of Biochimica and Chimica Medica, University of Bari, Bari, Italy. Received May 24, 1994; accepted December 12, 1994. Address reprint requests to: Gianluigi Vendemiale, MD, Istituto di Clinica Medica I, Universit& di Bari, Policlinico, P.zza G. Cesare 11, 70124 Bari, Italy. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2105-003153.00/0
cycle. 1 In particular, rat liver regeneration is a process characterized by a prereplicative phase, or retrodifferentiation, in which the hepatocytes of the remaining liver show drastic metabolic changes, followed by a replicative phase characterized by an active DNA synthesis. 1'2 In addition, it is well known that partial hepatectomy greatly increases the demand of the liver for energy that is required for biosynthesis of cellular components. 3 Because, in normal conditions, cellular energy is mainly supplied by the mitochondrial oxidative phosphorylation system, liver regeneration appears to be closely dependent on mitochondrial respiratory functions. Several studies have shown a decrease of mitochondrial adenosine triphosphatase activity during the early phase of rat liver regeneration. 4-8 One of the possible mechanisms for this finding may be an overproduction of oxygen free radicals occurring during rat liver regeneration. 3 This could result in alterations of proteins and lipids of the inner mitochondrial membrane. 79 Hepatic glutathione has an especially important relationship with lipid peroxidation because of the known characteristic of such a compound to combine with free radicals that may initiate peroxidation, as well as to reduce hydrogen peroxide formed in cells. 1°'11 Glutathione, which is intracellularly distributed in both the mitochondrial and cytosolic compartments, is not synthesized into mitochondria, and a transport system mediates its uptake from cytosol. ~2''3 A number of studies have elucidated the importance of glutathione and particularly of its mitochondrial pool for mitochondrial and cell functions. 14~7 However, the intracellular distribution of glutathione during rat liver regeneration has not been explored. This prompted us to evaluate the cytosolic and mitochondrial glutathione content during rat liver regeneration and to explore possible relationships between changes in mitochondrial glutathione levels and the rate of oxidative phosphorylation. MATERIALS AND METHODS Partial Hepatectomy. Male Wistar rats (300 g) were anesthetized with ether/oxygen, and the median and left lateral lobes of the liver (corresponding to about 70% of the whole liver) were excised. ~ Rats were then killed at different times (12, 24, 48, 72, and 96 hours) by decapitation, and the regen-
1450
HEPATOLOGYVol. 21, No. 5, 1995 erated liver was used to prepare the cytosol and mitochondria. All operations were carried out under sterile conditions. Sham-operated rats were subjected to the same treatment without excision of the liver. The study was approved by the state commission on animal experimentation. Preparation of Cytosol and Mitochondria. For the determination of total (GSH) and oxidized glutathione (GSSG), immediately after removal, the liver was cut into small pieces and homogenized in 10 volumes 0.44 mol/L of mannitol, 0.07 mol/L of sucrose, 5 mmol/L of 3-morpholinopropane sulfonic acid containing 0.1 mol/L of Na-ethylenediaminetetraacetic acid 50:1 (v/v). All subsequent manipulations were carried out at about 4°C. The homogenate was centrifuged at 700g for 10 minutes, and the supernatant at 7,000g for 10 minutes. The pellet was used for preparation of mitochondria by two centrifugations at 7,000g for 10 minutes. The supernatant was used for preparation of cytosol by ultracentrifugation at 105,000g. Cytosolic and mitochondrial fractions were treated with 15% (w/ v) sulfosalicylic acid containing 5 mmol/L of ethylenediaminetetraacetic acid. To determine the rate of adenosine triphosphate (ATP) synthesis and for immunodetection of fl-F1, rat liver mitochondria were prepared according to Pedersen. is The quality of mitochondria was determined by analysis of respiratory control ratio, which represents an index of coupling of the mitochondria. It was obtained by the ratio between the rate of oxygen consumption in phosphorylating conditions (presence of adenosine diphosphate and Pi or state 3) and rate of oxygen consumption in absence of adenosine diphosphate (or state 4) in succinate-supplemented mitochondria. 19 Both control and sham-operated animals showed a good coupling (RCI = 4.88 _+ 0.12 and 4.92 ± 0.14, respectively). Assays. To measure the rate of ATP synthesis, mitochondria (500 #g protein/mL) were suspended in 1 mL of 200 mmol/L of sucrose, 3 mmol/L of MgC12, 1 mmol/L of ethylenediaminetetraacetic acid, 10 mmol/L ofKPi (pH 7.4), 20 mmol/ L of glucose, 0.25% bovine serum albumine, 5 U ofhexokinase and 300 #mol/L ofpl,p5-Di[adenosine-S-] pentaphosphate (to inhibit adenylate kinase). After equilibration for 2 minutes, 20 mmot/L K-succinate was added. Five minutes later, 300 #mol/L Mg-adenosine diphosphate was added, and the ATP synthesized in a 3-minute interval was measured spectrophotometrically by determining glucose-6-P with glucose-6-P-dehydrogenase on a deproteinized sample of the incubation mixtureJ ° Assays of GSH and GSSG were performed enzymatically according to the methods of Tietze 2~ and Griffith, 22 respectively. Electrophoresis and Immunoblotting Procedures. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of mitochondria (50 #g protein) was performed on slab gels with a linear gradient polyacrylamide (14% to 20%). 23 Sodium dodecyl sulfate gels were subjected to immunoblot analysis using polyclonal antibodies against bovine F~23 (which cross-react with the fl-subunit of liver complex24). Nitrocellulose sheets were scanned at 590 nm. The quantity of antigen detected was evaluated from the computed peak areas and expressed in arbitrary units. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or represented the best available commercial grades. Statistics. Data are expressed as mean ± SEM of five animals. Statistical comparisons between groups were made by Student's unpaired t-test or by regression coefficients. Data were also analyzed by one-way ANOVA. RESULTS
F i g u r e 1A shows t h a t , after p a r t i a l h e p a t e c t o m y (PH), the r a t e of A T P s y n t h e s i s in m i t o c h o n d r i a , u s i n g
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Time after partial hepatectomy (h) FIG. 1. Changes in the rate of ATP synthesis and in the relative content of13-F1subunit ofATP synthase complexduring rat liver regeneration. PH, preparation of liver mitochondria, determination of rate of ATP synthesis (A), and immunoblot analysis of relative content of/% F1 subunit (B) were carried out as described in Materials and Methods. Figures reported in the picture are the mean _+SEM of five animals.
