Plasma HDLs are thought to protect against atherosclerosis via mechanisms related, in part, to reverse cholesterol transport (RCT) ( 1-3 ). This process involves removal of excess cholesterol from arterial foam-cells (i.e., cholesterol effl ux), followed by the transport of cholesterol to the liver for excretion in feces. Over-expression of apolipoprotein (apo) A-I, the major protein of HDL, in mice enhances RCT, inhibits atherosclerosis development, and stimulates regression of plaque lesions ( 4-8 ). Similar effects can be obtained upon infusion of apoA-I-phospholipid complexes in mice and humans, suggesting HDL may be useful therapeutically ( 9-11 ).Small peptides based on HDL apolipoproteins are being developed as alternatives to full-length recombinant proteins for therapeutic use (12)(13)(14). The mechanisms by which apolipoprotein mimetic peptides reduce atherosclerosis are not fully understood. The most widely studied 4F peptide reduces atherosclerosis in animal models via mechanisms related to binding of oxidized lipid ( 12,15 ). The latter anti-oxidant activity greatly exceeds that of apoA-I ( 16 ). The 18A/4F peptides can stimulate cholesterol effl ux from cells, albeit with far weaker activity Abstract Here, we report the creation of a single-helix peptide (ATI-5261) that stimulates cellular cholesterol effl ux with K m molar effi ciency approximating native apolipoproteins. Anti-atherosclerosis activity of ATI-5261 was evaluated in LDLR ؊ / ؊ and apolipoprotein (apo)E ؊ / ؊ mice ف 5-7 months of age, following 13-18 weeks on a high-fat Western diet (HFWD). Treatment of fat-fed LDLR؊ / ؊ mice with daily intraperitoneal injections of ATI-5261 (30 mg/kg) for 6 weeks reduced atherosclerosis by 30%, as judged by lesion area covering the aorta (7.9 ± 2 vs.11.3 ± 2.5% control, P = 0.011) and lipid-content of aortic sinus plaque (25 ± 5.8 vs. 33 ± 4.9% control, P = 0.014). In apoE ؊ / ؊ mice, the peptide administered 30 mg/kg ip on alternate days for 6 weeks reduced atherosclerosis by ف 45% (lesion area = 15 ± 7 vs. 25 ± 8% control, P = 0.00016; plaque lipid-content = 20 ± 6 vs. 32 ± 8% control, P < 0.0001). Similar reductions in atherosclerosis were achieved using ATI-5261:POPC complexes. Single intraperitoneal injection of ATI-5261 increased reverse cholesterol transport from macrophage foam-cells to feces over 24-48 h. In summary, relatively short-term treatment of mice with the potent cholesterol effl ux peptide ATI-5261 reduced substantial atherosclerosis. This was achieved using an L-amino acid peptide, in the presence of severe hypercholesterolemia/ HFWD, and did not require daily injections or formulation with phospholipids when administered via intraperitoneal
Previous studies from this laboratory identified excessive oxidative stress as an important mediator of age-related decline in steroid hormone production. Here, we investigated whether oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. To accomplish these studies, we employed a highly responsive mouse adrenocortical cell line, Y1-BS1 cells that secrete large quantities of steroids when stimulated with lipoprotein plus hormone. Treatment of these cells with superoxide, H 2 O 2 or 4-hydroxy-2-nonenal (HNE) significantly inhibited steroid production and increased phosphorylation and activation of p38 MAPK. None of the treatments altered the phosphorylation of either extracellular signal-regulated kinases or c-Jun N-terminal kinases (JNKs). Pretreatment of Y1-BS1 cells with MnTMPyP, a cell-permeable superoxide-dismutase/ catalase mimetic reactive oxygen species (ROS scavenger), completely prevented the superoxide-and H 2 O 2 -mediated inhibition of steroid production. Likewise, antioxidant N-acetylcysteine completely blocked the HNE-induced loss of steroidogenic response. Incubation of Y1-BS1 cells with either MnTMPyP or NAC also upregulated Bt 2 cAMP and Bt 2 cAMPChHDL 3 -stimulated steroid synthesis, indicating that endogenously produced ROS can inhibit steroidogenesis. Inhibition of p38 MAPK with SB203580 or SB202190 upregulated the basal steroid production and also prevented the oxidant-mediated inhibition of steroid production. mRNA measurements by qPCR indicated that Y1-BS1 adrenal cells predominantly express p38 MAPKa isoform, along with relatively low-level expression of p38 MAPKg. By contrast, little or no expression was detected for p38 MAPKb and p38 MAPKd isoforms in these cells. Transfection of Y1-BS1 cells with either caMKK3 or caMMK6 construct, the upstream p38 MAPK activators, decreased steroidogenesis, whereas transfection with dnMKK3 or dnMKK6 plasmid DNA increased steroidogenesis. Similarly, transfection of cells with a dnp38 MAPKa or dnp38 MAPKb construct also increased steroid hormone production; however, the effect was less pronounced after expression of either dnp38 MAPKg or dnp38 MAPKd construct. These results indicate that activated p38 MAPK mediates oxidant (excessive oxidative stress)-induced inhibition of adrenal steroidogenesis.
Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.
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