Coumestrol Induces Mitochondrial Biogenesis by Activating Sirt1 in Cultured Skeletal Muscle Cells

The mitochondrion is a central organelle in cellular energy homeostasis; thus, reduced mitochondrial activity has been associated with aging and metabolic disorders. This paper provides biological evidence that coumestrol, which is a natural isoflavone, activates mitochondrial biogenesis. In cultured myocytes, coumestrol activated the silent information regulator two ortholog 1 (Sirt1) through the elevation of the intracellular NAD⁺/NADH ratio. Coumestrol also increased the mitochondrial contents and induced the expression of key proteins in the mitochondrial electron transfer chain in cultured myocytes. A Sirt1 inhibitor and Sirt1-targeting siRNAs abolished the effect of coumestrol on mitochondrial biogenesis. Similar to an increase in mitochondrial content, coumestrol improved myocyte function with increased ATP concentration. Taken together, the data suggest that coumestrol is a novel inducer of mitochondrial biogenesis through the activation of Sirt1

An Ethanol Extract of <em>Artemisia iwayomogi</em> Activates PPARδ Leading to Activation of Fatty Acid Oxidation in Skeletal Muscle

<div><p>Although <em>Artemisia iwayomogi</em> (AI) has been shown to improve the lipid metabolism, its mode of action is poorly understood. In this study, a 95% ethanol extract of AI (95EEAI) was identified as a potent ligand of peroxisome proliferator-activated receptorδ (PPARδ) using ligand binding analysis and cell-based reporter assay. In cultured primary human skeletal muscle cells, treatment of 95EEAI increased expression of two important PPARδ-regulated genes, carnitine palmitoyl-transferase-1 (CPT1) and pyruvate dehydrogenase kinase isozyme 4 (PDK4), and several genes acting in lipid efflux and energy expenditure. Furthermore, 95EEAI stimulated fatty acid oxidation in a PPARδ-dependent manner. High-fat diet-induced obese mice model further indicated that administration of 95EEAI attenuated diet-induced obesity through the activation of fatty acid oxidation in skeletal muscle. These results suggest that a 95% ethanol extract of AI may have a role as a new functional food material for the prevention and/or treatment of hyperlipidermia and obesity.</p> </div

A 95EEAI attenuates body weight gain and adiposity in HFD-fed C57BL/6J mice.

<p>Male C57BL/6J mice were fed a normal diet and vehicle, HDF and vehicle or HFD and 200 mg/kg/day 95EEAI for 8 weeks (A) Body weight change. (B) Adipose tissue weight. Values are mean ± S.D. of nine mice. #, p<0.01 compared to normal diet/vehicle group; **, p<0.01; *, p<0.05 compared to HFD/vehicle group.</p

Physical and metabolic parameters in 95EEAI- and vehicle-treated mice.

<p>A 95EEAI (200 mg/kg) was orally administrated to mice once daily for 8 weeks.</p><p>Values are mean ± S.D. of nine mice.</p>#<p>, p<0.01 compared to normal diet/vehicle group;</p>**<p>, p<0.01;</p>*<p>, p<0.05 compared to HFD/vehicle group.</p

Ligand-induced binding of a coactivator derived peptide to PPARδ <i>in vitro</i>.

<p>Interaction of fluorescein-labeled coactivator peptide C33 and recombinant GST-PPARδ bound by a terbium-labeled anti-GST antibody was determined by TR-FRET. GW501516, WEAI, 50EEAI and 95EEAI were used at the concentrations indicated. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033815#s3" target="_blank">Results</a> are expressed as the ratio of fluorescence intensity at 520 nm (fluorescein emission excited by terbium emission) and 495 nm (terbium emission). All data points represent averages of triplicates (±S.D.).</p

A 95EEAI increases skeletal muscle fatty acid oxidation in C57BL/6J mice.

<p>Male C57BL/6J mice were fed a HDF and vehicle or HFD and 200 mg/kg/day 95EEAI for 8 weeks (A) fatty acid oxidation in skeletal muscle (B) Quantitative real-time PCR analysis in skeletal muscle. Values are mean ± S.D. of nine mice. ***, p<0.005; **, p<0.01; *, p<0.05 compared to HFD/vehicle group.</p

95EEAI-induced effects on PPARδ target gene expression in primary myotubes.

<p>Primary myotubes were transfected with PPARδ siRNA pool or control siRNA. At 48 h post-transfection, the cells were treated with 100 nM GW501516, 25 µg/ml 95EEAI or DMSO for 24 h. Cells were harvested for real-time quantitative PCR (A, C) or western blotting (B) GAPDH RNA was used as an internal control for calculating mRNA fold changes. Values are expressed mean ± S.D. from three independent experiments. **, p<0.01; *, p<0.05 for DMSO controls; #, p<0.05 for control siRNA versus PPARδ siRNA. One representative result is shown form three independent western blotting experiments.</p

Effects of 95EEAI on the transcriptional activity of PPARδ reporter cells.

<p>(A) Mock reporter cells (luciferase vector only) and the PPARδ reporter cells (PPARδ expression vector+PPARδ-responsive luciferase reporter vector) were cultured, treated with DMSO, GW501516 (1 µM), WEAI (100 µg/ml), 50EEAI (100 µg/ml) and 95EEAI (100 µg/ml) for 24 h before harvesting. (B) The effects of various ranges of 95EEAI concentrations were analyzed. Values represent averages of six independent experiments (± S.D.). ***, p<0.005; **, p<0.01 compared to vehicle control.</p

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