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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.


Inhibition of Akt phosphorylation in 3T3-L1 cells by baicalein.A, 3T3-L1 cells were differentiated into adipocytes for 60 min, 120 min or 6 days in DMEM containing MDI with baicalein or not (0 or 50 μM). Cell lysates (15 μg/lane) were subjected to SDS-PAGE and Western blot analysis for the detection of phosphorylated Akt (p-Akt), Akt, phosphorylated AMPK (p-AMPK) and AMPK proteins. β-actin was also detected as the internal control. The results are representative from at least three experiments. B, Ratio of band intensities of p-Akt/Akt. Band intensity was measured with MultiGauge software. The ratio of p-Akt and total Akt was shown.
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pone.0163640.g007: Inhibition of Akt phosphorylation in 3T3-L1 cells by baicalein.A, 3T3-L1 cells were differentiated into adipocytes for 60 min, 120 min or 6 days in DMEM containing MDI with baicalein or not (0 or 50 μM). Cell lysates (15 μg/lane) were subjected to SDS-PAGE and Western blot analysis for the detection of phosphorylated Akt (p-Akt), Akt, phosphorylated AMPK (p-AMPK) and AMPK proteins. β-actin was also detected as the internal control. The results are representative from at least three experiments. B, Ratio of band intensities of p-Akt/Akt. Band intensity was measured with MultiGauge software. The ratio of p-Akt and total Akt was shown.

Mentions: Some of natural compounds such as flavonoids are involved in the regulation of the early stage of adipogenesis [18], and control the insulin signaling in adipocytes [19, 20]. In addition, activation of Akt and AMPK is regulated by the insulin signaling [21]. So we examined whether the insulin signaling pathway was involved in the baicalein-inhibited adipogenesis or not. 3T3-L1 cells were differentiated into adipose cells for 6 days in DMEM containing baicalein, after which the expression levels of Akt and AMPK, and of their phosphorylated proteins were examined by Western blot analysis. Akt was expressed in the undifferentiated cells (0 h), and its expression level was slightly increased during adipocyte differentiation (Fig 7A and 7B). Akt was continuously phosphorylated during adipogenesis for 60 min. However, its phosphorylation was suppressed by baicalein at 60 min (1 h) after the starting of adipogenesis. In contrast, AMPK was slightly phosphorylated, but not altered in its phosphorylation level during adipogenesis for 60 min; although the expression of AMPK was detected in all samples (Fig 7A and 7B). In contrast, at 120 min and 6 days after the starting of adipogenesis, the transcription levels of Akt and AMPK, and of their phosphorylated forms were the same in both baicalein-treated and untreated differentiated cells (Fig 7A and 7B). These results mean that baicalein lowered the phosphorylation of Akt in the very early stage of adipogenesis of 3T3-L1 cells (at 1 h after the initiation of adipogenesis).


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
Inhibition of Akt phosphorylation in 3T3-L1 cells by baicalein.A, 3T3-L1 cells were differentiated into adipocytes for 60 min, 120 min or 6 days in DMEM containing MDI with baicalein or not (0 or 50 μM). Cell lysates (15 μg/lane) were subjected to SDS-PAGE and Western blot analysis for the detection of phosphorylated Akt (p-Akt), Akt, phosphorylated AMPK (p-AMPK) and AMPK proteins. β-actin was also detected as the internal control. The results are representative from at least three experiments. B, Ratio of band intensities of p-Akt/Akt. Band intensity was measured with MultiGauge software. The ratio of p-Akt and total Akt was shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036867&req=5

pone.0163640.g007: Inhibition of Akt phosphorylation in 3T3-L1 cells by baicalein.A, 3T3-L1 cells were differentiated into adipocytes for 60 min, 120 min or 6 days in DMEM containing MDI with baicalein or not (0 or 50 μM). Cell lysates (15 μg/lane) were subjected to SDS-PAGE and Western blot analysis for the detection of phosphorylated Akt (p-Akt), Akt, phosphorylated AMPK (p-AMPK) and AMPK proteins. β-actin was also detected as the internal control. The results are representative from at least three experiments. B, Ratio of band intensities of p-Akt/Akt. Band intensity was measured with MultiGauge software. The ratio of p-Akt and total Akt was shown.
Mentions: Some of natural compounds such as flavonoids are involved in the regulation of the early stage of adipogenesis [18], and control the insulin signaling in adipocytes [19, 20]. In addition, activation of Akt and AMPK is regulated by the insulin signaling [21]. So we examined whether the insulin signaling pathway was involved in the baicalein-inhibited adipogenesis or not. 3T3-L1 cells were differentiated into adipose cells for 6 days in DMEM containing baicalein, after which the expression levels of Akt and AMPK, and of their phosphorylated proteins were examined by Western blot analysis. Akt was expressed in the undifferentiated cells (0 h), and its expression level was slightly increased during adipocyte differentiation (Fig 7A and 7B). Akt was continuously phosphorylated during adipogenesis for 60 min. However, its phosphorylation was suppressed by baicalein at 60 min (1 h) after the starting of adipogenesis. In contrast, AMPK was slightly phosphorylated, but not altered in its phosphorylation level during adipogenesis for 60 min; although the expression of AMPK was detected in all samples (Fig 7A and 7B). In contrast, at 120 min and 6 days after the starting of adipogenesis, the transcription levels of Akt and AMPK, and of their phosphorylated forms were the same in both baicalein-treated and untreated differentiated cells (Fig 7A and 7B). These results mean that baicalein lowered the phosphorylation of Akt in the very early stage of adipogenesis of 3T3-L1 cells (at 1 h after the initiation of adipogenesis).

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.