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Mice lacking thyroid hormone receptor Beta show enhanced apoptosis and delayed liver commitment for proliferation after partial hepatectomy.

López-Fontal R, Zeini M, Través PG, Gómez-Ferrería M, Aranda A, Sáez GT, Cerdá C, Martín-Sanz P, Hortelano S, Boscá L - PLoS ONE (2010)

Bottom Line: Pharmacologically induced hypothyroidism yielded similar results.Loss of TR activity was associated with enhanced nitrosative stress in the liver remnant, due to an increase in the activity of the nitric oxide synthase (NOS) 2 and 3, caused by a transient decrease in the concentration of asymmetric dimethylarginine (ADMA), a potent NOS inhibitor.Taken together, these results indicate that TRbeta contributes significantly to the rapid initial round of hepatocyte proliferation following PH, and improves the survival of the regenerating liver at later times.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.

ABSTRACT

Background: The role of thyroid hormones and their receptors (TR) during liver regeneration after partial hepatectomy (PH) was studied using genetic and pharmacologic approaches. Roles in liver regeneration have been suggested for T3, but there is no clear evidence distinguishing the contribution of increased amounts of T3 from the modulation by unoccupied TRs.

Methodology/principal findings: Mice lacking TRalpha1/TRbeta or TRbeta alone fully regenerated liver mass after PH, but showed delayed commitment to the initial round of hepatocyte proliferation and transient but intense apoptosis at 48h post-PH, affecting approximately 30% of the remaining hepatocytes. Pharmacologically induced hypothyroidism yielded similar results. Loss of TR activity was associated with enhanced nitrosative stress in the liver remnant, due to an increase in the activity of the nitric oxide synthase (NOS) 2 and 3, caused by a transient decrease in the concentration of asymmetric dimethylarginine (ADMA), a potent NOS inhibitor. This decrease in the ADMA levels was due to the presence of a higher activity of dimethylarginineaminohydrolase-1 (DDAH-1) in the regenerating liver of animals lacking TRalpha1/TRbeta or TRbeta. DDAH-1 expression and activity was paralleled by the activity of FXR, a transcription factor involved in liver regeneration and up-regulated in the absence of TR.

Conclusions/significance: We report that TRs are not required for liver regeneration; however, hypothyroid mice and TRbeta- or TRalpha1/TRbeta-deficient mice exhibit a delay in the restoration of liver mass, suggesting a specific role for TRbeta in liver regeneration. Altered regenerative responses are related with a delay in the expression of cyclins D1 and E, and the occurrence of liver apoptosis in the absence of activated TRbeta that can be prevented by administration of NOS inhibitors. Taken together, these results indicate that TRbeta contributes significantly to the rapid initial round of hepatocyte proliferation following PH, and improves the survival of the regenerating liver at later times.

