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1.
J Lipid Res ; 57(4): 638-49, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26891736

ABSTRACT

Vaccenic acid (VA), the predominant ruminant-derivedtransfat in the food chain, ameliorates hyperlipidemia, yet mechanisms remain elusive. We investigated whether VA could influence tissue endocannabinoids (ECs) by altering the availability of their biosynthetic precursor, arachidonic acid (AA), in membrane phospholipids (PLs). JCR:LA-cprats were assigned to a control diet with or without VA (1% w/w),cis-9,trans-11 conjugated linoleic acid (CLA) (1% w/w) or VA+CLA (1% + 0.5% w/w) for 8 weeks. VA reduced the EC, 2-arachidonoylglycerol (2-AG), in the liver and visceral adipose tissue (VAT) relative to control diet (P< 0.001), but did not change AA in tissue PLs. There was no additive effect of combining VA+CLA on 2-AG relative to VA alone (P> 0.05). Interestingly, VA increased jejunal concentrations of anandamide and those of the noncannabinoid signaling molecules, oleoylethanolamide and palmitoylethanolamide, relative to control diet (P< 0.05). This was consistent with a lower jejunal protein abundance (but not activity) of their degrading enzyme, fatty acid amide hydrolase, as well as the mRNA expression of TNFα and interleukin 1ß (P< 0.05). The ability of VA to reduce 2-AG in the liver and VAT provides a potential mechanistic explanation to alleviate ectopic lipid accumulation. The opposing regulation of ECs and other noncannabinoid lipid signaling molecules by VA suggests an activation of benefit via the EC system in the intestine.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Arachidonic Acids/metabolism , Endocannabinoids/metabolism , Ethanolamines/metabolism , Intestinal Mucosa/metabolism , Intestines/drug effects , Metabolic Syndrome/metabolism , Oleic Acids/pharmacology , Polyunsaturated Alkamides/metabolism , Amidohydrolases/genetics , Amidohydrolases/metabolism , Animals , Anti-Inflammatory Agents/therapeutic use , Caco-2 Cells , Cytokines/genetics , Cytokines/metabolism , Dietary Supplements , Disease Models, Animal , Gene Expression Regulation, Enzymologic/drug effects , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Intestines/pathology , Intra-Abdominal Fat/drug effects , Intra-Abdominal Fat/metabolism , Liver/drug effects , Liver/metabolism , Male , Membrane Lipids/metabolism , Oleic Acids/therapeutic use , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats
2.
J Nutr ; 144(3): 252-7, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24368431

ABSTRACT

Dietary choline is required for proper structure and dynamics of cell membranes, lipoprotein synthesis, and methyl-group metabolism. In mammals, choline is synthesized via phosphatidylethanolamine N-methyltransferase (Pemt), which converts phosphatidylethanolamine to phosphatidylcholine. Pemt(-/-) mice have impaired VLDL secretion and developed fatty liver when fed a high-fat (HF) diet. Because of the reduction in plasma lipids, Pemt(-/-)/low-density lipoprotein receptor knockout (Ldlr(-/-)) mice are protected from atherosclerosis. The goal of this study was to investigate the importance of dietary choline in the metabolic phenotype of Pemt(-/-)/Ldlr(-/-) male mice. At 10-12 wk of age, Pemt(+/+)/Ldlr(-/-) (HF(+/+)) and half of the Pemt(-/-)/Ldlr(-/-) (HF(-/-)) mice were fed an HF diet with normal (1.3 g/kg) choline. The remaining Pemt(-/-)/Ldlr(-/-) mice were fed an HF diet supplemented (5 g/kg) with choline (HFCS(-/-) mice). The HF diet contained 60% of calories from fat and 1% cholesterol, and the mice were fed for 16 d. HF(-/-) mice lost weight and developed hepatomegaly, steatohepatitis, and liver damage. Hepatic concentrations of free cholesterol, cholesterol-esters, and triglyceride (TG) were elevated by 30%, 1.1-fold and 3.1-fold, respectively, in HF(-/-) compared with HF(+/+) mice. Choline supplementation normalized hepatic cholesterol, but not TG, and dramatically improved liver function. The expression of genes involved in cholesterol transport and esterification increased by 50% to 5.6-fold in HF(-/-) mice when compared with HF(+/+) mice. Markers of macrophages, oxidative stress, and fibrosis were elevated in the HF(-/-) mice. Choline supplementation normalized the expression of these genes. In conclusion, HF(-/-) mice develop liver failure associated with altered cholesterol metabolism when fed an HF/normal choline diet. Choline supplementation normalized cholesterol metabolism, which was sufficient to prevent nonalcoholic steatohepatitis development and improve liver function. Our data suggest that choline can promote liver health by maintaining cholesterol homeostasis.


