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Long-term topical skin care by traditional anti-melanogenic agents can raise several safety concerns. An understanding of the molecular mechanisms of active compounds on melanogenesis is, therefore, necessary to address pigmentation issues. Here we revealed that stimulation with 1 mM betaine, an abundant component in rice bran, significantly reduced 21% of intracellular melanin content by suppressing tyrosinase activity and microphthalmia-associated transcription factor (MITF) expression in B16-F1 murine melanocytes. The expression of MITF was suppressed at both mRNA and protein levels by 43 and 44%, respectively. Subsequently, the betaine-stimulated melanocytes showed inhibition of PKA-CREB signaling axis but activation of extracellular-signal-regulated kinase and AKT-GSK3β signaling pathways. This inhibition and activation led to downregulation of MITF expression at both the transcriptional and post-translational levels to suppress melanin synthesis. These findings collectively suggested that betaine is a potential anti-melanogenic compound for functional foods and cosmetics.

Mechanism of barley sprout extract (BS), which contains policosanols and polyphenols, on cholesterol and glucose metabolism was investigated. BS reduced the intracellular cholesterol concentrations in the HepG2 cells and the plasma cholesterol concentrations in the mice by the activation of AMPK and the subsequent phosphorylation inhibition of HMGCR. BS suppressed the nuclear translocation of SREBP2, reducing the transcription of HMGCR. AMPK activation with BS reduced the fasting glucose and hepatic triglyceride concentrations in mice by repressing the hepatic gluconeogenic genes, including fructose-1, 6-bisphosphatase and pyruvate carboxylase and the plasma levels of the proinflammatory cytokines tumor necrosis factor-α and interleukin-6. The activation of hepatic autophagy by BS was confirmed by induced protein expressions of LC3-II and LAMP. In conclusion, BS activates AMPK and hepatic autophagy and inhibits SREBP2, resulting in hypocholesterolemic and hypoglycemic activities and improvements in the symptoms of hepatic steatosis.

Intake of dietary aroma compounds may regulate cellular lipid metabolism. We demonstrated that trans-caryophyllene, a flavor compound in plant foods and teas, activates peroxisome proliferator-activated receptor (PPAR)-α through direct interaction with the ligand-binding domain of PPAR-α. The agonistic activity of trans-caryophyllene was investigated by the luciferase reporter assay, surface plasmon resonance, and time-resolved fluorescence resonance energy transfer assay. Following the stimulation of cells with trans-caryophyllene, intracellular triglyceride concentrations were significantly reduced by 17%, and hepatic fatty acid uptake was significantly increased by 31%. The rate of fatty acid oxidation was also significantly increased. The expressions of PPAR-α and its target genes and proteins in fatty acid uptake and oxidation were significantly up-regulated as well. In HepG2 cells transfected with small interfering RNA of PPAR-α, the effects of trans-caryophyllene on PPAR-α responsive gene expressions, intracellular triglyceride, fatty acid uptake and oxidation were disappeared. These results indicate that the aroma compound, trans-caryophyllene, is PPAR-α agonist thus regulates cellular lipid metabolism in PPAR-α dependent manners.

We investigated the hypotriglyceridemic mechanism of action of linalool, an aromatic monoterpene present in teas and fragrant herbs. Reporter gene and time-resolved fluorescence resonance energy transfer assays demonstrated that linalool is a direct ligand of PPARα. Linalool stimulation reduced cellular lipid accumulation regulating PPARα-responsive genes and significantly induced FA oxidation, and its effects were markedly attenuated by silencing PPARα expression. In mice, the oral administration of linalool for 3 weeks reduced plasma TG concentrations in Western-diet-fed C57BL/6J mice (31%, P < 0.05) and human apo E2 mice (50%, P < 0.05) and regulated hepatic PPARα target genes. However, no such effects were seen in PPARα-deficient mice. Transcriptome profiling revealed that linalool stimulation rewired global gene expression in lipid-loaded hepatocytes and that the effects of 1 mM linalool were comparable to those of 0.1 mM fenofibrate. Metabolomic analysis of the mouse plasma revealed that the global metabolite profiles were significantly distinguishable between linalool-fed mice and controls. Notably, the concentrations of saturated FAs were significantly reduced in linalool-fed mice. These findings suggest that the appropriate intake of a natural aromatic compound could exert beneficial metabolic effects by regulating a cellular nutrient sensor.

Flavonoids are abundant in fruits and vegetables and have various biological activities. A rapid, sensitive method for assessing cellular quercetin levels is necessary because cellular levels are often low. A new method for measuring cellular flavonoid levels is proposed using diphenylboric acid 2-aminoethyl ester (DPBA), a fluorescent probe, and quercetin, a representative flavonoid. This spectrofluorometry-based assay is simple and allows rapid quantification of the cellular quercetin content with high sensitivity. CHO-K1 cells were incubated with quercetin for 5 min, and then the cellular quercetin concentration was assayed using DPBA spectrofluorometry. Results, compared with high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), and conventional spectrophotometry measurements, were highly correlated with HPLC data (R>0.99). The method is more sensitive than TLC and conventional spectrophotometry. Spectrofluorometric measurement using DPBA is a simple, rapid, and sensitive method for assessing cellular quercetin levels.

We investigated the effect of cineole on the expression of genes related to reverse cholesterol transport and hepatic fatty acid metabolism. Cineole, a small aroma compound in teas and herbs, significantly stimulated the transactivation of liver X receptor modulator (LXR)-α and LXR-β. The mRNA and protein expression of LXRs and their target genes, including ABCA1 and ABCG1, was significantly increased in macrophages stimulated with cineole. This led to the subsequent removal of cholesterol from the cells. Interestingly, cineole showed tissue-selective LXR induction: hepatocytes stimulated with cineole showed significantly reduced expression of LXR-α and LXR-α-responsive genes, including FAS and SCD-1 (P

We investigated the in vitro effects of p-coumaric acid on melanogenesis. The melanin content in B16F1 cells stimulated with p-coumaric acid significantly decreased (68 % vs. control) through inhibition of tyrosinase enzyme activity assessed using both cell-free and cell-based assays (46 and 27 % compared with control, respectively). In addition, stimulating B16F1 cells with p-coumaric acid reduced cyclic adenosine monophosphate (cAMP)—responsive element-binding protein (CREB) protein phosphorylation (26 % vs. control), which in turn downregulated the expression of the microphthalmia-associated transcription factor (MITF) and its target gene tyrosinase (27 and 20 % vs. control, respectively). p-Coumaric acid has a hypopigmentation effect in melanocytes by both directly inhibiting tyrosinase enzyme activity and reducing CREB phosphorylation, which inhibits MITF and tyrosinase expression.

cope A natural carotenoid abundant in seafood, astaxanthin (AX), has hypolipidemic activity, but its underlying mechanisms of action and protein targets are unknown. We investigated the molecular mechanism of action of AX in hepatic hyperlipidemia by measuring peroxisome proliferator-activated receptors (PPAR) activity. Methods and results We examined the binding of AX to PPAR subtypes and its effects on hepatic lipid metabolism. AX binding activated PPAR-α, but inhibited PPAR-γ transactivation activity in reporter gene assay and time-resolved fluorescence energy transfer analyses. AX had no effect on PPARδ/β transactivation. AX bound directly to PPAR-α and PPAR-γ with moderate affinity, as assessed by surface plasmon resonance experiments. The differential effects of AX on PPARs were confirmed by measuring the expression of unique responsive genes for each PPAR subtype. AX significantly reduced cellular lipid accumulation in lipid-loaded hepatocytes. Transcriptome analysis revealed that the net effects of stimulation with AX (100 μM) on lipid metabolic pathways were similar to those elicited by fenofibrate and lovastatin (10 μM each), with AX rewiring the expression of genes involved in lipid metabolic pathways. Conclusion AX is a PPAR-α agonist and PPAR-γ antagonist, reduces hepatic lipid accumulation by rewiring the transcriptome in lipid-loaded hepatocytes.

cope Taurine, which is abundant in seafood, has antiatherogenic activities in both animals and humans; however, its molecular target has been elusive. We examined whether taurine could activate liver X receptor-α (LXR-α), a critical transcription factor in the regulation of reverse cholesterol transport in macrophages. Methods and results Taurine bound directly to LXR-α in a reporter gene assay, time-resolved fluorescence resonance energy transfer analysis, and limited protease digestion experiment. Macrophage cells incubated with taurine showed reduced cellular cholesterol and induced medium cholesterol in a dose-dependent manner with the induction of ATP-binding cassette transporter A1 and G gene and protein expression. In hepatocytes, taurine significantly induced Insig-2a levels and delayed nuclear translocation of the sterol regulatory element-binding protein 1 (SREBP-1) protein, resulting in a dose-dependent reduction in the cellular lipid levels without inducing the expression of fatty acid synthesis genes. Conclusion Taurine is a direct LXR-α ligand, represses cholesterol accumulation, and modulates the expression of genes involved in reverse cholesterol transport in macrophages, without inducing hepatic lipogenesis. The induction of Insig-2a suppressed the nuclear translocation of SREBP-1c.

We investigated the acute metabolic effects of isoflavones from Pueraria lobata (Willd.) Ohwi (IPL) in ovariectomized (OVX) mice. After 4 weeks of IPL feeding at 500 mg/day/kg body weight (OVX500), plasma 17β-estradiol concentrations were significantly higher (+25%, p

We investigated the hypolipidemic effects of Melissa officinalis essential oil (MOEO) in human APOE2 transgenic mice and lipid-loaded HepG2 cells. Plasma TG concentrations were significantly less in APOE2 mice orally administered MOEO (12.5 mg/d) for 2 wk than in the vehicle-treated group. Cellular TG and cholesterol concentrations were also significantly decreased in a dose- (400 and 800 mg/L) and time- (12 and 24 h) dependent manner in HepG2 cells stimulated with MOEO compared with controls. Mouse hepatic transcriptome analysis suggested MOEO feeding altered several lipid metabolic pathways, including bile acid and cholesterol synthesis and fatty acid metabolism. In HepG2 cells, the rate of fatty acid oxidation, as assessed using [1- 14 C]palmitate, was unaltered; however, the rate of fatty acid synthesis quantified with [1- 14 C]acetate was significantly reduced by treatment with 400 and 800 mg/L MOEO compared with untreated controls. This reduction was due to the decreased expression of SREBP-1c and its responsive genes in fatty acid synthesis, including FAS, SCD1, and ACC1. Subsequent chromatin immunoprecipitation analysis further demonstrated that the binding of p300/CBP-associated factor, a coactivator of SREBP-1c, and histone H3 lysine 14 acetylation at the FAS, SCD1, and ACC1 promoters were significantly reduced in the livers of APOE2 mice and HepG2 cells treated with MOEO compared with their controls. Additionally, MOEO stimulation in HepG2 cells induced bile acid synthesis and reduced the nuclear form of SREBP-2, a key transcription factor in hepatic cholesterol synthesis. These findings suggest that the intake of phytochemicals with pleasant scent could have beneficial metabolic effects. J. Nutr. 142: 432‐440, 2012.

Momilactone B (MB) is a terpenoid phytoalexin present in rice bran that exhibits several biological activities. MB reduced the melanin content in B16 melanocytes melanin content and inhibited tyrosinase activities. Using transcriptome analysis, the genes involved in protein kinase A (PKA) signaling were found to be markedly altered. B16 cells stimulated with MB had decreased concentrations of cAMP protein kinase A activity, and cAMP-response element-binding protein which is a key transcription factor for microphthalmia-associated transcription factor (MITF) expression. Accordingly, the expression of MITF and its target genes, which are essential for melanogenesis, were reduced. MB thus exhibits anti-melanogenic effects by repressing tyrosinase enzyme activity and inhibiting the PKA signaling pathway which, in turn, decreases melanogenic gene expression.

We investigated the effects of puerarin, the major isoflavone in Kudzu roots, on the regulation of autophagy in ethanol-treated hepatocytes. Incubation in ethanol (100 mM) for 24 h reduced cell viability by 20% and increased the cellular concentrations of cholesterol and triglycerides by 40% and 20%, respectively. Puerarin stimulation significantly recovered cell viability and reduced cellular lipid accumulation to a level comparable to that in untreated control cells. Ethanol incubation reduced autophagy significantly as assessed by microtubule-associated protein1 light chain 3 (LC3) expression using immunohistochemistry and immunoblot analysis. The reduced expression of LC3 was restored by puerarin in a dose-dependent manner in ethanol-treated cells. The effect of puerarin on mammalian targets of rapamycin (mTOR), a key regulator of autophagy, was examined in ethanol-treated hepatocytes. Immunoblotting revealed that puerarin significantly induced the phosphorylation of 5'AMP-activated protein kinase (AMPK), thereby suppressing the mTOR target proteins S6 ribosomal protein and 4E-binding protein 1. These data suggest that puerarin restored the viability of cells and reduced lipid accumulation in ethanol-treated hepatocytes by activating autophagy via AMPK/mTOR-mediated signaling.

The effects of wild type and UV-irradiated lemon balm (Melissa officinalis) ethanolic extracts (MOE and UMOE) on melanogenesis in vitro were examined. UMOE showed potent antioxidant activity and significantly inhibited the mushroom and melanocyte tyrosinase activity, and lowered cellular melanin content by 49% at 200 μg/mL in B16-F1 melanocytes. The key gene and protein expression of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 were reduced (−73% for TRP-1 protein at 200 μg/mL UMOE, p

Department of Biological Science, Konkuk UniversityAbstract We examined the in vitro mutagenic and antimutagenic effects of hexane-extracted hemp (Cannabis sativa L.subsp. sativa var. sativa) seed oil (HO) with and without S9-mediated metabolic activation, using the TA98 and TA100Salmonella Typhimurium strains. The MTT assay revealed no cytotoxicity in HepG2 cells for HO quantities

To investigate the hypocholesterolemic mechanism of barley in vivo, six-week-old C57BL/6J mice were fed a high-fat diet (HFD) or high-fat diet containing barley (HFD-B) for seven weeks. Total and LDL cholesterol concentrations were significantly reduced in the HFD-B group while fecal cholesterol and bile acid was increased. Real-time PCR and immunoblot analysis revealed the induction of FXR expression, which in turn suppressed the expression of ASBT and NPC1L1 in the HFD-B group compared with the controls. In the liver, the expression of HMG-CoA reductase was significantly reduced while LDL receptor expression was unaltered in the HFD-B group compared with the controls. Our data suggest that the hypocholesterolemic effects of barley are primarily the result of reduced dietary cholesterol uptake and bile acid resorption. Reduced expression of intestinal ASBT and NPC1L1 may play a key role in the regulation of dietary cholesterol and bile acid metabolism in mice consuming a diet containing barley.

