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Shikonin suppresses ERK 1/2 phosphorylation during the early stages of adipocyte differentiation in 3T3-L1 cells.

Gwon SY, Ahn JY, Jung CH, Moon BK, Ha TY - BMC Complement Altern Med (2013)

Bottom Line: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin.Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2.We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Functional Food Technology Research Group, Korea Food Research Institute, 516, Baekhyn-dong, Seongnam-si, Gyeonggi-do 463-746, Republic of Korea.

ABSTRACT

Background: The naphthoquinone pigment, shikonin, is a major component of Lithospermum erythrorhizon and has been shown to have various biological functions, including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we investigated the effect of shikonin on adipocyte differentiation and its mechanism of action in 3T3-L1 cells.

Methods: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the anti-adipogenic mechanism of shikonin, adipogenic transcription factors, the phosphorylation levels of ERK, and adipogenic gene expression were analyzed by Western blotting and quantitative real-time PCR. To further confirm that shikonin inhibits adipogenic differentiation through downregulation of ERK 1/2 activity, 3T3-L1 cells were treated with shikonin in the presence of FGF-2, an activator, or PD98059, an inhibitor, of the ERK1/2 signaling pathway.

Results: Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2. Adipocyte differentiation was mediated by ERK 1/2 phosphorylation, which was confirmed by pretreatment with PD98059 (an ERK 1/2 inhibitor) or FGF-2 (an ERK 1/2 activator). The phosphorylation of ERK1/2 during the early stages of adipogenesis in 3T3-L1 cells was inhibited by shikonin. We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

Conclusions: These results demonstrate that shikonin inhibits adipogenic differentiation via suppression of the ERK signaling pathway during the early stages of adipogenesis.

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Related in: MedlinePlus

Effect of shikonin on adipogenic transcription factor protein levels and gene expression in 3T3-L1 adipocytes. (A) Western blot analysis was performed with antibodies against PPARγ, C/EBPα, aP2, and β-actin. (B) The expression of PPARγ, C/EBPα, and aP2mRNAs from 3T3-L1 adipocytes was measured by qRT-PCR.
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Figure 2: Effect of shikonin on adipogenic transcription factor protein levels and gene expression in 3T3-L1 adipocytes. (A) Western blot analysis was performed with antibodies against PPARγ, C/EBPα, aP2, and β-actin. (B) The expression of PPARγ, C/EBPα, and aP2mRNAs from 3T3-L1 adipocytes was measured by qRT-PCR.

Mentions: Next, to examine whether shikonin inhibits adipocyte differentiation through the downregulation of adipogenic transcription factors and their target genes, we performed Western blotting and quantitative real-time PCR to analyze the protein and mRNA expression of PPARg, C/EBPa, and aP2. The protein levels of PPARγ, C/EBPα, and aP2 decreased with increasing dosages of shikonin in 3T3-L1 cells (Figure 2). Consistently, the mRNA expression of PPARγ, C/EBPα, and aP2 was also reduced by shikonin (Figure 2). These results demonstrate that shikonin inhibits adipogenesis through the downregulation of adipogenic transcription factors and their genes.


Shikonin suppresses ERK 1/2 phosphorylation during the early stages of adipocyte differentiation in 3T3-L1 cells.

Gwon SY, Ahn JY, Jung CH, Moon BK, Ha TY - BMC Complement Altern Med (2013)

Effect of shikonin on adipogenic transcription factor protein levels and gene expression in 3T3-L1 adipocytes. (A) Western blot analysis was performed with antibodies against PPARγ, C/EBPα, aP2, and β-actin. (B) The expression of PPARγ, C/EBPα, and aP2mRNAs from 3T3-L1 adipocytes was measured by qRT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750626&req=5

Figure 2: Effect of shikonin on adipogenic transcription factor protein levels and gene expression in 3T3-L1 adipocytes. (A) Western blot analysis was performed with antibodies against PPARγ, C/EBPα, aP2, and β-actin. (B) The expression of PPARγ, C/EBPα, and aP2mRNAs from 3T3-L1 adipocytes was measured by qRT-PCR.
Mentions: Next, to examine whether shikonin inhibits adipocyte differentiation through the downregulation of adipogenic transcription factors and their target genes, we performed Western blotting and quantitative real-time PCR to analyze the protein and mRNA expression of PPARg, C/EBPa, and aP2. The protein levels of PPARγ, C/EBPα, and aP2 decreased with increasing dosages of shikonin in 3T3-L1 cells (Figure 2). Consistently, the mRNA expression of PPARγ, C/EBPα, and aP2 was also reduced by shikonin (Figure 2). These results demonstrate that shikonin inhibits adipogenesis through the downregulation of adipogenic transcription factors and their genes.

Bottom Line: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin.Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2.We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Functional Food Technology Research Group, Korea Food Research Institute, 516, Baekhyn-dong, Seongnam-si, Gyeonggi-do 463-746, Republic of Korea.

ABSTRACT

Background: The naphthoquinone pigment, shikonin, is a major component of Lithospermum erythrorhizon and has been shown to have various biological functions, including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we investigated the effect of shikonin on adipocyte differentiation and its mechanism of action in 3T3-L1 cells.

Methods: To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the anti-adipogenic mechanism of shikonin, adipogenic transcription factors, the phosphorylation levels of ERK, and adipogenic gene expression were analyzed by Western blotting and quantitative real-time PCR. To further confirm that shikonin inhibits adipogenic differentiation through downregulation of ERK 1/2 activity, 3T3-L1 cells were treated with shikonin in the presence of FGF-2, an activator, or PD98059, an inhibitor, of the ERK1/2 signaling pathway.

Results: Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2. Adipocyte differentiation was mediated by ERK 1/2 phosphorylation, which was confirmed by pretreatment with PD98059 (an ERK 1/2 inhibitor) or FGF-2 (an ERK 1/2 activator). The phosphorylation of ERK1/2 during the early stages of adipogenesis in 3T3-L1 cells was inhibited by shikonin. We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.

Conclusions: These results demonstrate that shikonin inhibits adipogenic differentiation via suppression of the ERK signaling pathway during the early stages of adipogenesis.

Show MeSH
Related in: MedlinePlus