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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Thyrotoxicosis may be associated with a variety of abnormalities of liver function. The pathogenesis of hepatic dysfunction in thyrotoxicosis is unknown, but has been attributed to mitochondrial dysfunction. We studied the effect of altered thyroid function on the apoptotic index in rat liver. Extensive DNA fragmentation and significantly increased caspase-3 activity (P < .001) and caspase-9 activation (P < .005) were observed in hyperthyroid rat liver; cell death by apoptosis was confirmed. In hyperthyroid rat liver, 60% of mitochondria exhibited disruption of their outer membranes and a decrease in the number of cristae. These findings, along with significant translocation of cytochrome c and second mitochondria-derived activator of caspases to cytosol (P < .005), suggest activation of a mitochondrial-mediated pathway. However, no change in the expression levels of Bcl-2, Bax, and Bcl-xL were found in hyperthyroidism. For in vitro experiments, rat liver mitochondria were isolated and purified in sucrose density gradients and were treated with triiodothyronine (T3; 2–8 μM). T3 treatment resulted in an abrupt increase in mitochondrial permeability transition. Using a cell-free apoptosis system, the apoptogenic nature of proteins released from mitochondria was confirmed by observing changes in nuclear morphologic features and DNA fragmentation. Proteins released by 6 μM T3 contained significantly increased amounts of cytochrome c (P < .01) and induced apoptotic changes in 67% of nuclei. In conclusion, using in vivo and in vitro approaches, we provide evidence that excess T3 causes liver dysfunction by inducing apoptosis, as a result of activation of a mitochondria-dependent pathway. Thus, the results of this study provide an explanation for liver dysfunction associated with hyperthyroidism. (HEPATOLOGY 2004;39:1120–1130.)

Thyroid hormone (TH) affects all tissues and modulates the rate of metabolic activity. Liver damage in hyperthyroid patients has been extensively reported since Habershon's original report in 1874.1–5 Liver function becomes compromised in 45% to 90% of thyrotoxic patients; in most cases, the changes in the liver are characterized by some degree of fatty infiltration, and by cytoplasmic vacuolization, nuclear irregularity, and hyperchromatism in hepatocytes.6, 7 The pathogenesis of hepatic dysfunction in severe hyperthyroidism is unknown. Possible thyroid–liver interactions include liver damage secondary to the systemic effects of excess thyroid hormone, direct toxic effects of thyroid hormone on the liver, an association of intrinsic liver disease and intrinsic thyroid disease involving autoimmune mechanisms, changes in thyroid hormone metabolism secondary to intrinsic liver disease, and subclinical physiologic effects of thyroid hormone on functions of the liver.5 The hepatic injury associated with hyperthyroidism varies from mild liver dysfunction associated with nonspecific histologic changes to severe central hepatic ischemia.

Ultrastructural and functional changes in mitochondria, such as enlargement, a mass increase, and formation of megamitochondria, have been reported in the liver of hyperthyroid patients and in a rat model of hyperthyroidism.7–13 Decrease in mitochondrial transmembrane potential and proton motive force and altered cellular oxidation-reduction occur during mitochondrial-mediated apoptosis.14, 15 The changes reported in the liver mitochondria from thyrotoxic patients7–13 are similar to those found in the mitochondria of apoptotic cells.

Mitochondria contain several apoptogenic proteins in the intermembrane space.14, 16 Cytochrome c is a key mediator of apoptosis; its efflux into cytosol initiates the formation of apoptosome and activates the initiator, caspase-9, which leads to activation of a proteolytic cascade that results in cellular disassembly by the effector, caspase-3.15–17 Inhibitors of apoptosis proteins (IAPs) form heterodimers with caspases and inhibit their activation.18 Translocation of secondary mitochondria-derived activator of caspases (SMAC) to cytosol is one of the essential steps for downstream activation of caspase. It does so by binding to IAPs and releasing the caspases for activation.18, 19 Many proteins of the Bcl-2 family reside on the outer membrane of mitochondria, where they are anchored by a hydrophobic sequence of amino acids located within the COOH termini of proteins oriented toward the cytosol. An alteration in the ratio of proapoptotic to antiapoptotic proteins of the Bcl-2 family may modulate the release of apoptogenic proteins.15

Because of their biochemical functions, mitochondria are a natural target for the calorigenic effects of TH.20 The interaction between mitochondria and TH in relation to other physiological functions is not understood. TH-induced apoptosis has been demonstrated in cultured T lymphocytes and tadpole intestine in primary culture.21, 22 However, the mechanism of action of TH on mitochondria during this process is unknown. Adult liver, being a triiodothyronine (T3)-responsive tissue, is probably suitable for studying the role of T3 in initiating a mitochondrial pathway of apoptosis in hyperthyroidism.