We examined the effects of Lithospermum erythrorhizon Sieb. et Zucc. (LE) on cholesterol metabolism in vitro. The ethanolic LE extract (ELE) had total polyphenolic and flavonoid contents of 353 ± 7 and 285 ± 51 mg/dL, respectively. The ELE inhibited Cu2+-mediated LDL oxidation at

Vitamin C is a strong antioxidant that alters gene expression in cells, and its effects can be modified by cellular oxidative stress. We investigated the genome-wide effects of vitamin C on the in vivo transcriptome in the liver, which synthesizes various enzymes and proteins to defend against cellular oxidative stress. We fed mice vitamin C (0.056 mg/g of body weight) for 1 week and performed DNA microarray analysis with hepatic mRNA in fasting and refeeding states to mimic physiological conditions of oxidative stress. Significance analysis of microarray data identified approximately 6,000 genes differentially expressed in both fasting and refeeding states. In the fasting state, vitamin C induced overall energy metabolism as well as radical scavenging pathways. These were ameliorated in the refeeding state. These findings suggest that vitamin C has profound and immediate global effects on hepatic gene expression, which may help prevent oxidative stress, and that long-term treatment with vitamin C might reduce the risk of chronic disease.

Intake of saturated and trans-fatty acids is a strong risk factor for coronary heart disease. We investigated the inhibitory effects of 2 hexane extracts from white (WBE) and black soybeans (BBE) on cellular fatty acid uptake in vitro. Transcellular uptake of elaidic acid (t18:1), a major trans-fatty acid present in processed foods, in Caco-2 monolayers was significantly reduced by 28.3 and 16.7% 60 min after WBE and BBE treatment, respectively. Results of flow cytometry (FACS) analysis showed significant reductions in boron-dipyrromethene (BODIPY) fluorescence-labeled fatty acid uptake by 35.4 and 40.2% with WBE and BBE treatment, respectively. BBE treatment significantly reduced the expression of fatty acid transport protein-4 and CD36 in Caco-2 cells, as determined by quantitative real time-polymerase chain reaction (qRT-PCR). Similar trends were found in WBE treatment, although to a lesser degree. These observations suggest that soybean extract may reduce fatty acid uptake and cellular fat accumulation by altering fatty acid transporter expression.

We investigated the effects of puerarin (8-C-glucosyl-7,4′-dihydroxy isoflavone), an isoflavone found in Kudzu roots (Pueraria lobata), and its glycosides (enzymatically synthesised, water-soluble derivatives of puerarin) on melanogenesis in vitro. Puerarin and its glycosides reduced mushroom tyrosinase activity by 88 and 67% at 4.8 mM, respectively, in a concentration-dependent manner. The puerarin glycosides were less effective than puerarin at the same concentration but showed a comparable inhibitory effect at a concentration at which puerarin is insoluble in water. In cultured B16 melanocytes, the melanin content was reduced significantly; moreover, tyrosinase activity was inhibited significantly by both puerarin and its glycosides when added at a concentration of 480 μM. DNA microarray and RT-PCR analyses showed significant downregulation of the expression of microphthalmia-associated transcription factor (MITF) and its target genes. The protein expression of MITF and tyrosinase was also downregulated significantly by 40 and 50%, respectively. Our findings suggest that puerarin and its glycosides cause hypopigmentation via dual mechanisms: by inhibiting tyrosinase activity directly and by altering the expression of melanogenesis-related genes, such as MITF and tyrosinase. Therefore, puerarin and its glycosides may have potential for the development of functional cosmetics causing hypopigmentation.

Objective: We doubly coated Lactobacillus plantarumKCTC3928 with proteins and polysaccharide compounds to enhance its acid and bile resistance. The present study investigated the hypocholesterolemic effects of double-coated L. plantarum KCTC3928 in C57BL/6 mice fed a high-fat diet. The effects of live and dead bacteria were compared. Methods: Six-week-old C57BL/6 male mice were divided into three groups: the control group was fed no L. plantarum KCTC3928, and the two treatment groups were orally fed live or dead L. plantarum KCTC3928 daily. Plasma and liver cholesterol and lipid levels, fecal bile acid, and gene and protein expressions were measured. Results: Low-density lipoprotein cholesterol and plasma triacylglycerol levels were significantly lower in the group fed live bacteria, by 42% and 32%, respectively (P

Hypolipidemic effects of Agastache rugosa essential oil (AREO) were investigated. Gas chromatographymass spectrometry (GC-MS) analysis showed that the major compound in AREO is limonene (47%, w/w). AREO (1 mg/mL) markedly reduced low density lipoprotein (LDL) oxidation (−93%). After 3-week feeding of AREO, plasma cholesterol (−28%) and triglyceride levels (−26%) were significantly decreased in C57BL/6J mice. Mouse hepatic transcriptome profiling with oligonucleotide microarray revealed that AREO altered the expression of 2,524 genes. Notably, significant reductions in sterol regulatory element binding factor (SREBF)-1 and SREBF-2 mRNA levels were detected. Protein expression of HMG-CoA reductase, a major target for SREBP-2, was reduced in HepG2 cells (−36%) and in mouse liver (−35 %). AREO also significantly increased mRNA (+40%) and protein (+83%) expression of the LDL receptor in HepG2 cells and mouse liver, respectively. Our findings suggest that AREO may prevent atherosclerosis via the inhibition of LDL oxidation, down-regulation of SREBF-2 and HMG-CoA reductase expression, and up-regulation of LDL receptor expression.

