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Endocrinology Vol. 145, No. 11 5121-5128
Copyright © 2004 by The Endocrine Society

Thyroid Hormone Treatment of Hypothyroid Rats Restores the Regenerative Capacity and the Mitochondrial Membrane Permeability Properties of the Liver after Partial Hepatectomy

Loredana Moro, Ersilia Marra, Ferdinando Capuano and Margherita Greco

Institute of Biomembranes and Bioenergetics, National Research Council (L.M., E.M., M.G.); and Department of Medical Biochemistry and Biology, University of Bari (F.C.), I-70126 Bari, Italy

Address all correspondence and requests for reprints to: Dr. Margherita Greco, Institute of Biomembranes and Bioenergetics, National Research Council, Via Amendola 165/A, I-70126 Bari, Italy. E-mail: csmmmg14{at}area.area.ba.cnr.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the effect of thyroid hormone on recovery of liver mass and on the mitochondrial membrane permeability properties during rat liver regeneration after 70% partial hepatectomy (PH). In the euthyroid state, liver weight starts to recover 24 h after PH and is completely restored 96 h after PH. Cyclosporin A (CsA)-sensitive mitochondrial permeability transition (MPT) occurs 24 h after PH, and it has been suggested to act in the signaling mechanism for hepatocyte proliferation. In this study we show that hypothyroidism delays recovery of the liver mass, being only 50% of the initial weight 96 h after PH, and alters the duration and mode of MPT occurrence, first inducing a CsA-insensitive swelling 24 h after PH, followed by a CsA-sensitive swelling 96 h after PH. The occurrence of both CsA-sensitive and -insensitive swelling is shown to be associated with an increase in mitochondrial calcium content. Concurrent with mitochondrial swelling, external release of matrix proteins from mitochondria, such as aspartate aminotransferase and malate dehydrogenase, is shown to be CsA insensitive 24 h after PH and CsA sensitive 96 h after PH. After thyroid hormone administration to hypothyroid rats, the liver regenerative capacity is restored, and the duration and mode of MPT occurrence as well as changes in mitochondrial calcium content become similar to those observed in the euthyroid condition. The results of the present study suggest the involvement of a mitochondria-mediated pathway in regulation of the liver regenerative process by thyroid hormone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE (T3) plays a major role in mitochondrial maturation and function by regulating the expression of both mitochondrial and nuclear genes (1, 2, 3). Indeed, hypothyroidism is associated with a decrease in mitochondrial oxygen consumption and ATP synthesis (1). In contrast, injection of T3 in whole animals increases oxygen consumption and the metabolic rate (4). In addition, T3, a positive regulator in some tissues of the expression of the adenine nucleotide translocase (ANT) (5), a component of the mitochondrial permeability transition (MPT) pore (MPTP) (6, 7), can regulate the mitochondrial functions modulating the mitochondrial membrane permeability properties (8, 9, 10, 11).

Liver regeneration induced by 70% partial hepatectomy (PH) represents a model of tissue compensatory growth that may be suitable for studying T3-dependent alteration in mitochondrial functions associated with physiological cell proliferation. Hepatocyte proliferation after PH begins with priming of quiescent mature hepatocytes from a resting state (G0) to the prereplicative G1 phase of the cell cycle, which lasts for 12–14 h. The prereplicative phase is followed by DNA synthesis, which peaks 22–24 h after PH, and by mitosis 6–8 h later (12). During the prereplicative phase of liver regeneration, the mitochondrial function and structure of the liver are altered: ATP synthesis decreases (13), and mitochondrial oxidant production (13, 14), ultrastructure (15), and membrane permeability properties (15) are altered with the onset of calcium-induced MPT occurring in the absence of apoptosis (15). Thereafter, the liver mass is gradually recovered, and the regenerative process is complete 96 h after PH (15, 16). Progressive recovery of the liver mass is associated with recovery of normal mitochondrial function and structure (13, 14, 15, 16).

T3 is a powerful inducer of hepatocyte proliferation (17, 18, 19), and its mitogenic capacity has been used for gene therapy experiments and repopulation of hepatocytes (20, 21). It has been shown that T3 plays a role in the compensatory liver growth that follows surgical removal of the liver; its administration at the time of PH enhances the hepatic regenerative capacity (22). In contrast, either thyroidectomy or chemically induced hypothyroidism reduces the capacity of the liver mass to recover after PH (23, 24, 25, 26, 27). Reduction of the liver regenerative capacity by hypothyroidism has been correlated with a reduced efficiency of mitochondrial oxidative phosphorylation (26) and with decreased mitochondrial glutathione (GSH) concentration (27). Despite these findings, the involvement of mitochondria in the regulation of liver regeneration by thyroid hormone remains to be established.

In this study we have investigated the effect of administering T3 to hypothyroid animals on the regenerative capacity and the mitochondrial membrane permeability properties of the liver after PH, with special interest in MPT occurrence. Changes in the mitochondrial calcium content, the primary trigger for opening of the cyclosporin A (CsA)-sensitive MPTP (28, 29), were also examined in euthyroid, hypothyroid, and T3-treated hypothyroid rats during liver regeneration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
6-n-Propyl-2-thiouracil (PTU) and Arsenazo III were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All other chemicals were of high purity grade. CsA was a gift from Sandoz (Pharmaceutical Products, Milan, Italy).

Animals
Male Wistar rats (200–250 g) were housed in a temperature-controlled room (22 C) with food and water ad libitum. Rats were divided into three groups. The first group (group 1) was given ordinary drinking water (euthyroid); the second group (group 2) in addition was given 0.1% (wt/vol) PTU in drinking water for 21 d (hypothyroid) (9); and the third group (group 3), after 21 d of PTU treatment, was given 30 µg T3/100 g body weight for 3 d by ip injection (hyperthyroid) (9). Control PTU-treated animals received only the solvent for the same period of time. Twenty-four hours after the final administration, PH was performed on hypothyroid, hyperthyroid, and euthyroid rats as previously described (15), with removal of the median and left lateral lobes of the liver. After surgery, rats were kept on a standard diet until they were killed, as previously described (9). Blood samples were collected at the time that animals were killed for estimation of serum T3 levels (9). The livers were removed, weighed, and used for isolation of mitochondria. Sham-operated rats in the three groups, obtained after a small midline abdominal incision without excision of the liver, were used as a control. In all assays performed, no difference was observed between sham-operated rats and animals that did not receive any surgical operation as well as between hypothyroid animals 21 d after PTU treatment and hypothyroid rats injected with T3 solvent for 3 d after PTU treatment.

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.

Determination of T3
Blood was collected from hypothyroid and T3-treated rats 0–144 h after PH for T3 serum level analysis and compared with euthyroid samples. Serum T3 levels were determined as previously described (9). Briefly, the blood was quickly mixed with an equal volume of ice-cold 0.9% NaCl containing 0.24 mg EDTA/100 ml. Plasma was separated by centrifugation in the cold, and the samples were stored at –70 C until assayed. Plasma T3 was determined using commercial T3 luminescence immunoassay kits (Diagnostic Products BYK-Gulden, Cormano-Milan, Italy).

Preparation of mitochondria
Rat liver mitochondria were prepared at 4 C as described by Bustamante et al. (30) using a medium containing 0.25 M sucrose and 5 mM Tris-HCl (pH 7.4) as isolation buffer. In the preparation of mitochondria used for measurement of calcium content, 1.6 µM ruthenium red and 1 mM EGTA were added to the isolation buffer to restrict calcium movement during the subfractionation technique (31).

Protein concentration was determined using the Bio-Rad kit (Bio-Rad Laboratories, Milan, Italy) and albumin as standard.

Swelling assay
To analyze the mitochondrial swelling properties, mitochondria (0.35 mg protein/ml) were suspended in swelling medium (5 mM succinate-Tris, 10 mM morpholinepropanesulfonic acid-Tris, 0.2 M sucrose, 1 mM phosphate-Tris, 2 µM rotenone, and 1 µg/ml olygomycin, pH 7.4) and incubated at 25 C. During a 10-min incubation, the absorbance change ({Delta}A) in the mitochondrial suspension, which is an index of change in mitochondrial membrane permeability (32), was followed at 540 nm using a DU7400 spectrophotometer (Beckman Coulter, Palo Alto, CA) equipped with magnetic stirring and thermostatic control. Where indicated, 1 µM CsA was added to the medium.

