Minh Hien Hoang

Ursolic acid is a PPAR-α agonist that regulates hepatic lipid metabolism

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.

Cyanidin is an agonistic ligand for peroxisome proliferator-activated receptor-alpha reducing hepatic lipid.

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.

The natural carotenoid astaxanthin, a PPAR-α agonist and PPAR-γ antagonist, reduces hepatic lipid accumulation by rewiring the transcriptome in lipid-loaded hepatocytes

SCOPE 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.

Hepatic Lipid Accumulation Alters Global Histone H3 Lysine 9 and 4 Trimethylation in the Peroxisome Proliferator-Activated Receptor Alpha Network

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.

Fucosterol Is a Selective Liver X Receptor Modulator That Regulates the Expression of Key Genes in Cholesterol Homeostasis in Macrophages, Hepatocytes, and Intestinal Cells

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.

Taurine is a liver X receptor-α ligand and activates transcription of key genes in the reverse cholesterol transport without inducing hepatic lipogenesis.

SCOPE 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.

Barley intake induces bile acid excretion by reduced expression of intestinal ASBT and NPC1L1 in C57BL/6J mice

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.

Melissa officinalis Essential Oil Reduces Plasma Triglycerides in Human Apolipoprotein E2 Transgenic Mice by Inhibiting Sterol Regulatory Element-Binding Protein-1c–Dependent Fatty Acid Synthesis

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.

Induction of ABCA1 and ABCG1 expression by the liver X receptor modulator cineole in macrophages

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 <0.05). Accordingly, hepatocytes treated with cineole displayed reduced cellular lipid accumulation compared with control cells, as assessed by Oil Red O lipid staining and cholesterol quantification. These results suggest that cineole is a selective LXR modulator that regulates the expression of key genes in reverse cholesterol transport in macrophages without inducing lipogenesis in hepatocytes. This selective LXR modulator may have practical implications for the development of hypocholesterolemic or anti-atherosclerotic agents and also suggests.

Cyanidin, a natural flavonoid, is an agonistic ligand for liver X receptor alpha and beta and reduces cellular lipid accumulation in macrophages and hepatocytes

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.