6-Dihydroparadol, a Ginger Constituent, Enhances Cholesterol Efflux from THP-1-Derived Macrophages

Scope: Ginger is reported to be used for the prevention and treatment of cardiovascular diseases (CVD). Cholesterol efflux from macrophage foam cells is an important process in reverse cholesterol transport, whose increase may help to prevent or treat CVD. In this study, we investigated the effects of 6‐dihydroparadol from ginger on macrophage cholesterol efflux. Methods and results: We show that 6‐dihydroparadol concentration‐dependently enhances both apolipoprotein A1‐ and human plasma–mediated cholesterol efflux from cholesterol‐loaded THP‐1‐derived macrophages using macrophage cholesterol efflux assay. 6‐Dihydroparadol increases protein levels of both ATP‐binding cassette transporters A1 and G1 (ATP‐binding cassette transporter A1 [ABCA1] and ATP‐binding cassette transporter G1 [ABCG1]) according to Western blot analysis. The ABCA1 inhibitor probucol completely abolishes 6‐dihydroparadol‐enhanced cholesterol efflux. Furthermore, increased ABCA1 protein levels in the presence of 6‐dihydroparadol were associated with both increased ABCA1 mRNA levels and increased ABCA1 protein stability. Enhanced ABCG1 protein levels were only associated with increased protein stability. Increased ABCA1 protein stability appeared to be the result of a reduced proteasomal degradation of the transporter in the presence of 6‐dihydroparadol. Conclusion: We identified 6‐dihydroparadol from ginger as a novel promoter of cholesterol efflux from macrophages that increases both ABCA1 and ABCG1 protein abundance. This newly identified bioactivity might contribute to the antiatherogenic effects of ginger.© 2018 The Author

Glucose availability is a decisive factor for Nrf2-mediated gene expression

Activation of the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is one of the major cellular defense lines against oxidative and xenobiotic stress, but also influences genes involved in lipid and glucose metabolism. It is unresolved whether the cytoprotective and metabolic responses mediated by Nrf2 are connected or separable events in non-malignant cells. In this study we show that activation of Nrf2, either by the small molecule sulforaphane or knockout of the Nrf2 inhibitor Keap1, leads to increased cellular glucose uptake and increased glucose addiction in fibroblasts. Upon Nrf2 activation glucose is preferentially metabolized through the pentose phosphate pathway with increased production of NADPH. Interference with the supply of glucose or the pentose phosphate pathway and NADPH generation not only hampers Nrf2-mediated detoxification of reactive oxygen species on the enzyme level but also Nrf2-initiated expression of antioxidant defense proteins, such as glutathione reductase and heme-oxygenase1. We conclude that the Nrf2-dependent protection against oxidative stress relies on an intact pentose phosphate pathway and that there is crosstalk between metabolism and detoxification already at the level of gene expression in mammalian cells

Erythrodiol, an Olive Oil Constituent, Increases the Half-Life of ABCA1 and Enhances Cholesterol Efflux from THP-1-Derived Macrophages

Cholesterol efflux (ChE) from macrophages is an initial step of reverse cholesterol transport (RCT). The ATP-binding cassette transporter A1 (ABCA1) is a key transporter for ChE and its increased expression is regarded to attenuate atherosclerosis. Thus, the identification and characterization of molecules raising ABCA1 and thereby stimulating ChE is of pharmacological relevance. In this study, we tested dietary compounds from olive oil for their capacity of enhancing cellular ABCA1 protein level. We identified erythrodiol (Olean-12-ene-3β,28-diol) as an ABCA1 stabilizer and revealed its positive influence on ChE in THP-1-derived human macrophages. Among the nine tested compounds from olive oil, erythrodiol was the sole compound raising ABCA1 protein level (at 10 μM). None of the tested compounds impaired viability of THP-1 macrophages from 5 to 20 μM as determined by resazurin conversion. Western blot analyses of key membrane transporters contributing to ChE showed that the protein level of ABCG1 and scavenger receptor class B member 1 (SR-B1) remain unaffected by erythrodiol. Besides, erythrodiol (10 μM) did not influence the mRNA level of ABCA1, ABCG1, and SR-B1, as determined by quantitative reverse transcription PCR, but significantly inhibited the degradation of ABCA1 as evident by an increased half-life of the protein in the presence of cycloheximide, an inhibitor of de novo protein synthesis. Therefore, erythrodiol promotes ChE from THP-1-derived human macrophages by stabilizing the ABCA1 protein. This bioactivity makes erythrodiol a good candidate to be further explored for therapeutic or preventive application in the context of atherosclerosis.© 2017 Wang, Wesemann, Krenn, Ladurner, Heiss, Dirsch and Atanaso

