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Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis.

Kong X, Wang R, Xue Y, Liu X, Zhang H, Chen Y, Fang F, Chang Y - PLoS ONE (2010)

Bottom Line: Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes.Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis.

View Article: PubMed Central - PubMed

Affiliation: The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT

Background: Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results: Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (-407/-399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2)C(12) myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.

Conclusion: Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.

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PGC-1α regulates the expression of SIRT3.A, Differentiated C2C12 cells (left panel), primary hepatocytes isolated from mouse liver (middle panel) and HepG2 cells (right panel) were infected with adenoviruses expressing GFP (control) or PGC-1α. Cells were harvested 48 h after infection, and total RNA was extracted. The mRNA levels of Sirt3 were quantified by qPCR, normalized to β-actin, and expressed relative to the GFP-expressing control cells. B, C2C12 myotubes and primary hepatocytes were treated as described in Panel A, and SIRT3 protein level was determined by western blotting. Two specific bands of SIRT3 in C2C12 (left panel) and primary hepatocytes (right panel) were detected. L, long form of mSIRT3 (approximately 37 kDa). S, short form of mSIRT3 (approximately 28 kDa). C, left panel, C2C12 myoblasts were induced to differentiate into myotubes. Cells were harvested on day 0 (before differentiation) and day 5 (after differentiation), and total RNA was isolated. C, right panel, Total RNA was extracted from normal or fasted mouse livers. The mRNA levels of PGC-1α and Sirt3 were quantified by qPCR, normalized to β-actin and expressed relative to levels in the control. D, Differentiated C2C12 (left panel) and primary hepatocytes (right panel) were infected with adenovirus expressing luciferase shRNA (siControl) or PGC-1α shRNA (siPGC-1α). Cells were harvested 48 hours after infection, and total RNA was extracted. The mRNA levels of Sirt3 and PGC-1α were quantified by qPCR, normalized to β-actin, and expressed relative to the control cells. *, P<0.05.
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pone-0011707-g001: PGC-1α regulates the expression of SIRT3.A, Differentiated C2C12 cells (left panel), primary hepatocytes isolated from mouse liver (middle panel) and HepG2 cells (right panel) were infected with adenoviruses expressing GFP (control) or PGC-1α. Cells were harvested 48 h after infection, and total RNA was extracted. The mRNA levels of Sirt3 were quantified by qPCR, normalized to β-actin, and expressed relative to the GFP-expressing control cells. B, C2C12 myotubes and primary hepatocytes were treated as described in Panel A, and SIRT3 protein level was determined by western blotting. Two specific bands of SIRT3 in C2C12 (left panel) and primary hepatocytes (right panel) were detected. L, long form of mSIRT3 (approximately 37 kDa). S, short form of mSIRT3 (approximately 28 kDa). C, left panel, C2C12 myoblasts were induced to differentiate into myotubes. Cells were harvested on day 0 (before differentiation) and day 5 (after differentiation), and total RNA was isolated. C, right panel, Total RNA was extracted from normal or fasted mouse livers. The mRNA levels of PGC-1α and Sirt3 were quantified by qPCR, normalized to β-actin and expressed relative to levels in the control. D, Differentiated C2C12 (left panel) and primary hepatocytes (right panel) were infected with adenovirus expressing luciferase shRNA (siControl) or PGC-1α shRNA (siPGC-1α). Cells were harvested 48 hours after infection, and total RNA was extracted. The mRNA levels of Sirt3 and PGC-1α were quantified by qPCR, normalized to β-actin, and expressed relative to the control cells. *, P<0.05.

Mentions: Differentiated C2C12 and mouse primary hepatocytes were infected with adenovirus expressing PGC-1α or GFP (control). Total RNA was extracted from infected cells, and mRNAs of sirtuin family members were measured by real-time PCR. We observed that Sirt3 mRNA levels were increased by approximately 7-fold by PGC-1α in C2C12 myotubes compared to control cells (Fig. 1A). In addition, PGC-1α stimulated Sirt3 mRNA level by approximately 70-fold and 2.8-fold in mouse primary hepatocytes and HepG2 cells, respectively (Fig. 1A). These increases in Sirt3 mRNA were accompanied by potent up-regulation of the protein levels of mSIRT3 variants in C2C12 cells and mouse primary hepatocytes (Fig. 1B). In contrast to Sirt3, PGC-1α did not alter the expression of other sirtuin members (data not shown).


Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis.