succinate as r e s p i r a t o r y s u b s t r a t e , decreased. The lowest value w a s observed 24 h o u r s after PH. The decrease in the rate of A T P synthesis, observed in the first phase, was followed by a recovery of the activity in the
1452 VENDEMIALEET AL second phase of liver regeneration. The mitochondrial F1-Fo ATP synthase is composed of two sectors: the catalytic F1 sector, which consists of five nuclear encoded subunits (a,/3, % 5, c) and the membrane sector, F0, which consists of nine subunits. 23 The changes in the rate of ATP synthesis, during liver regeneration, were associated with comparable changes in the content of/3-F1 subunit of mitochondria estimated by iramunoblot raised against bovine F1 (Fig. 1B). The mitochondrial total glutathione concentration during rat liver regeneration is shown in Fig. 2A. Compared with control rats, a progressive and significant decrease of total GSH levels was observed during the early phase of liver regeneration. Mitochondrial GSH concentration reached the lowest value 24 hours after PH, started to recover during the second phase of liver regeneration and appeared to be completely restored 48 hours after PH. A different pattern was observed in the cytosol (Fig. 2B). Total GSH concentration was found to be comparable in the two groups during the first 24 hours, whereas after 48 hours and 72 hours, a significant increase of GSH was found in regenerating rat livers compared with those of control rats. Table 1 shows t h a t there were no changes in the concentration of mitochondrial GSSG after PH. It must be pointed out, however, t h a t 24 hours after PH mitochondria from regenerating rat livers do have higher GSSG/GSH ratio t h a n mitochondria from sham-operated rats. Figure 3 shows that, during the early phase (0 to 24 hours) of liver regeneration the decreases ofmitochondrial GSH and of ~-F1 subunit were linearly correlated (r = .925, P < .001). DISCUSSION
Our study shows t h a t the structural and functional changes of mitochondrial ATP synthase reported in this and other studies 4'6'19 are accompanied by remarkable alterations in the mitochondrial GSH levels during rat liver regeneration. The kinetics of these changes were very similar, showing a decrease in the early prereplicative phase and a recovery in the second replicative phase. The close kinetic relationship suggests a correlation between the two phenomena. It is well known t h a t GSH plays a critical role in a number of structural and functional processes of the cells and in several detoxification reactions9 5-27 Several investigations have provided evidence for a mitochondrial compartmentatio~ of GSH 2s'29 and have shown t h a t the mitochondrial p, : of GSH is essential for normal mitochondrial function. ~)Because mitochondria continually produce reactive oxygen species and contain no catalase, they would appear to be largely, if not entirely, dependent on the GSH-GSH peroxidase system. Thus, it would appear t h a t mitochondrial GSH is the principal functional antioxidant. ~3'3~ In spite of the importance of this mitochondrial compartmentation, mitochondria do not contain the enzyme activities required for GSH synthesis and effectively transport this tripeptide from the cytosolic compartment with an ATP-dependent mechanism. 12'13 Recently, an overproduction
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Time after partial hepatectomy (h) FIG. 2. Total glutathione (GSH) concentration in mitochondria (A) and cytosol (B) of rats during liver regeneration and controls (sham-operated). PH, preparation of mitochondria, and glutathione determination are described in Materials and Methods. Data represent the mean _+SEM of five animals.
