Liver fat metabolism of broilers regulated by Bacillus amyloliquefaciens TL via stimulating IGF-1 secretion and regulating the IGF signaling pathway

Abstract

Bacillus amyloliquefaciens TL (B.A-TL) is well-known for its capability of promoting protein synthesis and lipid metabolism, in particular, the abdominal fat deposition in broilers. However, the underlying molecular mechanism remains unclear. In our study, the regulations of lipid metabolism of broilers by B.A-TL were explored both in vivo and in vitro. The metabolites of B.A-TL were used to simulate in vitro the effect of B.A-TL on liver metabolism based on the chicken hepatocellular carcinoma cell line (i.e., LMH cells). The effects of B.A-TL on lipid metabolism by regulating insulin/IGF signaling pathways were investigated by applying the signal pathway inhibitors in vitro. The results showed that the B.A-TL metabolites enhanced hepatic lipid synthesis and stimulated the secretion of IGF-1. The liver transcriptome analysis revealed the significantly upregulated expressions of four genes (SI, AMY2A, PCK1, and FASN) in the B.A-TL treatment group, mainly involved in carbohydrate digestion and absorption as well as biomacromolecule metabolism, with a particularly prominent effect on fatty acid synthase (FASN). Results of cellular assays showed that B.A-TL metabolites were involved in the insulin/IGF signaling pathway, regulating the expressions of lipid metabolism genes (e.g., FASN, ACCα, LPIN, and ACOX) and the FASN protein, ultimately regulating the lipid metabolism via the IGF/PI3K/FASN pathway in broilers.

Keywords: Bacillus amyloliquefaciens TL; IGF signaling pathway; broilers; fatty acid synthase; insulin-like factor 1; lipid metabolism; liver.

Figure 1

The liver tissues stained with Oil Red O of broilers in the control and experimental groups at 21 days of age. “Con” = control group; “TL” = Bacillus amyloliquefaciens TL experimental group. The significance level for all analyses is set to p < 0.001 (***).

Figure 2

The Oil Red O staining of the LMH cells treated with Bacillus amyloliquefaciens TL fermentation supernatant, exopolysaccharide, and L-tyrosine, respectively. “Con” = control group; “FST” = fermentation supernatant group; “EPS” = exopolysaccharides group; “Tyr” = L-tyrosine group. The significance levels for all analyses were set at p < 0.05 (*), and p < 0.001 (***), respectively.

Figure 3

Functional annotation of differentially expressed genes (DEGs) identified in Bacillus amyloliquefaciens TL group of broilers in comparison to the control group. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs. (B) KEGG pathway enrichment analysis of downregulated DEGs. The vertical axis represents the name of the pathways, and the horizontal axis represents the Rich factor. The size of the color dot proportionally indicates the number of DEGs enriched in the pathway, while the color dot corresponds to the Q value range.

Figure 4

Correlation analysis of FASN with other genes and the relationships between FASN and other proteins. (A) Correlation analysis of FASN with other genes. (B) Interaction analysis of FASN with other proteins. Nodes (i.e., red and blue dots) indicate genes/transcripts, and dashed lines indicate the existence of the reciprocal relationship between two genes/transcripts. The size of a node is proportional to its connectivity (i.e., degree), i.e., the more edges connected to the node, the larger degree the node is, indicating the greater importance of the gene/transcript in the network.

Figure 5

Effects of fermentation supernatant (A,B), exopolysaccharides (C,D), and L-tyrosine (E,F) of different concentrations with different lengths of treatment time on LMH cell proliferation. “Con” = control group; “‘FST” = fermentation supernatant group; “EPS” = exopolysaccharides group; “Tyr” = L-tyrosine group. The significance levels for all analyses are set at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), respectively.

Figure 6

Effects of fermentation supernatant, exopolysaccharide, and L-tyrosine on the IGF-1 level of LMH cells. “Con” = control group; “FST” = fermentation supernatant group; “EPS” = exopolysaccharides group; “Tyr” = L-tyrosine group. The significance levels for all analyses are set at p < 0.05 (*), and p < 0.001 (***), respectively.

Figure 7

Relative mRNA levels of genes associated with fat synthesis (A) and fat oxidation (B) in LMH cells treated with fermentation supernatant, exopolysaccharide, and L-tyrosine, respectively. “Con” = control group; “FST” = fermentation supernatant group; “EPS” = exopolysaccharides group; “Tyr” = L-tyrosine group. The significance levels for all analyses are set at p < 0.05 (*), and p < 0.01 (***), respectively.

Figure 8

The FASN protein levels of LMH cells treated with fermentation supernatant, exopolysaccharide, and L-tyrosine, respectively. “Con” = control group; “FST” = fermentation supernatant group; “EPS” = exopolysaccharides group; “Tyr” = L-tyrosine group. FASN is used as a target protein; β-Actin is used as a loading standard. The significance levels for all analyses are set at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), respectively.

Figure 9

Relative mRNA levels of genes associated with fat synthesis (A) and fat oxidation (B) in LMH cells treated with fermentation supernatant and IGF-1R signaling inhibitor (i.e., picropodophyllin), exopolysaccharide and picropodophyllin, and L-tyrosine and picropodophyllin, respectively. DMSO is used as a toxicity control. “Con” = control group; “FST” = fermentation supernatant group; “EPS” = exopolysaccharides group; “Tyr” = L-tyrosine group. The significance levels for all analyses are set at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), respectively.

Figure 10

Effects of fermentation supernatant, exopolysaccharide, and L-tyrosine on LMH cells treated with different inhibitors including (A) IGF-1R inhibitor (picropodophyllin), (B) PI3K inhibitor (LY294002), and (C) MEK inhibitor (PD98059), respectively, at the FASN protein level. FASN is used as a target protein; β-Actin is used as a loading standard. “Con” = control group; “FST” = fermentation supernatant group; “EPS” = exopolysaccharides group; “Tyr” = L-tyrosine group. The signi?cance levels for all analyses are set at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), respectively.