Leucine Stimulates Peroxisome Proliferator Activator Receptors in Skeletal Muscle

Abstract

PURPOSE: Leucine has been shown to stimulate anabolic and catabolic processes in skeletal muscle, however little is known about the effects of leucine on peroxisome proliferator-activated receptor (PPAR) activity. This work characterized the effects of leucine on myotube expression of the PPAR superfamily, as well as related downstream targets that regulate cellular energetics and inflammation. METHODS: C2C12 myotubes were treated with leucine or valine (control) at 2mM for 24 hours. Protein expression of the PPAR superfamily and associated metabolic and inflammatory targets were measured via western blot and qRT-PCR. RESULTS: Leucine significantly increased PPARα and PPARβ/δ expression (53.3% ±28.2% and 55.9% ±15.1%, respectively). Leucine also stimulated markers of mitochondrial biogenesis (PGC-1α, NRF1, and TFAM) leading to increased cytochrome C (61.9% ±28.6%) and GLUT4 content (129.1% ±9.2%). Surprisingly, leucine simultaneously increased PPARγ expression (42.0% ±15.1%) promoting significantly elevated FAS (743.6% ±304.0%) but not SREBP-1c expression. Leucine also significantly induced TLR4 expression (707.1% ±36.3%), however p-P65-NFκB expression was significantly suppressed (by 59.1% ±7.2%) leading to reduced IL6 and enhanced IL10 mRNA expression. Finally, we explored the effects of leucine on other targets of TLR4 and found increased p-P38MAPK compared with valine controls with unaltered p-CREB content. CONCLUSION: Leucine induces PPAR expression, GLUT4 content, and mitochondrial biogenesis in vitro, suggesting leucine may increase substrate oxidation. Leucine-mediated enhanced glucose uptake may stimulate a compensatory response for cells to dissipate energy by (a) substrate oxidation (b) substrate/lipid storage, and (c) protein synthesis, thus providing a possible explanation for the simultaneous induction of the entire PPAR superfamily (anabolic and catabolic). Moreover, increased Akt and mTOR activity by leucine may promote TLR4 expression potentially sensitizing cells to inflammatory signals (such as cytokines), while concurrent increases in PPARγ may lead to reduced cellular inflammation (a hypothesis which requires further investigation).