Matrix protein release assay
For assay of the in vitro release of mitochondrial matrix proteins, isolated mitochondria (10 mg protein/ml) were suspended in the swelling medium described above and incubated at 25 C for 10 min. Where indicated, CsA (1.7 nmol/mg mitochondrial protein) was added to the incubation medium. After a 10-min incubation, mitochondria were precipitated by centrifugation at 8,000 x g for 40 sec. The supernatants were centrifuged for 2 min at 10,000 x g. Five microliters of the final supernatant were used for determination of mitochondrial aspartate aminotransferase (AAT) (33) and malate dehydrogenase (MDH) (34) activity. The activities of the two enzymes in isolated mitochondria were also determined.

Determination of mitochondrial calcium content
For determination of the endogenous mitochondrial calcium content, mitochondria (0.1 mg/ml) were suspended in isolation buffer (0.25 M sucrose and 5 mM Tris-HCl, pH 7.4) in the presence of 40 µM Arsenazo III. The {Delta}A at 675–685 nm was monitored by dual wavelength spectrophotometry, using a spectrophotometer ({lambda} 3B, PerkinElmer, Palo Alto, CA). After reading a baseline for 1 min, 0.2% Triton X-100 plus 3.3 µM sodium dodecyl sulfate was added to disrupt the mitochondrial membranes. The {Delta}A was calibrated by adding standard aliquots of CaCl2 to the incubation medium. A standard curve was obtained from the pooled results of five independent series of determinations and used for analysis of the mitochondrial calcium content (31).

Statistical analysis
The statistical significance of differences between groups was determined by one-way ANOVA, followed by Student-Newman-Keuls test. Comparison between independent means was performed using a t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum T3 levels and liver regenerative capacity after PH
Administration of PTU to euthyroid rats for 21 d induced hypothyroidism (9) and resulted in reduced liver weight (7.5 ± 0.4 g) compared with euthyroid rats (12.1 ± 0.8 g; P < 0.01). The switching from hypo- to hyperthyroidism was induced by administration of T3 for 3 d to PTU-treated rats (9) and caused an increase in liver weight (9.1 ± 0.15 g) compared with hypothyroid rats (P < 0.03).

We performed 70% PH in euthyroid, hypothyroid, and T3-treated hypothyroid rats and studied the kinetics of liver regeneration. The growth of remnant liver and serum T3 levels were monitored up to 6 d after surgery. In euthyroid rats, the liver mass progressively increased after an initial lag phase of about 24 h, reaching 100% of the initial mass 96 h after PH (Fig. 1Go), confirming results previously reported (15, 16). Serum T3 levels slightly decreased 24 h after PH (decrease of 20 ± 5% vs. sham-operated rats; P < 0.03) and thereafter steadily returned to normal levels (182 ± 11 ng/dl). In hypothyroid rats, recovery of the liver mass was significantly delayed compared with euthyroid controls; the liver weight was 49 ± 2% of the initial mass at 96 h (P < 0.001) and reached 60 ± 4% 144 h after PH (P < 0.001; Fig. 1Go). Hypothyroid rats recovered 100 ± 5% of the initial liver mass 3 wk after PH (not shown). Serum T3 levels, despite being lower (86 ± 5 ng/dl) than in euthyroid rats, remained constant during liver regeneration (not shown). In T3-treated hypothyroid rats, liver mass progressively increased after an initial lag phase of 24 h, reaching about 100% of the initial mass 96 h after PH (Fig. 1Go), and serum T3 levels, higher than in euthyroid rats (> 800 ng/dl), remained constant during liver regeneration (not shown). These results show that T3 administration to hypothyroid rats restores the regenerative capacity of the liver after PH.



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  		FIG. 1. Recovery of liver mass in euthyroid (N), hypothyroid (H), and T3-treated hypothyroid rats (H + T3) after PH. The mass of the liver after PH is expressed as a percentage of the weight of the liver in sham-operated rats. Data reported are the mean ± SEM of at least five different rats.