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Related in: MedlinePlus

TRβ is transiently downregulated after PH and liver regeneration is delayed in TRα1/TRβ double KO mice.(A,B) WT and TRα1/TRβ KO mice (‘KO’) were submitted to 70% PH, and the protein levels of TRα1 (55 kDa) and TRβ (47 kDa) were determined by Western blot, and expressed as percentage of the normalized band intensities (using β-actin as control) vs. sham operated animals at 0 h. (C) mRNA levels of TRα1 and TRβ were determined by quantitative real time RT-PCR. (D) The serum levels of T3 and T4 after PH were measured and expressed as percentage vs. sham operated animals at 0 h. The basal values were 7.4±0.5 and 36.5±4.2 µg/dl for T4 in WT and KO, respectively; 78±5 and 2965±307 ng/dl for T3 in WT and KO, respectively. (E) Liver mass recovery after PH was determined in these animals and in WT mice treated with MMI to pharmacologically induce hypothyroidism, and in one group of animals thyroid hormone was restituted by administration of T4. (F) Acute liver injury after PH was evaluated by measuring serum AST levels. Results show means ± SD of 6 to 8 animals per condition (B,C,E,F), 4 animals (D) or a representative blot of three (A). #P<0.01 vs. the corresponding condition at 0h (B,D); *P<0.05, **P<0.01 vs. WT condition or T4-untreated WT animals (E,F).
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pone-0008710-g001: TRβ is transiently downregulated after PH and liver regeneration is delayed in TRα1/TRβ double KO mice.(A,B) WT and TRα1/TRβ KO mice (‘KO’) were submitted to 70% PH, and the protein levels of TRα1 (55 kDa) and TRβ (47 kDa) were determined by Western blot, and expressed as percentage of the normalized band intensities (using β-actin as control) vs. sham operated animals at 0 h. (C) mRNA levels of TRα1 and TRβ were determined by quantitative real time RT-PCR. (D) The serum levels of T3 and T4 after PH were measured and expressed as percentage vs. sham operated animals at 0 h. The basal values were 7.4±0.5 and 36.5±4.2 µg/dl for T4 in WT and KO, respectively; 78±5 and 2965±307 ng/dl for T3 in WT and KO, respectively. (E) Liver mass recovery after PH was determined in these animals and in WT mice treated with MMI to pharmacologically induce hypothyroidism, and in one group of animals thyroid hormone was restituted by administration of T4. (F) Acute liver injury after PH was evaluated by measuring serum AST levels. Results show means ± SD of 6 to 8 animals per condition (B,C,E,F), 4 animals (D) or a representative blot of three (A). #P<0.01 vs. the corresponding condition at 0h (B,D); *P<0.05, **P<0.01 vs. WT condition or T4-untreated WT animals (E,F).

Mentions: Expression levels of TRα and TRβ were determined in liver extracts from animals that had undergone PH. TRα content did not change in the period after PH; however, TRβ decreased significantly 24–72 h post-PH, returning to control levels at 96 h (Fig. 1A–B). Western blot confirmed absence of TRα1 and TRβ from TR double KO mice. Consistent with the protein expression profile, TRβ mRNA expression increased 3-fold by 48–72 h (Fig. 1C). TRα1/TRβ double KO mice exhibit a marked hyperthyroidism that has been previously reported [17]. The T3 and T4 serum levels were determined after PH and both WT and TRα1/TRβ double KO mice exhibited a rapid decrease in thyroid hormone levels after PH that recovered at 72–96 h (Fig. 1D), presumably due to the rapid overexpression of deiodinase type 3 [8]. Interestingly, the basal thyroid hormone levels of the TRα1/TRβ double KO mice where higher than those of WT mice, in agreement with previous work [17]. Regarding liver regeneration, TRα1/TRβ double KO mice had a slower rate of liver mass recovery after PH than WT, reflected in a delayed increase in the regeneration index (Fig. 1E). A milder delay was also observed in hypothyroid animals treated with MMI, and the liver regeneration index was restored in these animals by administration of T4. Interestingly, serum AST activity in hepatectomized animals, a marker of liver injury, was about a third of the WT value in the double KO mice, reflecting a decrease in liver injury after PH (Fig. 1F). This unexpected protection against PH-induced liver injury was systematically observed in TRα1/TRβ double KO mice and in MMI treated mice, being lost after administration of T4 in the latter case. Despite the attenuated liver regeneration in TRα1+TRβ KO or MMI-treated mice, survival rates after PH in these animals were identical to those in non-treated wild-types (Fig. 2A). Animal death was usually due to post-surgery complications, and always occurred during the first 24 h after intervention. Determination of cell proliferation, by Ki67-positive cell count in liver sections, revealed delayed progression in the cell cycle in TRα1/TRβ KO and MMI-treated mice (Fig. 2B), while flow cytometry of disaggregated liver cells showed no significant differences in the ploidy distribution among the animal groups (Fig. 2C). The delayed hepatocyte replication in double-KO and MMI-treated mice was reflected in delayed upregulation in the expression of the cell-cycle markers PCNA and cyclins E and D1 (Fig. 2D). Moreover, the typical drop in hepatic levels of C/EBPα that normally follows PH, and which is required for progress of hepatocytes through the cell cycle [21], was delayed in double-KO and MMI-treated mice, whereas the increase in C/EBPβ levels was much lower in animals lacking functional TRs (Fig. 2D).


