Chitooligosaccharide Induces Mitochondrial Biogenesis and Increases Exercise Endurance through the Activation of Sirt1 and AMPK in Rats

<div><p>By catabolizing glucose and lipids, mitochondria produce ATPs to meet energy demands. When the number and activity of mitochondria are not sufficient, the human body becomes easily fatigued due to the lack of ATP, thus the control of the quantity and function of mitochondria is important to optimize energy balance. By increasing mitochondrial capacity? it may be possible to enhance energy metabolism and improve exercise endurance. Here, through the screening of various functional food ingredients, we found that chitooligosaccharide (COS) is an effective inducer of mitochondrial biogenesis. In rodents, COS increased the mitochondrial content in skeletal muscle and enhanced exercise endurance. In cultured myocytes, the expression of major regulators of mitochondrial biogenesis and key components of mitochondrial electron transfer chain was increased upon COS treatment. COS-mediated induction of mitochondrial biogenesis was achieved in part by the activation of silent information regulator two ortholog 1 (Sirt1) and AMP-activated protein kinase (AMPK). Taken together, our data suggest that COS could act as an exercise mimetic by inducing mitochondrial biogenesis and enhancing exercise endurance through the activation of Sirt1 and AMPK.</p> </div

Knockdown of Sirt1 or AMPK expression diminishes the mitochondriogenic effect of COS.

<p>C2C12 myotubes were transfected with scramble (sc), Sirt1, and AMPKα siRNA by using Lipofectamine™ 2000 reagent. After transfection, cells were treated with COS (100 µg/ml, and 500 µg/ml) for 12 h. Cells were rinsed with PBS and subjected for western blotting (<b>A</b>) or the measurement of mitochondrial density (n  = 3) (<b>B</b>). ** P<0.01 vs. sc/(-) (lane 1); *** P<0.001 vs. sc/(-) (lane 1); ## P<0.01 vs. sc/COS 100 (lane 2); ### P<0.001 vs. sc/COS 100 (lane 2); &&& P<0.001 vs. sc/COS 500 (lane 3). Relative expression level of PGC1, NDUFA9, and ATP5a is calculated in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040073#pone.0040073.s009" target="_blank">Figure S9G</a></b>.</p

COS activates Sirt1 and AMPK <i>in vivo</i>.

<p>COS-administered SD rats were sacrificed and proteins from skeletal muscle were subjected to the measurement of cellular NAD⁺/NADH ratio (n  = 6) (<b>A</b>) or western blotting (<b>B</b>). ** P<0.01 vs. vehicle. Densitometry of p-AMPK was shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040073#pone.0040073.s009" target="_blank">Figure S9H</a></b>.</p

COS increases mitochondrial content <i>in vivo</i>.

<p><b>A</b>. Electron microscopic image of skeletal muscle. White arrows indicate clusters of mitochondria. Bar  = 2 µM. <b>B.</b> Average time of exercise endurance (n  = 6). * P<0.05 vs. vehicle<b>. C</b>. Plasma lactate level before and after exercise (n  = 6). ** P<0.01 vs. pre-exercise vehicle (lane 1); ## P<0.01 vs. post-exercise vehicle (lane 2).</p

COS requires AMPK and Sirt1 to induce mitochondrial biogenesis.

<p>Differentiated C2C12 myocytes were pre-treated with nicotinamide (NAM, 1 mM) or Compound C (ComC; 10 µM) for 2 h prior to incubation with Res (11.4 µg/ml) or COS (500 µg/ml) for 12 h. Cells were rinsed with PBS and harvested for western blotting (<b>A</b>) or the measurement of mitochondrial density (n  = 3) (<b>B</b>). * P<0.05 vs. DMSO/(-) (lane 1); # P<0.05 vs. Res/(-) (lane 2); & P<0.05 vs. COS/(-) (lane 3). Relative protein expression level of PGC1, NDUFA9, and ATP5a is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040073#pone.0040073.s009" target="_blank">Figure S9E</a></b>.</p

Biological parameters of COS-treated rats.

a<p>P  = 0.19 vs. vehicle; <b>^b</b> P  = 0.08 vs. vehicle; * P<0.05 vs. vehicle; ^*** P<0.001 vs. vehicle.</p