 
Thyroid state and MPT occurrence during liver regeneration
Transient CsA-sensitive MPT has been shown to occur in euthyroid rats during liver regeneration, and it has been suggested that it could act in the signaling mechanism for cell proliferation (15, 16). To investigate whether serum T3 levels affected the onset of MPT during liver regeneration, the mitochondrial membrane permeability properties were examined by monitoring the swelling of mitochondria isolated from euthyroid (N-RLM), hypothyroid (H-RLM), and T3-treated hypothyroid rats (T3-H-RLM) 0, 24, and 96 h after PH in either the presence or the absence of CsA, the specific inhibitor of MPTP (35) (Fig. 2Go). Swelling was monitored by measuring the {Delta}A in the mitochondrial suspension, which is an index of change in mitochondrial membrane permeability (32). In agreement with our previous results (9), N-RLM exhibited no significant swelling 0 and 96 h after PH (Fig. 2AGo, traces a and c, respectively; {Delta}A after 10 min, 0.16 ± 0.02), but significantly swelled 24 h after PH (Fig. 2AGo, trace b; {Delta}A after 10 min, ~0.52 ± 0.02; P < 0.001 vs. N-RLM at both 0 and 96 h) in a CsA-sensitive manner (Fig. 2AGo, trace b'). H-RLM isolated from sham-operated rats exhibited no significant swelling up to 10 min of incubation (Fig. 2BGo, trace a; {Delta}A after 10 min, 0.15 ± 0.02). At 24 h after PH, H-RLM were found to swell (Fig. 2BGo, trace b), even though to a lesser extent than N-RLM ({Delta}A after 10 min, 0.27 ± 0.02; P < 0.001 vs. N-RLM at 24 h; P < 0.0001 vs. H-RLM at 0 h). CsA, which completely prevented swelling of N-RLM, had no significant effect on H-RLM swelling (Fig. 2BGo, trace b'; {Delta}A after 10 min, 0.23 ± 0.02 in the presence of CsA). At 96 h after PH, H-RLM still showed the capacity to swell (Fig. 2BGo, trace c; {Delta}A after 10 min, 0.25 ± 0.02; P < 0.002 vs. H-RLM 24 after PH), and CsA completely prevented it (P < 0.0001; Fig. 2BGo, trace c'). T3-H-RLM isolated from sham-operated rats showed swelling (Fig. 2CGo, trace a; {Delta}A after 10 min, 0.28 ± 0.03), and CsA completely inhibited it (P < 0.001; Fig. 2CGo, trace a'), confirming our previous results (9). At 24 h after PH, T3-H-RLM showed a higher capacity to swell (Fig. 2CGo, trace b; {Delta}A after 10 min, 0.64 ± 0.03) than either T3-H-RLM of sham-operated controls (P < 0.001) or H-RLM and N-RLM isolated 24 h after PH (P < 0.001 and P < 0.02, respectively), and CsA completely prevented it (P < 0.0001; Fig. 2CGo, trace b'). At 96 h after PH, the swelling properties of T3-H-RLM were comparable to those of T3-H-RLM isolated from sham-operated rats (Fig. 2CGo, traces c and c'; {Delta}A after 10 min, 0.29 ± 0.02 in the absence of CsA). These results show that T3 modulates the duration and mode of mitochondrial swelling, an index of MPT, during liver regeneration.



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  		FIG. 2. Mitochondrial permeabilization to sucrose in N-RLM, H-RLM, and T3-H-RLM during liver regeneration. N-RLM (A), H-RLM (B), and T3-H-RLM (C; 0.35 mg mitochondrial protein/ml), isolated 0 h (traces a), 24 h (traces b), and 96 h after PH (traces c), were added with swelling medium, and the absorbance change at 540 nm at 25 C was monitored as reported in Materials and Methods. Traces a', b', and c' show the same experiments run in the presence of 1 µM CsA, which was added to the suspension medium before mitochondria.

 
As additional confirmation that the thyroid state modulates MPT occurrence during liver growth after PH, the occurrence of CsA-sensitive release of certain mitochondrial proteins in the suspension medium was checked as previously described (9). We first measured the intramitochondrial activity of the matrix proteins AAT and MDH in N-RLM, H-RLM, and T3-H-RLM during liver regeneration (Fig. 3Go). A significant increase in the activity of these mitochondrial enzymes, expressed as enzymatic units per milligram of mitochondrial protein, was found in H-RLM isolated from sham-operated rats (0 h in Fig. 3Go) compared with N-RLM [increase of 30 ± 1% for AAT (P < 0.01); increase of 20 ± 2% for MDH (P < 0.02)], and T3 administration to hypothyroid rats restored the euthyroid levels, as previously reported (9). In N-RLM, the activities of the matrix enzymes, AAT and MDH, decreased 24 h after PH [29 ± 2% decrease for AAT (P < 0.01); 13 ± 1% decrease for MDH (P < 0.03)] and to steadily recover thereafter, confirming our previous results (16). H-RLM showed a biphasic pattern of decrease in mitochondrial AAT and MDH activities after PH, with peaks at 24 h [46 ± 1% decrease for AAT (P < 0.01) and 17 ± 2% decrease for MDH (P < 0.02), vs. sham-operated controls] and at 96 h [40 ± 0.5% decrease for AAT (P < 0.01) and 15 ± 1% decrease for MDH (P < 0.01), vs. sham-operated controls]. In T3-H-RLM, both AAT and MDH activities decreased 24 h after PH [36 ± 2% decrease for AAT (P < 0.01); 24 ± 3% decrease for MDH (P < 0.02)] and thereafter returned to the sham-operated values.



