Role of Leucine in Modulation of Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes

Mitochondrial dysfunction in skeletal muscle has been considered as a crucial step in the development of metabolic diseases, including insulin resistance syndrome, type 2 diabetes and cardiovascular diseases. Previous studies have demonstrated that dietary branched-chain amino acids, particularly leucine, protects against high-fat diet induced impairment of mitochondria and insulin resistance in skeletal muscle and adipose tissue through mTOR-dependent and independent pathways. In addition, previous ex vivo and in vitro approaches from this laboratory indicate that leucine and its metabolites (HMB and KIC) stimulate mitochondrial biogenesis and promote energy partitioning from adipocytes to muscle cells, partially through SIRT1 signaling. Moreover, recent work indicates that HMB promotes AMPK phosphorylation synergistically with metformin, resulting in increased insulin sensitivity and glucose tolerance in HFD mice. Therefore, it is hypothesized that leucine-induced activation of SIRT1 and AMPK are the central events that link to the up-regulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, SIRT1 activity, AMPK phosphorylation level, fatty acid oxidation, mitochondrial content and mitochondrial biogenesis related genes expressions were measured in C2C12 myotubes after incubated with leucine and controls. Furthermore, considering SIRT1 and AMPK share multiple downstream substrates and display mutual interactions in response to energy restriction, C2C12 myotubes were treated with SIRT1 inhibitor, EX527, and AMPK inhibitor, Compound C, to determine the respective roles of SIRT1 and AMPK in leucine effects on mitochondrial biogenesis. Additionally, a time-course experiment was conduced to elucidate the relationship between SIRT1 and AMPK. The results of this research confirm that leucine increased mitochondrial biogenesis and fatty acid oxidation in C2C12 myotubes via SIRT1 and AMPK dependent pathways, with SIRT1 activation being the primary event.