Enzymes: aspartate aminotransferase (EC 220.127.116.11); glutamate dehydrogenase (EC 18.104.22.168).
Changes in ultrastructure and the occurrence of permeability transition in mitochondria during rat liver regeneration
Article first published online: 4 AUG 2009
European Journal of Biochemistry
Volume 269, Issue 13, pages 3304–3312, July 2002
How to Cite
Guerrieri, F., Pellecchia, G., Lopriore, B., Papa, S., Esterina Liquori, G., Ferri, D., Moro, L., Marra, E. and Margherita Greco (2002), Changes in ultrastructure and the occurrence of permeability transition in mitochondria during rat liver regeneration. European Journal of Biochemistry, 269: 3304–3312. doi: 10.1046/j.1432-1033.2002.03010.x
Note: deceased in November 2000.
- Issue published online: 4 AUG 2009
- Article first published online: 4 AUG 2009
- (Received 8 February 2002, revised 20 May 2002, accepted 22 May 2002)
- liver regeneration;
- mitochondria ultrastructure;
- membrane permeability;
Mitochondrial bioenergetic impairment has been found in the organelles isolated from rat liver during the prereplicative phase of liver regeneration. To gain insight into the mechanism underlying this impairment, we investigated mitochondrial ultrastructure and membrane permeability properties in the course of liver regeneration after partial hepatectomy, with special interest to the role played by Ca2+ in this process. The results show that during the first day after partial hepatectomy, significant changes in the ultrastructure of mitochondria in situ occur. Mitochondrial swelling and release from mitochondria of both glutamate dehydrogenase and aspartate aminotransferase isoenzymes with an increase in the mitochondrial Ca2+ content were also observed. Cyclosporin-A proved to be able to prevent the changes in mitochondrial membrane permeability properties. At 24 h after partial hepatectomy, despite alteration in mitochondrial membrane permeability properties, no release of cytochrome c was found. The ultrastructure of mitochondria, the membrane permeability properties and the Ca2+ content returned to normal values during the replicative phase of liver regeneration. These results suggest that, during the prereplicative phase of liver regeneration, the changes in mitochondrial ultrastructure observed in liver specimens were correlated with Ca2+-induced permeability transition in mitochondria.
Seventy percent partial hepatectomy (PH) induces cell proliferation until the original mass of the liver is restored . The tissue regeneration process consists of two phases: the prereplicative phase, the duration of which depends on the age of the animal [2,3] as well as on hormones and dietary manipulation [2,4] and the replicative phase, during which a sharp increase in DNA synthesis occurs with active mitosis . In the light of early changes in ATP concentration found in liver after PH, before activation of cell proliferation [5,6], mitochondria were investigated as they are directly involved in the process of liver regeneration [4,7–16]. Many mitochondrial functions, including oxidative phosphorylation [11–13] and generation of reactive oxygen species [14,15], were investigated in some detail in the prereplicative phase of liver regeneration. In isolated mitochondria, a decrease in the respiratory control index , ATP synthesis, probably due to a decrease in the ATPsynthase complex content , and glutathione content  as well as an increase in malondialdehyde production  and oxidant production  were found. This suggests the occurrence in the prereplicative phase of liver regeneration of a transient mitochondrial oxidative stress in which mitochondria can also release proteins from the matrix . Despite this, mitochondria recover their functions in the replicative phase of liver regeneration [12,14–16].
In this paper, we investigated whether and how the mitochondrial structure can change in the prereplicative phase of liver regeneration and whether mitochondrial permeability properties are somehow affected in this phase of the process. In the prereplicative phase of liver regeneration, we found the occurrence of a number of mitochondria with dilated, paled and vacuolized matrix. The isolated mitochondria showed impairment in membrane permeability properties, which were prevented by cyclosporin-A (CsA). An increase in Ca2+ content was also observed. Despite alteration in mitochondrial membrane permeability properties, no release of cytochrome c was found during the prereplicative phase of liver regeneration. The mitochondrial ultrastructure, the membrane permeability properties and the Ca2+ content showed normal values during the replicative phase of liver regeneration when a progressive recovery of liver mass is observed.
