Influence of ceramide on lipid domain stability studied with small-angle neutron scattering: The role of acyl chain length and unsaturation

Ceramides and diacylglycerols are groups of lipids capable of nucleating and stabilizing ordered lipid domains, structures that have been implicated in a range of biological processes. Previous studies have used fluorescence reporter molecules to explore the influence of ceramide acyl chain structure on sphingolipid-rich ordered phases. Here, we use small-angle neutron scattering (SANS) to examine the ability of ceramides and diacylglycerols to promote lipid domain formation in the well-characterized domain- forming mixture DPPC/DOPC/cholesterol. SANS is a powerful, probe-free technique for interrogating membrane heterogeneity, as it is differentially sensitive to hydrogen\u27s stable isotopes protium and deuterium. Specifcally, neutron contrast is generated through selective deuteration of lipid species, thus enabling the detection of nanoscopic domains enriched in deuterated saturated lipids dispersed in a matrix of protiated un- saturated lipids. Using large unilamellar vesicles, we found that upon replacing 10 mol % DPPC with either C16:0 or C18:0 ceramide, or 16:0 diacylglycerol (dag), lipid domains persisted to higher temperatures. However, when DPPC was replaced with short chain (C6:0 or C12:0) or very long chain (C24:0) ceramides, or ceramides with unsaturated acyl chains of any length (C6:1(3), C6:1(5), C18:1, and C24:1), as well as C18:1-dag, lipid domains were destabilized, melting at lower temperatures than those in the DPPC/DOPC/cholesterol system. These results show how ceramide acyl chain length and unsaturation influence lipid domains, and have implications for how cell membranes might modify their function through the generation of different ceramide species

Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles

Ligand activation of the aryl hydrocarbon (AHR) has profound effects upon the immunological status of the gastrointestinal tract, establishing and maintaining signaling networks, which facilitate host-microbe homeostasis at the mucosal interface. However, the identity of the ligand(s) responsible for such AHR-mediated activation within the gut remains to be firmly established. Here, we combine in vitro ligand binding, quantitative gene expression, protein-DNA interaction and ligand structure activity analyses together with in silico modeling of the AHR ligand binding domain to identify indole, a microbial tryptophan metabolite, as a human-AHR selective agonist. Human AHR, acting as a host indole receptor may exhibit a unique bimolecular (2:1) binding stoichiometry not observed with typical AHR ligands. Such bimolecular indole-mediated activation of the human AHR within the gastrointestinal tract may provide a foundation for inter-kingdom signaling between the enteric microflora and the immune system to promote commensalism within the gut.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Nature Publishing Group. The published article can be found at: http://www.nature.com/articles/srep1268

Preliminary physiologically based pharmacokinetic models for benzo[a]pyrene and dibenzo[def,p]chrysene in rodents

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated as byproducts of natural and anthropogenic combustion processes. Despite significant public health concern, physiologically based pharmacokinetic (PBPK) modeling efforts for PAHs have so far been limited to naphthalene, plus simpler PK models for pyrene, nitropyrene, and benzo[a]pyrene (B[a]P). The dearth of published models is due in part to the high lipophilicity, low volatility, and myriad metabolic pathways for PAHs, all of which present analytical and experimental challenges. Our research efforts have focused upon experimental approaches and initial development of PBPK models for the prototypic PAH, B[a]P, and the more potent, albeit less studied transplacental carcinogen, dibenzo[def,p]chrysene (DBC). For both compounds, model compartments included arterial and venous blood, flow limited lung, liver, richly perfused and poorly perfused tissues, diffusion limited fat, and a two compartment theoretical gut (for oral exposures). Hepatic and pulmonary metabolism was described for both compounds, as were fractional binding in blood and fecal clearance. Partition coefficients for parent PAH along with their diol and tetraol metabolites were estimated using published algorithms and verified experimentally for the hydroxylated metabolites. The preliminary PBPK models were able to describe many, but not all, of the available data sets, comprising multiple routes of exposure (oral, intravenous) and nominal doses spanning several orders of magnitude. Supported by Award Number P42 ES016465 from the National Institute of Environmental Health Sciences. Published by Elsevier Inc.Keywords: Metabolic activation, Gastrointestinal absorption, Cytochrome P450, Mouse skin, Benzo[a]Pyrene, Rat mammary gland, Ultimate carcinogens, Aromatic hydrocarbons, Tumor initiating activity, Partition coefficient

Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction

The farnesoid X receptor (FXR) regulates bile acid, lipid and glucose metabolism. Here we show that treatment of mice with glycine-β-muricholic acid (Gly-MCA) inhibits FXR signalling exclusively in intestine, and improves metabolic parameters in mouse models of obesity. Gly-MCA is a selective high-affinity FXR inhibitor that can be administered orally and prevents, or reverses, high-fat diet-induced and genetic obesity, insulin resistance and hepatic steatosis in mice. The high-affinity FXR agonist GW4064 blocks Gly-MCA action in the gut, and intestine-specific Fxr-null mice are unresponsive to the beneficial effects of Gly-MCA. Mechanistically, the metabolic improvements with Gly-MCA depend on reduced biosynthesis of intestinal-derived ceramides, which directly compromise beige fat thermogenic function. Consequently, ceramide treatment reverses the action of Gly-MCA in high-fat diet-induced obese mice. We further show that FXR signalling in ileum biopsies of humans positively correlates with body mass index. These data suggest that Gly-MCA may be a candidate for the treatment of metabolic disorders.This is the publisher’s final pdf. The published article is copyrighted and published by the Nature Publishing Group. The published article can be found at: http://www.nature.com/ncomms/index.htm

Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction

The farnesoid X receptor (FXR) regulates bile acid, lipid and glucose metabolism. Here we show that treatment of mice with glycine-b-muricholic acid (Gly-MCA) inhibits FXR signalling exclusively in intestine, and improves metabolic parameters in mouse models of obesity. Gly-MCA is a selective high-affinity FXR inhibitor that can be administered orally and prevents, or reverses, high-fat diet-induced and genetic obesity, insulin resistance and hepatic steatosis in mice. The high-affinity FXR agonist GW4064 blocks Gly-MCA action in the gut, and intestine-specific Fxr-null mice are unresponsive to the beneficial effects of Gly-MCA. Mechanistically, the metabolic improvements with Gly-MCA depend on reduced biosynthesis of intestinal-derived ceramides, which directly compromise beige fat thermogenic function. Consequently, ceramide treatment reverses the action of Gly-MCA in high-fat diet-induced obese mice. We further show that FXR signalling in ileum biopsies of humans positively correlates with body mass index. These data suggest that Gly-MCA may be a candidate for the treatment of metabolic disorders

Implication of a Chromosome 15q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells from carcinogenic effects of alkylating agents; however, MGMT is silenced by promoter hypermethylation during carcinogenesis. A single nucleotide polymorphism (SNP) in an enhancer in the MGMT promoter was previously identified to be highly significantly associated with risk for MGMT methylation in lung cancer and sputum from smokers. To further genetic investigations, a genome-wide association and replication study was conducted in two smoker cohorts to identify novel loci for MGMT methylation in sputum that were independent of the MGMT enhancer polymorphism. Two novel trans-acting loci (15q15.2 and 17q24.3) that were identified acted together with the enhancer SNP to empower risk prediction for MGMT methylation. We found that the predisposition to MGMT methylation arising from the 15q15.2 locus involved regulation of the ubiquitin protein ligase E3 component UBR1. UBR1 attenuation reduced turnover of MGMT protein and increased repair of O6-methylguanine in nitrosomethylurea-treated human bronchial epithelial cells (HBEC), while also reducing MGMT promoter activity and abolishing MGMT induction. Overall, our results substantiate reduced gene transcription as a major mechanism for predisposition to MGMT methylation in the lungs of smokers, and support the importance of UBR1 in regulating MGMT homeostasis and DNA repair of alkylated DNA adducts in cells