Kong X, Wang R, Xue Y, Liu X, Zhang H, Chen Y, Fang F, Chang Y - PLoS ONE (2010)

PGC-1α regulates the expression of SIRT3.A, Differentiated C2C12 cells (left panel), primary hepatocytes isolated from mouse liver (middle panel) and HepG2 cells (right panel) were infected with adenoviruses expressing GFP (control) or PGC-1α. Cells were harvested 48 h after infection, and total RNA was extracted. The mRNA levels of Sirt3 were quantified by qPCR, normalized to β-actin, and expressed relative to the GFP-expressing control cells. B, C2C12 myotubes and primary hepatocytes were treated as described in Panel A, and SIRT3 protein level was determined by western blotting. Two specific bands of SIRT3 in C2C12 (left panel) and primary hepatocytes (right panel) were detected. L, long form of mSIRT3 (approximately 37 kDa). S, short form of mSIRT3 (approximately 28 kDa). C, left panel, C2C12 myoblasts were induced to differentiate into myotubes. Cells were harvested on day 0 (before differentiation) and day 5 (after differentiation), and total RNA was isolated. C, right panel, Total RNA was extracted from normal or fasted mouse livers. The mRNA levels of PGC-1α and Sirt3 were quantified by qPCR, normalized to β-actin and expressed relative to levels in the control. D, Differentiated C2C12 (left panel) and primary hepatocytes (right panel) were infected with adenovirus expressing luciferase shRNA (siControl) or PGC-1α shRNA (siPGC-1α). Cells were harvested 48 hours after infection, and total RNA was extracted. The mRNA levels of Sirt3 and PGC-1α were quantified by qPCR, normalized to β-actin, and expressed relative to the control cells. *, P<0.05.
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pone-0011707-g001: PGC-1α regulates the expression of SIRT3.A, Differentiated C2C12 cells (left panel), primary hepatocytes isolated from mouse liver (middle panel) and HepG2 cells (right panel) were infected with adenoviruses expressing GFP (control) or PGC-1α. Cells were harvested 48 h after infection, and total RNA was extracted. The mRNA levels of Sirt3 were quantified by qPCR, normalized to β-actin, and expressed relative to the GFP-expressing control cells. B, C2C12 myotubes and primary hepatocytes were treated as described in Panel A, and SIRT3 protein level was determined by western blotting. Two specific bands of SIRT3 in C2C12 (left panel) and primary hepatocytes (right panel) were detected. L, long form of mSIRT3 (approximately 37 kDa). S, short form of mSIRT3 (approximately 28 kDa). C, left panel, C2C12 myoblasts were induced to differentiate into myotubes. Cells were harvested on day 0 (before differentiation) and day 5 (after differentiation), and total RNA was isolated. C, right panel, Total RNA was extracted from normal or fasted mouse livers. The mRNA levels of PGC-1α and Sirt3 were quantified by qPCR, normalized to β-actin and expressed relative to levels in the control. D, Differentiated C2C12 (left panel) and primary hepatocytes (right panel) were infected with adenovirus expressing luciferase shRNA (siControl) or PGC-1α shRNA (siPGC-1α). Cells were harvested 48 hours after infection, and total RNA was extracted. The mRNA levels of Sirt3 and PGC-1α were quantified by qPCR, normalized to β-actin, and expressed relative to the control cells. *, P<0.05.
Mentions: Differentiated C2C12 and mouse primary hepatocytes were infected with adenovirus expressing PGC-1α or GFP (control). Total RNA was extracted from infected cells, and mRNAs of sirtuin family members were measured by real-time PCR. We observed that Sirt3 mRNA levels were increased by approximately 7-fold by PGC-1α in C2C12 myotubes compared to control cells (Fig. 1A). In addition, PGC-1α stimulated Sirt3 mRNA level by approximately 70-fold and 2.8-fold in mouse primary hepatocytes and HepG2 cells, respectively (Fig. 1A). These increases in Sirt3 mRNA were accompanied by potent up-regulation of the protein levels of mSIRT3 variants in C2C12 cells and mouse primary hepatocytes (Fig. 1B). In contrast to Sirt3, PGC-1α did not alter the expression of other sirtuin members (data not shown).

Bottom Line: Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes.Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis.

View Article: PubMed Central - PubMed

Affiliation: The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT

Background: Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results: Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (-407/-399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2)C(12) myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.

Conclusion: Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.

Show MeSH
Related in: MedlinePlus