Materials and Methods
Three-month-old male Wistar rats were anaesthetized with an ether/oxygen mix (at variable ratios) and the median and left lateral lobes of the liver were excised . After surgery, the rats were kept on a standard diet until they were sacrificed. The livers were removed, weighed, and processed as follow: one-third were cut into sections for electron microscopy studies and two-thirds were used for the isolation of mitochondria. Sham-operated rats, obtained after a small midline abdominal incision without excision of the liver, were used as a control and killed at 0, 24 and 96 h after the surgical operation. In all the assays reported, no difference between sham-operated and rats that did not receive any surgical operation was observed.
All operations were carried out under sterile conditions. The animals received humane care and the study was approved by the State Commission on animal experimentation.
Ultrastructural morphology of mitochondria was determined by electron microscopy. Liver specimens from control rats and from rats at 24 and 96 h after PH, were fixed with 4% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4 for 4 h at 4 °C. After fixation and an overnight wash in sodium cacodylate buffer at 4 °C, the specimens were postfixed with 1% osmium tetroxide in sodium cacodylate buffer for 1 h at 4 °C, dehydrated in alcohol and embedded in araldite resin (Taab Laboratories Equipment LTD, Aldermaston, Berkshire, England) and semithin sections (1 µm) were removed for optical microscopy. Ultra-thin sections were mounted on copper mesh grids and stained with uranyl acetate and lead citrate, according to Reynolds , before examination with a Zeiss EM 109 electron microscope. All tissue samples were first inspected on semithin sections by light microscopy. The ultrastructural morphology of mitochondria was evaluated on five rats for each experimental group (control, 24 and 96 h after PH) and 10 randomly selected electron micrographs of a hepatic lobule were observed in each animal (7000× magnification).
Five morphological groups of mitochondria were defined and divided into two types according to the observed conformation: normal and altered (*) (Fig. 1). For each animal the morphology of about 600 mitochondria in a hepatic lobule was examined.
Preparations of cytosolic fraction and mitochondria
Mitochondria were prepared according to Bustamante et al. using a medium containing 0.25 m sucrose and 5 mm Tris/HCl (pH 7.4) as isolation buffer. After precipitation of mitochondria, the supernatant was used for preparation of cytosol by ultracentrifugation at 105 000 g for 1 h. The final supernatant was used as cytosolic fraction. In the preparations used for measurements of mitochondrial Ca2+ content, 1.6 µm ruthenium red and 1 mm EDTA were added in the isolation buffer to restrict Ca2+ movement during the subfractionation technique. As preliminary analyses showed that there was no statistically significant difference in the Ca2+ content of mitochondria whether the buffers used for the subfractionation procedure contained either 1 mm EDTA alone, or 1 mm EDTA and 1.6 µm ruthenium red or 1 mm EGTA, for all subsequent preparations, 1 mm EDTA and 1.6 µm ruthenium red were included in the subfractionation buffers.
Protein concentration was determined using the Bio-Rad kit (Bio-Rad Laboratories Inc., Milan, Italy).
To monitor the mitochondrial swelling properties in sucrose solution, mitochondria (0.5 mg protein·mL−1) were suspended in a swelling medium [5 mm succinate/Tris, 10 mm Mops/Tris, 0.2 m sucrose, 1 mm phosphate/Tris, 2 µm rotenone and 1 µg·mL−1 oligomycin (pH 7.4)].
The absorbance was followed at 540 nm and at 25 °C, as described previously , using a spectrophotometer equipped with magnetic stirring and thermostatic control. Where indicated, 1 µm CsA (Sandoz Prodotti Farmaceutici, Milano, Italy) was added to the reaction medium.
Matrix proteins release assay
For the assay of the in vitro release of matrix proteins, mitochondria (10 mg protein·mL−1) were suspended in the swelling medium, above reported, and incubated at 25 °C for 8 min. After incubation, the mitochondria were precipitated by centrifugation at 8000 g for 40 s. The supernatants were then centrifuged for 10 min at 10 000 g. Five microliters of the final supernatants were used for SDS/PAGE analysis with a linear gradient of polyacrylamide (10–15%) . After the run, the gel was stained with Coomassie Brilliant Blue. Where indicated, mitochondrial aspartate-aminotransferase  (AAT) or glutamate-dehydrogenase (GDH)  activities were determined in the final supernatants. When indicated, CsA (1.7 nmol·mg−1 mitochondrial proteins) was added. The activities of the two enzymes were also determined in the mitochondrial and cytosolic fractions, and in the whole liver homogenate. The enzyme activity of mitochondrial AAT in the cytosol was determined as described by Greco et al.. Briefly, two aliquots of either cytosolic fraction or whole homogenate were incubated separately at 37 °C and 70 °C for 15 min, then AAT activity in both samples was determined. The AAT activity of the sample incubated at 37 °C was taken to be that of both isoenzymes (mitochondrial and cytosolic AAT), whereas that of the sample incubated at 70 °C was assumed to be solely due to cytosolic isoenzyme. In fact, under conditions where the cytosolic AAT was stable, there was a thermal instability of mitochondrial AAT at 70 °C . The activity of mitochondrial AAT was taken as the difference between the two values.
Determination of cytochrome c content
The amount of cytochrome c in cytosol and mitochondria during rat liver regeneration was determined by SDS polyacrylamide gel electrophoresis analysis, as described by Schaegger et al. . Mitochondrial (20 µg of protein) or cytosolic (90 µg of protein) preparations were loaded onto an SDS/polyacrylamide gel. Gels were then incubated in a medium containing tetrametylbenzidine in 10% isopropanol and 7% acetic acid. After 10 min, H2O2 30% was added and, after 1–2 min, the greenish-blue bands of heme-containing peptides, among which was cytochrome c, were developed, as described by Broger et al. . The bands were analyzed by laser densitometry at 595 nm, using a CAMAG TLC scanner II densitometer (Merck–Hitachi). Commercially purified horse cytochrome c (Sigma–Aldrich) was used as standard.
Determination of mitochondrial Ca2+ content
For determination of the endogenous Ca2+ content, mitochondria (0.1 mg protein·mL−1) were suspended in 0.25 m sucrose in the presence of 40 µm Arsenazo III (Sigma–Aldrich, Milan, Italy). The absorbance change at 675–685 nm, was monitored by dual wavelength spectrophotometry. After reading a baseline for 1 min, Triton X-100 (0.2%) plus 3.3 µm SDS were added to disrupt the mitochondrial membranes . The absorbance change was calibrated by addition of standard aliquots of Ca2+ to the medium. A standard curve was obtained from the pooled results of five independent series of determinations and used for analysis of mitochondrial Ca2+ content, which for the control was 8 ± 0.2 nmol per mg mitochondrial protein. No statistically significant differences in Ca2+ content were observed when the mitochondrial preparation was performed either in the presence or in the absence of ruthenium red and EDTA in isolation buffer.
Data are reported as the mean ± SEM of five experiments performed using liver sections or mitochondria and cytosol obtained from five different animals for each experimental group (control, 24 and 96 h after PH). Statistical analysis was performed using the Student's t-test.
