α1-COP modulates plasmodesmata function through sphingolipid enzyme regulation.

Callose, a ß-1,3-glucan plant cell wall polymer, regulates symplasmic channel size at plasmodesmata (PD) and plays a crucial role in a variety of plant processes. However, elucidating the molecular mechanism of PD callose homeostasis is limited. We screened and identified an Arabidopsis mutant plant with excessive callose deposition at PD and found that the mutated gene was α1-COP, a member of the coat protein I (COPI) coatomer complex. We report that loss of function of α1-COP elevates the callose accumulation at PD by affecting subcellular protein localization of callose degradation enzyme PdBG2. This process is linked to the functions of ERH1, an inositol phosphoryl ceramide synthase, and glucosylceramide synthase through physical interactions with the α1-COP protein. Additionally, the loss of function of α1-COP alters the subcellular localization of ERH1 and GCS proteins, resulting in a reduction of GlcCers and GlcHCers molecules, which are key sphingolipid (SL) species for lipid raft formation. Our findings suggest that α1-COP protein, together with SL modifiers controlling lipid raft compositions, regulates the subcellular localization of GPI-anchored PDBG2 proteins, and hence the callose turnover at PD and symplasmic movement of biomolecules. Our findings provide the first key clue to link the COPI-mediated intracellular trafficking pathway to the callose-mediated intercellular signaling pathway through PD.

Decrease of ceramides with long-chain fatty acids in psoriasis: Possible inhibitory effect of interferon gamma on chain elongation.

Reportedly, decreases in fatty acid (FA) chain length of ceramide (CER) are associated with interferon-γ (IFN-γ), which shows increased expression in psoriasis. However, the underlying mechanism of this association remains unclear. Therefore, in this study, we aimed to clarify this association between FA chain length of CER, IFN-γ, and the major transcriptional factors involving psoriasis. CER profiling according to FA chain length and class was performed in murine epidermis (n = 10 BALB/c mice topically treated with imiquimod, n = 10 controls) and human stratum corneum (SC) (n = 12 psoriasis, n = 11 controls). The expression of lipid synthetic enzymes, including elongases (ELOVLs), in murine epidermis was also measured using RT-PCR. Furthermore, the association of IFN-γ with various enzymes and transcription factors involved in the generation of long-chain CERs was also investigated using in vitro keratinocyte. A significant decrease in the percentage of long-chain CERs was observed in psoriasis-like murine epidermis and human psoriatic SC. Additionally, the expression levels of ELOVL1, ELOVL4, and ceramide synthase3 (CerS3) were significantly decreased in psoriasis-like murine epidermis and IFN-γ-treated keratinocyte. There was also a significant decrease in the expression of transcriptional factors, including peroxisome proliferator-activated receptor (PPAR), in IFN-γ treated keratinocyte. Thus, it could be suggested that IFN-γ may regulate ELOVL and CerS levels by down-regulating the transcriptional factors. Additionally, given the possible involvement of PPARs or liver X receptor agonist in the CER elongation process, they may serve as potential therapeutic agents for lengthening the CER FAs in psoriasis.

Sphingolipids Modulate Secretion of Glycosylphosphatidylinositol-Anchored Plasmodesmata Proteins and Callose Deposition.

Plasma membranes encapsulated in the symplasmic nanochannels of plasmodesmata (PD) contain abundant lipid rafts, which are enriched with sphingolipids (SLs) and sterols. Reduction of sterols has highlighted the role played by lipid raft integrity in the intercellular trafficking of glycosylphosphatidylinositol (GPI)-anchored PD proteins, particularly in affecting callose enhancement. The presence of callose at PD is strongly attributed to the regulation of callose accumulation and callose degradation by callose synthases and ß-1,3-glucanases (BGs), respectively. SLs are implicated in signaling and membrane protein trafficking; however, the underlying processes linking SL composition to the control of symplasmic apertures remain unknown. The wide variety of SLs in plants prompted us to investigate which SL molecules are important for regulating symplasmic apertures in Arabidopsis (Arabidopsis thaliana). We introduced several potential SL pathway inhibitors and genetically modified SL contents using two independent SL pathway mutants. We were able to modulate callose deposition to control symplasmic connectivity through perturbations of SL metabolism. Alteration in glucosylhydroxyceramides or related SL composition particularly disturbed the secretory machinery for the GPI-anchored PdBG2 protein, resulting in an overaccumulation of callose. Moreover, our results revealed that SL-enriched lipid rafts link symplasmic channeling to PD callose homeostasis by controlling the targeting of GPI-anchored PdBG2. This study elevates our understanding of the molecular linkage underlying intracellular trafficking and precise targeting of GPI-anchored PD proteins incorporating glucosyl SLs.

