Alliin inhibits adipocyte differentiation by downregulating Akt expression: Implications for metabolic disease

Abstract

Obesity is currently an important health problem and is associated with an increased likelihood of various diseases. The efficacies of various natural treatments have been assessed for their utility in treating obesity. Alliin (S-allyl-L-cysteine sulfoxides) is considered the major component of garlic and has a wide range of natural antioxidant properties. However, the direct effects of alliin on obesity have not been well clarified. The present study investigated the effects and possible mechanisms of alliin on adipocyte differentiation. The 3T3-L1 cells were treated with alliin (0-40 µg/ml) during adipogenic differentiation. The effect of alliin on lipid accumulation was evaluated by Oil red O staining. Reverse transcription-quantitative PCR was performed to investigate the expression levels of adipogenic differentiation-related genes. The accumulation of lipid droplets was markedly inhibited following alliin treatment. The expression levels of multiple adipogenic transcription markers, such as CCAAT/enhancer-binding protein (C/EBP) β, C/EBP α and peroxisome proliferation-activity receptor γ, were markedly decreased following treatment with alliin during adipogenic differentiation. Expression levels of several adipocyte-related genes were subsequently suppressed. Additionally, alliin suppressed PKB/Akt and PI3K expression. These results suggested that alliin exhibits anti-adipogenic activity by downregulating major adipogenic differentiation-related genes and Akt/PI3K expression. Alliin may have a potential therapeutic effect on metabolic disease.

Keywords: Akt; CCAAT/enhancer-binding protein β; adipogenesis; alliin; metabolic disease; peroxisome proliferation-activity receptor γ.

Figure 1

Effect of alliin on lipid droplet accumulation in 3T3-L1 cells. (A) Phase-contrast images of 3T3-L1 cells. (A-a and -b) 3T3-L1 cells exhibited fibroblastic morphology before adipogenic induction, (c-f) and then the cells changed to a round-shape after 7 days of induction. (A-g-j) Oil-red O staining revealed that the intracellular lipids were decreased in alliin-treated groups compared with the control. (B) Quantitative analysis demonstrated that the positively stained areas were decreased in the alliin treatment group. (C) Cell viability was tested on day 7. Alliin (10-40 µg/ml) exhibited no effect on cell viability. Scale bar, 100 µm. Data are presented as the mean ± SEM. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 2

Effect of alliin on adipogenic transcriptional markers during induction. The 3T3-L1 cells were treated with or without alliin during adipogenic induction. Reverse transcription-quantitative PCR revealed the expression levels of (A) C/EBP β, (B) C/EBP α and (C) PPAR γ in 3T3-L1 cells during induction. Data are presented as the mean ± SEM. ^*P<0.05, ^**P<0.01. C/EBP, CCAAT/enhancer-binding protein; PPAR γ, peroxisome proliferation-activity receptor γ.

Figure 3

Effect of alliin on adipocyte-related genes during induction. Alliin was administered at various concentrations during adipogenic differentiation. Reverse transcription-quantitative PCR analysis was used to evaluate the gene expression levels of (A) adiponectin, (B) Fabp4 and (C) leptin. Data are presented as the mean ± SEM. ^*P<0.05, ^**P<0.01. Fabp4, fatty acid binding protein 4.

Figure 4

Effect of alliin on the Akt and ERK 1/2 pathway-related genes during induction. The 3T3-L1 cells were treated with different concentration of alliin during adipogenesis. The expression levels of (A) PI3K, (B) Akt, (C) MAPK and (D) ERK were analyzed by reverse transcription-quantitative PCR. Data are presented as the mean ± SEM. ^*P<0.05, ^**P<0.01.

Figure 5

Schematic representation of the possible mechanism of the anti-adipogenic effect of alliin. Alliin (C₆H₁₁NO₃S) inhibits adipogenesis by downregulating PI3K and Akt activity, thereby attenuating the expression levels of C/EBP β, C/EBP α, PPAR γ and lipid metabolizing enzymes. C/EBP, CCAAT/enhancer-binding protein; PPAR γ, peroxisome proliferation-activity receptor γ.