Role of sirtuin 1 in the regulation of hepatic gene expression by thyroid hormone - PubMed (original) (raw)
Role of sirtuin 1 in the regulation of hepatic gene expression by thyroid hormone
Shalini Thakran et al. J Biol Chem. 2013.
Abstract
Sirtuin 1 (SIRT1) is a nuclear deacetylase that modulates lipid metabolism and enhances mitochondrial activity. SIRT1 targets multiple transcription factors and coactivators. Thyroid hormone (T(3)) stimulates the expression of hepatic genes involved in mitochondrial fatty acid oxidation and gluconeogenesis. We reported that T(3) induces genes for carnitine palmitoyltransferase (cpt1a), pyruvate dehydrogenase kinase 4 (pdk4), and phosphoenolpyruvate carboxykinase (pepck). SIRT1 increases the expression of these genes via the activation of several factors, including peroxisome proliferator-activated receptor α, estrogen-related receptor α, and peroxisome proliferator-activated receptor γ coactivator (PGC-1α). Previously, we reported that PGC-1α participates in the T(3) induction of cpt1a and pdk4 in the liver. Given the overlapping targets of T(3) and SIRT1, we investigated whether SIRT1 participated in the T(3) regulation of these genes. Resveratrol is a small phenolic compound whose actions include the activation of SIRT1. Addition of resveratrol increased the T(3) induction of the pdk4 and cpt1a genes in hepatocytes. Furthermore, expression of SIRT1 in hepatocytes mimicked resveratrol in the regulation of gene expression by T(3). The deacetylase activity of SIRT1 was required and PGC-1α was deacetylated following addition of T(3). We found that SIRT1 interacted directly with T(3) receptor (TRβ). Knockdown of SIRT1 decreased the T(3) induction of cpt1a and pdk4 and reduced the T(3) inhibition of sterol response element binding protein (srebp-1c) both in isolated hepatocytes and in rat liver. Our results indicate that SIRT1 contributes to the T(3) regulation of hepatic genes.
Figures
FIGURE 1.
Resveratrol and thyroid hormone (T3) alter mRNA abundance in hepatocytes. Rat hepatocytes were plated overnight in RPMI 1640 with 10% FBS. Cells were treated with 100 n
m
T3, 20 μ
m
resveratrol, or both for 24 h. RNA and protein were harvested and analyzed by real time PCR or Western blot, respectively, as described under “Materials and Methods.” The mRNA abundance measurements are as follows: A, cpt1a; B, pdk4; C, pepck; D, fasn; E, srebp-1c; F, _pgc-1_α; and G, sirt1. The changes are indicated relative to untreated control cells. H, a Western blot is shown for SIRT1 abundance in control and T3-treated hepatocytes. Values are mean ± S.E. of four independent culture experiments. Statistical significance was determined by t test (*, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p < 0.001).
FIGURE 2.
Resveratrol and T3 modulate mRNA levels in HepG2 cells. HepG2 hepatoma cells were treated with 100 n
m
T3, 20 μ
m
resveratrol, or both for 24 h as described in figure legend 1. The mRNA abundance was measured by real time PCR for: A, cpt1a; B, pdk4; C, pepck; and D, fasn. Values are mean ± S.E. of four or more independent experiments. Statistical significance was determined by t test (***, p < 0.001).
FIGURE 3.
SIRT1 enhances the T3 stimulation through gene promoters. HepG2 cells were transiently transfected with 2 μg of different luciferase constructs, 1 μg of SV40-TRβ and 0.1 μg of TK-Renilla. T3 and resveratrol were added at a concentration of 100 n
m
and 50 μ
m,
respectively, either separately or together for 24 h. Results are expressed as the relative induction by T3 and resveratrol compared with the untreated cells: A, −4495/+1240 _cpt1a_-luciferase; B, −1256/+78 _pdk4_-luciferase; C, −4495/+1240 and −4495/+19 _cpt1a_-luc were cotransfected with SV40-TRβ, TK-Renilla, and with either Flag-SIRT1 or Flag-SIRT1H363Y. Cells were treated with T3 as described above. D, −4495/+1240 _cpt1a_-luc wild type or TRE mutant were cotransfected with Flag-SIRT1, SV40-TRβ and TK-Renilla and treated with 100 n
m
T3 for 24 h. E, HepG2 cells were transfected with TREX2 SV40-luciferase as above. The cells were treated with 100 n
m
T3 or 50 μ
m
resveratrol. F, TREX2 SV40-luciferase vectors were transfected with Flag-SIRT1 and SV40-TRβ. Cells were treated with T3 as described above. All transfections were performed in duplicate and repeated three to six times. Luciferase activity was corrected for both protein content and Renilla activity. The significance was determined by t test (*, p = 0.01 to 0.05; **, p = 0.001 to 0.01).
FIGURE 4.