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  		FIG. 3. AAT and MDH enzyme activity in N-RLM, H-RLM, and T3-H-RLM during liver regeneration. AAT (A) and MDH (B) activities were measured in mitochondria isolated at different times after PH. The mean ± SEM of three different mitochondrial preparations are shown.

 
We then investigated whether N-RLM, H-RLM, and T3-H-RLM, isolated during liver regeneration and incubated in swelling medium in either the presence or the absence of CsA (1.7 nmol/mg mitochondrial protein), differentially lost the mitochondrial proteins AAT and MDH (Fig. 4Go). We found that after 10-min incubation in swelling medium, the release of matrix enzymes from mitochondria isolated from sham-operated rats (0 h in Fig. 4Go) was higher in N-RLM than in H-RLM [3.37 ± 0.1-fold for AAT (P < 0.01); 3.57 ± 0.1-fold for MDH (P < 0.01)], and that T3 administration to hypothyroid rats increased the CsA-sensitive release of matrix enzymes compared with N-RLM [2.5 ± 0.2-fold for AAT (P < 0.001); 2.3 ± 0.1-fold for MDH (P < 0.001)], confirming results previously reported (9). After PH, N-RLM showed a peak release of both AAT and MDH at 24 h (3.2 ± 0.2- and 2.9 ± 0.3-fold increases, respectively, vs. N-RLM isolated from sham-operated rats; P < 0.001); thereafter, the release decreased, reaching, levels comparable to those in sham-operated rats 96 h after PH, confirming previous results (16). H-RLM showed a first peak of release 24 h after PH (9.9 ± 0.3-fold increase for AAT and 9.1 ± 0.1-fold increase for MDH vs. H-RLM isolated from sham-operated rats; P < 0.002 and P < 0.001, respectively) and a second lower peak 96 h after PH (8.1 ± 0.3-fold increase for AAT and 6.5 ± 0.5-fold increase for MDH, vs. H-RLM isolated from sham-operated rats; P < 0.01 and P < 0.005, respectively). T3-H-RLM showed an increase in release of AAT and MDH 24 h after PH (1.7 ± 0.2- and 1.6 ± 0.1-fold increases, respectively, vs. T3-H-RLM isolated from sham-operated rats; P < 0.005 and P < 0.002, respectively). CsA, added to the mitochondrial suspensions before incubation, completely inhibited the release of enzymes from N-RLM isolated 24–48 h after PH (P < 0.001), from H-RLM isolated 96 h after PH (P < 0.001), and from T3-H-RLM isolated 0–144 h after PH (P < 0.001). However, CsA inhibited only a minor, not statistically significant, fraction of the release of matrix proteins from H-RLM isolated 24–72 h after PH.



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  		FIG. 4. Release of AAT and MDH in N-RLM, H-RLM, and T3-H-RLM isolated during liver regeneration. N-RLM (A), H-RLM (B), and T3-H-RLM (C; 10 mg mitochondrial protein/ml) were incubated in swelling medium in the absence (– CsA) or the presence (+ CsA) of 1.7 nmol/mg protein CsA. After 10-min incubation, 0.1-ml aliquots were taken, the mitochondria were precipitated, and the enzyme activities were determined in the supernatants. The amounts of AAT (upper panel) and MDH (bottom panels) activities released from mitochondria are shown as enzymatic units per milligram of incubated mitochondrial protein. The mean ± SEM of three different measurements of samples obtained from five different animals for each experimental group are reported.

 
Overall, these results show that 1) hypothyroidism causes a CsA-insensitive MPT during the first 72 h after PH and delays the occurrence of CsA-sensitive MPT from 24 to 96 h after PH; and 2) T3 administration to hypothyroid rats restores the occurrence, duration, and mode of MPT during liver regeneration.

