Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes

Abstract

Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, β-hydroxy-β-methyl butyrate (HMB), a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), EX527 (SIRT1 inhibitor, 25 μM), and Compound C (AMPK inhibitor, 25 μM) alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD+, SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.

The role of leucine and its metabolites in protein and energy metabolism

“…mTORC1, AMPK, and myokines are involved in the regulation of Leu and the cross-talk between protein and energy metabolism (Duan et al 2015a). Leu plays a key role in energy metabolism apart from its pivotal function in protein turnover (Liang et al 2014). It can promote energy partition from adipocytes to myocytes, causing reduced lipid storage in adipocytes and enhanced fatty acids utilization in muscle cells (Sun and Zemel 2007).…”

“…It can promote energy partition from adipocytes to myocytes, causing reduced lipid storage in adipocytes and enhanced fatty acids utilization in muscle cells (Sun and Zemel 2007). Activation of AMPK and SIRT1 by Leu is the major event that mediates fatty acid oxidation and mitochondrial biogenesis leading to increased energy expenditure in skeletal muscle cells (Liang et al 2014). However, it remains unclear whether energy metabolism is mediated directly by Leu itself, or if its metabolites α-KIC and/or HMB contribute to the observed effects.…”

“…The activation of AMP-activated protein kinase (AMPK) and silent information regulator transcript 1 (SIRT1) by Leu is the major event that mediates fatty acid oxidation and mitochondrial biogenesis in skeletal muscle (Liang et al 2014). Protein degradation and synthesis are equally important processes, but little is known about the mechanisms of Leu-regulated proteolysis (Nakashima et al 2005).…”

Introductory Chapter: Obesity—“OMICS” and Endocrinology

“…The Sirt1's involvement in the mitochondria theory of aging brings about a close link to mitochondrial dysfunction (i.e., Sirt1 downregulation), as well as an abnormal immune response [68,[84][85][86]. An essential requirement of some amino acids (e.g., leucine) [93] may sustain a nuclear and mitochondria p53-associated transport, along with an enhancement in the half-life of p53, as well as a blockage of immune dysfunction and development of NAFLD.…”

“…A series of drugs, which inhibit Sirt1 [92] have been developed, which prevent leucine-mediated activation of Sirt1 and xenobiotics (e.g., phthalates), modifying Sirt1 chromatin association, with an ensuing induction of p53-mediated apoptotic responses [91][92][93] in both the liver and adipose tissue.…”