of oxygen free radicals has been suggested during the early phase of rat liver regeneration, which might account for the reported decrease of adenosine triphosphatase and ATP synthase activities. 3 This hypothesis is supported by the decrease of the adenosine triphos-
HEPATOLOGYVol. 21, No. 5, 1995
VENDEMIALE ET AL
phatase activity during exposure of rat liver mitochondria to oxygen free radicals2 '32 It is therefore conceivable that the stress produced by liver resection could be responsible for a sharp increase of the production of mitochondrial oxygen free radicals 3 followed by a decrease of GSH levels and inactivation of the membrane-bound mitochondrial ATP synthase complex. Another possible explanation for the decrease of mitochondrial GSH during the early phase of liver regeneration could be an increased efflux of GSH outside of mitochondria; however, the finding of a very slow transport of GSH from mitochondria to cytoplasm 3° argues against this hypothesis and suggests that this transport mechanism functions to conserve mitochondrial GSH. An elevated rate of GSH oxidation as a cause of mitochondrial GSH depletion seems to be rather unlikely, because no changes of mitochondrial GSSG concentrations were observed in the current study; however, 24 hours after PH, regenerating livers do apparently have higher GSSG/GSH ratio than controls, which may be physiologically important. It is also possible that the reported efflux of GSSG from mitochondria, under conditions of severe oxidative stress, 33 could contribute to determine relatively low intramitochondrial GSSG levels. Because, as mentioned, GSH is not synthesized into mitochondria, being actively imported from the cytoSO112'13 by an ATP-dependent transport, our data could also suggest that during the first retrodifferential phase of liver regeneration, the ATP-dependent intramitochondrial transport of GSH might decrease after the fall of mitochondrial ATP synthesis. In fact, it should be observed that the cytosolic GSH does not fall in this first phase of liver regeneration (24 hours) and is even increased in the second phase. This apparently conflicting phenomenon could be explained by the fact that in the first phase of liver regeneration the synthesis of cytosolic GSH may use ATP produced by anaerobic glycolysis, which has been reported to be increased in this period. 1 In the second phase (48 hours after PH), the level of cytosolic GSH increased about two times with respect to sham-operated animals, which is in agreement with the study of Lee and Boyer34; this increment was accompanied by the resto-
TABLE 1. T i m e C o u r s e o f M i t o c h o n d r i a l G S S G Concentration and of GSSG/GSH Ratio After Partial Hepatectomy Partial Hepatectomy
Sham-Operated
Time (hr)
GSSG (pmol/mg prot)
GSSG/GSH (×10 3)
GSSG (pmol/mg prot)
GSSG/GSH (×10 3)
To 24 48 72 96
58_+8 56_+ 1 63_+ 1 51_+ 1 46± 8
16_+ 3 29-6" 15 ± 4 11 ± 1 10_+ 1
58_+8 42_+6 60 ÷ 6 67_+ 6 41_+ 4
16 ÷ 3 11+ 1 15 ÷ 1 16 + 1 10_+ 1
NOTE. Results are expressed as mean _+ SEM of five animals. * P < .01 when compared with controls.
1453
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FIG. 3. Linear relationship between decrease of relative amount of immunodetected ~-F1 subunit and of mitochondrial GSH during the early phase of liver regeneration. Data represent the percentage decrease of ~-F1 subunit and of total mitochondrial GSH at 12 and 24 hours after PH compared with their respective values at time 0 (time 0 - 100%).
ration of mitochondrial GSH content. This finding seems to exclude the possibility of an increased cytosolic GSH secondary to a lack of GSH transport into mitochondria. It could be noted that at this time (48 hours after PH) the rate of mitochondrial ATP synthesis did not change whereas the level of mitochondrial GSH and ~-F1 (which both require ATP for their synthesis and transport into mitochondria) increased. This observation suggests that in this interval (24 to 48 hours) after PH, the synthesis and the transport of these compounds should be carried out by the ATP produced by the anaerobic glycolysis. Further investigations are required to fully understand the underlying mechanism and the pathophysiological consequences of mitochondrial GSH depression during rat liver regeneration. GSH, however, has been shown to be essential for mitochondrial functioni3'3°'31; its depletion, moreover, seems to markedly enhance the susceptibility to mitochondrial dysfunction from oxidant stress. ~7 Therefore, it is conceivable that the decrease of mitochondrial GSH during the first 24 hours after PH might play a role in the remarkable metabolic changes occurring in the first retrodifferential phase of rat liver regeneration. 1.3 REFERENCES
1. Uriel J. Retrodifferentiation and the fetal patterns of gene expression in cancer. Adv Cancer Res 1979;29:127-174. 2. Michelopulos GK. Liver regeneration: molecular mechanisms of growth control. FASEB J 1990;4:176-187.
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VENDEMIALE ET AL
3. Tsai IL, King KL, Chang CC, Wei Y. Changes of mitochondrial respiratory functions and superoxide dismutase activity during liver regeneration. Biochem Int 1992;28:205-217. 4. Guerrieri F, Capozza G, Kalous M, Papa S. Age-related changes of mitochondrial Fo-F1 ATP synthase. Ann NY Acad Sci 1992; 395-402. 5. Buckle M, Guerrieri F, Papa S. Changes in activity and F1 content of mitochondrial H+ATPase in regenerating rat liver. FEBS Lett 1985;188:345-351. 6. Buckle M, Guerrieri F, Pazienza A, Papa S. Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial Fo-F~ H+ATPase in regenerating rat liver. Eur J Biochem 1986;155:439-445. 7. Sohal RS, Sohal BH. Hydrogen peroxide released by mitochondria increases during aging. Mech Ageing Dev 1991;57:187-202. 8. Nagy I. A membrane hypothesis of aging. J Theor Biol 1978; 75:189-195. 9. Zhang Y, Macillot O, Giulivi C, Ernesti L, Davies JA. The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J Biol Chem 1990;265:16330-16336. 10. Orrenius S, Jones DP. Functions of glutathione in drug metabolism. In: Sies H, Wendell A, eds. Functions of glutathione in liver and kidney. Berlin: Springer-Verlag, 1978:164-175. 11. Kaplowitz N. The importance and regulation of hepatic glutathione. Yale J Biol Med 1981;54:497-502. 12. Kurosawa K, Hayashi N, Sato N, Kamada T, Tagawa K. Transport of glutathione across the mitochondrial membranes. Biochem Biophys Res Commun 1990; 167:367-372. 13. Martensson J, Lai JC, Meister A. High affinity transport ofglutathione is part of a multicomponent system essential for mitochondrial function. Proc Natl Acad Sci U S A 1990;87:7185-7189. 14. Martensson J, Meister A. Mitochondrial damage in muscle occurs after marked depletion of glutathione and is prevented by giving glutathione monoesters. Proc Natl Acad Sci U S A 1989;86:471-475. 15. Martensson J, Tain A, Frayer W, Meister A. Glutathione metabolism in the lung: inhibition of its synthesis leads to lamellar body and mitochondrial defects. Proc Natl Acad Sci U S A 1989;86: 5296-5300. 16. Olafsdottir K, Pascoe GA, Reed DJ. Mitochondrial glutathione status during Ca2÷ ionophore-induced injury to isolated hepatocytes. Arch Biochem Biophys 1988;263:226-235. 17. Hirano T, Kaplowitz N, Tsukamoto H, Kamimura S. Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats. HEPATOLOGY1992;16: 1423-1427. 18. Pedersen PL. High yield preparative method for isolation of rat liver mitochondria. Anal Biochem 1977;80:401-408.
