CircGLIS3 Inhibits Intramuscular Adipogenesis and Alleviates Skeletal Muscle Fat Infiltration.

Background: Intramuscular fat (IMF) is a key determinant of meat quality. Excessive IMF deposition, commonly observed in human obesity and aging, negatively affects skeletal muscle function. Circular RNAs (circRNAs) play critical regulatory roles in muscle and fat development, as well as in the progression of related diseases. However, the specific functions and underlying mechanisms of circRNAs in IMF deposition have not been extensively studied.

Methods: We screened adipogenic differentiation-related circRNAs using circRNA-seq combined with WGCNA. Functional analyses, including loss-of-function and gain-of-function experiments, were performed to determine the role of circGLIS3 in regulating adipogenesis in bovine intramuscular preadipocytes and 3T3-L1 cells. A high-fat diet-induced mouse model was established to investigate the in vivo effects of circGLIS3 on skeletal muscle fat infiltration in mice (C57BL/6; n = 6 males). Mechanistic studies involved transcriptomic analysis, dual-luciferase reporter assays, RNA immunoprecipitation, and rescue experiments.

Results: We screened and characterized a novel circRNA, circGLIS3, which is highly expressed in bovine IMF tissue and in the skeletal muscle of mice exhibiting fat infiltration. Functionally, circGLIS3 overexpression inhibited triglyceride synthesis (-16.19%, p < 0.01) and lipid droplet accumulation (-42.44%, p < 0.01) in intramuscular preadipocytes, concurrently downregulating the expression of key adipogenesis-related genes and proteins (p < 0.05). CircGLIS3 knockdown promoted triglyceride synthesis (+28.32%, p < 0.05) and lipid droplet accumulation (+47.40%, p < 0.01) in intramuscular preadipocytes, accompanied by a significant upregulation of adipogenesis-related genes and proteins (p < 0.05). Notably, circGLIS3 exhibits more than 80% sequence homology among bovine, mouse, and human species. CircGLIS3 overexpression inhibited triglyceride synthesis (-28.84%, p < 0.01) and lipid droplet accumulation (-46.49%, p < 0.01) in 3T3-L1 cells, concurrently downregulating the expression of adipogenesis-related genes and proteins (p < 0.05). Overexpression of circGLIS3 reduced skeletal muscle fat infiltration induced by high-fat diet in mice (-57.88%, p < 0.05). Mechanistically, circGLIS3 acts as a sponge for miR-21-3p, increasing the expression of its target gene, LEPR (+56.05%, p < 0.01), and promoting the phosphorylation level of its downstream signalling protein, AMPKα (+68.68%, p < 0.01).

Conclusions: CircGLIS3 inhibits bovine intramuscular adipogenesis by regulating the miR-21-3p/LEPR/AMPK axis, reducing fat infiltration in mouse skeletal muscle. These findings suggest that circGLIS3 is a promising target for improving meat quality in livestock, and as a potential therapeutic marker for alleviating skeletal muscle fat infiltration associated with obesity.