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Supplemental material, sj-docx-1-npx-10.1177_1934578X231157145 for Chemical Constituents From the Marine Microalgae Thraustochytrium pachydermum by Nguyen Thi Thu Thuy, Hoang Thi Minh Hien, Nguyen Cam Ha and Le Thi Thom, Dang Diem Hong, Nguyen Van Thinh, Nguyen Trong Dan, Nguyen Dang Hoi, Vu Thi Loan, Hoang Duc Quang, Dan Thi Thuy Hang, Phan Van Kiem, Nguyen Tien Dat, Nguyen Xuan Nhiem in Natural Product Communications

Marine microalgae are an important source of raw materials for the extraction of highly bioactive substances for humans and domestic animals. In this study, the biological characteristics and the ability to grow biomass of green marine microalga of Dunaliella tertiolecta NY isolated from seawater of Namyit Island belonging to Spratly Islands, Vietnam (in May-June, 2021) was presented. Scientific name of the strain Dunaliella tertiolecta NY based on morphological characteristics and analysis of the sequence of rDNA ITS1-5.8S-ITS2 region was identified (with accession number of this gene region of this strain was supplied on the GenBank is OM 101011). At the best conditions for the growth (i.e. Walne medium, initial cell density of 2.0 x 106 cells/mL, growth temperature of 30oC, light intensity of 60 - 100 µmol/m2s, pH 7, salinity of 30-40‰), highest NY strain cell density of 8.63 x 106 cells/mL was obtained after 15 days of culture. The microalga D. tertiolecta NY was also successfully cultured on a pilot scale in the plastic bottles 10 L and closed photobioreactors 20 - 100 L resulting in a high biomass productivity of 285 mg/L/day and a biomass rich in polyunsaturated fatty acids such as gamma - linolenic acid (C18:3 ω-6; 5.07±0.59 % of total fatty acid - TFA), alpha - linolenic acid (ALA, C18:3 ω-3, 61.79±1.98 % of TFA) and stearidonic acid (SDA; C18: 4 ω-3, 1.59±0.65 % of TFA) qualified for the extraction of value bioactive compounds.

In this study, marine microalgal communities in 12 islands belonging to the sea water region of Truong Sa Islands (Spratly Islands), Vietnam in May-June and October-November 2021 was studied. There were 305 species of marine microalgae belonging to 7 phyla were identified in 12 islands in this sea water region of Truong Sa Islands (Spratly Islands) in 2021 in which with 39 dominant species, highest species number of 147 in Central (London) Reef (D1) and the lowest species number of 15 in Ambonya Cay (D4) were detected. Up to 2021, in the sea water region of the Truong Sa Islands (Spratly Islands), 14 genera of toxic harmful algae have appeared. The average value of biodiversity index (H') and the diversity value index (Dv) in all 12 studied islands in the Truong Sa Islands (Spratly Islands) in 2021 is 3.61±0.40 and 3.43±0.39, respectively, showing that the diversity of marine microalgae of this sea water region is rich and the quality of its sea water is generally good. It has been determined that two main factors NO3- and oil content, followed by DO, salinity, temperature and Hg content in seawater were affected the density of marine microalgal cells in the sea water of Truong Sa Islands (Spratly Islands) in 2021. In which, the marine microalgal cell density has a negative correlation with temperature, salinity, NO3- and Hg content of seawater and has a positive correlation with DO parameter was discovered.

The down-regulation of zeaxanthin (Zx) epoxidation is important for the regulation of Zx accumulation in xanthophyll cycle and for the development of non-photochemical quenching (NPQ). The NPQ development and Zx accumulation kinetics in rice, barley, and spinach leaves under light of different intensities were highly similar among the three plants. When the leaves were pre-treated with an inhibitor of Zx epoxidase (ZE), salicylaldoxime (SA), the two kinetics patterns in the leaves under low and moderately high light intensities became similar to those of high light intensity-treated leaves. Therefore, we propose that reversible down-regulation of Zx epoxidation plays an important role in plants, and this reversible down-regulation mechanism is a general mechanism in plants which occurs at room temperature under various light conditions as well as under different stress conditions in the presence of light. This reversible down-regulation is different from the irreversible down-regulation mechanism of ZE which involves ZE protein degradation together with D1 protein degradation under photooxidative conditions. There will be discussion on the mechanisms for the actual regulation of ZE activities involving phosphorylation/dephosphorylation of still unknown regulator(s) and/or by the redox regulation involving NADPH thioredoxin reductase C and thioredoxin m.

