Lipid-lowering activity of a novel class of cardiac-safe thyromimetics in dogs

The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/ 15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was required for the cell-mediated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/ 15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein ( Lipoxygenases are a class of enzymes that incorporate one molecular oxygen into unsaturated fatty acids giving rise to their hydroperoxy derivatives. There are 5-, 8-, 12-, and 15-lipoxygenases in mammalian tissues, named according to the number of carbon atoms of arachidonic acid to be oxygenated (1-4). The 12-lipoxygenase subfamily includes leukocyte, platelet, and epidermal isoforms. 15-Lipoxygenase-1 was first isolated from reticulocytes, and 15-lipoxygenase-2 was cloned from hair follicles (3). Because the leukocyte 12-lipoxygenase and 15-lipoxygenase-1 are highly related in their primary structures and enzymological properties and are abundant in various tissues of many species, these enzymes are called collectively 12/15-lipoxygenases (2, 5). Although the pathophysiological functions of the 12/15-lipoxygenases are still a subject of investigation and discussion, recent research progress has revealed the involvement of the enzymes in the development of atherosclerosis (6 -8).The oxidative modification of low density lipoprotein (LDL) We previously demonstrated that 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was involved essentially in the oxidation of LDL based upon the stereospecific oxygenation of esterified unsaturated fatty acid in LDL (21). This fact suggests direct interaction of the enzyme with LDL or the transfer of cellular lipids oxygenated by the enzyme to LDL. Secretion or leakage of the 12/15-lipoxygenase to the medium was ruled out (21). As the mechanism of the cell-mediated oxidation of extracellular LDL, we postulate that binding of native LDL to cell surface receptors is the first step in the 12/15-lipoxygenase-expressing cells. Among such receptors, the LDL receptor plays an important role in LDL metabolism in liver and steroidogenic tissues. However, the LDL receptor is not expressed in the intima of normal or atherosclerotic arteries (22). Importantly, the LDL receptor is not required in the cell-mediated LDL oxidation as shown by in vitro experiments (23) and LDL receptor-deficient mice studies (20,24). The native LDL also binds to LDL receptor-related protein

Section: Methodsmentioning

confidence: 99%

Low Density Lipoprotein Receptor-related Protein Is Required for Macrophage-mediated Oxidation of Low Density Lipoprotein by 12/15-Lipoxygenase

Xu¹,

Takahashi²,

Sakashita³

et al. 2001

“…DiD-labeled AcLDL Uptake Assays-DiD (1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindodicarbocyanine perchlorate)-labeled AcLDL was prepared from human LDL according to the methods previously described for 1,1Ј-dioctadecyl-3, 3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate labeling (17). CHO cells that stably expressed CFP-tagged LOX-1 (18) were grown on coverslips.…”

Section: Lc/esi//ms/ms-massmentioning

confidence: 99%

Lipid Peroxidation Generates Body Odor Component trans-2-Nonenal Covalently Bound to Protein in Vivo

Ishino

Wakita

Shibata

et al. 2010

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis-and trans-N ⑀ -3-[(hept-1-enyl)-4-hexylpyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis-and trans-HHP-lysine and confirmed that the 2-nonenallysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.

“…To determine whether the increased cellular cholesterol in the NPC mutants was due to defective regulation of LDL cholesterol uptake and/or de novo cholesterol synthesis, we performed the following studies. LDLr activity in the fibroblasts was measured using an established, quantitative assay that monitors the uptake of DiI-LDL, a fluorescent-tagged LDL, by flow cytometry (26,27). With this method, uptake of DiI-LDL was abolished in the presence of 80-fold excess of unlabeled LDL, demonstrating that the DiI-LDL uptake is a specific measure of LDLr activity (data not shown).…”

Section: Regulation Of Sterol Homeostatic Responses Is Defective Inmentioning

confidence: 99%

NPC1 and NPC2 Regulate Cellular Cholesterol Homeostasis through Generation of Low Density Lipoprotein Cholesterol-derived Oxysterols

Frolov

Zielinski

Crowley

et al. 2003

192

180

Mutations in the Niemann-Pick disease genes cause lysosomal cholesterol accumulation and impaired low density lipoprotein (LDL) cholesterol esterification. These findings have been attributed to a block in cholesterol movement from lysosomes to the site of the sterol regulatory machinery. In this study we show that Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) mutants have increased cellular cholesterol, yet they are unable to suppress LDL receptor activity and cholesterol biosynthesis. Cholesterol overload in both NPC1 and NPC2 mutants results from the failure of LDL cholesterol tobothsuppresssterolregulatoryelement-bindingproteindependent gene expression and promote liver X receptormediated responses. However, the severity of the defect in regulation of sterol homeostasis does not correlate with endoplasmic reticulum cholesterol levels, but rather with the degree to which NPC mutant fibroblasts fail to appropriately generate 25-hydroxycholesterol and 27-hydroxycholesterol in response to LDL cholesterol. Moreover, we demonstrate that treatment with oxysterols reduces cholesterol in NPC mutants and is able to correct the NPC1 I1061T phenotype, the most prevalent NPC1 disease genotype. Our findings support a role for NPC1 and NPC2 in the regulation of sterol homeostasis through generation of LDL cholesterol-derived oxysterols and have important implications for the treatment of NPC disease.