In Silico Workflow for the Discovery of Natural Products Activating the G Protein-Coupled Bile Acid Receptor 1

The G protein-coupled bile acid receptor (GPBAR1) has been recognized as a promising new target for the treatment of diverse diseases, including obesity, type 2 diabetes, fatty liver disease and atherosclerosis. The identification of novel and potent GPBAR1 agonists is highly relevant, as these diseases are on the rise and pharmacological unmet therapeutic needs are pervasive. Therefore, the aim of this study was to develop a proficient workflow for the in silico prediction of GPBAR1 activating compounds, primarily from natural sources. A protocol was set up, starting with a comprehensive collection of structural information of known ligands. This information was used to generate ligand-based pharmacophore models in LigandScout 4.08 Advanced. After theoretical validation, the two most promising models, namely BAMS22 and TTM8, were employed as queries for the virtual screening of natural product and synthetic small molecule databases. Virtual hits were progressed to shape matching experiments and physicochemical clustering. Out of 33 diverse virtual hits subjected to experimental testing using a reporter gene-based assay, two natural products, farnesiferol B (27) and microlobidene (28), were confirmed as GPBAR1 activators reaching more than 50% receptor activation at 20 μM with EC50s of 13.53 μM and 13.88 μM, respectively. This activity is comparable to that of the endogenous ligand lithocholic acid (1). Seven further virtual hits showed activity reaching at least 15% receptor activation either at 5 or 20 μM, including new scaffolds from natural and synthetic origin.© 2018 Kirchweger, Kratz, Ladurner, Grienke, Langer, Dirsch and Rollinge

Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5

Worldwide, metabolic diseases such as obesity and type 2 diabetes have reached epidemic proportions. A major regulator of metabolic processes that gained interest in recent years is the bile acid receptor TGR5 (Takeda G protein-coupled receptor 5). This G protein-coupled membrane receptor can be found predominantly in the intestine, where it is mainly responsible for the secretion of the incretins glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). The aim of this study was (i) to identify plant extracts with TGR5-activating potential, (ii) to narrow down their activity to the responsible constituents, and (iii) to assess whether the intestinal microbiota produces transformed metabolites with a different activity profile. Chenodeoxycholic acid (CDCA) served as positive control for both, the applied cell-based luciferase reporter gene assay for TGR5 activity and the biotransformation assay using mouse fecal slurry. The suitability of the workflow was demonstrated by the biotransformation of CDCA to lithocholic acid resulting in a distinct increase in TGR5 activity. Based on a traditional Tibetan formula, 19 plant extracts were selected and investigated for TGR5 activation. Extracts from the commonly used spices Syzygium aromaticum (SaroE, clove), Pimenta dioica (PdioE, allspice), and Kaempferia galanga (KgalE, aromatic ginger) significantly increased TGR5 activity. After biotransformation, only KgalE showed significant differences in its metabolite profile, which, however, did not alter its TGR5 activity compared to non-transformed KgalE. UHPLC-HRMS (high-resolution mass spectrometry) analysis revealed triterpene acids (TTAs) as the main constituents of the extracts SaroE and PdioE. Identification and quantification of TTAs in these two extracts as well as comparison of their TGR5 activity with reconstituted TTA mixtures allowed the attribution of the TGR5 activity to TTAs. EC50s were determined for the main TTAs, i.e., oleanolic acid (2.2 ± 1.6 μM), ursolic acid (1.1 ± 0.2 μM), as well as for the hitherto unknown TGR5 activators corosolic acid (0.5 ± 1.0 μM) and maslinic acid (3.7 ± 0.7 μM). In conclusion, extracts of clove, allspice, and aromatic ginger activate TGR5, which might play a pivotal role in their therapeutic use for the treatment of metabolic diseases. Moreover, the TGR5 activation of SaroE and PdioE could be pinpointed solely to TTAs.© 2017 Ladurner, Zehl, Grienke, Hofstadler, Faur, Pereira, Berry, Dirsch and Rollinge

Bilirubin Decreases Macrophage Cholesterol Efflux and ATP‐Binding Cassette Transporter A1 Protein Expression

Background: Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low‐density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Methods and Results: Cholesterol efflux from THP‐1 macrophages was assessed using plasma obtained from normo‐ and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3–17.1 μmol/L) exogenously added to plasma‐ or apolipoprotein A1–supplemented media also decreased macrophage cholesterol efflux in a concentration‐ and time‐dependent manner. We also showed reduced protein expression of the ATP‐binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1–mediated cholesterol efflux, in THP‐1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP‐1 macrophages. Conclusions: Cholesterol efflux from THP‐1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease.© 2017 The Author