Mitochondrial ultrastructure during liver regeneration after PH
In order to find out whether and how mitochondria structure changes occur during liver regeneration, 10 randomly selected electron micrographs of the same magnification (7000×) were examined from one hepatic lobule of five rats for each experimental group (control, 24 and 96 h after PH), and the morphology of about 600 mitochondria in a hepatic lobule of each animal was analyzed. The typical mitochondrial morphology of control liver is shown in Fig. 1A. Liver mitochondria of rats at 24 h after PH were quite variable in morphology and ultrastructure (Fig. 1B–D). Three different mitochondrial morphologies were observed: (a) normal mitochondria (Fig. 1B) characterized by the same basic architecture of the typical liver mitochondria with a folded internal membrane and a dense matrix; (b) altered mitochondria (*) with a marked decrease in the area of the inner membrane, reduction in the number of cristae, destructurization of the matrix compartment, a dilated and paled matrix, lack of dense granules (Fig. 1C); and (c) altered mitochondria (*) with clear vacuolization of the matrix compartment (Fig. 1D). No evident rupture of mitochondrial outer membrane integrity was observed in altered mitochondria. At 96 h after PH (Fig. 1E), mitochondria were nearly normal in morphology, cristae-rich, and with an electron-dense matrix. Quantitation of normal and altered mitochondria in control liver and in liver at 24 and 96 h after PH was performed. The majority of liver mitochondria from control rats presented a normal morphology; only a small fraction (3.0 ± 0.6%) belonged to the altered type. A large proportion (41.0 ± 6.6%) of mitochondria from liver at 24 h after PH showed alterations in mitochondrial ultrastructure. At 96 h after PH, only a small fraction (3.0 ± 0.05%) belonged to the altered type. The differences between the number of altered mitochondria at 24 h after PH and the number of altered mitochondria in control rats were statistically significant (P < 0.0001). Furthermore, in liver at 24 h after PH the total number of mitochondria, counted in 10 randomly selected electron micrographies of a hepatic lobule, was less than the total number present in either control liver (11% decrease; P = 0.001) or in liver at 96 h after PH (17% decrease; P < 0.001). The decrease in the mitochondria number corresponds to a decrease in the mitochondrial proportion of the cell volume at 24 h after PH. This was correlated with a decrease in the activity of the mitochondrial marker enzymes GDH and mAAT in the total liver homogenate at 24 h after PH (15% and 24% decrease for GDH and mAAT, respectively). Moreover, in the hepatocytes of liver at 24 h after PH, a small increase in the number of lysosomes and the presence of autophagosomes were also observed (data not shown). No significant change in the number of apoptotic nuclei was found with respect to control liver and liver at 96 h after PH (data not shown).
Mitochondrial membrane permeability during liver regeneration after PH
As the ultrastructure of 40% of liver mitochondria at 24 h after PH is suggestive of changes in membrane permeability of the organelles, we followed the swelling of mitochondria isolated during liver regeneration (0, 24, 96 h after PH) in isotonic sucrose medium supplemented with succinate and phosphate. Mitochondria were suspended in the swelling medium and the absorbance of the mitochondrial suspension as a function of time was monitored either in the absence or in the presence of CsA (1 µm), the specific inhibitor of the mitochondrial transition pore . Mitochondria isolated from control rats and at 96 h after PH, were found to swell at a low rate and extent in about 20 min (Fig. 2, traces a and c); mitochondria isolated at 24 h after PH showed, in contrast, a high rate and extent of swelling (Fig. 2, trace b). CsA was found to prevent swelling in every case (Fig. 2, traces a′, b′, c′). Liver mitochondria isolated from sham-operated rats at 0, 24 and 96 h after surgery were found to swell poorly in a manner similar to that found for control liver mitochondria (data not shown).
The CsA capability to prevent mitochondrial swelling is indicative of the occurrence of permeability transition in mitochondria during the prereplicative phase of liver regeneration. Thus we checked whether the isolated mitochondria could release matrix proteins into the external medium. Incubation of rat liver mitochondria, isolated at 24 h after PH, at 25 °C for 8 min in the swelling medium, resulted in an increased and nonspecific release of mitochondrial proteins in the suspension medium (Fig. 3A, lane c) compared to mitochondria isolated from control rats (Fig. 3A, lane b) and mitochondria isolated at 96 h after PH (Fig. 3A, lane d), as revealed by SDS/PAGE of the supernatants obtained after precipitation of mitochondria by centrifugation. This release of proteins at 24 h after PH was associated with the appearance, in the supernatant, of typical matrix enzyme activity, such as GDH (3.5 ± 0.26-fold increase vs. control mitochondria; 23 ± 2.5% of the total mitochondrial activity) and AAT (3.15 ± 0.23-fold increase vs. control mitochondria; 5.1 ± 0.1% of the total mitochondrial activity) (Fig. 3B, empty columns b). CsA, added to the mitochondrial suspensions before incubation, inhibited the release of enzyme activities (Fig. 3B, filled columns b). At 96 h after PH, the activities of the enzymes released in the supernatant (1.8 ± 0.1 and 0.8 ± 0.04% of the total mitochondrial activity of GDH and AAT, respectively), were as low as those found in the supernatant of mitochondria isolated from control rats (2.2 ± 0.1 and 0.8 ± 0.05% of the total mitochondrial activity of GDH and AAT, respectively) (Fig. 3B, columns a and c).
