ABCG1: The ATP binding cassette transporter G1 (ABCG1), expressed in macrophages, liver, and other tissues, has been implicated in the efflux of cholesterol to high density lipoprotein. The ABCG1 gene is transcriptionally activated by cholesterol loading and oxysterol activators of liver X receptors (LXR). In this study we show that ABCG1 mRNA is induced by LXR agonists not only in RAW264.7 macrophage cells but also in primary mouse hepatocytes and HepG2 hepatoma cells. We identify two evolutionarily highly conserved LXR response elements, LXRE-A and LXRE-B, that confer robust orientation- and position-independent responsiveness of ABCG1 promoter-directed luciferase gene constructs to LXR agonists or LXR plus RXR agonists in both RAW264.7 and HepG2 cells. Cotransfection of plasmids expressing LXRa/RXRa and LXRa/RXRa activated the ABCG1 promoter in the presence of LXRE-A or LXRE-B. Mutations in the DR4 core motif of LXRE-A or LXRE-B abolished LXR-agonist activation. In gel shift assays, LXRa/RXRa and LXRa/RXRa heterodimers bound to the LXRE-A and LXRE-B sequences but not to mutated sequences. Chromatin immunoprecipitation assays confirmed that LXR and RXR are bound to LXRE-A and -B regions in agonist-treated THP-1 macrophages. These studies identify DNA sequences responsible for the transcriptional upregulation of the ABCG1 gene by oxysterols in macrophages and liver, two key tissues where changes in ABCG1 gene expression may markedly affect cholesterol balance and atherogenesis.? ? ? Hepatic Lipase: To elucidate the separate contributions of the lipolytic versus ligand-binding functions of hepatic lipase (HL) to lipoprotein metabolism and atherosclerosis, and to investigate the role of the LDLr in these processes, we expressed catalytically active (HL-WT) and inactive (HL-S145G) HL in mice with no endogenous expression of HL or the LDLr (LDLr-KOxHL-KO). HL-WT and HL-S145G reduced (p<0.05; all) cholesterol (55% vs. 20%), non-HDL-cholesterol (63% vs. 22%) and apoB (34% vs. 16%) by enhancing the catabolism of autologous 125I-apoB-IDL/LDL (FCR in d-1; 6.07?0.25, LDLr-KOxHL-WT; 4.76?0.30, LDLr-KOxHL-S145G; 3.70?0.13, LDLr-KOxHL-KO); the lipolytic function of HL had a greater impact on the concentration, composition, particle size and catabolism of the apoB-containing lipoproteins (apoB-Lps) and HDL. Importantly, consistent with the changes in apoB-Lps, atherosclerosis was reduced by both HL-WT and HL-S145G (p<0.05; all; 71% vs. 51%). These data identify physiologically relevant but distinct roles for the lipolytic vs. ligand-binding functions of HL in apoB-Lp metabolism and atherosclerosis and demonstrate that their differential effects on these processes are mediated by changes in catabolism via non-LDLr pathways. These changes, evident even in the presence of apoE, establish an anti-atherogenic role of the ligand-binding function of HL in LDLr-deficient mice. ? ? ABCG5/G8: We have previously shown that ABCG5/G8 overexpression in liver enhances hepatobiliary cholesterol secretion and decreases plasma plant sterol levels but does not alter fractional intestinal cholesterol absorption, plasma lipid profile, or atherosclerosis in E-KO and LDLr-KO mice. The extra bile cholesterol secreted by the overexpressed ABCG5/G8 is reabsorbed by the intestine; thus cholesterol balance across the liver is unchanged. These findings indicate that reduced intestinal cholesterol absorption is essential for changes in cholesterol balance and atherosclerosis. To evaluate this hypothesis we fed LDLr-KO x G5/G8-Tg mice and controls 15 mg/kg/day Zetia for 9 and 24 wks. Zetia decreased intestinal cholesterol absorption to 5% in all mouse groups (p<0.01; all); hepatobiliary cholesterol secretion increased in LDLr-KO x G5/G8-Tg vs LDLr-KO (1426+257 vs 572+39 uM, 4.8+1.1 vs 1.8+0.2 um/hr/kg, p<0.01; all). Thus, in ABCG5/G8-Tg mice fed Zetia, mg cholesterol output to bile/day was greater than mg of cholesterol absorbed from intestine/day. Liver cholesterol concentration was similar in control and G5/G8-Tg mice in both study groups. Plasma lipids were decreased (p<0.01; all) in LDLr-KO x G5/G8-Tg vs LDLr-KO mice fed Zetia (TC 452+27 vs 617+26, PL 497+20 vs 604+19, FC 125+9 vs 182+10, CE 327+18 vs 436+18 mg/dl), reflecting reduced LDL cholesterol in LDLr-KO x G5/G8-Tg. Proximal aortic atherosclerosis was decreased in LDLr-KO x G5/G8-Tg vs LDLr-KO mice fed Zetia. These combined data demonstrates that in the absence of changes in fractional intestinal cholesterol absorption, increased secretion of sterols into bile induced by hepatic overexpression of ABCG5/G8 may not be sufficient to alter hepatic cholesterol balance. However, increased biliary secretion combined with reduced intestinal absorption enhances cholesterol removal from the body and alters atherogenic risk in liver G5/G8-Tg mice identifying distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.
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