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Cell  2013;155(1):200-214.
Macrophage-mediated inflammation is a major contributor to obesity-associated insulin resistance. The co-repressor NCoR interacts with inflammatory pathway genes in macrophages, suggesting that its removal would result in increased activity of inflammatory responses. Surprisingly, we find that macrophage-specific deletion of NCoR instead results in an anti-inflammatory phenotype along with robust systemic insulin sensitization in obese mice. We present evidence that de-repression of LXRs contributes to this paradoxical anti-inflammatory phenotype by causing increased expression of genes that direct biosynthesis of palmitoleic acid and ω3 fatty acids. Remarkably, the increased ω3 fatty acid levels primarily inhibit NF-κB-dependent inflammatory responses by uncoupling NF-κB binding and enhancer/promoter histone acetylation from subsequent steps required for pro-inflammatory gene activation. This provides a mechanism for the in vivo anti-inflammatory insulin sensitive phenotype observed in mice with macrophage-specific deletion of NCoR. Therapeutic methods to harness this mechanism could lead to a new approach to insulin sensitizing therapies.
PMCID: PMC4131699  PMID: 24074869
nuclear co-repressor; insulin resistance; obesity; macrophage; inflammation
3.  Lipidomic Profiling of Influenza Infection Identifies Mediators that Induce and Resolve Inflammation 
Cell  2013;154(1):213-227.
Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, LC/MS lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low pathogenicity strain, X31/H3N2, induced a pro-inflammatory response followed by a distinct anti-inflammatory response; infection by the high pathogenicity strain, PR8/H1N1, resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5 lipoxygenase metabolites correlated with the pathogenic phase of the infection whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-HODE, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009–2011 influenza seasons.
PMCID: PMC3753192  PMID: 23827684
4.  Modulation of p25 and inflammatory pathways by fisetin maintains cognitive function in Alzheimer’s disease transgenic mice 
Aging Cell  2013;13(2):379-390.
Alzheimer’s disease (AD) is the most common type of dementia. It is the only one of the top ten causes of death in the USA for which prevention strategies have not been developed. Although AD has traditionally been associated with the deposition of amyloid β plaques and tau tangles, it is becoming increasingly clear that it involves disruptions in multiple cellular systems. Therefore, it is unlikely that hitting a single target will result in significant benefits to patients with AD. An alternative approach is to identify molecules that have multiple biological activities that are relevant to the disease. Fisetin is a small, orally active molecule which can act on many of the target pathways implicated in AD. We show here that oral administration of fisetin to APPswe/PS1dE9 double transgenic AD mice from 3 to 12 months of age prevents the development of learning and memory deficits. This correlates with an increase in ERK phosphorylation along with a decrease in protein carbonylation, a marker of oxidative stress. Importantly, fisetin also reduces the levels of the cyclin-dependent kinase 5 (Cdk5) activator p35 cleavage product, p25, in both control and AD brains. Elevated levels of p25 relative to p35 cause dysregulation of Cdk5 activity leading to neuroinflammation and neurodegeneration. These fisetin-dependent changes correlate with additional anti-inflammatory effects, including alterations in global eicosanoid synthesis, and the maintenance of markers of synaptic function in the AD mice. Together, these results suggest that fisetin may provide a new approach to the treatment of AD.
PMCID: PMC3954948  PMID: 24341874
astrogliosis; ERK; eicosanoid; lipoxygenase; oxidative stress; prostaglandin
5.  Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses 
Cell  2012;151(1):138-152.
Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses observed in macrophage foam cells, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism and suppression of inflammatory response genes. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, pro-inflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.
PMCID: PMC3464914  PMID: 23021221
6.  The Human Plasma Lipidome 
The New England journal of medicine  2011;365(19):1812-1823.
PMCID: PMC3412394  PMID: 22070478
7.  High Sensitivity Quantitative Lipidomics Analysis of Fatty Acids in Biological Samples by Gas Chromatography-Mass Spectrometry 
Biochimica et biophysica acta  2011;1811(11):648-656.
Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids is achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography-mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples.
PMCID: PMC3205314  PMID: 21787881
8.  Maternal Immunization Affects In Utero Programming of Insulin Resistance and Type 2 Diabetes 
PLoS ONE  2012;7(9):e45361.