Integrated metabolomics and lipidomics reveals high accumulation of polyunsaturated lysoglycerophospholipids in human lung fibroblasts exposed to fine particulate matter.

Exposure to fine particulate matter (PM) comprising toxic compounds arising from air pollution is a major human health concern. It is linked to increased mortality and incidence of various lung diseases. However, the mechanisms underlying the toxic effects of PM on lung fibroblasts have not been fully explored. We used targeted quantitative metabolomics and lipidomics analysis along with cytotoxicity studies to comprehensively characterize the alterations in the metabolite profiles of human lung fibroblasts (HEL 299) upon exposure to PM2.5 and PM10. This exposure at 50 µg/mL for 72 h induced an abnormally high apoptotic response via triggering intracellular reactive oxygen species (ROS) production and mitochondrial dysfunction through an imbalance between pro- and anti-apoptotic signaling pathways. The cytotoxic effects of PM2.5 were more severe than those of PM10. Metabolomics and lipidomics analyses revealed that PM exposure triggered substantial changes in the cellular metabolite profile, which involved reduced mitochondria-related metabolites such as tricarboxylic acid (TCA) cycle intermediates, amino acids, and free fatty acids as well as increased lysoglycerophospholipids (LPLs) containing polyunsaturated fatty acids. The decrease in mitochondria-related metabolites suggested that PM exposure led to reduced TCA cycle capacity and energy production. Apoptotic and inflammatory responses as well as mitochondrial dysfunction were likely to be accelerated because of excessive accumulation of LPLs, contributing to the disruption of membrane rafts and Ca2+ homeostasis and causing increased mitochondrial ROS formation. These results provide valuable insights regarding the toxic effects of PM exposure. Our study also provides a new direction for research on PM exposure-related health disorders using different cell lines.

The impact of phenanthrene on membrane phospholipids and its biodegradation by Sphingopyxis soli.

The direct interactions of bacterial membranes and polycyclic aromatic hydrocarbons (PAHs) strongly influence the biological processes, such as metabolic activity and uptake of substrates due to changes in membrane lipids. However, the elucidation of adaptation mechanisms as well as membrane phospholipid alterations in the presence of phenanthrene (PHE) from α-proteobacteria has not been fully explored. This study was conducted to define the degradation efficiency of PHE by Sphingopyxis soli strain KIT-001 in a newly isolated from Jeonju river sediments and to characterize lipid profiles in the presence of PHE in comparison to cells grown on glucose using quantitative lipidomic analysis. This strain was able to respectively utilize 1-hydroxy-2-naphthoic acid and salicylic acid as sole carbon source and approximately 90% of PHE (50 mg/L) was rapidly degraded via naphthalene route within 1 day incubation. In the cells grown on PHE, strain KIT-001 appeared to dynamically change profiles of metabolite and lipid in comparison to cells grown on glucose. The levels of primary metabolites, phosphatidylethanolamines (PE), and phosphatidic acids (PA) were significantly decreased, whereas the levels of phosphatidylcholines (PC) and phosphatidylglycerols (PG) were significantly increased. The adaptation mechanism of Sphingopyxis sp. regarded mainly the accumulation of bilayer forming lipids and anionic lipids to adapt more quickly under restricted nutrition and toxicity condition. Hence, these findings are conceivable that strain KIT-001 has a good adaptive ability and biodegradation for PHE through the alteration of phospholipids, and will be helpful for applications for effective bioremediation of PAHs-contaminated sites.