Schizochytrium mangrovei strain PQ6 was investigated for coproduction of docosahexaenoic acid (C22: 6ω-3, DHA) and squalene using a 30-L bioreactor with a working volume of 15 L under various batch and fed-batch fermentation process regimes. The fed-batch process was a more efficient cultivation strategy for achieving higher biomass production rich in DHA and squalene. The final biomass, total lipid, unsaponifiable lipid content, and DHA productivity were 105.25 g · L-1 , 43.40% of dry cell weight, 8.58% total lipid, and 61.66 mg · g-1 · L-1 , respectively, after a 96 h fed-batch fermentation. The squalene content was highest at 48 h after feeding glucose (98.07 mg · g-1 of lipid). Differences in lipid accumulation during fermentation were correlated with changes in ultrastructure using transmission electron microscopy and Nile Red staining of cells. The results may be of relevance to industrial-scale coproduction of DHA and squalene in heterotrophic marine microalgae such as Schizochytrium.

When the leaf segments of rice (Oryza sativa L.) plants were subjected to chilling in the moderate light, zeaxanthin (Zx) formation was faster in a chilling-tolerant Dongjin-byeo (DJ) than in a chilling-sensitive IR841. Although the rate of Zx formation was accelerated by the treatment of 5 mM salicylaldoxime, an inhibitor of Zx epoxidase (ZE), there was almost no changes in DJ. A similar result was observed when leaf segments were treated with 50 mM sodium fluoride, a potent inhibitor of chloroplast phosphatase. The slow Zx epoxidation in IR841 during light-chilling was confirmed in leaf segments treated with 10 mM dithiothreitol, an inhibitor of violaxanthin de-epoxidase (VDE). However, the differences between the two cultivars were not observed at 25oC. These results suggest that compared with IR841 the higher rate of Zx formation in DJ is not due to the higher VDE activity in DJ but is due to more rapid down-regulation of ZE in DJ, possibly by its phosphorylation. Compared with DJ, IR841 accumulated more superoxide with PSI inactivation during light-chilling, which eliminates the possibility of increased ZE down-regulation in DJ leaves by photo-oxidation. In vitro study with alkaline phosphatase supports the idea of down-regulation of ZE by phosphorylation under light-chilling condition. We propose that this reversible down-regulation of Zx epoxidation possibly by the phosphorylation of ZE is an important regulation mechanism of violaxanthin cycle that confers chilling tolerance of a rice cultivar under chilling stress in the light with moderate intensities.

In this paper, we studied the mechanism of the triglyceride (TG)-lowering effect of kaempferol in vitro and in vivo. Kaempferol showed LXR agonistic activities without inducing TGs or the expression of several lipogenic genes in cultured cells. A luciferase and qPCR analysis showed that kaempferol increased the transactivation of PPARα and PPARδ and stimulated gene expression associated with fatty acid oxidation and uptake in hepatocytes. More importantly, kaempferol inhibited protein kinase B (Akt) activity and suppressed SREBP-1 activation via multiple mechanisms, including through increasing Insig-2a expression, reducing SREBP-1 phosphorylation, and increasing GSK-3 phosphorylation. Collectively, these actions inhibited the SREBP-1 activation process. Furthermore, as an Akt/mTOR pathway inhibitor, kaempferol led to the induction of hepatic autophagy and resulted in a decrease in lipid droplet formation in the mouse liver. These findings demonstrate that kaempferol exerts its TG-lowering effect via Akt inhibition and activation of PPARα and PPARδ. PRACTICAL APPLICATIONS: Kaempferol is a major dietary flavonoid in various plant-based foods, and it is used as a valuable ingredient in functional foods, with numerous beneficial properties such as anticancer, antioxidant, and anti-atherosclerotic activities. Kaempferol exerts its TG-lowering effect via Akt inhibition and activation of PPARα and PPARδ. Currently, the number of people with hyperlipidemia is rapidly growing in both developed and developing societies; thus, we propose that kaempferol could be used for therapeutic interventions aimed at the treatment of these individuals.

