Anti-Adipogenic Effects of Delphinidin-3- O- β-Glucoside in 3T3-L1 Preadipocytes and Primary White Adipocytes

Abstract

Delphinidin-3-O-β-glucoside (D3G) is a health-promoting anthocyanin whose anti-obesity activity has not yet been thoroughly investigated. We examined the effects of D3G on adipogenesis and lipogenesis in 3T3-L1 adipocytes and primary white adipocytes using real-time RT-PCR and immunoblot analysis. D3G significantly inhibited the accumulation of lipids in a dose-dependent manner without displaying cytotoxicity. In the 3T3-L1 adipocytes, D3G downregulated the expression of key adipogenic and lipogenic markers, which are known as peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBP1), CCAAT/enhancer-binding protein alpha (C/EBPα), and fatty acid synthase (FAS). Moreover, the relative protein expression of silent mating type information regulation 2 homolog 1 (SIRT1) and carnitine palmitoyltransferase-1 (CPT-1) were increased, alongside reduced lipid levels and the presence of several small lipid droplets. Furthermore, D3G increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), which suggests that D3G may play a role in AMPK and ACC activation in adipocytes. Our data indicate that D3G attenuates adipogenesis and promotes lipid metabolism by activating AMPK-mediated signaling, and, hence, could have a therapeutic role in the management and treatment of obesity.

Keywords: 3T3-L1 adipocytes; adipogenesis; anthocyanin; delphinidin-3-O-β-glucoside; differentiation; primary white adipocytes.

Figure 1

Effect of different delphinidin-3-O-β-glucoside (D3G) concentrations on the viability of 3T3-L1 preadipocytes. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. 0. All experiments were repeated at least three times and data represents the mean ± SD.

Figure 2

Effect of delphinidin-3-O-β-glucoside (D3G) on lipid accumulation in 3T3-L1 adipocytes. (A) Photomicrographs of lipid droplets in control (Con) and D3G (25, 50, and 100 µM)-treated cells measured by Oil Red O staining. (B) The percentage of lipid accumulation in D3G-treated and control cells. Data represent the mean ± SEM of three different experiments. *** p < 0.001 vs. 0. Scale bar (A) indicates 100 µm.

Figure 3

The effect of delphinidin-3-O-β-glucoside (D3G) treatment (25, 50, and 100 µM) on the mRNA expression of key adipogenic transcription factors: (A) peroxisome proliferator-activated receptor gamma (PPARγ), (B) sterol regulatory element-binding transcription factor 1 (SREBP1), and (C) CCAAT/enhancer-binding protein alpha (C/EBPα), as determined by quantitative real-time PCR. (D) Immunoblot analysis of PPARγ, SREBP1, and C/EBPα. (E) Relative protein expression of PPARγ, SREBP1, and C/EBPα in differentiating D3G-treated and control 3T3-L1 adipocytes. β-actin protein levels were used as an internal control. Data represent the mean ± SEM of three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. 0.

Figure 4

The effect of delphinidin-3-O-β-glucoside (D3G) treatment on the protein expression of (A) Fatty acid synthase (FAS), (B) carnitine palmitoyltransferase -1 (CPT-1), and (C) silent mating type information regulation 2 homolog 1 (SIRT1) in 3T3-L1 adipocytes. (D) Representative immunoblot image of FAS, CPT-1, and SIRT1 expression. β-actin protein levels were used as an internal control. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. 0. All experiments were repeated at least three times and data represent the mean ± SD.

Figure 5

The effects of delphinidin-3-O-β-glucoside (D3G) treatment on the expression and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in 3T3-L1 adipocytes. Western blotting revealed the upregulated expression of (A) p-AMPK/AMPK and (B) p-ACC/ACC. ** p < 0.01, and *** p < 0.001 vs. 0. Each experiment was repeated three times and the data represents the mean ± SD.

Figure 6

The effect of delphinidin-3-O-β-glucoside (D3G) treatment (25, 50, and 100 µM) on the expression and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in primary white adipocytes (PWATs). D3G treatment increased the expression of (A) p-AMPK/AMPK and (B) p-ACC/ACC in PWATs when compared to the control (0). * p < 0.05 vs. 0. Each experiment was repeated three times and the data represent the mean ± SD.

Figure 7

Effect of D3G on adenosine monophosphate-activated protein kinase (AMPK) activation in AMPK activator-treated and inhibitor-treated primary white adipocytes (PWATs). (A) p-AMPK/AMPK and (B) p-acetyl-CoA carboxylase (ACC)/ACC protein expression following D3G treatment in the presence of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; 10 µM) and dorsomorphin (10 µM) in PWATs. Protein expression levels were measured by Western blotting. Each experiment was repeated three times and data represented the mean ± SD.