Cardioprotective Effects of Palmitoleic Acid (C16:1n7) in a Mouse Model of Catecholamine-Induced Cardiac Damage Are Mediated by PPAR Activation

Palmitoleic acid (C16:1n7) has been identified as a regulator of physiological cardiac hypertrophy. In the present study, we aimed to investigate the molecular pathways involved in C16:1n7 responses in primary murine cardiomyocytes (PCM) and a mouse model of isoproterenol (ISO)-induced cardiac damage. PCMs were stimulated with C16:1n7 or a vehicle. Afterwards, RNA sequencing was performed using an Illumina HiSeq sequencer. Confirmatory analysis was performed in PCMs and HL-1 cardiomyocytes. For an in vivo study, 129 sv mice were orally treated with a vehicle or C16:1n7 for 22 days. After 5 days of pre-treatment, the mice were injected with ISO (25 mg/kg/d s. c.) for 4 consecutive days. Cardiac phenotyping was performed using echocardiography. In total, 129 genes were differentially expressed in PCMs stimulated with C16:1n7, including Angiopoietin-like factor 4 (Angptl4) and Pyruvate Dehydrogenase Kinase 4 (Pdk4). Both Angptl4 and Pdk4 are proxisome proliferator-activated receptor α/δ (PPARα/δ) target genes. Our in vivo results indicated cardioprotective and anti-fibrotic effects of C16:1n7 application in mice. This was associated with the C16:1n7-dependent regulation of the cardiac PPAR-specific signaling pathways. In conclusion, our experiments demonstrated that C16:1n7 might have protective effects on cardiac fibrosis and inflammation. Our study may help to develop future lipid-based therapies for catecholamine-induced cardiac damage

Liver X Receptor Agonist AZ876 Induces Beneficial Endogenous Cardiac Lipid Reprogramming and Protects Against Isoproterenol‐Induced Cardiac Damage

Background: It is known that dietary intake of polyunsaturated fatty acids may improve cardiac function. However, relatively high daily doses are required to achieve sufficient cardiac concentrations of beneficial omega-3 fatty acids. The liver X receptor (LXR) is a nuclear hormone receptor and a crucial regulator of lipid homeostasis in mammals. LXR activation has been shown to endogenously reprogram cellular lipid profiles toward increased polyunsaturated fatty acids levels. Here we studied whether LXR lipid reprogramming occurs in cardiac tissue and exerts cardioprotective actions. Methods and Results: Male 129SV mice were treated with the LXR agonist AZ876 (20 mu mol/kg per day) for 11 days. From day 6, the mice were injected with the nonselective beta-agonist isoproterenol for 4 consecutive days to induce diastolic dysfunction and subendocardial fibrosis while maintaining systolic function. Treatment with isoproterenol led to a marked impairment of global longitudinal strain and the E/e' ratio of transmitral flow to mitral annular velocity, which were both significantly improved by the LXR agonist. Histological examination showed a significant reduction in isoproterenol-induced subendocardial fibrosis by AZ876. Analysis of the cardiac lipid composition by liquid chromatography-high resolution mass spectrometry revealed a significant increase in cardiac polyunsaturated fatty acids levels and a significant reduction in saturated fatty acids by AZ876. Conclusions: The present study provides evidence that the LXR agonist AZ876 prevents subendocardial damage, improves global longitudinal strain and E/e' in a mouse model of isoproterenol-induced cardiac damage, accompanied by an upregulation of cardiac polyunsaturated fatty acids levels. Cardiac LXR activation and beneficial endogenous cardiac lipid reprogramming may provide a new therapeutic strategy in cardiac disease with diastolic dysfunction