Proteomic analysis of whole-body responses in medaka (Oryzias latipes) exposed to benzalkonium chloride.

Benzalkonium chloride (BAC) is a cationic surfactant commonly used as a disinfectant, and is discharged into the aquatic environment by various water sources such as wastewater. BAC may also interact with potentially toxic substances such as persistent organic chemicals. Although studies of BAC contamination toxicity and bioaccumulation have been widely reported, the biochemical responses to BAC toxicity remain incompletely understood, and the detailed molecular mechanisms are largely unknown. In this study, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry-based proteomic approaches were applied to investigate the protein profiles in Oryzias latipes (medaka) chronically exposed to BAC. Fish were exposed to three different concentrations of BAC, 0.05, 0.1, and 0.2 mg/L, for 21 days. A total of 20 proteins involved in the cytoskeleton, the oxidative stress response, the nervous and endocrine systems, signaling pathways, and cellular proteolysis were significantly upregulated by BAC exposure. The proteomic information obtained in the present study will be useful in identification of potential biomarkers for BAC toxicity, and begins to elucidate its molecular mechanisms, providing new insights into the ecotoxicity of BAC.

Deletion of PHO13 improves aerobic L-arabinose fermentation in engineered Saccharomyces cerevisiae.

Pentose sugars are increasingly being used in industrial applications of Saccharomyces cerevisiae. Although L-arabinose is a highlighted pentose that has been identified as next-generation biomass, arabinose fermentation has not yet undergone extensive development for industrial utilization. In this study, we integrated a heterologous fungal arabinose pathway with a deletion of PHO13 phosphatase gene. PHO13 deletion increased arabinose consumption rate and specific ethanol productivity under aerobic conditions and consequently depleted sedoheptulose by activation of the TAL1 gene. Global metabolite profiling indicated upregulation of the pentose phosphate pathway and downstream effects such as trehalose accumulation and downregulation of the TCA cycle. Our results suggest that engineering of PHO13 has ample potential for arabinose conversion to ethanol as an industrial source for biofuels.

Acetylshikonin is a novel non-selective cytochrome P450 inhibitor.

Acetylshikonin is a biologically active compound with anti-cancer and anti-inflammatory activity, which is isolated from the roots of Lithospermum erythrorhizoma. An inhibitory effect of acetylshikonin against CYP2J2 activity was discovered recently. Based on this result, this study was expanded to evaluate the inhibitory effects of acetylshikonin against nine different cytochrome P450 (P450) isoforms in human liver microsomes (HLMs) using substrate cocktails incubation assay. Acetylshikonin showed a strong inhibitory effect against all P450s tested with IC50 values of 1.4-4.0 µ m. Pre-incubation of acetylshikonin with HLMs and NADPH did not alter the inhibition potency, indicating that acetylshikonin is not a mechanism-based inhibitor. SKF-525A, a widely used non-specific P450 inhibitor, had no inhibitory activity against CYP1A2, 2A6, 2E1 and 2J2, while it showed an inhibitory effect against CYP2B6, CYP2C19 and 2D6 with IC50 values of 2.5, 3.6 and 0.5 µ m, respectively. Our findings indicate that acetylshikonin may be a novel general P450 inhibitor, which could replace SKF-525A.

Lipidomic platform for structural identification of skin ceramides with α-hydroxyacyl chains.

Skin ceramides are sphingolipids consisting of sphingoid bases, which are linked to fatty acids via an amide bond. Typical fatty acid acyl chains are composed of α-hydroxy fatty acid (A), esterified ω-hydroxy fatty acid (EO), non-hydroxy fatty acid (N), and ω-hydroxy fatty acid (O). We recently established a lipidomic platform to identify skin ceramides with non-hydroxyacyl chains using tandem mass spectrometry. We expanded our study to establish a lipidomic platform to identify skin ceramides with α-hydroxyacyl chains. Tandem mass spectrometry analysis of A-type ceramides using chip-based direct infusion nanoelectrospray-mass spectrometry showed the characteristic fragmentation pattern of both acyl and sphingoid units, which can be applied for structural identification of ceramides. Based on the tandem mass spectrometry fragmentation patterns of A-type ceramides, comprehensive fragmentation schemes were proposed. Our results may be useful for identifying A-type ceramides in the stratum corneum of human skin.