Dietary flavonoids have various biological functions. However, their cellular transport mechanisms are largely unknown. We have determined that the multidrug and toxic compound extrusion transporter-1 (MATE1) is a membrane transporter for flavonoids and has a high affinity for quercetin. HEK293T cells overexpressing MATE1 exhibited increased intracellular quercetin accumulation. This effect disappeared in the presence of a MATE1 inhibitor and after MATE1 gene knockdown. HepG2 cells expressed MATE1 significantly, with the uptake quercetin of which was dramatically reduced with MATE1 inhibition. On the basis of immunofluorescence analysis, MATE1 was highly expressed in peroxisomes and the endoplasmic reticulum (ER) as well as in plasma membranes in the liver and intestine, which suggests potential accumulation of quercetin in peroxisomes and the ER in these tissues. Fluorescent microscopic analysis confirmed selective accumulation of qurcetin in peroxisome. The effects of quercetin on cellular lipid reduction and glucose uptake were exaggerated with MATE1 overexpression. In conclusion, MATE1 is a membrane transporter for quercetin; its overexpression enhances the hypolipidemic activity of quercetin and cellular glucose transport. Considering the low bioavailability of quercetin, appropriate regulation of MATE1 expression may optimize cellular quercetin concentrations and promote health benefits.

cope This study investigated metabolic effects of ursolic acid (UA), a peroxisome proliferation-activated receptor (PPAR)-α activator, in vivo. Methods and results High-fat diet (HFD)-fed C57BL/6J mice were orally administered UA (50 or 200 mg/kg body weight) for 8 wk. UA reduced liver and adipose tissue mass, adipocyte size, and plasma leptin concentrations, plasma triglyceride and low-density-lipoprotein cholesterol concentrations, while it elevated the high-density-lipoprotein cholesterol and adiponectin concentrations significantly compared with controls. UA induced the expression of PPARα and its responsive genes involved in fatty acid uptake and β-oxidation in the livers, whereas genes involved in lipogenesis, including sterol regulatory element-binding proteins-1c, were downregulated. UA administration improved glucose tolerance and insulin sensitivity significantly compared with the HFD-fed control livers. UA administration also activated hepatic autophagy as assessed by the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II and other key proteins in the autophagy pathway. Conclusion Our findings suggest that UA ameliorates lipid and glucose metabolism in HFD-fed mice primarily by the activation of PPARα and induction of the hepatic autophagy pathway. Thus, intake of UA in the diet or in an isolated form may ameliorate lipid and glucose metabolism.

Kaempferol is a dietary flavonol previously shown to regulate cellular lipid and glucose metabolism. However, its molecular mechanisms of action and target proteins have remained elusive, probably due to the involvement of multiple proteins. This study investigated the molecular targets of kaempferol. Ligand binding of kaempferol to liver X receptors (LXRs) was quantified by time-resolved fluorescence resonance energy transfer and surface plasmon resonance analyses. Kaempferol directly binds to and induces the transactivation of LXRs, with stronger specificity for the β-subtype (EC50 = 0.33 μM). The oral administration of kaempferol in apolipoprotein-E-deficient mice (150 mg/day/kg body weight) significantly reduced plasma glucose and increased high-density lipoprotein cholesterol levels and insulin sensitivity compared with the vehicle-fed control. Kaempferol also reduced plasma triglyceride concentrations and did not cause liver steatosis, a common side effect of potent LXR activation. In immunoblotting analysis, kaempferol reduced the nuclear accumulation of sterol regulatory element-binding protein-1 (SREBP-1). Our results show that the suppression of SREBP-1 activity and the selectivity for LXR-β over LXR-α by kaempferol contribute to the reductions of plasma and hepatic triglyceride concentrations in mice fed kaempferol. They also suggest that kaempferol activates LXR-β and suppresses SREBP-1 to enhance symptoms in metabolic syndrome.

