Toxicity of oxidized phospholipids in cultured macrophages

Background: The interactions of oxidized low-density lipoprotein (LDL) and macrophages are hallmarks in the development of atherosclerosis. The biological activities of the modified particle in these cells are due to the content of lipid oxidation products and apolipoprotein modification by oxidized phospholipids. Results: It was the aim of this study to determine the role of short-chain oxidized phospholipids as components of modified LDL in cultured macrophages. For this purpose we investigated the effects of the following oxidized phospholipids on cell viability and apoptosis: 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and oxidized alkylacyl phospholipids including 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine (E-PGPC) and 1-O-hexadecyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (E-POVPC). We found that these compounds induced apoptosis in RAW264.7 and bone marrow-derived macrophages. The sn-2 carboxyacyl lipid PGPC was more toxic than POVPC which carries a reactive aldehyde function in position sn-2 of glycerol. The alkylacyl phospholipids (E-PGPC and E-POVPC) and the respective diacyl analogs show similar activities. Apoptosis induced by POVPC and its alkylether derivative could be causally linked to the fast activation of an acid sphingomyelinase, generating the apoptotic second messenger ceramide. In contrast, PGPC and its ether analog only negligibly affected this enzyme pointing to an entirely different mechanism of lipid toxicity. The higher toxicity of PGPC is underscored by more efficient membrane blebbing from apoptotic cells. In addition, the protein pattern of PGPC-induced microparticles is different from the vesicles generated by POPVC. Conclusions: In summary, our data reveal that oxidized phospholipids induce apoptosis in cultured macrophages. The mechanism of lipid toxicity, however, largely depends on the structural features of the oxidized sn-2 chain

Impaired Bile Acid Metabolism and Gut Dysbiosis in Mice Lacking Lysosomal Acid Lipase

Lysosomal acid lipase (LAL) is the sole enzyme known to be responsible for the hydrolysis of cholesteryl esters and triglycerides at an acidic pH in lysosomes, resulting in the release of unesterified cholesterol and free fatty acids. However, the role of LAL in diet-induced adaptations is largely unexplored. In this study, we demonstrate that feeding a Western-type diet to Lal-deficient (LAL-KO) mice triggers metabolic reprogramming that modulates gut-liver cholesterol homeostasis. Induction of ileal fibroblast growth factor 15 (three-fold), absence of hepatic cholesterol 7α-hydroxylase expression, and activation of the ERK phosphorylation cascade results in altered bile acid composition, substantial changes in the gut microbiome, reduced nutrient absorption by 40%, and two-fold increased fecal lipid excretion in LAL-KO mice. These metabolic adaptations lead to impaired bile acid synthesis, lipoprotein uptake, and cholesterol absorption and ultimately to the resistance of LAL-KO mice to diet-induced obesity. Our results indicate that LAL-derived lipolytic products might be important metabolic effectors in the maintenance of whole-body lipid homeostasis

Novel amino-β-lactam derivatives as potent cholesterol absorption inhibitors

Two new trans-(3R, 4R)-amino-β-lactam derivatives and their diastereoisomeric mixtures were synthesized as ezetimibe bioisosteres and tested in in vitro and in vivo experiments as novel β-lactam cholesterol absorption inhibitors. Both compounds exhibited low cytotoxicity in MDCKII, hNPC1L1/MDCKII, and HepG2 cell lines and potent inhibitory effect in hNPC1L1/MDCKII cells. In addition, these compounds markedly reduced cholesterol absorption in mice, resulting in reduced cholesterol concentrations in plasma, liver, and intestine. We determined the crystal structure of one amino-β-lactam derivative to establish unambiguously both the absolute and relative configuration at the new stereogenic centre C17, which was assigned to be S. The pKa values for both compounds are 9.35, implying that the amino-β-lactam derivatives and their diastereoisomeric mixtures are in form of ammonium salt in blood and the intestine. The IC50 value for the diastereoisomeric mixture is 60 μM. In vivo, it efficiently inhibited cholesterol absorption comparable to ezetimibe

Novel role of a triglyceride-synthesizing enzyme:DGAT1 at the crossroad between triglyceride and cholesterol metabolism

AbstractAcyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1−/−) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1−/− and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia

The PPARα agonist fenofibrate suppresses B-cell lymphoma in mice by modulating lipid metabolism