Here, we report that severe hyperthyroidism induces extensive apoptosis in rat liver; hypothyroidism inhibits apoptosis. Hyperthyroidism induces change in mitochondrial morphologic features and the release of cytochrome c from mitochondria, without altering the levels of Bcl-2, Bcl-xL, and Bax proteins. Using an in vitro apoptotic system, we also found that T3 acts directly on mitochondria and induces release of apoptogenic proteins from them.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In Vivo Studies

Animals.

Four-month-old male Sprague Dawley rats were used in all experiments. The animals were housed in a temperature- and light-controlled room (21-24°C, 12-hour:12-hour light:dark cycle); they were allowed a chow diet and water ad libitum. Rats were divided into three groups (n = 15 for each group). The first group (group 1) was given the chow diet and ordinary drinking water alone (control); the second group (group 2) in addition was given methimazole (Sigma, St. Louis, MO) at a concentration of 0.025 % (wt/vol) in drinking water for 8 weeks (hypothyroid)23; and the third group (group 3) in addition received not methimazole, but T3 (Sigma) 100 μg/100 g body weight subcutaneously for 10 days (hyperthyroid).24 The intake of water and food, and the body weight of each animal, were recorded daily. Blood samples were collected at the time that animals were sacrificed for estimation of TH, serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT). All animal procedures were undertaken in accordance with the institutional guidelines for animal care and research.

Serum Total T3 (TT3), Total T4, AST, and ALT.

Serum TT3 and total T4 were measured using standard radioimmunoassay kits (DPC kit, Los Angles, CA); AST and ALT were measured using standard kits according to the manufacturer's instructions (Merck Gmbh, Darmstadt, Germany).

DNA Fragmentation Analysis.

Liver (0.5 μg) was minced in 10 volumes of buffer A (20 mM Tris, pH 8.0, 10 mM ethylenediaminetetraacetic acid [EDTA], and 0.5% Triton-X-100) and incubated on ice for 2 hours. After centrifugation at 14,000g at 4°C for 20 minutes, the supernatant was extracted with phenol/chloroform. DNA was precipitated by ethanol and washed with 70% ethanol. The pellet resuspended in buffer B (10 mM Tris, pH 8.0, 1 mM EDTA), treated with RNase-A (100 μg/mL), and extracted with phenol/chloroform. DNA was precipitated as described above and was analyzed in a 2% agarose gel.25

Preparation of Cytosolic and Mitochondrial Fraction.

Liver tissue was minced in buffer C (0.32 M sucrose, 1 mM K-EDTA, 10 mM Tris HCl, pH 7.4, supplemented with a protease inhibitor cocktail [Roche Applied Sciences, Mannheim, Germany]), homogenized and centrifuged at 1300g for 10 minutes at 4°C. The supernatant was collected, and the pellet was resuspended in buffer C and centrifuged at 1300g for 10 minutes. The supernatant was pooled26 and centrifuged at 17000g for 15 minutes to collect the mitochondrial fraction. Assays for marker enzymes, such as lactate dehydrogenase and cytochrome oxidase, confirmed the supernatant to be a cytosolic fraction and the pellet a mitochondrial fraction. The mitochondrial pellet was resuspended in buffer C and the cytosolic fraction was aliquoted and stored at −80°C for subsequent analysis.26 The protein concentration was determined using a standard method.27

Western Blotting.

Cytosolic or mitochondrial fraction proteins (50 μg), or both, were subjected to 15% SDS-PAGE (sodium dodecyl sulfate-Polyacrylamide gel electrophoresis) and transferred onto a nitrocellulose membrane. Equal loading and transfer of proteins to membrane were confirmed by Ponceau S staining. Membranes were incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibody. The signals were detected using an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). Antibodies to caspase-9 and SMAC (gifts from Dr. X. Wang) and cytochrome c (7H8.2C12; gift from Dr. R. Jemmerson) were used. Antibodies against Bcl-xL, Bax, and Bcl-2 and secondary antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). The relative amount of each protein was determined quantitatively. Blots from all the three replicates were analyzed using an Alpha Imager microdensitometer (AlphaImager Corp., San Leandro, CA).

Colorimetric Assay for Caspase-3 Activation.

Caspase activity was quantitated using the ApoAlert Caspase-3 colorimetric assay kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions.

Electron Microscopy.

