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Suppression of Very Early Stage Of Adipogenesis by Baicalein, a Plant-Derived Flavonoid through Reduced Akt-C/EBP α -GLUT4 Signaling-Mediated Glucose Uptake in 3T3-L1 Adipocytes

View Article: PubMed Central - PubMed

ABSTRACT

Baicalein has been used as a Chinese medicine, and is an abundant plant flavonoid present in fruits and vegetables. Here, we examined the effects of baicalein in adipogenesis and investigated its molecular mechanism in adipocytes. Baicalein lowered the intracellular lipid accumulation and decreased the transcription levels of the adipocyte-specific genes in mouse 3T3-L1 adipocytes. Glucose uptake mediated by glucose transporter 4 (GLUT4) was reduced, causing down-regulation of the intracellular lipid accumulation. These reductions were also observed even when baicalein was added in only early stage of adipogenesis (0–2 days) of 6-day-adipogenesis. Chromatin immunoprecipitation assay showed that baicalein decreased the binding level of C/EBPα protein to the promoter region of the GLUT4 gene. Phosphorylation of Akt at 1 h after the initiation of adipogenesis was inhibited by the treatment with baicalein. Inhibition during only the first 1.5 h after the initiation of adipogenesis by baicalein or an Akt inhibitor was enough to decrease the lipid contents in the cells undergoing adipocyte differentiation for 6 days. These results indicate that baicalein decreased the intracellular lipid accumulation by down-regulation of glucose uptake via repression of Akt-C/EBPα-GLUT4 signaling in the very early stage of adipogenesis of 3T3-L1 adipocytes.

No MeSH data available.


Regulation of C/EBPα-mediated expression of GLUT4 gene in baicalein-treated 3T3-L1 cells.A, 3T3-L1 cells (undifferentiated cells: U; white columns) were differentiated into adipocytes (differentiated cells: D) for 6 days in DMEM without (gray columns) or with (50 μM; black columns) baicalein. The mRNA levels were quantified by qPCR. Data are presented as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. B, ChIP assay. 3T3-L1 cells (undifferentiated cells: U) were differentiated into adipocytes (differentiated cells: D) for 6 days in DMEM without or with baicalein (0 or 50 μM) during days 0–2 or 0–6, and the ChIP assay was then performed. The resultant PCR products were analyzed by agarose-gel electrophoresis. input (input control) means that a diluted aliquot before immunoprecipitation was used for PCR amplification. The results are representative from three experiments. Band intensity was measured with MultiGauge software.
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pone.0163640.g006: Regulation of C/EBPα-mediated expression of GLUT4 gene in baicalein-treated 3T3-L1 cells.A, 3T3-L1 cells (undifferentiated cells: U; white columns) were differentiated into adipocytes (differentiated cells: D) for 6 days in DMEM without (gray columns) or with (50 μM; black columns) baicalein. The mRNA levels were quantified by qPCR. Data are presented as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. B, ChIP assay. 3T3-L1 cells (undifferentiated cells: U) were differentiated into adipocytes (differentiated cells: D) for 6 days in DMEM without or with baicalein (0 or 50 μM) during days 0–2 or 0–6, and the ChIP assay was then performed. The resultant PCR products were analyzed by agarose-gel electrophoresis. input (input control) means that a diluted aliquot before immunoprecipitation was used for PCR amplification. The results are representative from three experiments. Band intensity was measured with MultiGauge software.

Mentions: To further analyze the molecular mechanism of the lipid accumulation decreased by baicalein in adipocytes, we investigated the glucose uptake and the mechanism of the transcriptional regulation of the GLUT4 gene in the baicalein-treated differentiated cells. Baicalein lowered the transcription levels of the C/EBPα and GLUT4 genes (Figs 2A, 3 and 5A). Moreover, it has been reported that the expression of the GLUT4 gene is regulated by C/EBPα in adipocytes [13, 15, 17]. Thus, we investigated whether the C/EBPα-GLUT4 signaling is involved in the control of the baicalein-decreased intracellular lipid accumulation. At first, we studied the time-course change in the mRNA levels of the C/EBPα and GLUT4 genes during adipogenesis. When 3T3-L1 cells were differentiated into adipose cells for 6 days in medium with or without baicalein, the mRNA level of the C/EBPα gene was gradually increased after 24 h of the initiation of adipogenesis (Fig 6A). However, when the cells were differentiated into adipose cells for 6 days in medium containing baicalein, its mRNA level was clearly suppressed (Fig 6A). Furthermore, almost the same expression profiles were found for the expression of the GLUT4 gene during adipogenesis in either the presence or absence of baicalein (Fig 6A).