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

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

Zugang zu 3-substituierten Kumarat-Derivaten

Phenylalanin-Ammoniak-Lyase (PAL) katalysiert die Deaminierung von L-Phenylalanin zu trans-Zimtsäure. Es ist das erste und das Schlüsselenzym der Phenylpropanoid-Biosynthese in höheren Pflanzen und hauptsächlich in Abwehrmechanismen involviert. Bei Mikroorganismen spielt PAL eine katabolische Rolle, indem sie ihnen erlaubt, L-Phenylalanin als eine Kohlenstoff- und Stickstoffquelle zu nutzen. Dieses Enzym kommt nicht nur in Mikroorganismen vor, sondern kann auch in Hefe gefunden werden, speziell in der roten Hefe-Basidiomyceten-Familie Rhodotorula. Einige von diesen Enzymen deaminieren L-Tyrosin zu p-Hydroxyzimtsäure, auch p-Kumarat genannt, welche große Anwendung im Gesundheitsbereich und in der Pharma- und Lebensmittelindustrie findet.Das Ziel dieser Arbeit war, die Reaktionsbedingungen der Biotransformation von L-Tyrosin zu p-Kumarat und Ammoniak durch PAL zu optimieren und das Substratspektrum des Enzyms zu untersuchen. Außerdem sollte PAL von R. glutinis in E. coli überexprimiert und mittels Ni-NTA Affinitätschromatographie das reine Enzym erhalten werden. Zuerst wurden aktive Zellen von Rhodotorula mucilaginosa und Rhodotorula glutinis angezüchtet, welche dafür bekannt sind PAL-Aktivität zu besitzen. PAL von R. glutinis wurde erfolgreich in E. coli überexprimiert und das Enzym mittels Ni-NTA Affinitätschromatographie aufgereinigt. Nach Optimierung der Reaktionsbedingungen mit dem Standardsubstrat L-Tyrosin wurden weitere dreizehn Substrate getestet, wovon zehn von PAL umgesetzt wurden.Folgende Kumarat-Derivate wurden synthetisiert und mittels 1H-NMR, wenn genügend Produkt vorhanden war teilweise auch mittels 13C-NMR, charakterisiert: p-Kumarat, trans-Zimtsäure, 4-Chlorzimtsäure, 3-Chlorkumarsäure, 4-Fluorzimtsäure, 3-Fluorzimtsäure, 4-Methoxyzimtsäure, 3,4-Dihydroxyzimtsäure, 3-Methylkumarsäure, 3-Methoxykumarsäure, 3-Fluorkumarsäure.Phenylalanine ammonia-lyase (PAL) catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid. It is the first and key enzyme of the phenyl propanoid pathway in higher plants and mainly involved in defense mechanisms. In microorganisms, it has a catabolic role, allowing them to use L-phenylalanine as a source of carbon and nitrogen. Among microorganisms, PAL can be found also in yeast, especially in the red yeast basidiomycetes family Rhodotorula. Some of these enzymes also deaminate L-tyrosine to p-hydroxycinnamic acid (p-coumarate), which has potential use in a variety of health, pharmaceutical, flavors and industrial applications.The aim of this diploma thesis was to optimize the reaction conditions of the biotransformation of L-tyrosine to p-coumarate and ammonia catalyzed by PAL and to investigate the substrate spectrum of the enzyme. Furthermore the PAL from R. glutinis should be overexpressed in E. coli and purified by Ni-NTA affinity chromatography. First active cells of Rhodotorula mucilaginosa and Rhodotorula glutinis were cultivated, which were known to possess PAL activity. R. glutinis PAL was successfully overexpressed in E. coli and the enzyme was purified by Ni-NTA affinity purification. After optimization of the reaction conditions with the standard-substrate L-tyrosine, thirteen substrates were additionally tested from which ten were accepted by PAL. The following coumarate-derivatives were synthesized and characterized by 1H-NMR and, if there was enough product, also by 13C-NMR: p-coumarate, trans-cinnamic acid, 4-chlorocinnamic acid, 3-chlorocinnamic acid, 4-fluorocinnamic acid, 3-fluorocinnamic acid, 4-methoxycinnamic acid, 3,4-dihydroxycinnamic acid, 3-methylcoumaric acid, 3-methoxycoumaric acid, 3-fluorocoumaric acid.vorgelegt von Madeleine GöritzerGraz, Univ., Dipl.-Arb., 2011(VLID)21269

2-Chlorohexadecanoic acid induces ER stress and mitochondrial dysfunction in brain microvascular endothelial cells

Peripheral leukocytes induce blood-brain barrier (BBB) dysfunction through the release of cytotoxic mediators. These include hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system of activated phagocytes. HOCl targets the endogenous pool of ether phospholipids (plasmalogens) generating chlorinated inflammatory mediators like e.g. 2-chlorohexadecanal and its conversion product 2-chlorohexadecanoic acid (2-ClHA). In the cerebrovasculature these compounds inflict damage to brain microvascular endothelial cells (BMVEC) that form the morphological basis of the BBB. To follow subcellular trafficking of 2-ClHA we synthesized a ‘clickable’ alkyne derivative (2-ClHyA) that phenocopied the biological activity of the parent compound. Confocal and superresolution structured illumination microscopy revealed accumulation of 2-ClHyA in the endoplasmic reticulum (ER) and mitochondria of human BMVEC (hCMEC/D3 cell line). 2-ClHA and its alkyne analogue interfered with protein palmitoylation, induced ER-stress markers, reduced the ER ATP content, and activated transcription and secretion of interleukin (IL)−6 as well as IL-8. 2-ClHA disrupted the mitochondrial membrane potential and induced procaspase-3 and PARP cleavage. The protein kinase R-like ER kinase (PERK) inhibitor GSK2606414 suppressed 2-ClHA-mediated activating transcription factor 4 synthesis and IL-6/8 secretion, but showed no effect on endothelial barrier dysfunction and cleavage of procaspase-3. Our data indicate that 2-ClHA induces potent lipotoxic responses in brain endothelial cells and could have implications in inflammation-induced BBB dysfunction

Myeloperoxidase and Septic Conditions Disrupt Sphingolipid Homeostasis in Murine Brain Capillaries In Vivo and Immortalized Human Brain Endothelial Cells In Vitro

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood–brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO−/− compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.ISSN:1422-006

Xanthohumol ameliorates atherosclerotic plaque formation, hypercholesterolemia, and hepatic steatosis in ApoE-deficient mice

SCOPE: Xanthohumol (XN), a prenylated antioxidative and anti-inflammatory chalcone from hops, exhibits positive effects on lipid and glucose metabolism. Based on its favorable biological properties, we investigated whether XN attenuates atherosclerosis in western-type diet-fed apolipoprotein-E-deficient (ApoE(-/-) ) mice. METHODS AND RESULTS: XN supplementation markedly reduced plasma cholesterol concentrations, decreased atherosclerotic lesion area, and attenuated plasma concentrations of the proinflammatory cytokine monocyte chemoattractant protein 1. Decreased hepatic triglyceride and cholesterol content, activation of AMP-activated protein kinase, phosphorylation and inactivation of acetyl-CoA carboxylase, and reduced expression levels of mature sterol regulatory element-binding protein (SREBP)-2 and SREBP-1c mRNA indicate reduced lipogenesis in the liver of XN-fed ApoE(-/-) mice. Concomitant induction of hepatic mRNA expression of carnitine palmitoyltransferase-1a in ApoE(-/-) mice-administered XN suggests increased fatty acid beta-oxidation. Fecal cholesterol concentrations were also markedly increased in XN-fed ApoE(-/-) mice compared with mice fed western-type diet alone. CONCLUSION: The atheroprotective effects of XN might be attributed to combined beneficial effects on plasma cholesterol and monocyte chemoattractant protein 1 concentrations and hepatic lipid metabolism via activation of AMP-activated protein kinase