Liver was fixed in phosphate buffered saline (PBS)-buffered 2.5% glutaraldehyde (Sigma) for 2 hours at 4°C. After dehydration in a series of diluted alcohols, blocks were placed in propylene oxide solutions and were embedded in epoxy resin. Five blocks from each animal were prepared, and semithin sections were stained with toluidine blue and were examined by light microscopy for orientation. Ultrathin sections of at least three blocks per animal were stained with uranyl acetate and lead citrate28 and were examined using a JEOL-1010 electron microscope (JEOL, Tokyo, Japan).

In Vitro Studies

Isolation of Mitochondria, Nuclei, and Cytosol.

Mitochondria from rat liver were isolated as described previously.12 Mitochondria were purified in 1.0 M/1.5 M sucrose density gradients and resuspended at a concentration of 10 mg protein/mL in buffer D (400 mM Mannitol, 50 mM Tris HCl, pH 7.4, bovine serum albumin (BSA) 5 mg/mL, KH2PO4).29 Liver nuclei were isolated using a previously described standard method.30 Purified nuclei were resuspended in buffer E (10 mM PIPES (piperazine-N,N′-bis [2-ethanesulfonic acid]), pH 7.4, 80 mM KCl, 20 mM NaCl, 5 mM Na-EGTA (ethylene glycol-bis [β-aminoethyl ether]-N,N,N′,N′-tetraacetic acid), 250 mM sucrose, and 1 mM DTT (dithiothreitol)) at a concentration of 8.5 × 107 nuclei/mL; the suspension was stored at −80°C in multiple aliquots. Cytosol from HeLa-S3 cells was prepared as described previously.31

Mitochondrial Swelling.

Mitochondria were washed in buffer F (0.175 M KCl, 0.025 M Tris HCl, pH 7.4) and were resuspended in the same buffer at a concentration of 10 mg protein/mL.32 The mitochondria were treated with different concentrations of T3; absorbance was recorded at 520 nm after different time intervals in a temperature-controlled cell (Hitachi Koki Co., Tokyo, Japan).

Electron Microscopy of Isolated Mitochondria.

The mitochondrial pellet was fixed in PBS-buffered 1% gluataraldehyde/1% paraformaldehyde. The pellet was washed in PBS, fixed by osmium tetroxide (OsO4), and embedded in epoxy resin.32 Ultrathin sections were examined using a JEOL-1010 electron microscope (Tokyo, Japan).

Nuclear Morphologic Features and DNA Fragmentation.

Aliquots of 50 μl of HeLa cytosol (250 μg), 6 μl of liver nuclei, 1 mM MgCl2, and 1 mM dATP (deoxyadenosine 5′-triphosphate) were incubated in the presence or absence of T3-treated mitochondrial supernatant at 37°C for 2 hours. After propidium iodide staining, nuclear morphologic features were assessed using a fluorescence microscope (Olympus, Tokyo, Japan). After incubation, an aliquot of 500 μl buffer G (100 mM Tris HCl, pH 8.5, 5 mM EDTA, 0.2 M NaCl, 0.2% SDS, and 0.2 mg/mL proteinase K) was added to each reaction mixture; after incubation of the mixtures at 37°C overnight, sodium chloride was added to achieve a final concentration of 1.5 M. Nuclear debris was centrifuged at 15,000g for 15 minutes. DNA was precipitated by ethanol, washed with 70% ethanol, and resuspended in buffer H (10 mM Tris HCl, pH 7.5, 1 mM Na-EDTA, and 200 μg/mL DNase-free-RNase-A). DNA was analyzed in a 2% agarose gel.31

In Vitro Assay for Cytochrome c Release.

Mitochondria (50 μg protein in 20 mL), resuspended in protein-release buffer I (220 mM mannitol, 68 mM sucrose, 20 mM HEPES KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM DTT, and 1 mM PMSF [phenylmethyl sulfonyl fluoride]) were treated with T3 at 30°C for 1 hour. The mixture was centrifuged at 14,000g for 10 min at 4°C to generate mitochondrial pellets. The presence of cytochrome c was detected in supernatants and pellets by Western blotting using an Enhanced Chemiluminescene (ECL) kit and spectroscopic scanning (Hitachi Koki Co).31

Statistical Analysis.

Results are expressed as mean ± SEM. Experimental groups were compared using a one-way ANOVA and Student's t test. P values of 0.05 or less were considered to be significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In Vivo Studies

Induction of Hypothyroidism and Hyperthyroidism.

