Linoleic Acid Induced Angiopoietin‐Like Protein 4 Impairs Skeletal Muscle Differentiation

Abstract

Skeletal muscle differentiation is a pivotal process in embryonic development as well as regeneration/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. Thus, the objective of the current study is to evaluate the effects of fatty acids on skeletal muscle differentiation in vitro. Skeletal muscle differentiation is known to be regulated by Wnt/β‐catenin signaling, and Wnt/β‐catenin signaling is inhibited by angiopoietin‐like protein 4 (ANGPTL4), a protein that is induced by free fatty acids (FFA). Therefore, the hypothesis of the current study is that FFA‐induced ANGPTL4 inhibits skeletal muscle differentiation by suppressing Wnt/β‐catenin signaling. We used C2C12 murine myoblasts and treated with various FFAs, recombinant ANGPTL4 protein, or siRNA knockdown of ANGPTL4 during cell differentiation. Among FFAs, 18:2n6 linoleic acid (LA) significantly inhibited myotube formation, showing decreased myoblast fusion. LA significantly lowered the protein expression of myogenic regulatory factors, including MyoD and MyoG and increased Pax7 during cell differentiation. We also confirmed that ANGPTL4 knockdown at day 2 and ‐6 of differentiation restored myotube formation in the presence of LA. Next, we performed an RNA‐sequencing analysis to determine which pathways were involved in the mechanism of LA‐ and ANGPTL4‐mediated inhibition of skeletal muscle differentiation at day 2 and ‐6 of differentiation. LA at day 2 and ‐6 and ANGPTL4 at day 2 downregulated Wnt/β‐catenin signaling as well as genes involved in skeletal muscle differentiation. We further found that ANGPTL4 reduced β‐catenin protein expression in nucleus, indicating that ANGPTL4 blocks β‐catenin translocation into nucleus from cytoplasm, and it was restored with ANGPTL4 knockdown. Taken together, these results demonstrate that LA‐induced ANGPTL4 inhibits skeletal muscle differentiation by suppressing Wnt/β‐catenin signaling in C2C12 myoblasts.