Table 1.
Composition of COS.
Table 2.
Biological parameters of COS-treated rats.
Figure 1.
COS increases mitochondrial content in vivo.
A. Electron microscopic image of skeletal muscle. White arrows indicate clusters of mitochondria. Bar = 2 µM. B. Average time of exercise endurance (n = 6). * P<0.05 vs. vehicle. C. 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).
Figure 2.
COS increases the mitochondrial content in myocytes.
A. Differentiated C2C12 cells were treated with resveratrol (Res; 2.28 µg/ml and 11.4 µg/ml) or chitoologosaccharide (COS; 10 µg/ml, 100 µg/ml, and 500 µg/ml) for 24 h. After incubation, the mitochondrial density was measured (n = 3). B and C. C2C12 myocytes were treated with Res (10 µM and 50 µM) or COS (10 µg/ml, 100 µg/ml, and 500 µg/ml) for 24 h. After rinsing with PBS, cells were harvested and proteins were blotted with antibodies against NDUFA9, SDHA, UQCRC2, COX1, ATP5a (B), or PGC1 and NRF1 (C). β-actin was used as a loading control. The densitometry of each band in panel B and C is shown in Figure S9A and S9B, respectively.
Figure 3.
A. Sirt1 activity was examined. Relative Sirt1 activity is shown as a bar graph (n = 4). B. C2C12 myocytes were treated with Res (2.28 µg/ml and 11.4 µg/ml) or COS (10 µg/ml, 100 µg/ml, and 500 µg/ml) for 12 h. The intracellular NAD+/NADH ratio was calculated (n = 4). * P<0.05 vs. (-); ** P<0.01 vs. (-); *** P<0.001 vs. (-). C. Differentiated C2C12 cells were incubated with Res (10 µM and 50 µM) or COS (10 µg/ml, 100 µg/ml, 500 µg/ml) for 6 h. After incubation, cells were rinsed with PBS and the protein lysates were immunoprecipitated with anti-Ac-Lys antibody and subjected to western blotting. D. Differentiated C2C12 myocytes were incubated with Res (11.4 µg/ml) or COS (10 µg/ml, 100 µg/ml, and 500 µg/ml) for 24 h. Then cells were rinsed with PBS two times. Total cell lysates were subjected to western blot analysis using p-AMPK, AMPK, p-ACC, and ACC antibodies. The average band intensities of panel C and D are shown in Figure S9C and S9D, respectively.
Figure 4.
COS requires AMPK and Sirt1 to induce mitochondrial biogenesis.
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 (A) or the measurement of mitochondrial density (n = 3) (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 Figure S9E.
Figure 5.
Knockdown of Sirt1 or AMPK expression diminishes the mitochondriogenic effect of COS.
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 (A) or the measurement of mitochondrial density (n = 3) (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 Figure S9G.
Figure 6.
COS activates Sirt1 and AMPK in vivo.
COS-administered SD rats were sacrificed and proteins from skeletal muscle were subjected to the measurement of cellular NAD+/NADH ratio (n = 6) (A) or western blotting (B). ** P<0.01 vs. vehicle. Densitometry of p-AMPK was shown in Figure S9H.