Serum levels of T3 were significantly lower (P < .05) in the hypothyroid group and significantly higher (P < .001) in the hyperthyroid group than in the control group (Fig. 1A). The weights of the hypothyroid and control rats remained similar throughout the experimental period, whereas the weights of hyperthyroid rats decreased (P < .05; Fig. 1B).

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Figure 1. Change in serum biochemistry and body weight in hypothyroid and hyperthyroid rats. (A) Serum TT3: Serum TT3 was determined in blood samples collected from control, hypothyroid, and hyperthyroid rats (n = 15 in each group) using a standard RIA method. *P < .05 control versus hypothyroid; θ P < .001 control versus hyperthyroid. (B) Weight of the rats in control, hypothyroid, and hyperthyroid groups after treatment. *P < .05 control versus hyperthyroid. (C,D) Serum ALT and AST levels: Blood was collected from rats at the end of the experimental period (n = 5 in each group). *P < .05 control versus hypothyroid; θ P < .001 control versus hyperthyroid. ψ P < .001 control versus hyperthyroid; data are expressed as means ± SEM.

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Markers of Liver Toxicity.

Serum ALT and AST are known markers of liver injury. We found several-fold increases in the levels of ALT and AST in the hyperthyroid group (P < .001; Fig. 1C,D). In the hypothyroid group, ALT levels decreased (P < .001), whereas AST levels remained unchanged (Fig. 1D).

Hyperthyroidism and DNA Fragmentation.

To examine whether hyperthyroidism affects liver function by inducing apoptosis, we assessed DNA fragmentation, a typical marker of apoptosis. Dose-dependent extensive DNA fragmentation occurred in liver tissue from rats treated with T3; it was detected after even low doses of T3 (Fig. 2, lanes 3–6). Maximum DNA fragmentation was observed in rats treated with 100 μg T3/100 g body weight; this dose was used in further studies. DNA fragmentation was not detected in the hypothyroid and control groups (Fig. 2).

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Figure 2. Hyperthyroidism and DNA fragmentation in rat liver. Rats were given normal saline (control), 12.5, 25, 50 or 100 μg T3/100 g body weight subcutaneously for 10 days (hyperthyroid), or methimazole in drinking water (hypothyroid). DNA was extracted from liver tissue as described in the Materials and Methods and analyzed in a 2% agarose gel (lane M, 1 kbp DNA ladder; lane1, control; lane2, hypothyroid; lanes 3–6, 12.5, 25, 50, 100 μg T3/100 g body weight, respectively). The experiment was repeated four times.

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Hyperthyroidism and Caspase-3 and Caspase-9 Activity.

Caspase-3 is the main effector caspase that is involved in apoptosis. To confirm that apoptosis is induced by hyperthyroidism, we applied a colorimetric assay for caspase-3 to the cytosolic fraction prepared from rat liver of all three groups. Caspase-3 activity was significantly higher in the hyperthyroid group (P < .001) than in the control group (Fig. 3A). Interestingly, we found less caspase-3 activity in the hypothyroid group than in the control group (P < .01; Fig. 3A).

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Figure 3. (A) Hyperthyroidism and caspase-3 activation. Cytosolic fractions were prepared from control, hypothyroid, and hyperthyroid rat liver. Caspase-3 activity was determined using the ApopAlert caspase-3 assay kit (Clontech). There was a substantial increase in caspase-3 activity in hyperthyroid rat liver; low levels were detected in hypothyroid group. The experiments were undertaken on three subgroups; each subgroup consisted of five rats in each group. Results are expressed as mean ± SEM. *P < .05 control versus hypothyroid; P < .001 control versus hyperthyroid. (B) Hyperthyroidism and caspase-9 activation. Cytosolic fractions (50 g) from liver of control (C), hypothyroid (M), and hyperthyroid (H) rats were subjected to 15% SDS-PAGE followed by Western blotting with a caspase-9 antibody that recognizes procaspase-9 and its cleaved subunits. Cleaved subunits of caspase-9 were significantly increased in hyperthyroid rats. Equal loading and transfer of proteins on membrane was confirmed by Ponceau S staining (lower panel). The experiment was repeated three times.

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In hyperthyroidism, both the alteration in mitochondrial morphologic features reported previously7–13 and the increased caspase-3 activity found in this study strongly suggest that initiator caspase-9 is activated via the mitochondrial pathway. Accordingly, we undertook Western blot analysis of caspase-9 activation using a polyclonal antibody that recognizes the procaspase and cleaved subunits. A protein band of 35 kd, corresponding to procaspase-9, with no detectable cleavage products was observed in both euthyroid and hypothyroid groups. In the hyperthyroid group, the intensity of the procaspase-9 band was not significantly greater than that in the control group (Fig. 3B). However, high levels of cleaved subunits of 20 kd and 12 kd were found in the hyperthyroid group (P < .01).

