Novel Bacillus ginsengihumi CMRO6 Inhibits Adipogenesis via p38MAPK/Erk44/42 and Stimulates Glucose Uptake in 3T3-L1 Pre-Adipocytes through Akt/AS160 Signaling

Abstract

The health benefits of probiotics have been known for decades, but there has only been limited use of probiotics in the treatment of obesity. In this study, we describe, for the first time, the role of cell-free metabolites (CM) from Bacillus ginsengihumi-RO6 (CMRO6) in adipogenesis and lipogenesis in 3T3-L1 pre-adipocytes. The experimental results show that CMRO6 treatment effectively reduced lipid droplet accumulation and the expression of CCAAT/enhancer-binding protein α and β (C/EBPα and C/EBPβ), peroxisome proliferator-activated receptor γ (PPAR-γ), serum regulatory binding protein 1c (SREBP-1c), fatty acid-binding protein 4 (FABP4), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), phosphorylated p38MAPK, and Erk44/42. Additionally, CMRO6 treatment significantly increased glucose uptake and phosphorylated Akt (S473), AS160, and TBC1D1 protein expressions. Considering the results of this study, B. ginsengihumi may be a novel probiotic used for the treatment of obesity and its associated metabolic disorders.

Keywords: 3T3-L1; Bacillus ginsengihumi; cell-free metabolites; glucose uptake; lipid; probiotic.

Figure 1

Cytotoxic effects of cell-free metabolites of B. ginsengihumi-RO6 (CMRO6) on 3T3-L1 pre-adipocytes. The cells were treated with different concentrations of CMRO6 (0.98–500 μg/mL) and incubated under normal cell culture conditions. After that, cell viabilities were determined after 24 h and 48 h using EZ-cytox reagent. (a) The percentage of viable cells in the experimental groups at 24 h; (b) the percentage of viable cells in the experimental groups at 48 h. The data are presented as the mean ± STD for six replicates (n = 6). Different letters within the figure indicate significant differences between groups (p < 0.05).

Figure 2

Impact of CMRO6 at different concentrations on lipid accumulation and differentiation. Cells were seeded in cell culture plates and incubated at 37 °C with 5% CO₂. The differentiation was induced by dexamethasone, IBMX, and insulin for 48 h and then media was replaced by insulin medium for another 48 h. CMRO6 at different concentrations was added to the cell when differentiation began. Differentiated cells were then monitored under an Evos microscope and lipid deposits were stained with Oil Red O stain. The differentiated cells were photographed on the 5th and 10th day of differentiation. The stained lipids were extracted using isopropyl alcohol and lipid levels were measured. (a,b) Microscopic views of lipid accumulation in differentiated cells at 5th and 10th day of differentiation; (c,d) the percentage of lipids in experimental adipocytes at 5th and 10th day of differentiation. The data are represented as the mean ± STD of six replicates (n = 6). Different letters within the figure indicate significant differences between groups (p < 0.05).

Figure 3

Comparative studies between rosiglitazone (RGZ) and CMRO6 on adipocyte differentiation. The cells were treated with either CMRO6 (100 ug/mL) or RGZ (1 μM), or RGZ with CMRO6, when differentiation began. Adipocytes treated with RGZ differentiated faster and accumulated more fat, while cells treated with CMRO6 showed a significant reduction in fat deposition compared to controls. CMRO6 also attenuated the RGZ-induced lipid accumulation as compared to cells treated with RGZ alone. (a) Control cells; (b) RGZ-treated cells; (c) RGZ + CMRO6-treated cells; (d) CMRO6-treated cells; (e) PPAR-γ protein expression in the experimental cells on day 10; (f) fat deposition in the experimental cells on day 10, determined by Oil Red O staining method. The data are represented as the mean ± STD of six replicates (n = 6). Different letters within the figure indicate significant differences (p < 0.05).

Figure 4

(a) Effect of CMRO6 on key transcriptional factors and their downstream targets. The proteins were extracted with extraction buffer on day 10 in the presence of protease and phosphatase inhibitors and quantified by the BCA method. Proteins were then separated by SDS-PAGE. The expression of C/CEBβ, C/CEBα, PPAR-γ2, SREBP-1c, FAS, ACC, and FABP4 proteins was detected with specific antibodies by the immunoblot method. The protein intensity was quantified by ImageJ software. CMRO6 treatment during differentiation reduced the translation level of C/CEBβ, C/CEBα, PPAR-γ2, SREBP-1c, FAS, ACC, and FABP4. Results are expressed as the mean ± STD, n = 3, * p values significant between control and treatment by an independent t-test. (b) Changes in Erk44/42 and P38MAPK signaling pathways in response to CMRO6 treatment. The proteins were extracted with extraction buffer on day 10 in the presence of protease and phosphatase inhibitors and quantified by the BCA method. Proteins were then separated by SDS-PAGE. The phosphorylating levels of Erk44/42 and p38MAPK were determined with specific antibodies by the Western blot method. The protein intensity was quantified by ImageJ software. Results are expressed as the mean ± STD of three replicates, n = 3, * p values (0.02-0.027), ** p values (0.001), control vs treatment by an independent t-test.

Figure 4

(a) Effect of CMRO6 on key transcriptional factors and their downstream targets. The proteins were extracted with extraction buffer on day 10 in the presence of protease and phosphatase inhibitors and quantified by the BCA method. Proteins were then separated by SDS-PAGE. The expression of C/CEBβ, C/CEBα, PPAR-γ2, SREBP-1c, FAS, ACC, and FABP4 proteins was detected with specific antibodies by the immunoblot method. The protein intensity was quantified by ImageJ software. CMRO6 treatment during differentiation reduced the translation level of C/CEBβ, C/CEBα, PPAR-γ2, SREBP-1c, FAS, ACC, and FABP4. Results are expressed as the mean ± STD, n = 3, * p values significant between control and treatment by an independent t-test. (b) Changes in Erk44/42 and P38MAPK signaling pathways in response to CMRO6 treatment. The proteins were extracted with extraction buffer on day 10 in the presence of protease and phosphatase inhibitors and quantified by the BCA method. Proteins were then separated by SDS-PAGE. The phosphorylating levels of Erk44/42 and p38MAPK were determined with specific antibodies by the Western blot method. The protein intensity was quantified by ImageJ software. Results are expressed as the mean ± STD of three replicates, n = 3, * p values (0.02-0.027), ** p values (0.001), control vs treatment by an independent t-test.

Figure 5

Effect of CMRO6 on glucose uptake and insulin-related signaling pathways. (a) Glucose uptake was measured in differentiated adipocytes with a Promega glucose uptake-Glo assay kit. (b) Phosphorylation levels of insulin-stimulated signaling pathways (Akt, AS160, and TBC1D1) and adiponectin expression, which is related to glucose uptake, in response to insulin and CMRO6 treatment. Results are presented as the mean ± STD of three replicates. Different letters within the figure indicate significant differences (p < 0.05).