HEPATOLOGYMay 1995 19. Guerrieri F, Kalous M, Capozza G, Muolo L, Drahota Z, Papa S. Age dependent changes in mitochondrial F0-F1 ATP synthase in regenerating rat-liver. Biochem Mol Biol Int 1994;33:117-129. 20. Papa S, Tager JM, Guerrieri F, Quagliariello E. Effect of monovalent cations on oxidative phosphorylation in submitochondrial particles. Biochem Biophys Acta 1969; 172:184-186. 21. Tietze F. Enzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969;27: 502-522. 22. Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinyl-pyridine. Anal Biochem 1980;106:207-212. 23. Guerrieri F, Kopecky J, Zanotti F. Functional and immunological characterization of mitochondrial FoFI ATP synthase. In: Tager JM, Azzi A, Papa S, et al, eds. Organelles in eukaryotic cells: molecular structure and interactions. New York, London: Plenum Publ. Co., 1989:197-208. 24. Guerrieri F, Capuano F, Buckle M, Papa S. Alteration of mitochondrial H+ATPase complex in tissue regeneration and neoplasia. In: Gorrod JM, Albano O, Papa S, eds. Molecular aspects of human disease. London: Ellis Horwood Ltd., 1989:108-114. 25. Kosower ND, Kosower EM. The glutathione status of cells. Int Rev Cytol 1978;54:104-160. 26. Reed DJ, Beatty PW. Biosynthesis and regulation of glutathione: toxicological implications. In: Hodgson E, Bend JR, Philpot RM, eds. Reviews in Biochemical Toxicology 2. New York: Elsevier, 1980:213-241.
27. Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983;52:711-760. 28. Jocelyn PC, Kamminga A. The non-proteinthiol of rat liver mitochondria. Biochim Biophys Acta 1974;343:356-362. 29. Meredith MJ, Reed DJ. Status of mitochondrial pool of glutathione in the isolated hepatocyte. J Biol Chem 1982;257:3747-3753. 30. Griffith OW, Meister A. Origin and turnover of mitochondrial glutathione. Proc Nat] Acad Sci U S A 1985;82:4668-4672. 31. Masini A, Ceccarelli D, Trenti T, Gallesi D, Muscatello U. Mitochondrial inner membrane permeability changes induced by octadecadienoic acid hydroperoxide: role of mitochondrial GSH pool. Biochim Biophys Acta 1992; 1101:84-89. 32. Guerrieri F, Capozza G, Fratello A, Zanotti F, Papa S. Functional and molecular changes in Fo-F1ATP-synthase of cardiac muscle during aging. Cardioscience 1993;4:93-98. 33. Brodie AE, Reed DJ. Glutathione disulfide reduction in tumor mitochondria after t-Butyl-Hydroperoxide treatment. Chem Biol Interact 1992;84:125-132. 34. Lee SJ, Boyer TD. The effect of hepatic regeneration on the expression of the glutathione S-transferases. Biochem J 1993; 293:137-142.