Hyperthyroidism and Translocation of Apoptotic Molecules From Mitochondria to Cytosol.

Activation of caspase-9 in hyperthyroidism strongly suggests an involvement of mitochondria. Accordingly, we studied the translocation of apoptogenic molecules from mitochondria to cytosol.14, 18, 19 Cytochrome c was localized largely in the mitochondrial fraction in all of the experimental groups (Fig. 4A). However, a significant amount of cytochrome c was translocated to cytosol in the hyperthyroid group (P < .001). At an early stage of apoptosis, SMAC is released from mitochondria into the cytosol, where it binds with IAPs and inhibits their antiapoptotic activity.14, 18, 19 To investigate the role of SMAC, we also studied its translocation. SMAC was localized in the cytosolic fraction in both the hypothyroid and hyperthyroid groups. In the control group SMAC was detected in neither mitochondrial pellets nor cytosol (Fig. 4B).

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Figure 4. Altered thyroid function and translocation of cytochrome c and SMAC from mitochondria into cytosol. Cytosolic and mitochondrial fractions were prepared from rat liver (control, C; hypothyroid: M; and hyperthyroid: H). The fractions were subjected to 15% SDS-PAGE and probed with anti-cytochrome c (A) and anti-SMAC (B). Release of cytochrome c from mitochondria into cytosol was detected in hyperthyroid rats; expression and translocation of SMAC occurred in both hypothyroid and hyperthyroid rats. Equal loading was confirmed by Ponceau S staining (C). The experiment was repeated three times.

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Hyperthyroidism, Mitochondrial Integrity, and Levels of Proteins of the Bcl-2 Family.

The Bcl-2 family of proteins are located on outer membrane of mitochondria; they maintain mitochondrial integrity14, 17 and control the release of apoptogenic proteins that initiate the caspase pathway. To study the role of proteins of the Bcl-2 family, we assessed expression of Bcl-2, Bcl-xL, and Bax by Western blotting. The levels of antiapoptotic proteins, such as Bcl-2 and Bcl-xL, and the proapoptotic protein, Bax, were not significantly changed either in either the hypothyroid or hyperthyroid group (Fig. 5).

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Figure 5. Altered thyroidal function and levels of Bcl-2, Bcl-xL, and Bax in rat liver. Cytosolic fractions from liver (control, lane C; hypothyroid, lane M; and hyperthyroid, lane H) were subjected to 15% SDS-PAGE followed by Western blotting with antibodies to Bcl-2, Bcl-xL, and Bax. No alteration in the levels of Bcl-2, Bcl-xL, and Bax were associated with altered thyroid function. Equal loading and transfer of proteins to membrane was confirmed by Ponceau S staining (lower panel). The experiment was repeated three times.

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To examine whether higher levels of T3 may affect mitochondrial integrity directly, electron microscopic studies were undertaken. Mitochondria from the control group showed intact morphologic features; the cristae and outer and inner membranes appeared normal. In sections of liver from hyperthyroid rats, the mitochondria appeared enlarged, their outer membranes were disrupted, and the number of cristae was decreased (Fig. 6A). The number of mitochondria having altered morphologic features was significantly higher (P < .001) in the hyperthyroid group (Fig. 6B).

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Figure 6. Ultrastructure of hepatic mitochondria and hyperthyroidism. (A) Mitochondrial morphologic features. For electron microscopy, liver tissue from control and hyperthyroid rats, fixed in phosphate buffered saline-buffered 2.5% glutaraldehyde, was dehydrated in a series of diluted alcohols and embedded in epoxy resin. Three ultrathin sections from three rats in each control and hyperthyroid group were stained with uranyl acetate and lead citrate and were examined under a JEOL-1010 electron microscope (magnification, ×20,000). Disrupted outer membranes and decreased numbers of cristae were found in mitochondria of hyperthyroid rat liver. The electron micrographs are representative of three experiments. (B) The number of mitochondria in control and hypothyroid groups that had altered morphologic features were counted in five different fields of view in sections from three different rats in each group. As many as 60% of mitochondria exhibited abnormal morphologic features in hyperthyroid rat liver; the corresponding figure for controls was 17%. The results are expressed as means ± SEM. *P < .001 control versus hyperthyroid.

