Cocoa tea (Camellia ptilophylla) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes

Abstract

Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. Previously we found that cocoa tea demonstrated a beneficial effect against high-fat diet induced obesity, hepatic steatosis, and hyperlipidemia in mice. The present study aimed to investigate the anti-adipogenic effect of cocoa tea in vitro using preadipocytes 3T3-L1. Adipogenic differentiation was confirmed by Oil Red O stain, qPCR and Western blot. Our results demonstrated that cocoa tea significantly inhibited triglyceride accumulation in mature adipocytes in a dose-dependent manner. Cocoa tea was shown to suppress the expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPAR γ) and CCAAT/enhancer binding protein (C/EBP α). The tea extract was subsequently found to reduce the expressions of adipocyte-specific genes such as sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), fatty acid translocase (FAT) and stearoylcoenzyme A desaturase-1 (SCD-1). In addition, JNK, ERK and p38 phosphorylation were inhibited during cocoa tea inhibition of 3T3-L1 adipogenic differentiation. Taken together, this is the first study that demonstrates cocoa tea has the capacity to suppress adipogenesis in pre-adipocyte 3T3-L1 similar to traditional green tea.

Figure 1. Effect of CTE and GTE on cell viability in 3T3-L1 cells.

Cell viability was determined by MTT assay. CTE: Cocoa tea extract; GTE: Green tea extract.

Figure 2. Anti-adipogenic effects of CTE and GTE on 3T3-L1 cells.

(A) Effects of CTE and GTE on the differentiation of 3T3-L1 cells. 3T3-L1 cells were cultured in adipocyte differentiation cocktail media with or without the treatment of CTE or GTE, After 8 days culture, the cells were stained with Oil-red O, and then photographed under the microscope (Magnification: 40 × ), (B) The relative lipid content in different treatment groups. CTE: cocoa tea extract; GTE: green tea extract. Tro: troglitazone. The results are presented as the mean ± SD of triplicate tests. Significant difference: *p < 0.05, **p < 0.01, ***p < 0.001 as compared to control group.

Figure 3. Effect of CTE and GTE on the triglyceride deposition in differentiated 3T3-L1 cells.

3T3-L1 cells were cultured in adipocyte differentiation cocktail media with or without the treatment of CTE or GTE. CTE: Cocoa tea extract; GTE: green tea extract. The results are presented as the mean ± SD of triplicate tests. Significant difference: ***p < 0.001 as compared to control group.

Figure 4. Effect of CTE and GTE on the gene expressions of the key adipogenic transcription factors, PPAR γ and C/EBP α associated with (A) PPAR γ; (B) C/EBP α; CTE: cocoa tea extract; GTE: green tea extract.

All values are expressed as mean ± SEM of triplicate tests. *p < 0.05; **p < 0.01; ***p < 0.001 as compared to control group.

Figure 5. Effect of CTE and GTE on the protein expressions of the key adipogenic transcription factors, PPAR γ and C/EBP α on day 3 and 8.

The cells were induced to differentiate into adipocytes in MDI medium with or without CTE and GTE. The experiment was performed three to four times using independently prepared cell lysates, and representative blots were shown.

Figure 6. Effect of CTE and GTE on gene expressions of the adipocyte-specific genes of 3T3-L1 adipocyte differentiation.

The cells were induced to differentiate into adipocytes in MDI medium with or without CTE and GTE. mRNA were extracted and the expression of SREBP-1c, FAS, ACC, FAT and SCD-1 genes were detected using one-step RT-PCR. CTE: cocoa tea extract; GTE: green tea extract. Each value represents the Mean ± SEM of triplicate test. *p < 0.05; **p < 0.01; ***p < 0.001 as compared to control group.

Figure 7. Cocoa tea inhibits the protein expressions of the adipocyte-specific genes of 3T3-L1 adipocyte differentiation.

The cells were induced to differentiate into adipocytes in MDI medium with or without CTE and GTE. The proteins were extracted and the expressions of SREBP-1c, FAS, ACC, FAT and SCD-1 genes were detected using Western blot assay. The western blot was performed three to four times using independently prepared cell lysates, and representative blots were shown.

Figure 8. Cocoa tea inhibits JNK, ERK and p38 phosphorylation during its inhibition of 3T3-L1 adipocyte differentiation.

The cells were induced to differentiate into adipocytes in MDI medium with or without CTE OR GTE for 15 min, 30 min, 1 h and 2 h, the protein were extracted and the phosphorylation of JNK, ERK and p38 were detected using Western Blot. Immunoblotting was performed three to four times using independently prepared cell lysates, and representative blots were shown.