Marine microalga Isochrysis sp. was isolated from Hai Phong area of Vietnam coastal zone. Their pure cultures were raised in the laboratory for culture optimization, molecular identification and biochemical analysis. Based on the morphological studies the newly isolated species was identified as Isochrysis galbana Parke and was confirmed by 18S rRNA gene analysis. The nucleotide sequence of partial 18S rRNA gene was submitted to the GENEBANK with accession number FJ536744. Optimum conditions for growth of the species was f/2 medium at temperature 25 ± 1°C, salinity 30 ± 1 psu, pH 7.0 and light intensity of 100 μmol m−2 s−1. Under these optimum conditions, the content of protein, carbohydrate and total lipid were 27.981 ± 0.892%, 25.263 ± 0.789% and 9.780 ± 0.120% of dry cell weight, respectively (equivalent 38.768 ± 0.892%, 35.000 ± 0.789% and 13.550 ± 0.120% of ash free dry weight). I. galbana Parke, strain HP has the highest content of docosahexanenoic acid (DHA), up to 14.7% of total fatty acid and maximal polyunsaturated fatty acids (PUFAs) values at the early stationary phase in f/2 medium. The n-3 and n-6 PUFA ratio (n-3/n-6) of strain HP was from 4.50 to 5.69 at different phases of growth curve. These results suggest that biomass of this strain can be used as live feed in aquaculture.

Polysaccharides from seaweed have different biological activities. Two types of sulfated polysaccharides (SPs) were purified from Monostroma nitidum (MF1 and MF2) and investigated for biological activities in vitro. The MF1 and MF2 fractions exhibited strong antioxidant activities assessed using superoxide dismutase (SOD) assays. Stimulation of lipid-loaded hepatocytes by the MF1 and MF2 fractions significantly (p

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 mechanisms underlying the antiobesogenic properties of black-colored rice (BCR; Oryza sativa L. cv. Heugkwangbyeo) with high levels of anthocyanins. Stimulation of cultured adipocytes with BCR extract caused a significant decrease in cellular lipid reduction, and C57BL/6J mice fed BCR extract for 8 weeks exhibited significant reductions in body weight and body fat. Plasma concentrations of leptin and insulin decreased, while levels of adiponectin increased in BCR group. These metabolic alterations were mediated by the activation of peroxisome proliferator-activated receptors (PPARs), activating PPAR-α while inhibiting PPAR-γ. These changes were accompanied by down-regulation and up-regulation of the genes involved in hepatic fatty acid synthesis and fatty acid oxidation, respectively. Evaluation of gene expression in adipocytes also suggested that those involved in lipid accumulation were reduced, with a concomitant induction of the genes involved in lipid hydrolysis. The plasma metabolites in mice rendered obese by intake of a high-fat-diet were analyzed using GC-TOF-MS and multivariate analysis after being fed different diets for 8 weeks: control, Garcinia cambogia, control rice (O. sativa L. cv. Ilpumbyeo), and BCR. The BCR group could be distinguished from the others after 4 weeks of the intervention using multivariate analysis. The major components related to this distinction were mannose, isoleucine, threonine, and phenylalanine. Also, body weight, glucose, and total cholesterol were positively correlated with the levels of propionic acid, butyric acid, cholesterol, alanine, ribitol, glucose, and galactose, whereas the high-density lipoprotein (HDL)/non-HDL ratio was negatively correlated with those of stearic acid, cholesterol, valine, leucine, glucose, and galactose.

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

The heterotrophic marine microalga, Schizochytrium mangrovei PQ6, synthesizes large amounts of polyunsaturated fatty acids (PUFAs) with possible nutritional applications. We characterized the transcriptome of S. mangrovei PQ6, focusing on lipid metabolism pathways throughout growth. Cell growth, total lipid, and docosahexaenoic acid (DHA, 22:6n-3) contents of S. mangrovei PQ6 in 500 ml batch cultures rapidly increased on day 1 in cultivation and reached their maximum levels on day 5. Maximum lipid accumulation in 500 ml batch cultures occurred on day 5, with total lipid and DHA contents reaching 33.2 ± 1.25% of dry cell weight (DCW) and 136 mg/g DCW, respectively. 11,025 unigenes, 28,617 unigenes and 18,480 unigenes from the transcriptomes of samples collected on day 1, 3, and 5 in cultivation were identified, respectively. These unigenes of the three samples were further assembled into 30,782 unigenes with an average size of 673 bp and N50 of 950 bp, and a total of 9,980 unigenes were annotated in public protein databases. 93 unigenes involved in lipid metabolism in which expression patterns corresponded with total lipid and DHA accumulation patterns were identified. The possible roles of PUFAs pathways, such as those mediated by fatty acid synthase, polyketide synthase, and desaturase/elongase, co-exist in S. mangrovei PQ6.