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In Vitro Studies

Activation of caspase-9 and alterations of mitochondrial morphologic features strongly suggest a direct action of T3 on mitochondria to initiate apoptosis. We used an in vitro approach to study whether there was a cause-and-effect relationship between these variables. Isolated rat liver mitochondria from control animals were treated with a high concentration of T3 to determine whether T3 had a direct action on mitochondria.

T3, Mitochondrial Permeability Transition (PT), and Changes in Mitochondrial Morphologic Features.

To determine whether T3 leads to mitochondrial PT, mitochondria were exposed to different concentrations of T3, using conditions that had previously been shown to be conducive to swelling.17 T3 induced rapid swelling of isolated liver mitochondria. Maxima were attained after incubation for 12 minutes; swelling was sustained thereafter (Fig. 7A).

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Figure 7. (A) T3 and permeability transition in isolated mitochondria. Mitochondria isolated from rat liver were treated with different concentrations of T3 (0, 2, 4, 6, and 8 μM) at 20°C. Mitochondrial swelling was observed by measuring decrease in optical density at 520 nm with time. Optical density normalized to 100 and was plotted against time. A significant increase in mitochondrial permeability transition (PT) occurred at T3 concentrations 4, 6, and 8 μM; a maximal increase in PT occurred at a T3 concentration of 6 μM. The data shown are representative of four separate experiments.*P < .01; P < .001. (B) Ultrastructural changes in T3-treated mitochondria. Mitochondria isolated from rat liver were treated with T3 (6 μM); morphologic features were assessed by electron microscopy (magnification, ×18,000). T3-treated mitochondria exhibited less cristae and more swelling and rupturing of outer membranes than normal mitochondria. These data are representative of four separate experiments.

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To examine whether a change in PT results in structural alterations, electron microscopic assessment was undertaken. Mitochondria treated with T3 (6 μM) exhibited maximum change in PT. Untreated mitochondria exhibited regular cristae and integrated morphologic features. Mitochondria treated with 6 μM T3 were swollen; the number of cristae was reduced and outer membranes were enlarged and ruptured. The experiment was undertaken in duplicate, and approximately 80% of mitochondria exhibited abnormal morphologic features (Fig. 7B).

Nuclear Morphologic Features and DNA Fragmentation.

The induction in PT suggests that proteins are released from mitochondria. To examine the apoptogenic nature of such proteins, liver nuclei were incubated with proteins released from mitochondria that had been treated with 6 μM T3, in the presence of HeLa cell cytosol and dATP. The nuclei underwent dramatic morphologic changes and irregular condensations of chromatin were observed (Fig. 8A). As soon as we had confirmed the release of apoptogenic proteins, the assay was repeated after treatments at other concentrations of T3. The percentage of nuclei undergoing apoptosis also was maximal after exposure to 6 μM T3; 67% of such nuclei exhibited apoptotic morphologic features (Fig. 8B). We found that morphologic changes in nuclei and fragmentation of chromatin DNA occurred simultaneously (Fig. 8C). DNA fragmentation also increased as the T3 concentration increased, up to a maximum at 6 μM; it decreased at even higher concentrations of T3.

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Figure 8. (A) Triiodothyronine-induced release of proteins and the initiation of apoptosis in vitro. Mitochondria isolated from rat liver were treated with different concentrations of T3 (0, 2, 4, 6, and 8 μM) at 30°C for 1 hour. After treatment, reaction mixtures were centrifuged and supernatants containing proteins that were released from mitochondria were collected. Aliquots of HeLa-cytosol (250 μg in 50 L) and 6 μL of liver nuclei were incubated at 37°C for 2 hours either in the absence (a) or presence (c–f) of supernatant or cytochrome c (b). Nuclei were stained with propidium iodide (PI) and were examined under a fluorescence microscope. Apoptotic morphologic changes in nuclei were assessed. (a) Normal morphologic features; (b) positive control; (c–f) various stages of apoptosis in nuclei in the presence of supernatant after treatment with 6 μM T3. The data are representative of four separate experiments. (B) The number of apoptotic nuclei in the presence of supernatant was counted in five different fields of view. A significant increase in the number of apoptotic nuclei was found in the presence of supernatants correlated with the concentrations of T3 used before and was found in the presence of supernatant collected after treatment with T3; the magnitude of the increase in apoptotic nuclei correlated with the T3 concentration was used. The results are expressed as means ± SEM. The experiment was repeated three times. *P < .001. (C) T3-induced release of proteins from mitochondria and DNA fragmentation. DNA was extracted from rat liver nuclei of untreated (nuclei + cytosol, lane 1); negative control (nuclei + cytosol + supernatant from untreated mitochondria, lane 2); positive control (nuclei + cytosol + cytochrome c, lane 3), and treated (nuclei + cytosol + supernatant collected from mitochondria treated with 2, 4, 6, and 8 μM T3, lanes 4–7, respectively). The DNA was extracted by a salt precipitation method and was analyzed in a 2% agarose gel. Appreciable DNA fragmentation occurred in DNA extracted from T3-treated nuclei (lanes 4–7).