Mice lacking thyroid hormone receptor Beta show enhanced apoptosis and delayed liver commitment for proliferation after partial hepatectomy.

López-Fontal R, Zeini M, Través PG, Gómez-Ferrería M, Aranda A, Sáez GT, Cerdá C, Martín-Sanz P, Hortelano S, Boscá L - PLoS ONE (2010)

TRβ is transiently downregulated after PH and liver regeneration is delayed in TRα1/TRβ double KO mice.(A,B) WT and TRα1/TRβ KO mice (‘KO’) were submitted to 70% PH, and the protein levels of TRα1 (55 kDa) and TRβ (47 kDa) were determined by Western blot, and expressed as percentage of the normalized band intensities (using β-actin as control) vs. sham operated animals at 0 h. (C) mRNA levels of TRα1 and TRβ were determined by quantitative real time RT-PCR. (D) The serum levels of T3 and T4 after PH were measured and expressed as percentage vs. sham operated animals at 0 h. The basal values were 7.4±0.5 and 36.5±4.2 µg/dl for T4 in WT and KO, respectively; 78±5 and 2965±307 ng/dl for T3 in WT and KO, respectively. (E) Liver mass recovery after PH was determined in these animals and in WT mice treated with MMI to pharmacologically induce hypothyroidism, and in one group of animals thyroid hormone was restituted by administration of T4. (F) Acute liver injury after PH was evaluated by measuring serum AST levels. Results show means ± SD of 6 to 8 animals per condition (B,C,E,F), 4 animals (D) or a representative blot of three (A). #P<0.01 vs. the corresponding condition at 0h (B,D); *P<0.05, **P<0.01 vs. WT condition or T4-untreated WT animals (E,F).
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pone-0008710-g001: TRβ is transiently downregulated after PH and liver regeneration is delayed in TRα1/TRβ double KO mice.(A,B) WT and TRα1/TRβ KO mice (‘KO’) were submitted to 70% PH, and the protein levels of TRα1 (55 kDa) and TRβ (47 kDa) were determined by Western blot, and expressed as percentage of the normalized band intensities (using β-actin as control) vs. sham operated animals at 0 h. (C) mRNA levels of TRα1 and TRβ were determined by quantitative real time RT-PCR. (D) The serum levels of T3 and T4 after PH were measured and expressed as percentage vs. sham operated animals at 0 h. The basal values were 7.4±0.5 and 36.5±4.2 µg/dl for T4 in WT and KO, respectively; 78±5 and 2965±307 ng/dl for T3 in WT and KO, respectively. (E) Liver mass recovery after PH was determined in these animals and in WT mice treated with MMI to pharmacologically induce hypothyroidism, and in one group of animals thyroid hormone was restituted by administration of T4. (F) Acute liver injury after PH was evaluated by measuring serum AST levels. Results show means ± SD of 6 to 8 animals per condition (B,C,E,F), 4 animals (D) or a representative blot of three (A). #P<0.01 vs. the corresponding condition at 0h (B,D); *P<0.05, **P<0.01 vs. WT condition or T4-untreated WT animals (E,F).
Mentions: Expression levels of TRα and TRβ were determined in liver extracts from animals that had undergone PH. TRα content did not change in the period after PH; however, TRβ decreased significantly 24–72 h post-PH, returning to control levels at 96 h (Fig. 1A–B). Western blot confirmed absence of TRα1 and TRβ from TR double KO mice. Consistent with the protein expression profile, TRβ mRNA expression increased 3-fold by 48–72 h (Fig. 1C). TRα1/TRβ double KO mice exhibit a marked hyperthyroidism that has been previously reported [17]. The T3 and T4 serum levels were determined after PH and both WT and TRα1/TRβ double KO mice exhibited a rapid decrease in thyroid hormone levels after PH that recovered at 72–96 h (Fig. 1D), presumably due to the rapid overexpression of deiodinase type 3 [8]. Interestingly, the basal thyroid hormone levels of the TRα1/TRβ double KO mice where higher than those of WT mice, in agreement with previous work [17]. Regarding liver regeneration, TRα1/TRβ double KO mice had a slower rate of liver mass recovery after PH than WT, reflected in a delayed increase in the regeneration index (Fig. 1E). A milder delay was also observed in hypothyroid animals treated with MMI, and the liver regeneration index was restored in these animals by administration of T4. Interestingly, serum AST activity in hepatectomized animals, a marker of liver injury, was about a third of the WT value in the double KO mice, reflecting a decrease in liver injury after PH (Fig. 1F). This unexpected protection against PH-induced liver injury was systematically observed in TRα1/TRβ double KO mice and in MMI treated mice, being lost after administration of T4 in the latter case. Despite the attenuated liver regeneration in TRα1+TRβ KO or MMI-treated mice, survival rates after PH in these animals were identical to those in non-treated wild-types (Fig. 2A). Animal death was usually due to post-surgery complications, and always occurred during the first 24 h after intervention. Determination of cell proliferation, by Ki67-positive cell count in liver sections, revealed delayed progression in the cell cycle in TRα1/TRβ KO and MMI-treated mice (Fig. 2B), while flow cytometry of disaggregated liver cells showed no significant differences in the ploidy distribution among the animal groups (Fig. 2C). The delayed hepatocyte replication in double-KO and MMI-treated mice was reflected in delayed upregulation in the expression of the cell-cycle markers PCNA and cyclins E and D1 (Fig. 2D). Moreover, the typical drop in hepatic levels of C/EBPα that normally follows PH, and which is required for progress of hepatocytes through the cell cycle [21], was delayed in double-KO and MMI-treated mice, whereas the increase in C/EBPβ levels was much lower in animals lacking functional TRs (Fig. 2D).