As shown in Fig. 4, the total activities of the matrix enzymes GDH and AAT were found to decrease in mitochondria isolated 24 h after PH, with respect to mitochondria isolated from control rats (Fig. 4, columns b) (3.07 ± 0.85-fold decrease for GDH and 1.67 ± 0.3-fold decrease for AAT). An increase in enzymatic activities in the corresponding cytosol (Fig. 4, columns b′) with respect to cytosol isolated from control rats (Fig. 4, columns a′) was observed (4.75 ± 0.59-fold increase for GDH and 2.28 ± 0.13-fold increase for AAT). Mitochondria and cytosols obtained 96 h after PH show a pattern similar to that of mitochondria and cytosols obtained from control rats (Fig. 4, columns c, c′).
Ca2+ content in mitochondria during liver regeneration after PH
The occurrence of mitochondrial permeability transition is due to an increase in mitochondrial Ca2+ content . Consistently, Ca2+ pulse to mitochondria isolated before PH or from sham-operated rats and suspended in an isotonic sucrose medium supplemented with succinate and phosphate, caused mitochondrial swelling (Fig. 5A), which reflects a change in mitochondrial membrane permeability . Such a mitochondrial swelling was inhibited by the addition to the mitochondrial suspension of CsA (Fig. 5A), the specific inhibitor of the permeability transition pore of mitochondria . This change in permeability of the inner mitochondrial membrane due to Ca2+ loading was accompanied by a nonspecific release of mitochondrial proteins in the suspension medium  with the appearance, in the supernatants, of typical matrix enzyme activities, such as mitochondrial AAT, the release of which was also inhibited by the addition of CsA (Fig. 5B).
As the mitochondrial permeability transition is dependent on the Ca2+ content of mitochondria, we checked whether the mitochondrial Ca2+ content could change during liver regeneration (Fig. 6). The mitochondrial Ca2+ content in sham-operated rats was about 8 ± 0.2 nmol·mg−1 protein; this amount remained constant up to 6 h after PH. No difference in liver mitochondrial Ca2+ content was observed between sham-operated rats and animals that did not receive any surgical intervention (data not shown). A large increase in Ca2+ content (17.7 ± 0.4 nmol·mg−1 protein) was found at 24 h after PH. The Ca2+ content at 72–96 h after PH was the same as the control (Fig. 6). The increase in liver weight after PH showed a biphasic pattern. A low rate of increase was measured up to 24 h. After this interval the liver weight increased linearly with the time (Fig. 6) .
Following PH, the remaining mature hepatocytes enter a complex process, known as liver regeneration, which after an initial prereplicative phase reconstitutes the original mass of the liver [1,2]. The residual hepatocytes re-enter the cell cycle while the normal homeostatic mechanisms that couple cell cycle re-entry to cell death are suspended [29,30].
The present study shows that after surgical removal of two-thirds of the mass of rat liver, mitochondria in the remaining hepatocytes undergo, in the first 24 h after hepatectomy, i.e. in the prereplicative phase, ultrastructural changes. These are associated with enhancement of the mitochondrial Ca2+ content and increase of CsA-sensitive permeability to sucrose of the mitochondria isolated from the residual liver mass.
Analysis of the structural and functional state of mitochondria in the liver mass which is reconstituted in the successive 96 h, shows, on the other hand, normal mitochondrial ultrastructure, return of mitochondrial Ca2+ content and CsA-sensitive sucrose permeability to the normal values observed in the liver before hepatectomy or in sham-operated rats.
