Leucine treatment enhances oxidative capacity through complete carbohydrate oxidation and increased mitochondrial density in skeletal muscle cells

Abstract

Leucine has been largely implicated for increasing muscle protein synthesis in addition to stimulating mitochondrial biosynthesis. Limited evidence is currently available on the effects and potential benefits of leucine treatment on skeletal muscle cell glycolytic and oxidative metabolism. This work identified the effects of leucine treatment on oxidative and glycolytic metabolism as well as metabolic rate of human and murine skeletal muscle cells. Human rhabdomyosarcoma cells (RD) and mouse myoblast cells (C2C12) were treated with leucine at either 100 or 500 μM for 24 or 48 h. Glycolytic metabolism was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate. Peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α), an important stimulator of mitochondrial biosynthesis, was quantified using flow cytometry and verified by immunofluorescent confocal microscopy. Mitochondrial content was quantified using mitochondrial and cytochrome C staining measured by flow cytometry and confirmed with confocal microscopy. Treatment with leucine significantly increased both basal and peak oxidative metabolism in both cell models. Leucine treated cells also exhibited significantly greater mitochondrial proton leak, which is associated with heightened energy expenditure. Basal ECAR was significantly reduced in both cell models following leucine treatment, evidence of reduced lactate export and more complete carbohydrate oxidation. In addition, both PGC-1α and cytochrome C expression were significantly elevated in addition to mitochondrial content following 48 h of leucine treatment. Our observations demonstrated few dose-dependent responses induced by leucine; however, leucine treatment did induce a significant dose-dependent expression of PGC-1α in both cell models. Interestingly, C2C12 cells treated with leucine exhibited dose-dependently reduced ATP content, while RD ATP content remain unchanged. Leucine presents a potent dietary constituent with low lethality with numerous beneficial effects for increasing oxidative preference and capacity in skeletal muscle. Our observations demonstrate that leucine can enhance oxidative capacity and carbohydrate oxidation efficiency, as well as verify previous observations of increased mitochondrial content.

Leucine stimulates PPARβ/δ-dependent mitochondrial biogenesis and oxidative metabolism with enhanced GLUT4 content and glucose uptake in myotubes

“…We observed a significant increase in proadipogenic PPARγ, SREBP-1c, and FAS expression, suggesting leucine stimulates lipid synthesis in addition to increased oxidative metabolism (likely from glucose oxidation) [10,11]. These observations are not surprising given mTOR is a known regulator of PPARγ and SREBP-1c [20].…”

“…Stock L-leucine from Sigma (St. Louis, MO) was dissolved in culture media to a final concentration of 2mM prior to treating cells for 24 hours (as determined through pilot data and previous observations) [11,12,18]. Similar observations in C2C12 myoblasts and cultured adipocytes have discerned the metabolic effects of leucine to be a unique result of characteristics not found in similar isonitrogenous branched-chain amino acids such as valine [12].…”

“…Leucine has previously been shown to increase muscle protein synthesis through stimulation of mammalian target of rapamycin (mTOR) and Akt phosphorylation [1][2][3][4][5]. Despite this anabolic function, leucine has also been shown to promote several processes involved in catabolic metabolism including increased expression of fatty acid oxidation genes [6][7][8][9], heightened oxygen consumption [10,11], and increased mitochondrial content in both skeletal muscle and adipocytes [8,9,11,12]. Biochemical investigations have repeatedly demonstrated that leucine stimulates peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) expression leading to increased mitochondrial biosynthesis and heightened metabolic rate [11][12][13][14].…”

“…Despite this anabolic function, leucine has also been shown to promote several processes involved in catabolic metabolism including increased expression of fatty acid oxidation genes [6][7][8][9], heightened oxygen consumption [10,11], and increased mitochondrial content in both skeletal muscle and adipocytes [8,9,11,12]. Biochemical investigations have repeatedly demonstrated that leucine stimulates peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) expression leading to increased mitochondrial biosynthesis and heightened metabolic rate [11][12][13][14]. While the ability of leucine to stimulate PGC-1α expression has been reproducible, the mechanism(s) through which leucine acts to increase mitochondrial content and PGC-1α activation have not been completely elucidated.…”

Irisin, a unique non-inflammatory myokine in stimulating skeletal muscle metabolism

“…Extracellular acidification, an indirect measure of glycolytic capacity, and oxygen consumption, a measure of oxidative metabolism, were measured using the SeaHorse XF24 Extracellular Analyzer from SeaHorse Bioscience (Billerica, MA). SeaHorse XF24 Extracellular Analyzer was run using 8-min cyclic protocol commands (mix for 3 min, let stand 2 min, and measure for 3 min) in triplicate as previously performed [45].…”