Sulforaphane enhanced muscle growth by promoting lipid oxidation through modulating key signaling pathways

Abstract

Sulforaphane (SFN) has shown diverse effects on human health and diseases. SFN was administered daily to C57BL/6J mice at doses of 1 mg/kg (SFN1) and 3 mg/kg (SFN3) for 8 weeks. Both doses of SFN accelerated body weight increment. The cross-sectional area and diameter of Longissimus dorsi (LD) muscle fibers were enlarged in SFN3 group. Triglyceride (TG) and total cholesterol (TC) levels in LD muscle were decreased in SFN groups. RNA sequencing results revealed that 2455 and 2318 differentially expressed genes (DEGs) were found in SFN1 and SFN3 groups, respectively. Based on GO enrichment analysis, 754 and 911 enriched GO terms in the SFN1 and SFN3 groups, respectively. KEGG enrichment analysis shown that one KEGG pathway was enriched in the SFN1 group, while six KEGG pathways were enriched in the SFN3 group. The expressions of nine selected DEGs validated with qRT-PCR were in line with the RNA sequencing data. Furthermore, SFN treatment influenced lipid and protein metabolism related pathways including AMPK signaling, fatty acid metabolism signaling, cholesterol metabolism signalling, PPAR signaling, peroxisome signaling, TGFβ signaling, and mTOR signaling. In summary, SFN elevated muscle fibers size and reduced TG and TC content of in LD muscle by modulating protein and lipid metabolism-related signaling pathways.

Keywords: Sulforaphane; fiber size; lipid; metabolism; muscle growth; protein.

Figure 1. Effect of SFN on muscle size and lipid content in mice

(A) Body weight of mice over eight weeks and values with different lowercase letters (a, b, and c) at the same week and uppercase letters (A, B, and C) in the same group were significantly different from each other (P<0.05). (B) H&E staining of LD muscle. (C,D) Cross-section area (C) and diameter (D) of muscle fibre. (E,F) TG (E) and TC (F) content in LD muscle. Data were shown as the mean ± SD, n=7, **P<0.01, ***P<0.001.

Figure 2. RNA-seq applied in LD muscle

(A) Work flow for RNA-seq. (B) DEGs in SFN1 and SFN3 groups. (C,D) Up- and down-regulated DEGs in SFN1 (C) and SFN3 (D) vs. Ctrl group. (E,F) Venn diagrams of up-regulated genes (E), and down-regulated genes (F) in SFN1 and SFN3 groups. Data are derived from RNA-seq analysis of one pooled total RNA sample for each group (n=1).

Figure 3. Enrichment analysis of RNA-seq data

(A,B) GO (A) and KEGG (B) pathway enrichment for DEGs in SFN1 groups. (C,D) GO (C) and KEGG (D) pathway enrichment for DEGs in SFN3 groups. Data are derived from RNA-seq analysis of one pooled total RNA sample for each group (n=1).

Figure 4. Heatmaps for gene expression in lipid and protein metabolism pathway

(A) AMPK signaling pathway. (B) Fatty acid metabolism. (C) Cholesterol metabolism. (D) PPAR signaling pathway. (E) Peroxisome. (F) Peroxisome. (G) mTOR signaling pathway. Data are derived from RNA-seq analysis of one pooled total RNA sample for each group (n=1).

Figure 5. qRT-PCR verification of selected DEGs relative mRNA expression

(A–I) Acox1, Acsl1, Acadm, Ppara, Npy, Acacb, Prkaa2, Irs1, and Mapk1. n=3 and *P<0.05, **P<0.01, ***P<0.001.

Figure 6. Protein–protein interaction network for DEGs in lipid and protein signaling pathway

The nodes mean genes. The edges indicate both functional and physical associations. Distinct colors stand for different clusters.