[Show abstract][Hide abstract] ABSTRACT: Syringaresinol exists either exclusively as one enantiomer or enantiomeric mixtures in plant foods. We found that (+)-syringaresinol, but not (-)-syringaresinol, upregulates silent information regulator two ortholog 1 (SIRT1) gene expression, and thus, Panax ginseng berry with predominantly high contents of (+)-syringaresinol exhibits higher activity in inducing SIRT1 gene expression than Acanthopanax senticosus Harms stem with almost equal proportion of the two enantiomers. These findings highlight the importance of the absolute configuration of syringaresinol for the biological activity.
[Show abstract][Hide abstract] ABSTRACT: Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the -1209/-1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes.
Full-text · Article · Aug 2013 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Knockdown effect of Sirt1 and AMPKα siRNAs in myocytes. Differentiated C2C12 cells were transfected with non-specific siRNA (sc; 100 pmol), Sirt1 siRNA (Sirt1; 100 pmol), or AMPKα siRNA (AMPK; 100 pmol). After transfection, cells were washed with PBS twice and harvested. A. mRNAs were isolated and cDNA was synthesized. The relative mRNA level of Sirt1 and AMPK was measured by qPCR and normalized to GAPDH (n = 2). * P<0.05 vs. control siRNA. B. proteins were subjected to Western Blot to detect the expression level of Sirt1 or AMPKα. β-actin expression was measured as a loading control. Average band intensity is shown in Figure S9F. Based on the changes of mRNA and protein expression level after siRNA transfection, the estimated knockdown efficiency of each siRNA is approximately 50% (Sirt1) and 60% (AMPK), respectively.
[Show abstract][Hide abstract] ABSTRACT: COS reduces plasma TG and cholesterol levels. Graphs show plasma profiles of TG (A) and cholesterol (B) of before (white bar) and after (black bar) exercise (n = 6). * P<0.05 vs. pre-exercise vehicle (lane 1); # P<0.05 vs. post-exercise vehicle (lane 2).
[Show abstract][Hide abstract] ABSTRACT: COS increases mRNA expression of mitochondrial transcription factors. Differentiated C2C12 cells 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 24 h and washed with PBS two times, and RNAs were isolated and cDNAs were synthesized. Relative mRNA expression of PGC1α (A), PGC1β (B), NRF1 (C), and tfam (D) was measured by using qPCR and normalized to GAPDH (n = 3). * P<0.05 vs. (-); ** P<0.01 vs. (-).
[Show abstract][Hide abstract] ABSTRACT: Densitometry of Western Blot results. Band intensity of each band in the Figure 2B (A), Figure 2C (B), Figure 3C (C), Figure 3D (D), Figure 4A (E), Figure S4B (F), Figure 5A (G), Figure 6B (H), and Figure S8B (I) was measured by Multiguage software (Fujifilm) and showed in bar graphs. * P<0.05 vs. (-); ** P<0.01 vs. (-); *** P<0.001 vs. (-).
[Show abstract][Hide abstract] ABSTRACT: AMPK and Sirt1 inhibitor prevents COS-induced mRNA expression of mitochondrial biogenesis-related genes. 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 qPCR analysis. cDNAs were subjected to qPCR analysis to detect the relative mRNA expression of PGC1α (A), PGC1β (B), NRF1 (C), and tfam (D) (n = 3). * P<0.05 vs. DMSO-treated (-) (lane 1); ** P<0.01 vs. DMSO-treated (-); *** P<0.001 vs. DMSO-treated (-); # P<0.05 vs. DMSO-treated Res; ## P<0.01 vs. DMSO-treated Res; & P<0.05 vs. DMSO-treated COS; && P<0.01 vs. DMSO-treated COS.
[Show abstract][Hide abstract] ABSTRACT: Small components of COS do not activate Sirt1 and AMPK.
A. Sirt1 activity using small constituents of COS (LOW; monomer to tetramer) (n = 3). * P<0.05 vs. (-); ** P<0.01 vs. (-); *** P<0.001 vs. (-). B. Differentiated C2C12 myocytes were treated with COS (500 µg/ml), glucosamine-lactate (1; 500 µg/ml), or mixture of dimer, trimer, and tetramer (2–4; 500 µg/ml) for 24 hours. Proteins were separated and hybridized with p-AMPK, PGC1, NDUFA9, and β-actin antibodies respectively. The band intensity of each band is shown in Figure S9I.
[Show abstract][Hide abstract] ABSTRACT: Sequences of qPCR primers. The qPCR primers for mitochondria-related genes (PGC1α, PGC1β, NRF1, tfam, and CPT1b) were used to examine the effect of COS on mitochondrial biogenesis. The qPCR primers for AMPKα1 and Sirt1 were used to determine the knock-down efficiency of AMPKα and Sirt1 siRNAs, respectively. GAPDH primer was used to normalize the relative mRNA expression of each gene. All qPCR primers were purchased from Bioneer Co.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: COS does not cause excess fatigue in spite of enhanced exercise endurance. Rats after endurance exercise were sacrificed and plasma LDH (A) and CK (B) activity was measured by using LDH and CK measuring kit (Bayer), respectively (n = 6). No statistical significance was observed among groups.
[Show abstract][Hide abstract] ABSTRACT: COS augments fatty acid oxidation. Differentiated C2C12 myocytes were treated with Res (11.4 µg/ml) or COS (10 µg/ml, 100 µg/ml, and 500 µg/ml) for 24 h (n = 10 in each group). After incubation, cells were rinsed with PBS and incubated in α-MEM (Lonza) containing 0.1 mmol/l palmitate (9,10-[3H]palmitate, 5 mCi/ml, PerkinElmer Life, Boston, MA) and 2% BSA for 24 h. The medium was then precipitated with an equal volume of 10% trichloroacetic acid (Sigma) by centrifugation at 12,000 rpm for 10 min. The supernatants were transferred to open 1.5 ml microcentrifuge tubes, placed in a scintillation vial containing 0.5 ml water, and incubated at 55°C for 12 h. After the removal of tubes containing the precipitated medium, the 3H2O contents were measured in a scintillation counter (PerkinElmer Life) in the presence of an enhancer solution (PerkinElmer Life). * P<0.05 vs. (-).
[Show abstract][Hide abstract] ABSTRACT: Major chemical compounds in 95% ethanol extracts of Artemisia iwayomogi tested identified by GC-MS. The 95% ethanol extracts of Artemisia iwayomogi were analyzed using the Thermo Scientific TRACE GC Ultra™ gas chromatograph. It was fitted with a split-splitless injector and connected to an MS PolarisQ-Quadrupole Ion Trap (Thermo Electron) fused silica column VB5 (5% phenyl, 95% methylpolyxiloxane, 30 m with 0.25 mm i.d. film thickness 0.25 µm) (J & W Scientific Fisons, Folsom, CA, USA). The injector and interface were operated at 250 and 300°C, respectively. The oven temperature was programmed as follows: 50°C raised to 250°C (4°C/min) and held for 3 min. Helium was the carrier gas at 1 ml/min. The sample (1 µl) was injected in the split mode (1∶20). MS conditions were as follows: ionization voltage EI of 70 eV, mass range 10–350 amu. The components were identified by comparing their relative retention times and mass spectra with those of authentic samples (analytical standards from data base).
[Show abstract][Hide abstract] ABSTRACT: Although Artemisia iwayomogi (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.
[Show abstract][Hide abstract] ABSTRACT: COS increases mRNA expression of mitochondria-related genes. mRNAs were isolated from skeletal muscle of animals described in Figures 1 and 5 and RNAs were isolated and cDNAs were synthesized using Trizol™ Reagent (Invitrogen) and RevertAid™ First Strand cDNA Synthesis Kit (Fermentas), respectively. The relative mRNA level of PGC1a, NRF1, and CPT1b was measured by qPCR and normalized to GAPDH (n = 6). * P<0.05 vs. vehicle; ** P<0.01 vs. vehicle. The primer sequences are listed in Table S2.