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Presence of Cytochrome c in Released Factors.

Cytochrome c is a protein that is known to be released in response to apoptotic stimuli.14 We applied an in vitro cytochrome c release assay. The intensity of the cytochrome c band increased in the supernatant and the intensity of the cytochrome c band in the mitochondrial pellet decreased as concentrations of T3 increased. The intensity of the cytochrome c band in supernatants after exposure to T3 concentrations in the range of 2 to 8 μM was significantly greater than that in basal supernatants in the absence of exposure to T3 (P < .01; Fig. 9A). Spectroscanning also confirmed the presence of cytochrome c in supernatants (Fig. 9B).

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Figure 9. (A) T3 and cytochrome c release from rat liver mitochondria. Mitochondrial supernatants and pellets were prepared as described. Release of cytochrome c was assessed by Western blotting. Significant release of cytochrome c was detected in supernatants after mitochondria had been treated with 2, 4, 6, and 8 μM T3 (lanes 2–5). Lane 1 shows basal release of cytochrome c; lane C shows a positive control in which purified cytochrome cwas loaded. Equal loading and transfer of proteins on membrane were confirmed by Ponceau S staining (lower panel). The experiment was repeated three times. (B) Absorption spectrum of cytochromec. Mitochondria isolated from rat liver were treated with T3 (6 μM). After centrifugation, the supernatant was analyzed for the presence of cytochrome c by spectroscopic scanning. The supernatant had peaks of absorption at 415, 520, and 549 nm, a pattern characteristic of the reduced form of cytochrome c.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

There is increasing evidence that apoptosis plays a major role in various pathologic conditions. Earlier, morphologic changes in the liver of patients with thyrotoxicosis were attributed to necrotic cell death.3, 4 However, at the time, the process of apoptosis had not been investigated. Here, we provide experimental evidence that high levels of TH activate the mitochondrial pathway of apoptosis in liver tissue.

To investigate the pathogenic effect of TH on liver, we induced hypothyroidism and hyperthyroidism in rats and studied the mechanism of apoptosis in the liver. The changes in serum levels of TT3 indicated that severe hyperthyroidism and hypothyroidism had been induced (Fig. 1). In hyperthyroid rats, we found increased DNA fragmentation, which suggested increased apoptosis (Fig. 2). Caspases, which are cysteine proteases, cleave different intracellular targets, which results in cell shrinkage, chromatin condensation, and DNA fragmentation.16 To confirm that hyperthyroidism induce apoptosis, we also studied caspase-3 activation. We observed significant increases in caspase-3 activity in hyperthyroid rats (Fig. 3A), suggesting that apoptosis is caspase dependent.

Caspase-3 is activated by either death receptor or a mitochondria-mediated pathway.18 However, widely recognized calorigenic effects of TH in mammals32 and other previously reported effects of TH on liver mitochondria4–10 prompted us to study mitochondrial metabolism in hyperthyroidism. The activation of initiator caspase-9 in hyperthyroid rats strongly suggests an involvement of a mitochondrial pathway (Fig. 3B). During apoptosis, mitochondrial proteins located in the intermembrane space are released into the cytosol, where they activate the caspase-9 pathway, as found in this study. Efflux of cytochrome c into cytosol is a primary event that leads to the formation of apoptosomes and activation of the caspase cascade. The release of cytochrome c was observed only in hyperthyroid rats (Fig. 4). In hypothyroid rats, the absence of release of cytochrome c and DNA fragmentation, and a significant decrease in ALT levels, indicate that hypothyroidism inhibits apoptosis. SMAC in cytosol (Fig. 4) inactivate the IAPs that inhibit apoptosis, thereby rendering hepatic cells liable to undergo apoptosis.18, 19 We observed the release of SMAC in both hypothyroid and hyperthyroid rats. Translocation of cytochrome c and SMAC, together with caspase-3 and caspase-9 activation, supports our hypothesis that an excess of TH causes massive apoptosis via a mitochondria-mediated pathway. In hypothyroid rats, however, release of SMAC from mitochondria may not be sufficient to induce apoptosis, as no release of cytochrome c was apparent. Evidence presented here and our earlier reports33, 34 clearly suggest that adequate levels of TH are required for hepatocytes and other cell types to mediate their differentiated functions and to prevent them from undergoing changes that make apoptosis liable to occur.

