Inhibitory Effect of Bacterial Lysates Extracted from Pediococcus acidilactici on the Differentiation of 3T3-L1 Pre-Adipocytes

Abstract

Postbiotics, including bacterial lysates, are considered alternatives to probiotics. The aim of the current study was to investigate the effect of bacterial lysates (BLs) extracted from Pediococcus acidilactici K10 (K10 BL) and P. acidilactici HW01 (HW01 BL) on the differentiation of 3T3-L1 pre-adipocytes. Both K10 and HW01 BLs significantly reduced the accumulation of lipid droplets and the amounts of cellular glycerides in 3T3-L1 cells (p < 0.05). However, another postbiotic molecule, peptidoglycan of P. acidilactici K10 and P. acidilactici HW01, moderately inhibited the accumulation of lipid droplets, whereas heat-killed P. acidilactici did not effectively inhibit the lipid accumulation. The mRNA and protein levels of the transcription factors, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, responsible for the differentiation of 3T3-L1 cells, were significantly inhibited by K10 BL and HW01 BL (p < 0.05). Both K10 and HW01 BLs decreased adipocyte-related molecules, adipocyte fatty acid-binding protein and lipoprotein lipase, at the mRNA and protein levels. Furthermore, both K10 and HW01 BLs also downregulated the mRNA expression of leptin, but not resistin. Taken together, these results suggest that P. acidilactici BLs mediate anti-adipogenic effects by inhibiting adipogenic-related transcription factors and their target molecules.

Keywords: Pediococcus acidilactici; adipogenesis; bacterial lysates; postbiotics.

Figure 1

Inhibitory effect of P. acidilactici BLs on lipid accumulation in 3T3-L1 cells. Cells were treated with various concentrations (0, 5, 10, and 20 μg/mL) of P. acidilactici K10 BL (A) or P. acidilactici HW01 BL (B). At day 12, lipid accumulation in 3T3-L1 cells was assessed by Oil Red O staining. Representative microscopic images of Oil Red O-stained cells are shown. Data are expressed as the mean ± standard deviation. Statistical significance between groups was determined by ANOVA when p < 0.05. Significant differences between treatment with K10 BL or HW01 BL are expressed with different letters (a, b, c, d).

Figure 2

Inhibitory effect of P. acidilactici BLs on cellular triglycerides in 3T3-L1 cells. Cells were treated with various concentrations (0, 5, 10, and 20 μg/mL) of P. acidilactici K10 BL (A) or P. acidilactici HW01 BL (B). At day 12, the amounts of cellular triglyceride were assessed using a commercial triglyceride assay kit. Data are expressed as the mean ± standard deviation. Statistical significance between groups was determined by ANOVA when p < 0.05. Significant differences between treatment with K10 BL or HW01 BL are expressed with different letters (a, b, c, d).

Figure 3

Effect of P. acidilactici PGNs and heat-killed P. acidilactici on lipid accumulation in 3T3-L1 cells. Cells were treated with various concentrations (0, 10, and 20 μg/mL) of P. acidilactici K10 PGN (A) or P. acidilactici HW01 PGN (B). Cells were treated with various concentrations (0, 10⁷ and 10⁸ CFU/mL) of heat-killed P. acidilactici K10 (C) or P. acidilactici HW01 (D). At day 12, lipid accumulation in 3T3-L1 cells was assessed by Oil Red O staining. Data are expressed as the mean ± standard deviation. Statistical significance between groups was determined by ANOVA when p < 0.05. Significant differences between treatment with P. acidilactici PGN (A,B) or heat-killed P. acidilactici (C,D) are expressed with different letters (a, b, c).

Figure 4

Inhibitory effect of P. acidilactici BLs on the expressions of adipogenic transcription factors in 3T3-L1 cells. Cells were treated with 20 μg/mL of P. acidilactici K10 BL (A) or P. acidilactici HW01 BL (B). At days 3 and 12, the mRNA expressions of Pparg, Cebpa, Cebpb, and Srebf1 genes were measured by real-time quantitative reverse-transcription PCR. Cells were treated with 20 μg/mL of P. acidilactici K10 BL (C) or P. acidilactici HW01 BL (D). At days 3 and 12, cell lysates were collected and subjected to Western blot analysis to determine the protein levels of PPARγ and C/EBPα. Data are expressed as the mean ± standard deviation. Statistical significance between groups was determined by ANOVA when p < 0.05. Significant differences between treatment with K10 BL or HW01 BL are expressed with different letters (a, b, c). NT denotes not treated.

Figure 5

Inhibitory effect of P. acidilactici BLs on aP2 and LPL in 3T3-L1 cells. Cells were treated with 20 μg/mL of P. acidilactici K10 BL or P. acidilactici. At day 12, the mRNA expressions of Fabp4 (A) and Lpl (B) genes were measured by real-time quantitative reverse-transcription PCR. (C) At day 12, cell lysates were collected and subjected to Western blot analysis to determine the protein levels of aP2 and LPL. Data are expressed as the mean ± standard deviation. Statistical significance between groups was determined by ANOVA when p < 0.05. Significant differences between treatment with K10 BL or HW01 BL are expressed with different letters (a, b). NT denotes not treated.

Figure 6

Inhibitory effect of P. acidilactici BLs on the mRNA expressions of adipokines in 3T3-L1 cells. Cells were treated with 20 μg/mL of P. acidilactici K10 BL or P. acidilactici HW01 BL. At day 12, the mRNA expressions of Retn (A) and Lep (B) genes were measured by real-time quantitative reverse-transcription PCR. Data are expressed as the mean ± standard deviation. Statistical significance between groups was determined by ANOVA when p < 0.05. Significant differences between treatment with K10 BL or HW01 BL are expressed with different letters (a, b). NT denotes not treated.