The rate of mitochondrial oxidative phosphorylation and the cytosolic and mitochondrial total and oxidized glutathione concentrations were studied in regenerating rat livers after partial (70%) hepatectomy. The rate of mitochondrial oxidative phosphorylation progressively decreased during the early prereplicative phase of liver regeneration. This was accompanied by a progressive decrease in mitochondrial, but not cytosolic, glutathione concentration. Twenty.four hours after partial hepatectomy, both the rate of adenosine triphosphate (ATP) synthesis and the amount of mitochondrial glutathione were depressed by 50% with respect to controls (sham. operated animals). During the second replicative phase, both the oxidative phosphorylation rate and mitochon. drial glutathione concentration were recovered; however, the kinetics of the recovery were different, being the total amount of mitochondrial glutathione completely restored 48 hours after partial hepatectomy, whereas 72 hours were needed for the recovery of oxidative phosphorylation. The decrease in the rate of oxidative phosphorylation, during the early phase of liver regeneration, appeared to be secondary to the decreased content of the catalytic subunit fl-F, of the ATP synthase complex, which in turn was shown to be linearly related to the decrease of intramitochondrial glutathione. These observations suggest that the two phenomena may be due to the previously reported increased free radical production during the early phase of liver regeneration. The depression of mitochondrial glutathione after partial hepatectomy may play a contributory role in structural and functional alterations of mitochondria occurring in the first retrodifferential phase of liver
regeneration. (HEPATOLOGY1995;21:1450-1454.) The liver is a useful organ for the study of growth regulation in mammals. Under different experimental conditions, such as regeneration or neoplasia, the hepatocytes of adult animals can easily enter the replicative Abbreviations: GSH, total glutathione; GSSG, oxidized glutathione; ATP, adenosine triphosphate; PH, partial hepatectomy. From the 1Institute of Clinica Medica I, and the 2 Institute of Biochimica and Chimica Medica, University of Bari, Bari, Italy. Received May 24, 1994; accepted December 12, 1994. Address reprint requests to: Gianluigi Vendemiale, MD, Istituto di Clinica Medica I, Universit& di Bari, Policlinico, P.zza G. Cesare 11, 70124 Bari, Italy. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2105-003153.00/0
cycle. 1 In particular, rat liver regeneration is a process characterized by a prereplicative phase, or retrodifferentiation, in which the hepatocytes of the remaining liver show drastic metabolic changes, followed by a replicative phase characterized by an active DNA synthesis. 1'2 In addition, it is well known that partial hepatectomy greatly increases the demand of the liver for energy that is required for biosynthesis of cellular components. 3 Because, in normal conditions, cellular energy is mainly supplied by the mitochondrial oxidative phosphorylation system, liver regeneration appears to be closely dependent on mitochondrial respiratory functions. Several studies have shown a decrease of mitochondrial adenosine triphosphatase activity during the early phase of rat liver regeneration. 4-8 One of the possible mechanisms for this finding may be an overproduction of oxygen free radicals occurring during rat liver regeneration. 3 This could result in alterations of proteins and lipids of the inner mitochondrial membrane. 79 Hepatic glutathione has an especially important relationship with lipid peroxidation because of the known characteristic of such a compound to combine with free radicals that may initiate peroxidation, as well as to reduce hydrogen peroxide formed in cells. 1°'11 Glutathione, which is intracellularly distributed in both the mitochondrial and cytosolic compartments, is not synthesized into mitochondria, and a transport system mediates its uptake from cytosol. ~2''3 A number of studies have elucidated the importance of glutathione and particularly of its mitochondrial pool for mitochondrial and cell functions. 14~7 However, the intracellular distribution of glutathione during rat liver regeneration has not been explored. This prompted us to evaluate the cytosolic and mitochondrial glutathione content during rat liver regeneration and to explore possible relationships between changes in mitochondrial glutathione levels and the rate of oxidative phosphorylation. MATERIALS AND METHODS Partial Hepatectomy. Male Wistar rats (300 g) were anesthetized with ether/oxygen, and the median and left lateral lobes of the liver (corresponding to about 70% of the whole liver) were excised. ~ Rats were then killed at different times (12, 24, 48, 72, and 96 hours) by decapitation, and the regen-
1450
HEPATOLOGYVol. 21, No. 5, 1995 erated liver was used to prepare the cytosol and mitochondria. All operations were carried out under sterile conditions. Sham-operated rats were subjected to the same treatment without excision of the liver. The study was approved by the state commission on animal experimentation. Preparation of Cytosol and Mitochondria. For the determination of total (GSH) and oxidized glutathione (GSSG), immediately after removal, the liver was cut into small pieces and homogenized in 10 volumes 0.44 mol/L of mannitol, 0.07 mol/L of sucrose, 5 mmol/L of 3-morpholinopropane sulfonic acid containing 0.1 mol/L of Na-ethylenediaminetetraacetic acid 50:1 (v/v). All subsequent manipulations were carried out at about 4°C. The homogenate was centrifuged at 700g for 10 minutes, and the supernatant at 7,000g for 10 minutes. The pellet was used for preparation of mitochondria by two centrifugations at 7,000g for 10 minutes. The supernatant was used for preparation of cytosol by ultracentrifugation at 105,000g. Cytosolic and mitochondrial fractions were treated with 15% (w/ v) sulfosalicylic acid containing 5 mmol/L of ethylenediaminetetraacetic acid. To determine the rate of adenosine triphosphate (ATP) synthesis and for immunodetection of fl-F1, rat liver mitochondria were prepared according to Pedersen. is The quality of mitochondria was determined by analysis of respiratory control ratio, which represents an index of coupling of the mitochondria. It was obtained by the ratio between the rate of oxygen consumption in phosphorylating conditions (presence of adenosine diphosphate and Pi or state 3) and rate of oxygen consumption in absence of adenosine diphosphate (or state 4) in succinate-supplemented mitochondria. 