Cyanidin, a natural flavonoid abundant in fruits and vegetables, is known to regulate cellular lipid metabolism; however, its underlying mechanism of action and protein targets remain unknown. Here, the ligand binding activity of cyanidin on liver X receptors (LXRs) was investigated utilizing surface plasmon resonance and time-resolved fluorescence energy transfer (TR-FRET) analyses. LXRs are nuclear receptors which function as critical transcription factors in the regulation of cellular lipid and glucose metabolism. This includes the stimulation of high-density-lipoprotein synthesis and activation of reverse cholesterol transport. The present findings show that cyanidin induces the transactivation of LXRs and binds directly to the ligand-binding domain of both LXRα and LXRβ with dissociation constants of 2.2 and 73.2μM, respectively. Cell-free FRET analysis demonstrated that cyanidin induces the recruitment of co-activator peptide for LXRα and LXRβ with EC50 of 3.5μM and 125.2μM, respectively. In addition, intracellular cholesterol and triglyceride (TG) concentrations were reduced in macrophages following cyanidin stimulation. In cultured hepatocytes, cyanidin mildly induced SREBP1c gene expression but marginally affected cellular TG concentrations as well as reduced cellular cholesterol accumulations which activated the expression of genes for reverse cholesterol transport. Two cyanidin metabolites, procatechic acid and phloroglucinaldehyde, did not directly bind or activate LXRs. These results demonstrate that cyanidin is a direct ligand for both LXRα and LXRβ, suggesting that cyanidin may operate, at least in part, through modulation of cellular LXR activity.

We demonstrated that ombuin-3-O-β-D-glucopyranoside (ombuine), a flavonoid from Gynostemma pentaphyllum, is a dual agonist for peroxisome proliferator-activated receptors (PPARs) α and δ/β. Using surface plasmon resonance (SPR), time-resolved fluorescence resonance energy transfer (FRET) analyses, and reporter gene assays, we showed that ombuine bound directly to PPARα and δ/β but not to PPARγ or liver X receptors (LXRs). Cultured HepG2 hepatocytes stimulated with ombuine significantly reduced intracellular concentrations of triglyceride and cholesterol and downregulated the expression of lipogenic genes, including sterol regulatory element binding protein-1c (SREBP1c) and stearoyl-CoA desaturase-1 (SCD-1), with activation of PPARα and δ/β. Activation of LXRs by ombuine was confirmed by reporter gene assays, however, SPR and cell-based FRET assays showed no direct binding of ombuine to either of the LXRs suggesting LXR activation by ombuine may be operated via PPARα stimulation. Ombuine-stimulated macrophages showed significantly induced transcription of ATP binding cassette cholesterol transporter A1 (ABCA1) and G1 (ABCG1), the key genes in reverse cholesterol transport, which led to reduced cellular cholesterol concentrations. These results suggest that ombuine is a dual PPAR ligand for PPARα and δ/β with the ability to decrease lipid concentrations by reducing lipogenic gene expression in hepatocytes and inducing genes involved in cholesterol efflux in macrophages.

The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

The present study reports a novel liver X receptor (LXR) activator, ethyl 2,4,6-trihydroxybenzoate (ETB), isolated from Celtis biondii. Using a reporter gene assay, time-resolved fluorescence resonance energy transfer (TR-FRET), and surface plasmon resonance (SPR) analysis, we showed that ETB directly bound to and stimulated the transcriptional activity of LXR-α and LXR-β. In macrophages, hepatocytes, and intestinal cells, ETB suppressed cellular cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes. Notably, ETB did not induce lipogenic gene expression or cellular triglyceride accumulation in hepatocytes. These results suggest that ETB is a dual-LXR modulator that regulates the expression of key genes in cholesterol homeostasis in multiple cells without inducing lipid accumulation in HepG2 cells.

To investigate the underlying mechanism of targets of cyanidin, a flavonoid, which exhibits potent anti-atherogenic activities in vitro and in vivo, a natural chemical library that identified potent agonistic activity between cyanidin and peroxisome proliferator-activated receptors (PPAR) was performed. Cyanidin induced transactivation activity in all three PPAR subtypes in a reporter gene assay and time-resolved fluorescence energy transfer analyses. Cyanidin also bound directly to all three subtypes, as assessed by surface plasmon resonance experiments, and showed the greatest affinity to PPARα. These effects were confirmed by measuring the expression of unique genes of each PPAR subtype. Cyanidin significantly reduced cellular lipid concentrations in lipid-loaded steatotic hepatocytes. In addition, transcriptome profiling in lipid-loaded primary hepatocytes revealed that the net effects of stimulation with cyanidin on lipid metabolic pathways were similar to those elicited by hypolipidemic drugs. Cyanidin likely acts as a physiological PPARα agonist and potentially for PPARβ/δ and γ, and reduces hepatic lipid concentrations by rewiring the expression of genes involved in lipid metabolic pathways.

A novel liver X receptor (LXR) modulator, iristectorigenin B isolated from Belamcanda chinensis, stimulated the transcriptional activity of both LXR-α and LXR-β. In macrophages, iristectorigenin B suppressed cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes, ATP-binding cassette transporters A1 and G1. It did not induce hepatic lipid accumulation nor the expression of the lipogenesis genes sterol regulatory element-binding protein-1c, fatty acid synthase, and stearoyl-CoA desaturase-1. Iristectorigenin B thus is a dual-LXR agonist that regulates the expression of key genes in cholesterol homeostasis in macrophage cells without inducing hepatic lipid accumulation.