Fucosterol, a sterol that is abundant in marine algae, has hypocholesterolemic activity, but the mechanism underlying its effect is not clearly understood. Because data suggest that fucosterol can increase plasma high-density lipoprotein concentrations, we investigated whether it could activate liver X receptors (LXRs), critical transcription factors in reverse cholesterol transport. Fucosterol dose-dependently stimulated the transcriptional activity of both LXR-α and -β in a reporter gene assay, responses that were attenuated by the LXR antagonist As(2)O(3). Fucosterol also activated co-activator recruitment in cell-free time-resolved fluorescence resonance energy transfer analysis. In THP-1-derived macrophages, it induced the transcriptional activation of ABCA1, ABCG1, and ApoE, key genes in reverse cholesterol transport, and thereby significantly increased the efflux of cholesterol. Fucosterol also regulated intestinal NPC1L1 and ABCA1 in Caco-2 cells. Notably, fucosterol did not induce cellular triglyceride accumulation in HepG2 cells, primarily because of its upregulation of Insig-2a, which delays nuclear translocation of SREBP-1c, a key hepatic lipogenic transcription factor. These results suggest that fucosterol is a dual-LXR agonist that regulates the expression of key genes in cholesterol homeostasis in multiple cell lines without inducing hepatic triglyceride accumulation.

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.

Red yeast (Monascus purpureus) fermented over rice has a limited hypoglycemic activity. To enhance its glucose-lowering effect, we fermented red yeast over waxy barley, a hypoglycemic grain with high levels of fibers and β-glucans, and investigated the metabolic effects of red yeast barley (RYB) in high-fat-fed hyperglycemic db/db mice for 6 weeks. The fasting glucose levels were significantly reduced in the RYB group at 6 weeks by 25% (p

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.

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.

Peroxisome proliferator-activated receptor-alpha (PPARα) is a nuclear receptor that has critical roles in the treatment of atherosclerosis, hyperlipidemia and hepatic steatosis. Squalene is a novel nature PPARα agonist, identified from reporter gene assay and qPCR analysis. Cultured hepatocytes stimulated with squalene exhibited significantly decreased cellular triacylglycerols and cholesterol concentrations, while cellular uptake of fatty acids was increased. Quantitative PCR analysis revealed that expression of genes related to fatty acid uptake, fatty acid oxidation, ketogenesis and reverse cholesterol transport metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed in cell treated with squalene. Squalene is hypolipidemic by activation of PPARα via a ligand-mediated mechanism that regulates the expression of lipid metabolism genes in hepatocytes.

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.

Today microalgae represent a viable alternative source of squalene for commercial application. The species Schizochytrium mangrovei, a heterotrophic microalga, has been widely studied and provides a high amount of squalene, polyunsaturated fatty acids and has good profiles for biodiesel production. Our work was aimed at examining the squalene contents in Vietnam's heterotrophic marine microalga S. mangrovei PQ6 biomass and residues of the biodiesel process from this strain. Thin-layer chromatography and high-performance liquid chromatography (HPLC) methods were successfully applied to the determination of squalene in S. mangrovei PQ6. The squalene content and production of S. mangrovei PQ6 reached 33.00 ± 0.02 and 33.04 ± 0.03 mg g(-1) of dry cell weight; and 0.992 g L(-1) and 1.019 g L(-1) in 30 and 150 L bioreactors, respectively after 96 h of fermentation. In addition, squalene was also detected in spent biomass (approximately 80.10 ± 0.03 mg g(-1) of spent biomass) from the S. mangrovei PQ6 biodiesel production process. The structure of squalene in residues of the biodiesel process was confirmed from its nuclear magnetic resonance spectra. The results obtained from our work suggest that there is tremendous potential in the exploitation of squalene as a value-added by-product besides biodiesel from S. mangrovei PQ6 to reduce biodiesel price.