Thyroid state and mitochondrial calcium content during liver regeneration
Because we have previously shown that opening the CsA-sensitive MPTP during the prereplicative phase of liver regeneration is due to a rise in mitochondrial calcium content (15), we investigated whether alteration in the MPT process in hypothyroidism and its normalization after T3 treatment are associated with changes in mitochondrial calcium levels. Figure 5Go shows that the calcium content in rat liver mitochondria isolated from sham-operated rats (0 h in the figure) was about 7.8 ± 1 nmol/mg protein in N-RLM, 20.3 ± 1.8 nmol/mg protein in H-RLM, and 12.1 ± 1 nmol/mg protein in T3-H-RLM, confirming results previously reported (9). These amounts increased 24 h after PH in N-RLM (17.4 ± 1.9 nmol/mg protein; an increase of 55%; P < 0.01), in H-RLM (40.7 ± 4.5 nmol/mg protein; an increase of 50%; P < 0.001), and in T3-H-RLM (25 ± 2 nmol/mg protein; an increase of 52%; P < 0.001). Thereafter, the mitochondrial calcium content returned to the levels in sham-operated rats in N-RLM and T3-H-RLM, but in H-RLM reached a second peak at 72–96 h after PH (28.5–29 nmol/mg protein; 30% increase vs. H-RLM isolated from sham-operated rats; P < 0.01), lower than at 24 h, then gradually decreased. These results show that T3 affects the mitochondrial calcium content during the regenerative process.



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  		FIG. 5. Mitochondrial calcium content in N-RLM, H-RLM, and T3-H-RLM during liver regeneration. N-RLM, H-RLM, and T3-H-RLM (0.1 mg mitochondrial protein/ml) were suspended in an isolation buffer containing 40 µM Arsenazo III, and the calcium content was determined as described in Materials and Methods. The mean ± SEM of three different measurements of the samples obtained from five different animals for each experimental group are reported.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Three novelties can be highlighted from this study: 1) T3 administration to hypothyroid rats restores liver regenerative capacity; 2) hypothyroidism alters the occurrence, duration, and mode of MPT during liver regeneration, and T3 administration to hypothyroid rats restores euthyroid MPT; and 3) recovery of mitochondrial permeability properties by T3 during liver regeneration involves modulation of the mitochondrial calcium content.

It has been reported that the liver regeneration rate after PH is reduced in thyroidectomized (23) and chemically induced (25, 26, 27) hypothyroid rats vs. euthyroid controls. Consistent with these findings, we show that hypothyroid rats are able to regenerate their liver mass completely, but the process is significantly delayed, with a recovery of about 50% of the initial liver mass 96 h after PH, when regeneration is already complete in the euthyroid group. We demonstrate for the first time that T3 administration to hypothyroid rats restores the liver regenerative capacity. Hypothyroid animals are also GH deficient (19); however, because T3 has been shown to play the essential regulatory role in the process of hepatocyte proliferation (19), it is conceivable that the increase in the regenerative capacity of the liver after T3 administration to hypothyroid rats is mainly dependent on the direct action of T3 on liver cells.

Mitochondria are known to play important roles in controlling cell proliferation (36, 37) and liver regeneration (13, 14, 15, 16, 38) and to be a direct target of T3 (11). It has been reported that the occurrence of CsA-sensitive MPT is involved in the liver regenerative process (15, 16). We and others have shown that T3 regulates the mitochondrial membrane permeability properties (8, 9, 10, 11) and that hypothyroidism per se does not make mitochondria prone to undergoing permeability transition (PT) (9). In this study we show that during liver regeneration, H-RLM become able to undergo PT, but differently from N-RLM. In fact, hypothyroidism first induces a CsA-insensitive mitochondrial swelling and then, 96 h after PH, a CsA-sensitive swelling of lower magnitude than that occurring 24 h after PH in euthyroid controls. At 24 h after PH, the release of matrix proteins from H-RLM, occurring through a CsA-insensitive MPTP, is comparable with that occurring in N-RLM through a CsA-sensitive MPTP, notwithstanding the fact that the capacity of H-RLM to swell is about 50% less than that of N-RLM. These results suggest that the CsA-insensitive MPTP induced in H-RLM has a higher capacity and/or selectivity for matrix proteins than the CsA-sensitive MPTP occurring in N-RLM. T3-H-RLM, which before PH show a basal CsA-sensitive swelling vs. N-RLM (9), undergo large amplitude swelling 24 h after PH, with release of matrix proteins. The mitochondrial permeability properties return to the pre-PH condition 96 h after PH. Because T3 regulates the expression of ANT (5), a component of the CsA-sensitive MPTP (6, 7), it is conceivable that the absence of large amplitude, CsA-sensitive swelling in hypothyroidism could be due to down-regulation of ANT expression, whereas the occurrence of a CsA-sensitive swelling of larger magnitude in T3-H-RLM than in N-RLM might be due to up-regulated expression of ANT.