Synthesis of (plus or minus) [5-{sup 3}H] N'-Nitrosoanatabine, a tobacco-specific nitrosamine

Tobacco-specific N'-nitrosamines (TSNA) are a unique class of systemic organ-specific carcinogens. The TSNA are formed by N-nitrosation of nicotine and of the minor tobacco alkaloids after harvesting of tobacco and during smoking. The N-nitrosation of anatabine leads to N'-nitrosoanatabine (NAT; 1-nitroso-1,2,3,4-tetrahydro-2,3'-bipyridyl) which requires in-depth assays in laboratory animals other than the rat. Furthermore, delineation of its tissue distribution and metabolism is needed for structure:activity comparisons with other TSNA and for the assessment of potential human risk from this TSNA. We have, therefore, synthesized (+)[5-3H]NAT. 5-Bromo-3-pyridine-carboxaldehyde was condensed with ethyl carbamate prior to Diels-Alder reaction with 1,4-butadiene to give the racemic anatabine ring system. Hydrolysis followed by reduction with LiAlT4 and nitrosation, led to (+)[5-3H]NAT (60 percent yield, specific activity 266 mCi/mmol, radiochemical purity of >99 percent)

Targeting Cell Survival Proteins for Cancer Cell Death

Escaping from cell death is one of the adaptations that enable cancer cells to stave off anticancer therapies. The key players in avoiding apoptosis are collectively known as survival proteins. Survival proteins comprise the Bcl-2, inhibitor of apoptosis (IAP), and heat shock protein (HSP) families. The aberrant expression of these proteins is associated with a range of biological activities that promote cancer cell survival, proliferation, and resistance to therapy. Several therapeutic strategies that target survival proteins are based on mimicking BH3 domains or the IAP-binding motif or competing with ATP for the Hsp90 ATP-binding pocket. Alternative strategies, including use of nutraceuticals, transcriptional repression, and antisense oligonucleotides, provide options to target survival proteins. This review focuses on the role of survival proteins in chemoresistance and current therapeutic strategies in preclinical or clinical trials that target survival protein signaling pathways. Recent approaches to target survival proteins-including nutraceuticals, small-molecule inhibitors, peptides, and Bcl-2-specific mimetic are explored. Therapeutic inventions targeting survival proteins are promising strategies to inhibit cancer cell survival and chemoresistance. However, complete eradication of resistance is a distant dream. For a successful clinical outcome, pretreatment with novel survival protein inhibitors alone or in combination with conventional therapies holds great promise

Accumulation of Linoleic Acid by Altered Peroxisome Proliferator-Activated Receptor-α Signaling Is Associated with Age-Dependent Hepatocarcinogenesis in <i>Ppara</i> Transgenic Mice

Long-term ligand activation of PPARα in mice causes hepatocarcinogenesis through a mechanism that requires functional PPARα. However, hepatocarcinogenesis is diminished in both Ppara-null and PPARA-humanized mice, yet both lines develop age-related liver cancer independently of treatment with a PPARα agonist. Since PPARα is a master regulator of liver lipid metabolism in the liver, lipidomic analyses were carried out in wild-type, Ppara-null, and PPARA-humanized mice treated with and without the potent agonist GW7647. The levels of hepatic linoleic acid in Ppara-null and PPARA-humanized mice were markedly higher compared to wild-type controls, along with overall fatty liver. The number of liver CD4+ T cells was also lower in Ppara-null and PPARA-humanized mice and was negatively correlated with the elevated linoleic acid. Moreover, more senescent hepatocytes and lower serum TNFα and IFNγ levels were observed in Ppara-null and PPARA-humanized mice with age. These studies suggest a new role for PPARα in age-associated hepatocarcinogenesis due to altered lipid metabolism in Ppara-null and PPARA-humanized mice and the accumulation of linoleic acid as part of an overall fatty liver that is associated with loss of CD4+ T cells in the liver in both transgenic models. Since fatty liver is a known causal risk factor for liver cancer, Ppara-null and PPARA-humanized mice are valuable models for examining the mechanisms of PPARα and age-dependent hepatocarcinogenesis