Highlights: Black-Right-Pointing-Pointer Catalposide is a novel ligand for PPAR{alpha}. Black-Right-Pointing-Pointer Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid {beta}-oxidation and synthesis. Black-Right-Pointing-Pointer Catalposdie reduces hepatic triacylglycerides. Black-Right-Pointing-Pointer Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPAR{alpha}) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPAR{alpha} agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPAR{alpha} agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPAR{alpha}. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while thatmore » of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPAR{alpha} via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.« less

The present study reports a novel liver X receptor (LXR) activator, ethyl 2,4,6-trihydroxybenzoate (ETB), isolated from Celtis biondii. Using a reporter gene assay, time-resolved fluorescence resonance energy transfer (TR-FRET), and surface plasmon resonance (SPR) analysis, we showed that ETB directly bound to and stimulated the transcriptional activity of LXR-α and LXR-β. In macrophages, hepatocytes, and intestinal cells, ETB suppressed cellular cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes. Notably, ETB did not induce lipogenic gene expression or cellular triglyceride accumulation in hepatocytes. These results suggest that ETB is a dual-LXR modulator that regulates the expression of key genes in cholesterol homeostasis in multiple cells without inducing lipid accumulation in HepG2 cells.

The consumption of soy protein and fiber reduces body fat accumulation; however, the mechanism of this effect has not been clearly understood. We investigated the antiobesogenic effect of soy protein and fiber in two different mouse models. Normolipidemic nonobese C57BL/6J and hyperlipidemic obese human apolipoprotein E2 transgenic mice were fed either delipidated soybean (DLSB) containing soy protein and fiber or a control diet. The DLSB-fed mice showed a significant reduction in body weight gain and adiposity compared with controls, in both C57BL/6J and apoE2 mice. All metabolic parameters were significantly improved in the DLSB group compared with controls: total cholesterol, low-density lipoprotein cholesterol, insulin, and leptin levels were significantly reduced. Adiponectin concentrations were significantly elevated, and glucose tolerance was improved. In both types of DLSB-fed mice, the specific induction of PPAR-δ protein expression was evident in muscle and adipose tissues. The expression of PPAR-δ target genes in the DLSB-fed mice was also significantly altered. Acetyl-CoA carboxylase-1 and fatty acid synthase levels in adipose tissue were downregulated, and uncoupling protein-2 in muscle was upregulated. Intestinal expression of fatty acid transport protein-4, cluster of differentiation-36, and acyl-CoA synthetase were significantly downregulated. We propose that marked activation of PPAR-δ is the primary mechanism mediating the antiobesogenic effect of soybean and that PPAR-δ has multiple actions: induction of thermogenesis in muscle, reduction of fatty acid synthesis in adipose tissue, and reduction of fatty acid uptake in intestinal tissue.

In this study, we confirmed that ursolic acid, a plant triterpenoid, activates peroxisome proliferator-activated receptor (PPAR)-α in vitro. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses do not show direct binding of ursolic acid to the ligand-binding domain of PPAR-α; however, ursolic acid enhances the binding of PPAR-α to the peroxisome proliferator response element in PPAR-α-responsive genes, alters the expression of key genes in lipid metabolism, significantly reducing intracellular triglyceride and cholesterol concentrations in hepatocytes. Thus, ursolic acid is a PPAR-α agonist that regulates the expression of lipid metabolism genes, but it is not a direct ligand of PPAR-α.

Scope: Enzymatically modified rice starch (ERS) synthesized with 4-a-glucanotransferase has a longer structure than rice starch, which could delay digestion, similar to dietary fiber. We investigated the effects of ERS on glucose and lipid metabolism with mice fed a high-fat diet containing ERS (HFD-ERS). Method and results: Four weeks of ERS feeding showed hypoglycemic effects with a significant reduction in fasting glucose (46%), insulin (57%), and leptin (83%) levels; improved glucose tolerance (20% in AUC of oral glucose tolerance test); and increased adiponectin concentrations (127%) compared to the HFD group. Notably, phosphorylation of AMP kinase (AMPK) was markedly induced in the HFD-ERS livers compared to HFD livers. Additionally, ERS significantly reduced total cholesterol concentrations with induction of fecal bile acid excretion (121%, Po0.05) in the HFD-ERS group compared to the HFD group. The mRNA and protein expressions of hepatic LDL receptors were significantly induced. However, cholesterol 7 alpha-hydroxylase (CYP7A1) expression was downregulated possibly due to induction of intestinal farnesoid X receptor (FXR; 12.4-fold, po0.05) and fibroblast growth factor-15 (FGF-15; 12.2-fold, po0.01). Conclusion: Our data suggest that ERS feeding may have hypoglycemic and hypocholesterolemic effects via a mechanism similar to that of dietary fiber.