Maternal immunization with oxidized lipoproteins prior to pregnancy protects against atherogenic in utero programming by gestational hypercholesterolemia and enhances beneficial lymphocyte-dependent immune responses in offspring. To determine whether in utero programming and immunomodulation also affect insulin resistance (IR) and type 2 diabetes, we investigated the effects of immunization on glucose and insulin responses in LDL receptor-deficient mice fed regular or 60% sucrose diets, as well as in offspring fed 0.5% cholesterol or 60% sucrose diets. IR was assessed by fasting glucose and insulin levels, oral glucose tolerance tests, glucose clamps, pancreatic immunohistochemistry and plasma free fatty acid concentrations. Immunizations improved glucose responses in both genders and protected both immunized mice and their offspring against IR and type 2 diabetes. Protection occurred even under euglycemic conditions, but was greatest in obese males exposed to very obesogenic/diabetogenic conditions. Hyperinsulinemic euglycemic clamps confirmed that maternal immunization protected mainly by reducing IR, but pancreatic immunocytochemistry also indicated some protection against beta cell damage. Maternal immunization was associated with marked regulation in offspring of 4 genes relevant to diabetes and 19 genes of importance for oxidative stress, as well as increased hepatic activities of key antioxidant enzymes. These findings establish that targeted immunomodulation may be used to protect immunized subjects and their offspring against IR and type 2 diabetes, and thus to reduce cardiovascular risk. They also support the notion that in utero programming influences offspring disease not by a single mechanism, but by multiple systemic effects.
PMCID: PMC3445481  PMID: 23028961
9.  Effect of gestational hypercholesterolemia and maternal immunization on offspring plasma eicosanoids 
American journal of obstetrics and gynecology  2011;205(2):156.e15-156.e25.
Maternal immunization with oxidized low-density-lipoprotein prior to pregnancy prevents pathogenic in utero programming by gestational hypercholesterolemia, but it is unknown whether gestational hypercholesterolemia and maternal immunization affect similar pathways.
Study Design
A lipidomic approach was used for unbiased plasma eicosanoid profiling in adult offspring of immunized and non-immunized normo- or hypercholesterolemic rabbit mothers.
Gestational hypercholesterolemia was associated with increased levels of some eicosanoids formed by the cyclooxygenase and 12-lipoxygenase pathways only (including TXB2, PGF2α, PGE2, and PGD2). Immunization of hypercholesterolemic or normocholesterolemic mothers reduced 9 of 14 eicosanoids of the cyclooxygenase pathway, 21 of 23 eicosanoids of the 5- and 12-lipoxygenase pathways (e.g. 5-HETE, HXB3, 12-HETE), 8 of 19 eicosanoids of the cytochrome P-450 pathway and all metabolites of the nonenzymatic pathway.
Maternal immunization not only counteracts in utero programming by gestational hypercholesterolemia but reduces a broad range of eicosanoid modulators of immunity and inflammation in offspring.
PMCID: PMC3175501  PMID: 21621186
Cardiovascular disease; developmental programming; eicosanoids; immunization; inflammation; rabbits
10.  TLR-4 mediated group IVA phospholipase A2 activation is phosphatidic acid phosphohydrolase 1 and protein kinase C dependent # 
Biochimica et biophysica acta  2009;1791(10):975-982.
Group IVA phospholipase A2 (GIVA PLA2) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA2. We also studied the effect of PAP-1 and PKC on Ca+2 induced and synergy enhanced GIVA PLA2 activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo2-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca+2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA2 synergistic activation by stimulation with Kdo2–Lipid A and ATP.
PMCID: PMC2743746  PMID: 19230851
11.  Lipidomics Analysis of Essential Fatty Acids in Macrophages 
The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides the tools for the analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over thirty fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards that permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.
PMCID: PMC2643973  PMID: 18996688
12.  Oxidized LDL reduces monocyte CCR2 expression through pathways involving peroxisome proliferator–activated receptor γ 
The CCR2-mediated recruitment of monocytes into the vessel wall plays an important role in all stages of atherosclerosis. In recent studies, we have shown that lipoproteins can modulate CCR2 expression and have identified native LDL as a positive regulator. In contrast, oxidized LDL (OxLDL), which is mainly formed in the aortic intima, reduces CCR2 expression, promotes monocyte retention, and may cause pathological accumulation of monocytes in the vessel wall. We now provide evidence that OxLDL reduces monocyte CCR2 expression by activating intracellular signaling pathways that may involve peroxisome proliferator–activated receptor γ (PPARγ). Receptor-mediated uptake of the lipoprotein particle was required and allows for delivery of the exogenous ligand to the nuclear receptor. The suppression of CCR2 expression by OxLDL was mediated by lipid components of OxLDL, such as the oxidized linoleic acid metabolites 9-HODE and 13-HODE, known activators of PPARγ. Modified apoB had no such effect. Consistent with a participation of the PPARγ signaling pathway, BRL49653 reduced CCR2 expression in freshly isolated human monocytes ex vivo and in circulating mouse monocytes in vivo. These results implicate PPARγ in the inhibition of CCR2 gene expression by oxidized lipids, which may help retain monocytes at sites of inflammation, such as the atherosclerotic lesion.
PMCID: PMC381395  PMID: 10995790

Results 1-12 (12)