AbstractObesity is associated with an increased risk for malignant lymphoma development. We used Bcr/Abl transformed B cells to determine the impact of aggressive lymphoma formation on systemic lipid mobilization and turnover. In wild-type mice, tumor size significantly correlated with depletion of white adipose tissues (WAT), resulting in increased serum free fatty acid (FFA) concentrations which promote B-cell proliferation in vitro. Moreover, B-cell tumor development induced hepatic lipid accumulation due to enhanced hepatic fatty acid (FA) uptake and impaired FA oxidation. Serum triglyceride, FFA, phospholipid and cholesterol levels were significantly elevated. Consistently, serum VLDL/LDL-cholesterol and apolipoprotein B levels were drastically increased. These findings suggest that B-cell tumors trigger systemic lipid mobilization from WAT to the liver and increase VLDL/LDL release from the liver to promote tumor growth. Further support for this concept stems from experiments where we used the peroxisome proliferator-activated receptor α (PPARα) agonist and lipid-lowering drug fenofibrate that significantly suppressed tumor growth independent of angiogenesis and inflammation. In addition to WAT depletion, fenofibrate further stimulated FFA uptake by the liver and restored hepatic FA oxidation capacity, thereby accelerating the clearance of lipids released from WAT. Furthermore, fenofibrate blocked hepatic lipid release induced by the tumors. In contrast, lipid utilization in the tumor tissue itself was not increased by fenofibrate which correlates with extremely low expression levels of PPARα in B-cells. Our data show that fenofibrate associated effects on hepatic lipid metabolism and deprivation of serum lipids are capable to suppress B-cell lymphoma growth which may direct novel treatment strategies. This article is part of a Special Issue entitled Lipid Metabolism in Cancer

Lysosomal acid lipase regulates VLDL synthesis and insulin sensitivity in mice

AIMS/HYPOTHESIS: Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal (-/-) ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). METHODS: We studied metabolic adaptations in Lal (-/-) mice. RESULTS: Despite loss of adipose tissue, Lal (-/-) mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [(3)H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal (-/-) mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal (-/-) mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal (-/-) mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal (-/-) mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal (-/-) mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. CONCLUSIONS/INTERPRETATION: Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal (-/-) mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply

Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover

Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of cholesteryl esters and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study, we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with RE gavage exhibited largely reduced post-prandial circulating RE content, indicating that LAL is required for efficient nutritional vitamin A availability. In summary, our results indicate that LAL is the major acid RE hydrolase and required for functional retinoid homeostasis

Activated prostaglandin D2 receptors on macrophages enhance neutrophil recruitment into the lung

BackgroundProstaglandin (PG) D2 is an early-phase mediator in inflammation, but its action and the roles of the 2 D-type prostanoid receptors (DPs) DP1 and DP2 (also called chemoattractant receptor–homologous molecule expressed on TH2 cells) in regulating macrophages have not been elucidated to date.ObjectiveWe investigated the role of PGD2 receptors on primary human macrophages, as well as primary murine lung macrophages, and their ability to influence neutrophil action in vitro and in vivo.MethodsIn vitro studies, including migration, Ca2+ flux, and cytokine secretion, were conducted with primary human monocyte-derived macrophages and neutrophils and freshly isolated murine alveolar and pulmonary interstitial macrophages. In vivo pulmonary inflammation was assessed in male BALB/c mice.ResultsActivation of DP1, DP2, or both receptors on human macrophages induced strong intracellular Ca2+ flux, cytokine release, and migration of macrophages. In a murine model of LPS-induced pulmonary inflammation, activation of each PGD2 receptor resulted in aggravated airway neutrophilia, tissue myeloperoxidase activity, cytokine contents, and decreased lung compliance. Selective depletion of alveolar macrophages abolished the PGD2-enhanced inflammatory response. Activation of PGD2 receptors on human macrophages enhanced the migratory capacity and prolonged the survival of neutrophils in vitro. In human lung tissue specimens both DP1 and DP2 receptors were located on alveolar macrophages along with hematopoietic PGD synthase, the rate-limiting enzyme of PGD2 synthesis.ConclusionFor the first time, our results show that PGD2 markedly augments disease activity through its ability to enhance the proinflammatory actions of macrophages and subsequent neutrophil activation

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Changes in intra-and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Forster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging

Impaired Rho GTPase activation abrogates cell polarization and migration in macrophages with defective lipolysis

Infiltration of monocytes and macrophages into the site of inflammation is critical in the progression of inflammatory diseases such as atherosclerosis. Cell migration is dependent on the continuous organization of the actin cytoskeleton, which is regulated by members of the small Rho GTPase family (RhoA, Cdc42, Rac) that are also important for the regulation of signal transduction pathways. We have recently reported on reduced plaque formation in an atherosclerotic mouse model transplanted with bone marrow from adipose triglyceride lipase-deficient (Atgl−/−) mice. Here we provide evidence that defective lipolysis in macrophages lacking ATGL, the major enzyme responsible for triacylglycerol hydrolysis, favors an anti-inflammatory M2-like macrophage phenotype. Our data implicate an as yet unrecognized principle that insufficient lipolysis influences macrophage polarization and actin polymerization, resulting in impaired macrophage migration. Sustained phosphorylation of focal adhesion kinase [due to inactivation of its phosphatase by elevated levels of reactive oxygen species (ROS)] results in defective Cdc42, Rac1 and RhoA activation and in increased and sustained activation of Rac2. Inhibition of ROS production restores the migratory capacity of Atgl−/− macrophages. Since monocyte and macrophage migration are a prerequisite for infiltrating the arterial wall, our results provide a molecular link between lipolysis and the development of atherosclerosis