19 Both control and sham-operated animals showed a good coupling (RCI = 4.88 _+ 0.12 and 4.92 ± 0.14, respectively). Assays. To measure the rate of ATP synthesis, mitochondria (500 #g protein/mL) were suspended in 1 mL of 200 mmol/L of sucrose, 3 mmol/L of MgC12, 1 mmol/L of ethylenediaminetetraacetic acid, 10 mmol/L ofKPi (pH 7.4), 20 mmol/ L of glucose, 0.25% bovine serum albumine, 5 U ofhexokinase and 300 #mol/L ofpl,p5-Di[adenosine-S-] pentaphosphate (to inhibit adenylate kinase). After equilibration for 2 minutes, 20 mmot/L K-succinate was added. Five minutes later, 300 #mol/L Mg-adenosine diphosphate was added, and the ATP synthesized in a 3-minute interval was measured spectrophotometrically by determining glucose-6-P with glucose-6-P-dehydrogenase on a deproteinized sample of the incubation mixtureJ ° Assays of GSH and GSSG were performed enzymatically according to the methods of Tietze 2~ and Griffith, 22 respectively. Electrophoresis and Immunoblotting Procedures. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of mitochondria (50 #g protein) was performed on slab gels with a linear gradient polyacrylamide (14% to 20%). 23 Sodium dodecyl sulfate gels were subjected to immunoblot analysis using polyclonal antibodies against bovine F~23 (which cross-react with the fl-subunit of liver complex24). Nitrocellulose sheets were scanned at 590 nm. The quantity of antigen detected was evaluated from the computed peak areas and expressed in arbitrary units. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or represented the best available commercial grades. Statistics. Data are expressed as mean ± SEM of five animals. Statistical comparisons between groups were made by Student's unpaired t-test or by regression coefficients. Data were also analyzed by one-way ANOVA. RESULTS
F i g u r e 1A shows t h a t , after p a r t i a l h e p a t e c t o m y (PH), the r a t e of A T P s y n t h e s i s in m i t o c h o n d r i a , u s i n g
VENDEMIALE ET AL 1451 ,%'. ~ A ~ 350 m E c 3OO
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Time after partial hepatectomy (h) FIG. 1. Changes in the rate of ATP synthesis and in the relative content of13-F1subunit ofATP synthase complexduring rat liver regeneration. PH, preparation of liver mitochondria, determination of rate of ATP synthesis (A), and immunoblot analysis of relative content of/% F1 subunit (B) were carried out as described in Materials and Methods. Figures reported in the picture are the mean _+SEM of five animals.
succinate as r e s p i r a t o r y s u b s t r a t e , decreased. The lowest value w a s observed 24 h o u r s after PH. The decrease in the rate of A T P synthesis, observed in the first phase, was followed by a recovery of the activity in the
1452 VENDEMIALEET AL second phase of liver regeneration. The mitochondrial F1-Fo ATP synthase is composed of two sectors: the catalytic F1 sector, which consists of five nuclear encoded subunits (a,/3, % 5, c) and the membrane sector, F0, which consists of nine subunits. 23 The changes in the rate of ATP synthesis, during liver regeneration, were associated with comparable changes in the content of/3-F1 subunit of mitochondria estimated by iramunoblot raised against bovine F1 (Fig. 1B). The mitochondrial total glutathione concentration during rat liver regeneration is shown in Fig. 2A. Compared with control rats, a progressive and significant decrease of total GSH levels was observed during the early phase of liver regeneration. Mitochondrial GSH concentration reached the lowest value 24 hours after PH, started to recover during the second phase of liver regeneration and appeared to be completely restored 48 hours after PH. A different pattern was observed in the cytosol (Fig. 2B). Total GSH concentration was found to be comparable in the two groups during the first 24 hours, whereas after 48 hours and 72 hours, a significant increase of GSH was found in regenerating rat livers compared with those of control rats. Table 1 shows t h a t there were no changes in the concentration of mitochondrial GSSG after PH. It must be pointed out, however, t h a t 24 hours after PH mitochondria from regenerating rat livers do have higher GSSG/GSH ratio t h a n mitochondria from sham-operated rats. Figure 3 shows that, during the early phase (0 to 24 hours) of liver regeneration the decreases ofmitochondrial GSH and of ~-F1 subunit were linearly correlated (r = .925, P < .001). DISCUSSION
Our study shows t h a t the structural and functional changes of mitochondrial ATP synthase reported in this and other studies 4'6'19 are accompanied by remarkable alterations in the mitochondrial GSH levels during rat liver regeneration. The kinetics of these changes were very similar, showing a decrease in the early prereplicative phase and a recovery in the second replicative phase. The close kinetic relationship suggests a correlation between the two phenomena. It is well known t h a t GSH plays a critical role in a number of structural and functional processes of the cells and in several detoxification reactions9 5-27 Several investigations have provided evidence for a mitochondrial compartmentatio~ of GSH 2s'29 and have shown t h a t the mitochondrial p, : of GSH is essential for normal mitochondrial function. ~)Because mitochondria continually produce reactive oxygen species and contain no catalase, they would appear to be largely, if not entirely, dependent on the GSH-GSH peroxidase system. Thus, it would appear t h a t mitochondrial GSH is the principal functional antioxidant. ~3'3~ In spite of the importance of this mitochondrial compartmentation, mitochondria do not contain the enzyme activities required for GSH synthesis and effectively transport this tripeptide from the cytosolic compartment with an ATP-dependent mechanism. 12'13 Recently, an overproduction
HEPATOLOGYMay 1995
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Time after partial hepatectomy (h) FIG. 2. Total glutathione (GSH) concentration in mitochondria (A) and cytosol (B) of rats during liver regeneration and controls (sham-operated). PH, preparation of mitochondria, and glutathione determination are described in Materials and Methods. Data represent the mean _+SEM of five animals.