Suppression of Very Early Stage Of Adipogenesis by Baicalein, a Plant-Derived Flavonoid through Reduced Akt-C/EBP α -GLUT4 Signaling-Mediated Glucose Uptake in 3T3-L1 Adipocytes
Regulation of C/EBPα-mediated expression of GLUT4 gene in baicalein-treated 3T3-L1 cells.A, 3T3-L1 cells (undifferentiated cells: U; white columns) were differentiated into adipocytes (differentiated cells: D) for 6 days in DMEM without (gray columns) or with (50 μM; black columns) baicalein. The mRNA levels were quantified by qPCR. Data are presented as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. B, ChIP assay. 3T3-L1 cells (undifferentiated cells: U) were differentiated into adipocytes (differentiated cells: D) for 6 days in DMEM without or with baicalein (0 or 50 μM) during days 0–2 or 0–6, and the ChIP assay was then performed. The resultant PCR products were analyzed by agarose-gel electrophoresis. input (input control) means that a diluted aliquot before immunoprecipitation was used for PCR amplification. The results are representative from three experiments. Band intensity was measured with MultiGauge software.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5036867&req=5

pone.0163640.g006: Regulation of C/EBPα-mediated expression of GLUT4 gene in baicalein-treated 3T3-L1 cells.A, 3T3-L1 cells (undifferentiated cells: U; white columns) were differentiated into adipocytes (differentiated cells: D) for 6 days in DMEM without (gray columns) or with (50 μM; black columns) baicalein. The mRNA levels were quantified by qPCR. Data are presented as the means ± S.D. from three experiments. **p<0.01, as indicated by the brackets. B, ChIP assay. 3T3-L1 cells (undifferentiated cells: U) were differentiated into adipocytes (differentiated cells: D) for 6 days in DMEM without or with baicalein (0 or 50 μM) during days 0–2 or 0–6, and the ChIP assay was then performed. The resultant PCR products were analyzed by agarose-gel electrophoresis. input (input control) means that a diluted aliquot before immunoprecipitation was used for PCR amplification. The results are representative from three experiments. Band intensity was measured with MultiGauge software.
Mentions: To further analyze the molecular mechanism of the lipid accumulation decreased by baicalein in adipocytes, we investigated the glucose uptake and the mechanism of the transcriptional regulation of the GLUT4 gene in the baicalein-treated differentiated cells. Baicalein lowered the transcription levels of the C/EBPα and GLUT4 genes (Figs 2A, 3 and 5A). Moreover, it has been reported that the expression of the GLUT4 gene is regulated by C/EBPα in adipocytes [13, 15, 17]. Thus, we investigated whether the C/EBPα-GLUT4 signaling is involved in the control of the baicalein-decreased intracellular lipid accumulation. At first, we studied the time-course change in the mRNA levels of the C/EBPα and GLUT4 genes during adipogenesis. When 3T3-L1 cells were differentiated into adipose cells for 6 days in medium with or without baicalein, the mRNA level of the C/EBPα gene was gradually increased after 24 h of the initiation of adipogenesis (Fig 6A). However, when the cells were differentiated into adipose cells for 6 days in medium containing baicalein, its mRNA level was clearly suppressed (Fig 6A). Furthermore, almost the same expression profiles were found for the expression of the GLUT4 gene during adipogenesis in either the presence or absence of baicalein (Fig 6A).

View Article: PubMed Central - PubMed

ABSTRACT

Baicalein has been used as a Chinese medicine, and is an abundant plant flavonoid present in fruits and vegetables. Here, we examined the effects of baicalein in adipogenesis and investigated its molecular mechanism in adipocytes. Baicalein lowered the intracellular lipid accumulation and decreased the transcription levels of the adipocyte-specific genes in mouse 3T3-L1 adipocytes. Glucose uptake mediated by glucose transporter 4 (GLUT4) was reduced, causing down-regulation of the intracellular lipid accumulation. These reductions were also observed even when baicalein was added in only early stage of adipogenesis (0&ndash;2 days) of 6-day-adipogenesis. Chromatin immunoprecipitation assay showed that baicalein decreased the binding level of C/EBP&alpha; protein to the promoter region of the GLUT4 gene. Phosphorylation of Akt at 1 h after the initiation of adipogenesis was inhibited by the treatment with baicalein. Inhibition during only the first 1.5 h after the initiation of adipogenesis by baicalein or an Akt inhibitor was enough to decrease the lipid contents in the cells undergoing adipocyte differentiation for 6 days. These results indicate that baicalein decreased the intracellular lipid accumulation by down-regulation of glucose uptake via repression of Akt-C/EBP&alpha;-GLUT4 signaling in the very early stage of adipogenesis of 3T3-L1 adipocytes.

No MeSH data available.