Simultaneous evaluation of substrate-dependent CYP3A inhibition using a CYP3A probe substrates cocktail.

Cytochrome P450 (P450) 3A (CYP3A) is an enzyme responsible for the metabolism of therapeutic drugs such as midazolam, nifedipine, testosterone and triazolam. It is involved in 40% of all cases of P450-mediated metabolism of marketed drugs. Therefore, it is important to evaluate the CYP3A-mediated drug interaction potential of new chemical entities (NCEs). In the past, one P450 isoform-specific probe substrate has been used at a time to evaluate the degree of inhibition of P450 isoforms by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, CYP3A enzymes have been shown to have a multi-substrate binding site. Therefore, multiple CYP3A substrates should be used to evaluate precisely the drug interaction potential of NCEs with the enzyme CYP3A. In this study, a method of screening NCEs for their potential to inhibit by CYP3A enzyme activity was developed. It involves the employment of a CYP3A substrate cocktail (including midazolam, testosterone and nifedipine). The concentration of each CYP3A probe substrate in vitro was optimized (0.1 µm for midazolam, 2 µm for testosterone and 2 µm for nifedipine) to minimize mutual drug interactions among probe substrates. The method was validated by comparing inhibition data obtained from the incubation of CYP3A with each individual substrate with data from incubation with a cocktail of all three substrates. The CYP3A inhibition profiles from the substrate cocktail approach were similar to those from the individual substrates approach. This new method could be an effective tool for the robust and accurate screening of the CYP3A inhibition potential of NCEs in drug discovery. Copyright © 2016 John Wiley & Sons, Ltd.

Danazol Inhibits Cytochrome P450 2J2 Activity in a Substrate-independent Manner.

Cytochrome P450 2J2 (CYP2J2) is an enzyme responsible for the metabolism of endogenous substrates including arachidonic acid, as well as therapeutic drugs such as albendazole, astemizole, ebastine, and terfenadine. Selective inhibitors of CYP2J2 are essential for P450 reaction phenotyping studies. To find representative CYP2J2 index inhibitors, we evaluated the inhibitory potential of danazol, hydroxyebastine, telmisartan, and terfenadone against CYP2J2 activity for four representative CYP2J2 substrates (albendazole, astemizole, ebastine, and terfenadine) using recombinant CYP2J2. Of these four CYP2J2 inhibitors, danazol strongly inhibited CYP2J2-mediated albendazole, astemizole, ebastine, and terfenadine metabolism in a substrate-independent manner, with IC50 values of 0.05, 0.07, 0.18, and 0.34 µM, respectively. Danazol noncompetitively inhibited CYP2J2-mediated astemizole O-demethylation activities with a Ki value of 0.06 µM. Terfenadone strongly inhibited CYP2J2-mediated albendazole, astemizole, and terfenadine metabolism (IC50 < 0.21 µM), whereas it showed weak inhibition against CYP2J2-catalyzed ebastine hydroxylase activity (IC50 = 6.04 µM). Telmisartan had no inhibitory effect on CYP2J2-mediated ebastine and terfenadine hydroxylation (IC50 > 20 µM). Taken together, these data suggest that danazol may be used as a CYP2J2 index inhibitor in reaction phenotyping studies.

