Linoleic acid-induced ANGPTL4 inhibits C2C12 skeletal muscle differentiation by suppressing Wnt/β-catenin

Skeletal muscle differentiation is an essential process in embryonic development as well as regeneration and repair throughout the lifespan. It is well-known that dietary fat intake impacts biological and physiological function in skeletal muscle, however, understanding of the contribution of nutritional factors in skeletal muscle differentiation is limited. Therefore, the objective of the current study was to evaluate the effects of free fatty acids (FFAs) on skeletal muscle differentiation in vitro. We used C2C12 murine myoblasts and treated them with various FFAs, which revealed a unique response of angiopoietin-like protein-4 (ANGPTL4) with linoleic acid (LA) treatment that was associated with reduced differentiation. LA significantly inhibited myotube formation and lowered the protein expression of myogenic regulatory factors, including MyoD and MyoG and increased Pax7 during cell differentiation. Next, recombinant ANGPTL4 protein or siRNA knockdown of ANGPTL4 was employed to examine its role in skeletal muscle differentiation, and we confirmed that ANGPTL4 knockdown at day two and six of differentiation restored myotube formation in the presence of LA. RNA-sequencing analysis revealed that ANGPTL4-mediated inhibition of skeletal muscle differentiation at day two as well as LA at day two or -6 led to a reduction in Wnt/β-catenin signaling pathways. We confirmed that LA reduced Wnt11 and Axin2 while increasing expression of the Wnt inhibitor, Dkk2. ANGPTL4 knockdown increased β-catenin protein in the nucleus in response to LA and increased Axin2 and Wnt11 expression. Taken together, these results demonstrate that LA induced ANGPTL4 inhibits C2C12 differentiation by suppressing Wnt/β-catenin signaling.