To understand further the mechanism of hyperthyroidism-induced release of cytochrome c and SMAC, we studied factors controlling the integrity of mitochondria. The Bcl-2 family of proteins are known to regulate mitochondrial-mediated apoptosis.11, 13 However, we found no change in the expression of either antiapoptotic Bcl-2 and Bcl-xL proteins or the proapoptotic Bax proteins in hyperthyroidism, suggesting that T3 may act directly on mitochondria to induce apoptotic effects (Fig. 5). The alteration in mitochondrial morphologic features observed in hyperthyroid rat liver in this study, is consistent with earlier observations (Fig. 6)7, 11–13 and further supports our hypothesis of a direct action of T3 on mitochondria.

Our in vivo observations suggest that T3 may act directly on mitochondria, altering its structure and inducing release of apoptogenic proteins. However, the mechanism of action of T3 on liver cells in vivo is complicated; T3 acts on both the nucleus and the mitochondria. Accordingly, an in vitro system became our method of choice to study direct actions of T3 on liver mitochondria. In many aspects of cell biology and metabolism, in vitro reconstitution of complicated pathways has facilitated considerably the assessment of cause-and-effect relationships between various cellular components. A higher concentration of substrate is required for a direct effect to occur in a cell-free system.31 Therefore, we used higher concentrations of T3, which previously have been shown to induce effects in isolated mitochondria.12, 32 Our finding that T3 induced an increase in mitochondrial PT (Fig. 7A) is in agreement with the findings of others.11–13 The increase in mitochondrial PT is accompanied by electron-microscopic changes in mitochondrial morphologic features (Fig. 7B). The T3-induced mitochondrial swelling reported here is analogous to the increase in mitochondrial size observed histologically in liver tissue from hyperthyroid rats in this study and liver biopsies from hyperthyroid patients reported previously.7

The change in PT results in the release of apoptogenic proteins from mitochondria.14 We investigated the release and nature of those proteins in vitro. T3 induces release of cytochrome c (Fig. 9A,B) and other proteins, having molecular weights in the range 20 kd to 50 kd (data not shown), that orchestrate the process of apoptosis (Fig. 8B,C). The formation of condensed chromatin bodies observed in vitro in liver nuclei exposed to proteins released by T3-treated mitochondria (Fig. 8A,B) seems to be analogous to nuclear irregularity and ballooning of nuclei reported previously in liver histologic results of thyrotoxic patients.1–8 The in vitro observations support our hypothesis of a direct action of T3 on mitochondria, and also corroborate our in vivo findings of T3-induced disruption of mitochondrial integrity and release of apoptogenic proteins by mitochondria. In addition, the results of this study support an earlier hypothesis that an intrinsic pathway of apoptosis prevails in hepatocytes.35

This study has provided insights into the pathogenesis of liver dysfunction associated with severe hyperthyroidism. No firm evidence has been provided on the nature of the hepatic injury in existing literature on hyperthyroidism.1–8 Data from this study suggest that hyperthyroidism induces apoptosis in the liver. Our results provide evidence that excess TH may be directly toxic to the liver. However, the study does not rule out the possibility of liver damage occurring secondary to the systemic effects of excess TH. Our in vitro and in vivo data together indicate that T3 acts directly on mitochondria; it induces PT and release of proteins that lead to activation downstream of the caspase cascade, which, in turn, leads to apoptosis. However, the mechanisms of the change in PT and the release of apoptotic proteins from T3-treated mitochondria currently are not understood. Adenine nucleotide translocator, a unit of the PT pore complex, was reported to bind T3 with high affinity.36, 37 TH receptors also have been identified in the matrix of rat liver mitochondria.38 Whether binding of T3 to mitochondrial T3 receptors and adenine nucleotide translocator regulates the PT and release of apoptogenic proteins has not yet been investigated. Further studies should enable us to broaden our understanding of action of T3, not only on the liver, but also on other organs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr. X. Wang (University of Southwestern Texas Medical Centre, Dallas, TX) for the kind gift of antibodies against SMAC and caspase-9, Dr. R. Jemmerson (University of Minnesota) for anti-cytochrome c, and Dr. Shashi Wadhwa (All India Institute of Medical Sciences, New Delhi, India) for providing the electron microscope facility. We also thank Dr. Cliona Stapleton, NIEHS, NIH, and Dr. S.K. Yaccha, SGPGIMS, Lucknow, for their critical evaluation of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References