ABSTRACT
BACKGROUND AND AIMS: Members of the family of ABC transporters are involved in different processes of sterol metabolism, and ABCA1 was recently identified as a key regulator of high-density lipoprotein (HDL) metabolism. Our aim was to further analyze the role of ABCA1 in cholesterol metabolism. METHODS: ABCA1-deficient mice (ABCA1-/-) and wild-type mice were compared for different aspects of sterol metabolism. Intestinal cholesterol absorption was determined by a dual stable isotope technique, and analysis of fecal, plasma, and tissue sterols was performed by gas chromatography/mass spectrometry. Key regulators of sterol metabolism were investigated by Northern and Western blot analyses or enzyme activity assays. RESULTS: ABCA1-disrupted sv129/C57BL/6 hybrid mice showed a significant reduction in intestinal cholesterol absorption. The decrease in cholesterol absorption was followed by an enhanced fecal loss of neutral sterols, whereas fecal bile acid excretion was not affected. Total body cholesterol synthesis was significantly increased, with enhanced 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase observed in adrenals and spleen. In addition, ABCA1-/- mice showed markedly increased concentrations of cholesterol precursors in the plasma, lung, intestine, and feces. Reduced HMG-CoA reductase messenger RNA and enzyme activity in the liver suggest that enhanced cholesterol synthesis in ABCA1-/- mice occurs in peripheral tissues rather than the liver. CONCLUSIONS: The metabolism of cholesterol and cholesterol precursors is markedly affected by a lack of ABCA1 function.
Subject(s)
ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Cholesterol/biosynthesis , Cholesterol/pharmacokinetics , Intestinal Absorption/physiology , Membrane Proteins , Receptors, Immunologic , Receptors, Lipoprotein , ATP Binding Cassette Transporter 1 , Animals , Bile Acids and Salts/analysis , CD36 Antigens/biosynthesis , Cholesterol/blood , Eating , Feces/chemistry , Intestinal Mucosa/metabolism , Liver/metabolism , Lung/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Scavenger , Scavenger Receptors, Class BABSTRACT
Tangier disease (TD) and familial HDL deficiency (FHA) have recently been linked to mutations in the human ATP-binding cassette transporter 1 (hABCA1), a member of the ABC superfamily. Both diseases are characterized by the lowering or lack of high-density lipoprotein cholesterol (HDL-C) and low serum cholesterol. The murine ABCA1-/- phenotype corroborates the human TD linkage to ABCA1. Similar to TD in humans, HDL-C is virtually absent in ABCA1-/- mice accompanied by a reduction in serum cholesterol and lipid deposition in various tissues. In addition, the placenta of ABCA1-/- mice is malformed, resulting in severe embryo growth retardation, fetal loss, and neonatal death. The basis for these defects appears to be altered steroidogenesis, a direct result of the lack of HDL-C. By 6 months of age, ABCA1-/- animals develop membranoproliferative glomerulonephritis due to deposition of immunocomplexes followed by cardiomegaly with ventricular dilation and hypertrophy, ultimately succumbing to congestive heart failure. This murine model of TD will be very useful in the study of lipid metabolism, renal inflammation, and cardiovascular disease and may reveal previously unsuspected relationships between them.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Cholesterol, HDL/deficiency , Disease Models, Animal , Glomerulonephritis/etiology , Placenta/abnormalities , Tangier Disease/etiology , ATP Binding Cassette Transporter 1 , Animals , Animals, Newborn , Blotting, Northern , Blotting, Southern , Cholesterol, HDL/blood , DNA/analysis , DNA Mutational Analysis , DNA Primers/chemistry , Female , Fetal Death , Glomerulonephritis/metabolism , Glomerulonephritis/pathology , In Situ Hybridization , Male , Mice , Mice, Knockout , Mutation , Placenta/metabolism , Placenta/pathology , Polymerase Chain Reaction , Pregnancy , RNA, Messenger/metabolism , Tangier Disease/metabolism , Tangier Disease/pathologyABSTRACT
In this work, we address the question of whether replicative senescence can be induced in immortal nontumorigenic human fibroblasts. The immortal fibroblasts used in this study were derived from two Li-Fraumeni (LF) patients who carry in their germ line one wild-type and one mutant p53 allele. Both immortal lines have lost the wtp53 allele and express no detectable p16INK4a protein, although they carry the p16INK4a gene. In contrast to immortal human fibroblasts, senescent human fibroblasts have a low content of 5-methyl-cytosine in their DNA. This observation suggested the possibility that a demethylating agent could revert the immortal phenotype and induce replicative senescence in the immortal cell lines. Cells of the two LF lines were exposed to the demethylating agent 5-aza-2'-deoxycytidine. Within 6 days, all cells were growth arrested and showed the enlarged and flat morphology characteristic of senescent cells, an accumulation of lipofuscin granules and senescence-associated beta-galactosidase activity at pH6, both biomarkers for senescence. Immunoblots of 5-aza-2'-deoxycytidine-treated cells showed a greatly increased expression of p16INK4a protein but no detectable change in the expression of p21CIP1, a gene known to be strongly expressed in senescent normal human fibroblasts. In two other experimental series, cells of the two LF lines were infected with retroviral constructs encoding either p16INK4a or p21CIP1. Each of the transduced genes induced senescence without affecting the expression of the other endogenous gene. The results show that induction of senescence in immortal LF fibroblasts can occur by different pathways: (a) by demethylation-dependent pathways that induce the expression of p16INK4a; and (b) by demethylation-independent pathways involving the expression of p21CIP1. The induction of senescence by p16INK4a and p21CIP1 occurred equally in the two human immortal fibroblast lines, which differed in the length of their telomeres and the activity of their telomerase.