Subject(s)
Cholesterol/metabolism , Choline/administration & dosage , Diet, High-Fat/adverse effects , Liver/drug effects , Liver/metabolism , Animals , Cholesterol Esters/metabolism , Fatty Liver/drug therapy , Fatty Liver/etiology , Lipid Metabolism/drug effects , Liver/pathology , Male , Mice , Mice, Knockout , Non-alcoholic Fatty Liver Disease , Phosphatidylethanolamine N-Methyltransferase/blood , Receptors, LDL/blood , Triglycerides/metabolism
3.
Biochim Biophys Acta ; 1821(3): 396-404, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22179027

ABSTRACT

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1(-/-) fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1(-/-) fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2(-/-) cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1(-/-) and NPC2(-/-) fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1(-/-) fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2(-/-) fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1(-/-) cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Carrier Proteins/metabolism , Cholesterol/metabolism , Glycoproteins/metabolism , Lysosomes/metabolism , Membrane Glycoproteins/metabolism , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/genetics , Animals , Carrier Proteins/genetics , Cells, Cultured , Cholesterol Esters/metabolism , Esterification , Fibroblasts/enzymology , Fibroblasts/metabolism , Gene Expression , Glycoproteins/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Hydroxymethylglutaryl CoA Reductases/genetics , Hydroxymethylglutaryl CoA Reductases/metabolism , Intracellular Signaling Peptides and Proteins , Lipoproteins, HDL/biosynthesis , Lipoproteins, LDL/physiology , Membrane Glycoproteins/genetics , Mice , Niemann-Pick C1 Protein , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vesicular Transport Proteins
4.
Circ Res ; 92(3): 272-8, 2003 Feb 21.
Article in English | MEDLINE | ID: mdl-12595338

ABSTRACT

Endothelial cell proteinase activated receptors (PARs) belong to a family of heterotrimeric G protein-coupled receptors that are implicated in leukocyte accumulation and potentiation of reperfusion injury. We characterized the effect and the signal transduction pathways recruited after stimulation of endothelial PAR2. We used von Willebrand Factor (vWF) release and monolayer permeability to peroxidase to report Weibel-Palade body (WPB) exocytosis and pore formation, respectively. Human umbilical vein endothelial cells (HUVECs) were stimulated with the selective PAR2 agonist peptide SLIGRL-NH2 or PAR1 agonist peptide TFLLR-NH2. PAR2 stimulation resulted in WPB exocytosis like PAR1 stimulation but, unlike PAR1, failed to increase monolayer permeability. BAPTA-AM inhibited PAR2-induced exocytosis, indicating a PAR2 calcium-dependent signal in ECs. Moreover, PAR2-like PAR1-stimulated exocytosis requires actin cytoskeleton remodeling, because vWF release is inhibited if the cells were pretreated with Jasplakinolide. Rho-GTPase activity is required for PAR-stimulated exocytosis, because inactivation of this family of actin-regulatory proteins with Clostridium difficile toxin B blocked exocytosis. Expression of dominant-negative mutant Cdc42(17N) inhibited exocytosis whereas neither dominant-negative Rac(17N) expression nor C3 exotoxin treatment affected vWF release. PAR2 stimulated RhoA-GTP weakly compared with the PAR1 agonist. We conclude that both PAR2 and PAR1 elicit WP body exocytosis in a calcium and Cdc42 GTPase-dependent manner. In contrast, the differential effect of PAR1 versus PAR2 activation to increase monolayer permeability correlates with weak RhoA activation by the PAR2 agonist.


Subject(s)
Bacterial Proteins , Depsipeptides , Egtazic Acid/analogs & derivatives , Endothelium, Vascular/metabolism , Exocytosis/physiology , Receptors, Thrombin/metabolism , Bacterial Toxins/pharmacology , Calcium/metabolism , Cells, Cultured , Chelating Agents/pharmacology , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Egtazic Acid/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Exocytosis/drug effects , Humans , Membranes, Artificial , Nucleic Acid Synthesis Inhibitors/pharmacology , Peptides/pharmacology , Peptides, Cyclic/pharmacology , Permeability/drug effects , Receptor, PAR-1 , Receptor, PAR-2 , Receptors, Thrombin/agonists , Signal Transduction/drug effects , Signal Transduction/physiology , Weibel-Palade Bodies/metabolism , cdc42 GTP-Binding Protein/metabolism , rho GTP-Binding Proteins/antagonists & inhibitors , rho GTP-Binding Proteins/metabolism , von Willebrand Factor/metabolism
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