Besides the classic MPT, activated by calcium and inhibited by CsA (39, 40), several inducers may promote increases in mitochondrial permeability that are CsA insensitive (41, 42, 43, 44). Calcium is a primary trigger of CsA-sensitive MPT (28) and, in some cases, of CsA-insensitive MPT (43), and T3 has been shown to regulate the intracellular calcium content (45, 46). We show for the first time that T3 modulates the mitochondrial calcium content, the increase in which is directly correlated to the occurrence and amplitude of MPT during liver regeneration in N-RLM, H-RLM, and T3-H-RLM. Indeed, the massive CsA-insensitive MPT, found exclusively in H-RLM, occurs 24 h after PH, when the mitochondrial calcium content reaches the highest levels. The high mitochondrial calcium content in hypothyroidism might have an independent effect on opening of the MPTP, inducing oxygen radical formation by mitochondria (47) that leads to protein cross-linking, misfolding, and generation of amphipathic pore-forming clusters that are CsA-insensitive (44). The effect of an excess of mitochondrial calcium content on the onset of MPT could be enhanced in hypothyroid rats by reduced levels of mitochondrial GSH (27), an antioxidant against excess free radicals. Thus, T3, through regulation of the mitochondrial calcium content, apart from ANT expression (5) and GSH levels (27), could modulate the opening of both CsA-sensitive and -insensitive MPTP during liver regeneration. However, other factors, activated during the regenerative process and at present unknown, must be involved in the occurrence of both CsA-sensitive and -insensitive MPT in hypothyroid animals, because mitochondria isolated from sham-operated hypothyroid rats are not able to undergo MPT, even in the presence of calcium overload (9).

It is known that a transient MPT can cause the release of mitochondrial calcium, which is involved in modulation of the expression of nuclear genes encoding for a variety of proteins that modulate cell proliferation and mitochondria biogenesis (48, 49). Thus, it is conceivable that in the hypothyroid state, a prolonged and/or altered MPT during liver regeneration could cause the release of factors, including calcium, that in excess could negatively regulate the turnover of whole mitochondria as well as hepatocyte proliferation, thus delaying liver regeneration. According to this hypothesis, T3 administration to hypothyroid rats, restoring the euthyroid MPT properties, would positively regulate mitochondria biogenesis and cell proliferation, thus promoting liver regeneration. Ascertaining which factors, released through MPT, modulate hepatocyte proliferation could be a good goal to pursue.

In conclusion, the results reported in this study provide evidence for the involvement of a mitochondria-mediated pathway in regulation of the liver regenerative process by thyroid hormone and suggest that regulation of serum T3 levels at the time of PH could be a valuable therapeutic tool in liver transplant surgery.


   Acknowledgments
 
We thank Mr. R. Lusardi for linguistic consultation.


   Footnotes
 
This work was supported in part by a grant from MURST (Piani di Potenziamento della rete Scientifica e Tecnologica-Cluster 03; to E.M.).

Abbreviations: {Delta}A, Absorbance change; AAT, aspartate aminotransferase; ANT, adenine nucleotide translocase; CsA, cyclosporin A; GSH, glutathione; H-RLM, liver mitochondria isolated from hypothyroid rats; MDH, malate dehydrogenase; MPT, mitochondrial permeability transition; MPTP, mitochondrial permeability transition pore; N-RLM, liver mitochondria isolated from euthyroid rats; PH, partial hepatectomy; PT, permeability transition; PTU, 6-n-propyl-2-thiouracil; T3-H-RLM, liver mitochondria isolated from T3-treated hypothyroid rats.

Received July 14, 2004.

Accepted for publication August 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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