[Show abstract][Hide abstract] ABSTRACT: Chitooligosaccharides (COS), a kind of oligosaccharide made from chitin or chitosan, have been used a popular remedy for hangovers. In this study we investigated the in vitro effect of COS lactate salt on ethanol-induced cytotoxicity and the in vivo effect of short-term COS lactate salt feeding on ethanol-induced hangover. Pretreatment of HepG2 cells with COS lactate salt significantly reduced ethanol-induced cytotoxicity and suppressed generation of reactive oxygen species. In addition, COS lactate salt dose-dependently increased acetaldehyde dehydrogenase (ALDH) activity in vitro and reversed the ALDH inhibition induced by daidzin. Furthermore, oral administration of COS lactate salt (200 mg/kg) for 5 days significantly decreased the blood levels of alcohol and acetaldehyde in ethanol-treated mice. It was also demonstrated that hepatic mitochondrial ALDH activity was significantly increased in COS lactate salt-treated mice. Taken together, these findings indicate that COS lactate salt may have efficacy for the management of alcoholic hangovers.
No preview · Article · Oct 2010 · Journal of medicinal food
[Show abstract][Hide abstract] ABSTRACT: Chitooligosaccharides (COS), oligosaccharides composed of two to seven glucosamine residues, are known to exhibit various biological activities. In this study, we investigated the effects of COS in an in vivo mouse sleep deprivation-induced fatigue model in an effort to develop a functional food with anti-fatigue efficacy. Male Balb/c mice were orally administered 500 mg (kg d)(-1) of COS lactate or COS HCl for 2 weeks, and severe fatigue was induced by sleep deprivation. To evaluate the extent of fatigue, the swimming time, representing the immobility time, was measured in a forced swim test. As a result, oral intake of COS lactate-manifested anti-fatigue effects could be observed by the attenuation of fatigue-induced body weight loss and shorter immobility period. In addition, COS lactate was shown to alleviate the fatigue-induced increase in cortisol and lipid peroxidation and a decrease in superoxide dismutase (SOD) activity. Of particular note, the oral administration of COS lactate increased the mitochondrial membrane potential and the mitochondrial number significantly, indicating that COS lactate may enhance mitochondrial function. In support of this, COS lactate increased the expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) and cytochrome c (Cyt C) mRNA, indicating that it may increase mitochondrial biogenesis. These results suggest that COS lactate can be an effective anti-fatigue functional food, and this anti-fatigue effect may result from, at least in part, the enhancement of mitochondrial biogenesis and the inhibition of free radical generation.
[Show abstract][Hide abstract] ABSTRACT: The regulation of adipocyte lipolysis is increasingly believed to influence insulin resistance, in a process that may be associated with mitochondrial dysfunction. However, the molecular basis of the relationship between mitochondrial protein expression, lipolytic responsiveness, and insulin resistance remains unknown. A set of proteins that shows altered abundances in the mitochondria of untreated and treated 3T3-L1 adipocytes with TNF-alpha or isoproterenol was identified. These include the proteins associated with energy production, including fatty acid oxidation, TCA cycle, and oxidative phosphorylation. Proteins associated with oxidative stress dissipation were down-regulated in lipolytically stimulated adipocytes. Lipolytic stimulation with isoproterenol and TNF-alpha, which is also a potent proinflammatory cytokine, showed some noticeable differences in mitochondrial protein expression. For example, isoproterenol markedly enhanced the expression of prohibitin which is involved in the integrity of mitochondria but TNF-alpha did not. These results provide valuable information on mitochondrial dysfunction associated with oxidative stress induced by lipolytic stimulation.
No preview · Article · Feb 2009 · Journal of Cellular Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Cidec is a lipid droplet-associated protein, which inhibits lipolysis, leading to the accumulation of triglycerides in adipocytes. However, the transcriptional regulation of Cidec in adipocyte remains unknown. In the present study we investigated that the mouse Cidec transcript is regulated by PPARgamma2. After the differentiation of adipocyte, the expression pattern of Cidec was similar to that of PPARgamma2. In the presence of a PPARgamma agonist, the level of Cidec mRNA was highly increased. In addition, putative PPRE sites were identified in the Cidec promoter. By chromatin immunoprecipitation assay and reporter assay, we observed the binding of PPARgamma2 to the promoter of Cidec. Gel shift assay and the mutagenesis study were showed that the -219/-207 region of the Cidec promoter could function as a PPRE of the Cidec promoter. These results suggest that PPARgamma2 is required for the transcriptional activity of Cidec during adipogenesis, which could be contributed to understand the molecular mechanism of lipid droplet formation in adipocytes.
No preview · Article · Nov 2008 · Biochemical and Biophysical Research Communications