of oxygen free radicals has been suggested during the early phase of rat liver regeneration, which might account for the reported decrease of adenosine triphosphatase and ATP synthase activities. 3 This hypothesis is supported by the decrease of the adenosine triphos-
HEPATOLOGYVol. 21, No. 5, 1995
VENDEMIALE ET AL
phatase activity during exposure of rat liver mitochondria to oxygen free radicals2 '32 It is therefore conceivable that the stress produced by liver resection could be responsible for a sharp increase of the production of mitochondrial oxygen free radicals 3 followed by a decrease of GSH levels and inactivation of the membrane-bound mitochondrial ATP synthase complex. Another possible explanation for the decrease of mitochondrial GSH during the early phase of liver regeneration could be an increased efflux of GSH outside of mitochondria; however, the finding of a very slow transport of GSH from mitochondria to cytoplasm 3° argues against this hypothesis and suggests that this transport mechanism functions to conserve mitochondrial GSH. An elevated rate of GSH oxidation as a cause of mitochondrial GSH depletion seems to be rather unlikely, because no changes of mitochondrial GSSG concentrations were observed in the current study; however, 24 hours after PH, regenerating livers do apparently have higher GSSG/GSH ratio than controls, which may be physiologically important. It is also possible that the reported efflux of GSSG from mitochondria, under conditions of severe oxidative stress, 33 could contribute to determine relatively low intramitochondrial GSSG levels. Because, as mentioned, GSH is not synthesized into mitochondria, being actively imported from the cytoSO112'13 by an ATP-dependent transport, our data could also suggest that during the first retrodifferential phase of liver regeneration, the ATP-dependent intramitochondrial transport of GSH might decrease after the fall of mitochondrial ATP synthesis. In fact, it should be observed that the cytosolic GSH does not fall in this first phase of liver regeneration (24 hours) and is even increased in the second phase. This apparently conflicting phenomenon could be explained by the fact that in the first phase of liver regeneration the synthesis of cytosolic GSH may use ATP produced by anaerobic glycolysis, which has been reported to be increased in this period. 1 In the second phase (48 hours after PH), the level of cytosolic GSH increased about two times with respect to sham-operated animals, which is in agreement with the study of Lee and Boyer34; this increment was accompanied by the resto-
TABLE 1. T i m e C o u r s e o f M i t o c h o n d r i a l G S S G Concentration and of GSSG/GSH Ratio After Partial Hepatectomy Partial Hepatectomy
Sham-Operated
Time (hr)
GSSG (pmol/mg prot)
GSSG/GSH (×10 3)
GSSG (pmol/mg prot)
GSSG/GSH (×10 3)
To 24 48 72 96
58_+8 56_+ 1 63_+ 1 51_+ 1 46± 8
16_+ 3 29-6" 15 ± 4 11 ± 1 10_+ 1
58_+8 42_+6 60 ÷ 6 67_+ 6 41_+ 4
16 ÷ 3 11+ 1 15 ÷ 1 16 + 1 10_+ 1
NOTE. Results are expressed as mean _+ SEM of five animals. * P < .01 when compared with controls.
1453
_~ 80 O L. t-
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/
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FIG. 3. Linear relationship between decrease of relative amount of immunodetected ~-F1 subunit and of mitochondrial GSH during the early phase of liver regeneration. Data represent the percentage decrease of ~-F1 subunit and of total mitochondrial GSH at 12 and 24 hours after PH compared with their respective values at time 0 (time 0 - 100%).
ration of mitochondrial GSH content. This finding seems to exclude the possibility of an increased cytosolic GSH secondary to a lack of GSH transport into mitochondria. It could be noted that at this time (48 hours after PH) the rate of mitochondrial ATP synthesis did not change whereas the level of mitochondrial GSH and ~-F1 (which both require ATP for their synthesis and transport into mitochondria) increased. This observation suggests that in this interval (24 to 48 hours) after PH, the synthesis and the transport of these compounds should be carried out by the ATP produced by the anaerobic glycolysis. Further investigations are required to fully understand the underlying mechanism and the pathophysiological consequences of mitochondrial GSH depression during rat liver regeneration. GSH, however, has been shown to be essential for mitochondrial functioni3'3°'31; its depletion, moreover, seems to markedly enhance the susceptibility to mitochondrial dysfunction from oxidant stress. ~7 Therefore, it is conceivable that the decrease of mitochondrial GSH during the first 24 hours after PH might play a role in the remarkable metabolic changes occurring in the first retrodifferential phase of rat liver regeneration. 1.3 REFERENCES
1. Uriel J. Retrodifferentiation and the fetal patterns of gene expression in cancer. Adv Cancer Res 1979;29:127-174. 2. Michelopulos GK. Liver regeneration: molecular mechanisms of growth control. FASEB J 1990;4:176-187.