Screening of non-steroidal anti-inflammatory drugs for inhibitory effects on the activities of six UDP-glucuronosyltransferases (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) using LC-MS/MS.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used widely to relieve pain and to decrease inflammation. Several clinical studies have reported that NSAIDs inhibit uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. Therefore, the study evaluated the inhibitory potential of 15 NSAIDs on the activities of six UGT isoforms (i.e. UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes (HLMs). Among the 15 NSAIDs tested here, mefenamic acid and diclofenac inhibited all UGTs tested in this study. Piroxicam and niflumic acid inhibited UGT1A9 activity (IC50 = 73.8 µm and 0.38 µm, respectively) and naproxen selectively inhibited UGT2B7 activity (IC50 = 53.1 µm), whereas it did not inhibit the other UGTs tested (IC50 > 200 µm). Diflunisal inhibited the UGT1A1 (IC50 = 33.0 µm) and UGT1A9 (IC50 = 19.4 µm). Acetaminophen, fenoprofen, ibuprofen, ketoprofen, meloxicam, phenylbutazone, salicylic acid and sulindac showed negligible inhibitory effects on the six UGTs (IC50 > 100 µm). These results suggest that some NSAIDs have the potential to inhibit UGTs in vitro.

In vitro metabolism of an estrogen-related receptor γ modulator, GSK5182, by human liver microsomes and recombinant cytochrome P450s.

GSK5182 (4-[(Z)-1-[4-(2-dimethylaminoethyloxy)phenyl]-hydroxy-2-phenylpent-1-enyl]phenol) is a specific inverse agonist for estrogen-related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N-desmethyl-GSK5182 and GSK5182 N-oxide, respectively, on the basis of liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. M2 was suggested to be hydroxy-GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N-demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N-oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo.

Urine and serum metabolite profiling of rats fed a high-fat diet and the anti-obesity effects of caffeine consumption.

In this study, we investigated the clinical changes induced by a high fat diet (HFD) and caffeine consumption in a rat model. The mean body weight of the HFD with caffeine (HFDC)-fed rat was decreased compared to that of the HFD-fed rat without caffeine. The levels of cholesterol, triglycerides (TGs), and free fatty acid, as well as the size of adipose tissue altered by HFD, were improved by caffeine consumption. To investigate the metabolites that affected the change of the clinical factors, the urine and serum of rats fed a normal diet (ND), HFD, and HFDC were analyzed using ultra performance liquid chromatography quadruple time-of-flight mass spectrometry (UPLC-Q-TOF-MS), gas chromatography (GC-TOF-MS), and linear trap quadruple mass spectrometry (LTQ-XL-MS) combined with multivariate analysis. A total of 68 and 52 metabolites were found to be different in urine and serum, respectively. After being fed caffeine, some glucuronide-conjugated compounds, lysoPCs, CEs, DGs, TGs, taurine, and hippuric acid were altered compared to the HFD group. In this study, caffeine might potentially inhibit HFD-induced obesity and we suggest possible biomarker candidates using MS-based metabolite profiling.

A lipidomic platform establishment for structural identification of skin ceramides with non-hydroxyacyl chains.

The stratum corneum (SC) is the outermost layer of skin that functions as a barrier and protects against environmental influences and transepidermal water loss. Its unique morphology consists of keratin-enriched corneocytes embedded in a distinctive mixture of lipids containing mainly ceramides, free fatty acids, and cholesterol. Ceramides are sphingolipids consisting of sphingoid bases, which are linked to fatty acids by an amide bond. Typical sphingoid bases in the skin are composed of dihydrosphingosine (dS), sphingosine (S), phytosphingosine (P), and 6-hydroxysphingosine (H), and the fatty acid acyl chains are composed of non-hydroxy fatty acid (N), α-hydroxy fatty acid (A), ω-hydroxy fatty acid (O), and esterified ω-hydroxy fatty acid (E). The 16 ceramide classes include several combinations of sphingoid bases and fatty acid acyl chains. Among them, N-type ceramides are the most abundant in the SC. Mass spectrometry (MS)/MS analysis of N-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, which could be applied to structural identification of ceramides. Based on the MS/MS fragmentation patterns of N-type ceramides, comprehensive fragmentation schemes were proposed. In addition, mass fragmentation patterns, which are specific to the sphingoid backbone of N-type ceramides, were found in higher m/z regions of tandem mass spectra. These characteristic and general fragmentation patterns were used to identify N-type ceramides in human SC. Based on established MS/MS fragmentation patterns of N-type ceramides, 52 ceramides (including different classes of NS, NdS, NP, and NH) were identified in human SC. The MS/MS fragmentation patterns of N-type ceramides were characterized by interpreting their product ion scan mass spectra. This information may be used to identify N-type ceramides in the SC of human, rat, and mouse skin.