The potential toxicity of fucoidan from Undaria pinnatifida was investigated in vitro and in vivo. By the Ames test, fucoidan showed no mutagenicity up to 500 microL/plate, and inhibited the mutagenicity induced by 4-nitro-quinoline-1-oxide, by up to 71%, compared with controls. In the bone marrow micronucleus test, fucoidan, at all levels tested, did not change the micronucleated polychromatic erythrocyte percentage ratio in mouse bone marrow cells. As an acute in vivo toxicity test, fucoidan from 0 to 2000 mg/kg body weight per day was administered orally to Sprague-Dawley rats for 28 days. No significant toxicological change was induced by fucoidan treatment up to 1000 mg/kg body weight per day in biochemical analyses, hematological analyses, necropsy and liver histopathology. The plasma ALT level was slightly, but significantly, increased in male rats at 2000 mg/kg/day. The consumption of fucoidan from Undaria pinnatifida, up to 1000 mg/kg body weight per day, may be safe in rodents, with no sign of toxicity after up to 28 days of daily administration.

Furan is a colorless, volatile compound that is found in heat-treated foods, such as canned and jarred foods, at levels up to 100 ppb. When animals ingest high doses, furan metabolites, such as cis-2-butene-1,4-dial, cause severe hepatotoxicity and carcinogenicity. However, the levels and effects of furan on humans are not known. Therefore, we measured urinary furan in 100 healthy individuals consumed normal diet (49 men, 51 women) using solid phase micro-extraction–gas chromatography–mass spectrometry (SPME–GCMS). Urinary furan was detected in 56 subjects (31 males, 25 females) and ranged up to 3.14 ppb. In individuals with detectable urinary furan, the level of c-glutamyltranspeptidase (c-GT), a marker for liver damage, was strongly correlated with the urinary furan concentration (r = 0.56, p < 0.0001). Linear regression analysis indicated that the urinary furan level was significantly associated with c-GT in both univariate (p < 0.0001) and multiple (p = 0.0001) models including age, sex, body weight, and blood pressure as covariates. To our knowledge, this is the first study to measure detectable levels of furan in human urine. These levels of urinary furan, which may be dietary origin, could be hepatotoxic in humans; therefore; the metabolic fates and potential toxicity of dietary furan in humans should be investigated further. 2008 Elsevier Ltd. All rights reserved.

Monocyte adhesion to vascular endothelium is an initial step in atherogenesis. To quantify this, we incubated monocytes with cultured endothelial cells, and quantified the adhered live monocytes using a colorimetric assay. Endothelium activated with lipopolysaccharide attracted monocytes in a dose-dependent manner and the adhesion was attenuated with post-treatments with L-ascorbic acid (53%), alpha- (40%) and gamma-tocopherol (39%), resveratrol (39%), and Lithospermum erythrorhizon root extract (45%). This non-radioactive, colorimetric assay may be useful for screening anti-atherogenic compounds in early atherogenesis.

Genistin has antioxidant activities; however, its insolubility in water often limits its biological availability in vivo. Using a novel transglycosylation process, the solubility of genistin glycosides was increased 1000 to 10000-fold, but it was not known whether these modified genistin glycosides maintained antioxidant activity. We found that both genistin and its glycosides similarly up-regulated the transcription of several metallothionein (MT) antioxidant genes (MT1A, MT2A, MT1E, and MT1X), as well as the glucose 6-phosphate dehydrogenase (G6PD) gene in HepG2 cells. This gene induction was mediated by the sequestration of zinc in the cytosol, which up-regulated the metal-responsive transcription factor-1 (MTF-1) that induced MT gene expression. Although not as effective as ascorbic acid, genistin glycosides possessed slightly greater reducing power than genistin. We concluded that genistin and genistin glycosides have a direct antioxidant effect and an indirect antioxidant effect, perhaps via induction of MT by activity of MTF-1.

Wormwood ( Artemisia princeps ) due to the abundance of antioxidant in its essential oils (EO), has been used as a traditional drug and health food in Korea. Oxidative stress plays an important role in the etiology of atherosclerosis thus antioxidative chemicals improves hepatic lipid metabolism partly by reducing oxysterol formation. The antioxidant activity was assessed using two methods, human low-density lipoprotein (LDL) oxidation and the anti-DPPH free radical assays. It was found that the antioxidant activity of EO with vitamin E higher than EO alone. To study mechanisms accounting for the antiatherosclerotic properties of this wormwood EO, we examined the expression of key genes in cholesterol metabolism such as the LDL receptor, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and sterol regulatory element binding proteins. The induction was increased up to twofold at 0.05 mg/mL of EO treatment in HepG2 cells for 24 h. When EO (0.2 mg/mL) was co-incubated with vitamin E, interestingly, the LDL receptor was dramatically induced by 5–6-folds. HMG-CoA reductase did not change. However, treatment with the higher concentration resulted in cytotoxicity. Our data suggest that wormwood EO with vitamin E may be anti-atherogenic due to their inhibition of LDL oxidation and upregulation of the LDL receptor.

We investigated hypocholesterolemic mechanisms of linalool, an aromatic anti-oxidative monoterpene, which is abundant in teas and essential oils. Oral administration of linalool to mice for 6weeks significantly lowered total and low-density lipoprotein cholesterol concentrations, and HMG-CoA reductase protein expression (−46%; P