Previous electron microscopy studies [15,31–33] had revealed changes in the residual hepatocytes after PH but less attention was paid to elucidating the correlation between the changes occurring in the ultrastructure of mitochondria and biochemical parameters during liver regeneration. The present electron microscopy study shows that the general organization of the mitochondrial inner membrane cristae into the typical transverse alignment in control animals was absent in about 40% of the mitochondria in the hepatocytes at 24 h after PH. These mitochondria were characterized by highly fractured and degenerated cristae and a clear vacuolation. This suggests that the decrease in ATP synthesis rate observed in mitochondria isolated during the prereplicative phase of liver regeneration  is probably a result of the decrease in the surface area of the inner membrane.
The ultrastructural changes observed in liver mitochondria at 24 h after PH are consistent with the changes found in the membrane permeability properties of the mitochondria isolated from the residual liver mass. The in vitro experiments show, in fact, that mitochondria isolated from rat liver at 24 h after PH exhibit high CsA-sensitive permeability to sucrose. It has been suggested that permeabilization of the inner mitochondrial membrane could be required for the turnover of matrix proteins . A release of mitochondrial AAT into the extramitochondrial phase has been observed following oxygen radical injury of mitochondria during hypoxic liver reoxygenation . Our data show a release of the mitochondrial matrix enzymes GDH and AAT into the cytosol of liver at 24 h after PH. A CsA-sensitive release of the same matrix enzymes can be observed in vitro, following swelling of mitochondria, isolated 24 h after PH. This suggests an involvement of the inner mitochondrial membrane transition pore in the release of matrix enzymes in vivo.
Our study shows that, during the prereplicative phase of liver regeneration, the mitochondrial Ca2+ content increases, reaching a maximum (17.75 nmol·mg−1 of protein) at 24 h after PH, when oxidative alteration of mitochondria is also observed [14,15]. Following PH, an increase in cell Ca2+ content has been observed during the prereplicative phase of liver regeneration . HGF, the most important in vitro mitogen for primary hepatocytes and whose plasma level increases within 1 h upon PH [29,36], has been shown to induce Ca2+ entry across the hepatocyte plasma membrane . Furthermore, some hormones, that are known to modulate liver regeneration acting as mitogens or comitogens [29,36], raise the liver cytosolic Ca2+ concentration and cause an increase in the mitochondrial matrix volume as a consequence of Ca2+ entry from cytosol into mitochondria .
Both mitochondrial Ca2+ accumulation and oxidative stress increase the probability that changes in the mitochondrial membrane permeability occur [25,38,39]. Oxidative stress, Ca2+ uptake and opening of the transition pore in mitochondria are signals for cell death [40–42]. However, only a transient small increase in the number of apoptotic cells (≈ 5%) has been reported at 1 h after PH . Three to six hours after PH, the level of apoptotic cells was as low as that observed in control liver and no increase in apoptosis was observed at 24 h after PH . The present ultrastructural analysis does not show any detectable alteration in mitochondrial outer membrane integrity at 24 h after PH. The increase in the number of lysosomes, even if at a low extent, the presence of autophagosomes and the reduction in the number of mitochondria that we observe in hepatocytes at 24 h after PH, suggest that autophagic processes could occur in the prereplicative phase of liver regeneration.
It has been proposed that if the permeability transition occurs only for brief periods, its activity would not create survival problems for mitochondria and cells . The mitochondria in intact cells may undergo permeability transition and swelling in a fully reversible manner without progressing to cell death [44–46]. Furthermore, it has been observed that mitochondrial swelling is not sufficient to affect cytochrome c release, and thus to trigger apoptosis processes . We show here that no release of cytochrome c occurs in the prereplicative phase of liver regeneration. This finding is in agreement with the electron microscopy observations showing that neither evident breakage of the mitochondrial outer membrane nor increased number of apoptotic nuclei are present at 24 h after PH. We suggest that the mitochondrial permeability transition occurring in the prereplicative phase of liver regeneration is a transient event and that, with the exception of irreparably damaged mitochondria that could be eliminated by autophagy, a great proportion of mitochondria undergoing permeability transition recover in a fully reversible manner. Future studies will be needed to ascertain the fate of mitochondrial subpopulations during liver regeneration.
This work was partially supported by a grant within the National Research Project PRIN: ‘Bioenergetics and Membrane Transport’ of Murst, Italy.
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