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VENDEMIALE ET AL
3. Tsai IL, King KL, Chang CC, Wei Y. Changes of mitochondrial respiratory functions and superoxide dismutase activity during liver regeneration. Biochem Int 1992;28:205-217. 4. Guerrieri F, Capozza G, Kalous M, Papa S. Age-related changes of mitochondrial Fo-F1 ATP synthase. Ann NY Acad Sci 1992; 395-402. 5. Buckle M, Guerrieri F, Papa S. Changes in activity and F1 content of mitochondrial H+ATPase in regenerating rat liver. FEBS Lett 1985;188:345-351. 6. Buckle M, Guerrieri F, Pazienza A, Papa S. Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial Fo-F~ H+ATPase in regenerating rat liver. Eur J Biochem 1986;155:439-445. 7. Sohal RS, Sohal BH. Hydrogen peroxide released by mitochondria increases during aging. Mech Ageing Dev 1991;57:187-202. 8. Nagy I. A membrane hypothesis of aging. J Theor Biol 1978; 75:189-195. 9. Zhang Y, Macillot O, Giulivi C, Ernesti L, Davies JA. The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J Biol Chem 1990;265:16330-16336. 10. Orrenius S, Jones DP. Functions of glutathione in drug metabolism. In: Sies H, Wendell A, eds. Functions of glutathione in liver and kidney. Berlin: Springer-Verlag, 1978:164-175. 11. Kaplowitz N. The importance and regulation of hepatic glutathione. Yale J Biol Med 1981;54:497-502. 12. Kurosawa K, Hayashi N, Sato N, Kamada T, Tagawa K. Transport of glutathione across the mitochondrial membranes. Biochem Biophys Res Commun 1990; 167:367-372. 13. Martensson J, Lai JC, Meister A. High affinity transport ofglutathione is part of a multicomponent system essential for mitochondrial function. Proc Natl Acad Sci U S A 1990;87:7185-7189. 14. Martensson J, Meister A. Mitochondrial damage in muscle occurs after marked depletion of glutathione and is prevented by giving glutathione monoesters. Proc Natl Acad Sci U S A 1989;86:471-475. 15. Martensson J, Tain A, Frayer W, Meister A. Glutathione metabolism in the lung: inhibition of its synthesis leads to lamellar body and mitochondrial defects. Proc Natl Acad Sci U S A 1989;86: 5296-5300. 16. Olafsdottir K, Pascoe GA, Reed DJ. Mitochondrial glutathione status during Ca2÷ ionophore-induced injury to isolated hepatocytes. Arch Biochem Biophys 1988;263:226-235. 17. Hirano T, Kaplowitz N, Tsukamoto H, Kamimura S. Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats. HEPATOLOGY1992;16: 1423-1427. 18. Pedersen PL. High yield preparative method for isolation of rat liver mitochondria. Anal Biochem 1977;80:401-408.
HEPATOLOGYMay 1995 19. Guerrieri F, Kalous M, Capozza G, Muolo L, Drahota Z, Papa S. Age dependent changes in mitochondrial F0-F1 ATP synthase in regenerating rat-liver. Biochem Mol Biol Int 1994;33:117-129. 20. Papa S, Tager JM, Guerrieri F, Quagliariello E. Effect of monovalent cations on oxidative phosphorylation in submitochondrial particles. Biochem Biophys Acta 1969; 172:184-186. 21. Tietze F. Enzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969;27: 502-522. 22. Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinyl-pyridine. Anal Biochem 1980;106:207-212. 23. Guerrieri F, Kopecky J, Zanotti F. Functional and immunological characterization of mitochondrial FoFI ATP synthase. In: Tager JM, Azzi A, Papa S, et al, eds. Organelles in eukaryotic cells: molecular structure and interactions. New York, London: Plenum Publ. Co., 1989:197-208. 24. Guerrieri F, Capuano F, Buckle M, Papa S. Alteration of mitochondrial H+ATPase complex in tissue regeneration and neoplasia. In: Gorrod JM, Albano O, Papa S, eds. Molecular aspects of human disease. London: Ellis Horwood Ltd., 1989:108-114. 25. Kosower ND, Kosower EM. The glutathione status of cells. Int Rev Cytol 1978;54:104-160. 26. Reed DJ, Beatty PW. Biosynthesis and regulation of glutathione: toxicological implications. In: Hodgson E, Bend JR, Philpot RM, eds. Reviews in Biochemical Toxicology 2. New York: Elsevier, 1980:213-241.
27. Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983;52:711-760. 28. Jocelyn PC, Kamminga A. The non-proteinthiol of rat liver mitochondria. Biochim Biophys Acta 1974;343:356-362. 29. Meredith MJ, Reed DJ. Status of mitochondrial pool of glutathione in the isolated hepatocyte. J Biol Chem 1982;257:3747-3753. 30. Griffith OW, Meister A. Origin and turnover of mitochondrial glutathione. Proc Nat] Acad Sci U S A 1985;82:4668-4672. 31. Masini A, Ceccarelli D, Trenti T, Gallesi D, Muscatello U. Mitochondrial inner membrane permeability changes induced by octadecadienoic acid hydroperoxide: role of mitochondrial GSH pool. Biochim Biophys Acta 1992; 1101:84-89. 32. Guerrieri F, Capozza G, Fratello A, Zanotti F, Papa S. Functional and molecular changes in Fo-F1ATP-synthase of cardiac muscle during aging. Cardioscience 1993;4:93-98. 33. Brodie AE, Reed DJ. Glutathione disulfide reduction in tumor mitochondria after t-Butyl-Hydroperoxide treatment. Chem Biol Interact 1992;84:125-132. 34. Lee SJ, Boyer TD. The effect of hepatic regeneration on the expression of the glutathione S-transferases. Biochem J 1993; 293:137-142.