Crosstalk between TM4SF5 and GLUT8 regulates fructose metabolism in hepatic steatosis.

OBJECTIVE: Transmembrane 4 L six family member 5 (TM4SF5) is likely involved in non-alcoholic steatohepatitis, although its roles and cross-talks with glucose/fructose transporters in phenotypes derived from high-carbohydrate diets remain unexplored. Here, we investigated the modulation of hepatic fructose metabolism by TM4SF5. METHODS: Wild-type or Tm4sf5-/- knockout mice were evaluated via different diets, including normal chow, high-sucrose diet, or high-fat diet without or with fructose in drinking water (30% w/v). Using liver tissues and blood samples from the mice or hepatocytes, the roles of TM4SF5 in fructose-mediated de novo lipogenesis (DNL) and steatosis via a crosstalk with glucose transporter 8 (GLUT8) were assessed. RESULTS: Tm4sf5 suppression or knockout in both in vitro and in vivo models reduced fructose uptake, DNL, and steatosis. Extracellular fructose treatment of hepatocytes resulted in an inverse relationship between fructose-uptake activity and TM4SF5-mediated translocalization of GLUT8 through dynamic binding at the cell surface. Following fructose treatment, TM4SF5 binding to GLUT8 transiently decreased with translocation to the plasma membrane (PM), where GLUT8 separated and became active for fructose uptake and DNL. CONCLUSIONS: Overall, hepatic TM4SF5 modulated GLUT8 localization and activity through transient binding, leading to steatosis-related fructose uptake and lipogenesis. Thus, TM4SF5 and/or GLUT8 may be promising treatment targets against liver steatosis resulting from excessive fructose consumption.

In Vitro Metabolism Study of Seongsanamide A in Human Liver Microsomes Using Non-Targeted Metabolomics and Feature-Based Molecular Networking.

Seongsanamide A is a bicyclic peptide with an isodityrosine residue discovered in Bacillus safensis KCTC 12796BP which exhibits anti-allergic activity in vitro and in vivo without significant cytotoxicity. The purpose of this study was to elucidate the in vitro metabolic pathway and potential for drug interactions of seongsanamide A in human liver microsomes using non-targeted metabolomics and feature-based molecular networking (FBMN) techniques. We identified four metabolites, and their structures were elucidated by interpretation of high-resolution tandem mass spectra. The primary metabolic pathway associated with seongsanamide A metabolism was hydroxylation and oxidative hydrolysis. A reaction phenotyping study was also performed using recombinant cytochrome P450 isoforms. CYP3A4 and CYP3A5 were identified as the major metabolic enzymes responsible for metabolite formation. Seongsanamide A did not inhibit the cytochrome P450 isoforms commonly involved in drug metabolism (IC50 > 10 µM). These results will contribute to further understanding the metabolism and drug interaction potential of various bicyclic peptides.

Plasma Lipidomics Reveals Insights into Anti-Obesity Effect of Chrysanthemum morifolium Ramat Leaves and Its Constituent Luteolin in High-Fat Diet-Induced Dyslipidemic Mice.