We investigated the hypolipidemic effects of Goami-3 rice (GR; Oryza sativa L. cv. Goami-3), a newly developed strain with high levels of amylose and fibers. Diet-induced obese mice were fed three types of isocaloric diets for 8 weeks: a high-fat diet, a high-fat diet with GR or control rice (CR; O. sativa L. cv. Ilpumbyeo). Mice fed GR exhibited a significant reduction in body fat (–23%), total cholesterol (–20%) and triglyceride concentrations (–30%) compared to mice fed CR. The mice fed GR showed induction of peroxisome proliferator-activated receptor (PPAR)-α and inhibition of γ expressions in the liver and adipose tissue. The reduced adiposity of mice fed GC was supported by changes in the expression of genes related to lipid accumulation and hydrolysis in adipose tissues and the plasma concentrations of insulin, adiponectin and leptin. Principal components analysis with gas chromatography–time-of-flight mass spectrometry-based metabolomic data revealed that the average level of specific plasma metabolites in the GR group was statistically different from that in the other groups after 4 weeks. These metabolites included propionic acid, valine, leucine and proline. Based on partial least-squares analysis, the plasma concentrations of valine were inversely correlated with the high-density lipoprotein (HDL) to non-HDL and HDL to total cholesterol ratios. In conclusion, GR feeding for 8 weeks significantly improved dyslipidemia and adiposity in diet-induced obese mice by regulating gene expression of PPARs and its target genes. Key plasma metabolites (including valine) were significantly altered by the hypolipidemic effects of GR.

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 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

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 μg/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.

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.

Recent data suggest that the etiology of several metabolic diseases is closely associated with transcriptome alteration by aberrant histone methylation. We performed DNA microarray and ChIP-on-chip analyses to examine transcriptome profiling and trimethylation alterations to identify the genomic signature of nonalcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease. Transcriptome analysis showed that steatotic livers in high-fat diet-fed apolipoprotein E2 mice significantly altered the expression of approximately 70% of total genes compared with normal diet-fed control livers, suggesting that hepatic lipid accumulation induces dramatic alterations in gene expression in vivo. Also, pathway analysis suggested that genes encoding chromatin-remodeling enzymes, such as jumonji C-domain-containing histone demethylases that regulate histone H3K9 and H3K4 trimethylation (H3K9me3, H3K4me3), were significantly altered in steatotic livers. Thus, we further investigated the global H3K9me3 and H3K4me3 status in lipid-accumulated mouse primary hepatocytes by ChIP-on-chip analysis. Results showed that hepatic lipid accumulation induced aberrant H3K9me3 and H3K4me3 status in peroxisome proliferator-activated receptor alpha and hepatic lipid catabolism network genes, reducing their mRNA expression compared with non-treated control hepatocytes. This study provides the first evidence that epigenetic regulation by H3K9me3 and H3K4me3 in hepatocytes may be involved in hepatic steatosis and the pathogenesis of NAFLD. Thus, control of H3K9me3 and H3K4me3 represents a potential novel NAFLD prevention and treatment strategy.

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 0.05), whereas plasma triglyceride levels were significantly lower in OVX mice (-15%, p 0.05) compared with controls. Abdominal adipose tissue weight was marginally reduced in IPL-fed groups compared with OVX controls and the plasma levels of liver enzymes were unchanged. In addition, IPL significantly inhibited the reduction of bone mineral density in the femurs of OVX mice (OVX200, +22%; OVX500, +26%; p 0.05) compared with controls after 4 weeks of IPL feeding. In quantitative polymerase chain reaction analysis the expression of aromatase was significantly suppressed and SULT1E1 was increased by IPL feeding, showing that IPL feeding may not alter the risk for breast cancer in mice. Our results suggest that IPL could ameliorate menopausal symptoms in mice. Further studies will confirm the effects of IPL in humans.

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.

The report gives study results assessing the change in productivity of some staple food crops in Vietnam and assessing the influence and losses caused by climatic disturbances in agricultural production.