Bottom Line: Pharmacologically induced hypothyroidism yielded similar results.Loss of TR activity was associated with enhanced nitrosative stress in the liver remnant, due to an increase in the activity of the nitric oxide synthase (NOS) 2 and 3, caused by a transient decrease in the concentration of asymmetric dimethylarginine (ADMA), a potent NOS inhibitor.Taken together, these results indicate that TRbeta contributes significantly to the rapid initial round of hepatocyte proliferation following PH, and improves the survival of the regenerating liver at later times.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.

ABSTRACT

Background: The role of thyroid hormones and their receptors (TR) during liver regeneration after partial hepatectomy (PH) was studied using genetic and pharmacologic approaches. Roles in liver regeneration have been suggested for T3, but there is no clear evidence distinguishing the contribution of increased amounts of T3 from the modulation by unoccupied TRs.

Methodology/principal findings: Mice lacking TRalpha1/TRbeta or TRbeta alone fully regenerated liver mass after PH, but showed delayed commitment to the initial round of hepatocyte proliferation and transient but intense apoptosis at 48h post-PH, affecting approximately 30% of the remaining hepatocytes. Pharmacologically induced hypothyroidism yielded similar results. Loss of TR activity was associated with enhanced nitrosative stress in the liver remnant, due to an increase in the activity of the nitric oxide synthase (NOS) 2 and 3, caused by a transient decrease in the concentration of asymmetric dimethylarginine (ADMA), a potent NOS inhibitor. This decrease in the ADMA levels was due to the presence of a higher activity of dimethylarginineaminohydrolase-1 (DDAH-1) in the regenerating liver of animals lacking TRalpha1/TRbeta or TRbeta. DDAH-1 expression and activity was paralleled by the activity of FXR, a transcription factor involved in liver regeneration and up-regulated in the absence of TR.

Conclusions/significance: We report that TRs are not required for liver regeneration; however, hypothyroid mice and TRbeta- or TRalpha1/TRbeta-deficient mice exhibit a delay in the restoration of liver mass, suggesting a specific role for TRbeta in liver regeneration. Altered regenerative responses are related with a delay in the expression of cyclins D1 and E, and the occurrence of liver apoptosis in the absence of activated TRbeta that can be prevented by administration of NOS inhibitors. Taken together, these results indicate that TRbeta contributes significantly to the rapid initial round of hepatocyte proliferation following PH, and improves the survival of the regenerating liver at later times.

Show MeSH
Related in: MedlinePlus