The Chrysanthemum morifolium Ramat (CM) is widely used as a traditional medicine and herbal tea by the Asian population for its health benefits related to obesity. However, compared to the flowers of CM, detailed mechanisms underlying the beneficial effects of its leaves on obesity and dyslipidemia have not yet been elucidated. Therefore, to investigate the lipidomic biomarkers responsible for the pharmacological effects of CM leaf extract (CLE) in plasma of mice fed a high-fat diet (HFD), the plasma of mice fed a normal diet (ND), HFD, HFD plus CLE 1.5% diet, and HFD plus luteolin 0.003% diet (LU) for 16 weeks were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with multivariate analysis. In our analysis, the ND, HFD, CLE, and LU groups were clearly differentiated by partial least-squares discriminant analysis (PLS-DA) score plots. The major metabolites contributing to this differentiation were cholesteryl esters (CEs), lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs), ceramides (CERs), and sphingomyelins (SMs). The levels of plasma CEs, LPCs, PCs, SMs, and CERs were significantly increased in the HFD group compared to those in the ND group, and levels of these lipids recovered to normal after administration of CLE or LU. Furthermore, changes in hepatic mRNA expression levels involved in the Kennedy pathway and sphingolipid biosynthesis were also suppressed by treatment with CLE or LU. In conclusion, this study examined the beneficial effects of CLE and LU on obesity and dyslipidemia, which were demonstrated as reduced synthesis of lipotoxic intermediates. These results may provide valuable insights towards evaluating the therapeutic effects of CLE and LU and understanding obesity-related diseases.

6,8-Diprenylorobol Induces Apoptosis in Human Hepatocellular Carcinoma Cells via Activation of FOXO3 and Inhibition of CYP2J2.

6,8-Diprenylorobol is a phytochemical derived from the roots of Glycyrrhiza uralensis Fisch. 6,8-Diprenylorobol exhibits several biological activities, but the effects of 6,8-diprenylorobol on cancers have been hardly investigated. This study is aimed at elucidating the anticancer effect and working mechanism of 6,8-diprenylorobol in HepG2 and Huh-7, two kinds of human hepatocellular carcinoma (HCC) cell lines. WST-1, cell counting, and colony formation assays and morphological change analysis showed that 6,8-diprenylorobol treatment decreased the cell viability and proliferation rate. Cell cycle analysis indicated that 6,8-diprenylorobol treatment increased the population of the G1/0 stage. Annexin V/PI double staining and TUNEL analysis showed that 6,8-diprenylorobol treatment increased the apoptotic cell population and DNA fragmentation. Western blot analysis showed that 6,8-diprenylorobol treatment increased the expression of cleaved PARP1, cleaved caspase-3, FOXO3, Bax, Bim, p21, and p27 but decreased the expression of Bcl2 and BclXL. Interestingly, 6,8-diprenylorobol inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities with K i values of 9.46 and 2.61 µM, respectively. CYP2J2 siRNA transfection enhanced the anticancer effect of 6,8-diprenylorobol in HepG2 and Huh-7 cells through the downregulation of CYP2J2 protein expression and upregulation of FOXO3. Taken together, this study proposes that 6,8-diprenylorobol treatment may be a useful therapeutic option against HCC by targeting CYP2J2 and FOXO3.

Chrysanthemum Leaf Ethanol Extract Prevents Obesity and Metabolic Disease in Diet-Induced Obese Mice via Lipid Mobilization in White Adipose Tissue.

This study aimed to elucidate the molecular mechanism of Chrysanthemum morifolium Ramat. against obesity and diabetes, by comparing the transcriptional changes in epididymal white adipose tissue (eWAT) with those of the bioactive compound in C. morifolium, luteolin (LU). Male C57BL/6J mice were fed a normal diet, high-fat diet (HFD), and HFD supplemented with 1.5% w/w chrysanthemum leaf ethanol extract (CLE) for 16 weeks. Supplementation with CLE and LU significantly decreased the body weight gain and eWAT weight by stimulating mRNA expressions for thermogenesis and energy expenditure in eWAT via lipid mobilization, which may be linked to the attenuation of dyslipidemia. Furthermore, CLE and LU increased uncoupling protein-1 protein expression in brown adipose tissue, leading to energy expenditure. Of note, CLE and LU supplements enhanced the balance between lipid storage and mobilization in white adipose tissue (WAT), in turn, inhibiting adipocyte inflammation and lipotoxicity of peripheral tissues. Moreover, CLE and LU attenuated hepatic steatosis by suppressing hepatic lipogenesis, thereby ameliorating insulin resistance and dyslipidemia. Our data suggest that CLE helps inhibit obesity and its comorbidities via the complex interplay between liver and WAT in diet-induced obese mice.