The Dietary Constituent Falcarindiol Promotes Cholesterol Efflux from THP-1 Macrophages by Increasing ABCA1 Gene Transcription and Protein Stability

We report increased cholesterol efflux from macrophages in the presence of falcarindiol, an important dietary constituent present in commonly used vegetables and medicinal plants. Falcarindiol (3–20 μM) increased cholesterol efflux from THP-1-derived macrophages. Western blot analysis showed an increased protein level of ABCA1 upon falcarindiol exposure. Quantitative real-time PCR revealed that also ABCA1 mRNA level rise with falcarindiol (10 μM) treatment. The effect of falcarindiol on ABCA1 protein as well as mRNA level were counteracted by co-treatment with BADGE, an antagonist of PPARγ. Furthermore, falcarindiol significantly inhibited ABCA1 protein degradation in the presence of cycloheximide. This post-translational regulation of ABCA1 by falcarindiol occurs most likely by inhibition of lysosomal cathepsins, resulting in decreased proteolysis and extended protein half-life of ABCA1. Taken together, falcarindiol increases ABCA1 protein level by two complementary mechanisms, i.e., promoting ABCA1 gene expression and inhibiting ABCA1 protein degradation, which lead to enhanced cholesterol efflux.© 2017 Wang, Palme, Schilcher, Ladurner, Heiss, Stangl, Bauer, Dirsch and Atanaso

Novel interactomics approach identifies ABCA1 as direct target of evodiamine, which increases macrophage cholesterol efflux

Evodiamine, a bioactive alkaloid from the fruits of the traditional Chinese medicine Evodia rutaecarpa (Juss.) Benth. (Fructus Evodiae, Wuzhuyu), recently gained attention as a dietary supplement for weight loss and optimization of lipid metabolism. In light of its use by patients and consumers, there is an urgent need to elucidate the molecular targets affected by this natural product. Using a novel interactomics approach, the Nematic Protein Organisation Technique (NPOT), we report the identification of ATP-binding cassette transporter A1 (ABCA1), a key membrane transporter contributing to cholesterol efflux (ChE), as a direct binding target of evodiamine. The binding of evodiamine to ABCA1 is confirmed by surface plasmon resonance (SPR) experiments. Examining the functional consequences of ABCA1 binding reveals that evodiamine treatment results in increased ABCA1 stability, elevated cellular ABCA1 protein levels, and ultimately increased ChE from THP-1-derived human macrophages. The protein levels of other relevant cholesterol transporters, ABCG1 and SR-B1, remain unaffected in the presence of evodiamine, and the ABCA1 mRNA level is also not altered.© The Author(s) 201

Indirubin-3'-Monoxime Blocks Vascular Smooth Muscle Cell Proliferation by Inhibition of Signal Transducer and Activator of Transcription 3 Signaling and Reduces Neointima Formation In Vivo.

Objective-Our goal was to examine the influence of indirubin-3 '-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. Methods and Results-I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G(0)/G(1) phase of the cell cycle as assessed by 5-bromo-2 '-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38(MAPK) was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-gamma-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [C-14]L-arginine/[C-14]L-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells. Conclusion-I3MO represses PDGF-and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis. (Arterioscler Thromb Vasc Biol. 2010;30:2475-2481.

Linked magnolol dimer as a selective PPARγ agonist – Structure-based rational design, synthesis, and bioactivity evaluation

The nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and its hetero-dimerization partner retinoid X receptor α (RXRα) are considered as drug targets in the treatment of diseases like the metabolic syndrome and diabetes mellitus type 2. Effort has been made to develop new agonists for PPARγ to obtain ligands with more favorable properties than currently used drugs. Magnolol was previously described as dual agonist of PPARγ and RXRα. Here we show the structure-based rational design of a linked magnolol dimer within the ligand binding domain of PPARγ and its synthesis. Furthermore, we evaluated its binding properties and functionality as a PPARγ agonist in vitro with the purified PPARγ ligand binding domain (LBD) and in a cell-based nuclear receptor transactivation model in HEK293 cells. We determined the synthesized magnolol dimer to bind with much higher affinity to the purified PPARγ ligand binding domain than magnolol (K i values of 5.03 and 64.42 nM, respectively). Regarding their potency to transactivate a PPARγ-dependent luciferase gene both compounds were equally effective. This is likely due to the PPARγ specificity of the newly designed magnolol dimer and lack of RXRα-driven transactivation activity by this dimeric compound.© The Author(s) 201