Sirtuin 1 and TRβ physically interact. A, His-tagged TRβ was prepared in BL21 E. coli. HeLa cells were transfected with a vector that expresses Flag-SIRT1, and cell lysates were prepared. His-TRβ and cell lysates were allowed to interact, and the interacting proteins were precipitated with nickel (Ni) resin. The interacting proteins were resolved by Western analysis with antibodies for the Flag epitope, TRβ, and SIRT1. B, His-tagged SIRT1 was prepared in BL21 E. coli. HeLa cells were transfected with a Flag-TRβ expression vector, and the protein interactions were tested as described above. C, His-TRβ was allowed to interact with either purified GST-SIRT1 or GST protein, and the interacting proteins were pulled down with glutathione-Sepharose 4B beads. Proteins were detected with TRβ and GST antibodies. D, purified GST-SIRT1 protein was mixed with His-TRβ; E, His-tagged DNA binding (DBD) and ligand binding domains (LBD) of TRβ. Proteins were pulled down with nickel (Ni) resin and assayed with SIRT1 and His tag antibodies. F, rat hepatocytes were treated with T3 for 24 h and cross-linked. The chromatin was sheared and immunoprecipitated with IgG or SIRT1 antibodies. The immunoprecipitated DNA was amplified with primers for cpt1a TRE, first intron, and upstream region.
FIGURE 5.
SIRT1 enhances the T3 induction of gene expression in hepatocytes. Rat hepatocytes were infected with either SIRT1-expressing adenoviruses (AdSIRT1), the catalytic dead SIRT1-expressing adenoviruses (AdSIRT1H355A), or nonspecific adenoviruses (AdNC) for 16 h and treated with 1 μ
m
T3 for the next 24 h. The mRNA abundance of the indicated genes was measured by real time PCR: A, cpt1a; B, pdk4; C, pepck; D, fasn; E, srebp_-1c_; and F, sirt1 mRNA abundance are shown. The experiments were repeated three or four times. The data are expressed as the mean of the fold induction ± S.E. of mRNA abundance relative to untreated control cells (*, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p < 0.001). G, a Western blot is shown for SIRT1 overexpression in AdSIRT1 and AdSIRT1H355A infected hepatocytes compared with nonspecific adenovirus-infected cells. H, HeLa cells were transfected with the expression vectors for PGC-1α, TRβ, or SIRT1. T3 was added for 16 h. Levels of acetylated PGC-1α were assessed with antibody that recognizes acetyl lysine residues.
FIGURE 6.
SIRT1 elevates the T3 stimulation of gene expression in HepG2 cells. HepG2 cells were infected with SIRT1 expressing adenoviruses (AdSIRT1) or nonspecific adenoviruses (AdNC) for 16 h and exposed to 1 μ
m
T3 for 24 h as described in the legend to Fig. 5. The mRNA abundance of specific genes was measured by real time PCR: A, CPT1a; B, PDK4; C, PEPCK; D, FASN. The data are shown as the mean of the fold induction ± S.E. of mRNA abundance relative to untreated control cells (*, p = 0.01 to 0.05).
FIGURE 7.
SIRT1 knockdown blocks the T3 induction of gene expression in rat hepatocytes. Rat hepatocytes were infected with adenoviral vectors expressing shRNA for SIRT1 (AdshSIRT1) or scrambled shRNA (AdScr) overnight. Hepatocytes were treated with 1 μ
m
T3 for 24 h. The mRNA abundance was measured by real time PCR: A, cpt1a; B, pdk4; C, pepck; D, fasn; E, srebp-1c mRNA; and F, fold decrease of sirt1 mRNA by AdshSIRT1 adenovirus compared with untreated hepatocytes infected with AdScr adenovirus. The data are expressed as the mean of the fold induction by T3 ± S.E. of mRNA abundance relative to untreated hepatocytes (*, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p < 0.001). G, a Western blot is shown for SIRT1 knockdown with AdshSIRT1-infected hepatocytes compared with AdScr-infected cells.
FIGURE 8.
SIRT1 knockdown reduces the T3 induction of gene expression in rat liver. Hypothyroid rats were infected with 1011 pfu/kg AdshSIRT1 and AdScr adenovirus via the tail vein. T3 (0.33 mg/kg) was administered for two consecutive days before harvesting the liver. The mRNA abundance in liver was measured by real time PCR: A, cpt1a; B, pdk4; C, pepck; D, fasn; E, srebp-1c mRNA; and F, fold decrease of sirt1 mRNA by AdshSIRT1 adenovirus compared with untreated rats infected with AdScr adenovirus. G, Western blot is shown for SIRT1 and CPT1a protein abundance in AdshSIRT1- and AdScr-infected rat liver with and without T3 treatment. H, the protein abundance of SIRT1; and I, CPT1a was measured by densitometry of the Western blots. The data are expressed as the mean of the fold induction by T3 ± S.E. of mRNA abundance relative to untreated rat liver. The induction by T3 treatment compared with its respective control or the effect of SIRT1 knockdown is indicated as fold induction, and the inhibition is indicated in percent inhibition. (*, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p < 0.001).
References
- Feng X., Jiang Y., Meltzer P., Yen P. M. (2000) Thyroid hormone regulation of hepatic genes in vivo detected by complementary DNA microarray. Mol. Endocrinol. 14, 947–955 - PubMed
- Liu Y., Xia X., Fondell J. D., Yen P. M. (2006) Thyroid hormone-regulated target genes have distinct patterns of coactivator recruitment and histone acetylation. Mol. Endocrinol. 20, 483–490 - PubMed
- Yen P. M. (2001) Physiological and molecular basis of thyroid hormone action. Physiol. Rev. 81, 1097–1142 - PubMed
- Jansen M. S., Cook G. A., Song S., Park E. A. (2000) Thyroid hormone regulates carnitine palmitoyltransferase Iα gene expression through elements in the promoter and first intron. J. Biol. Chem. 275, 34989–34997 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases