High plasma apoB-containing lipoprotein (B-lp) levels enhance the risk for atherosclerosis. Our long-term goal is to explain molecular mechanisms of B-lp biosynthesis to find novel ways of reducing their concentrations. Previously, we described apoB-dependent and -independent pathways for free and esterified cholesterol transport by the intestine and defined the roles of MTP, ABCA1, ACAT2, apoAI, and apoAIV in these pathways. Concurrently, we observed that MTP-deficient abetalipoproteinemia patients and mice deficient in hepatic and intestinal MTP (L,I-Mttp-/-) have markedly lower plasma ceramide and sphingomyelin levels compared with controls but have normal hexosylceramide and lactosylceramide. Moreover, hepatic ABCA1 deficiency in mice and total deficiency in Tangier patients significantly reduced plasma sphingomyelin and hexosylceramide with no effect on lactosylceramide. Hence, we hypothesize that (1) MTP and ABCA1 are the major determinants of plasma ceramide and sphingomyelin; (2) ceramide secretion depends on MTP-mediated B-lp assembly; and (3) sphingomyelin transport involves both MTP- and ABCA1-dependent pathways.
Aim 1 : To determine the role of hepatic and intestinal MTP and ABCA1 in the control of plasma and tissue sphingolipids, we will use mice deficient in one or both of these genes in specific tissues. We will (1) measure total and individual species of ceramide, sphingomyelin, hexosylceramide, and lactosylceramide in the plasma, liver, and intestine of fed and fasted male L-Mttp-/- and I-Mttp-/- mice; (2) quantify these lipids in the plasma, liver, and intestine of fed and fasted Abca1-/-, L-Abca1-/-, and I-Abca1-/- mice; and (3) evaluate tissue-specific contributions of both MTP and ABCA1 using I-Mttp-/-;I-Abca1-/- and L-Mttp-/-;L-Abca1-/- double knockout mice.
Aim 2 : To explain biochemical and molecular mechanisms of sphingolipid transport to the plasma, we will (1) determine whether MTP is required for ceramide and sphingomyelin secretion; (2) find out if their secretion with B-lp is regulated by different faty acids that regulate B-lp secretion and ceramide synthesis; (3) ascertain if MTP transfers these lipids; and (4) elucidate mechanisms by which ABCA1 transports sphingolipids and study regulation of their secretion with HDL by liver X receptor (LXR) using agonists and Lxr deficient mice.
Aim 3 : To examine the role of human MTP and ABCA1 in sphingolipid transport, we will (1) study their role in the secretion of sphingolipids using human liver and intestinal cells; and (2) analyze the regulation of their secretion with B-lp and HDL by fatty acids and LXR. We expect these studies to (1) establish that sphingolipid transport involves different mechanisms; (2) show that MTP and ABCA1 are the major determinants of plasma sphingolipids; and (3) quantify the contributions of intestinal and hepatic B-lps and HDL to plasma sphingolipids. It is likely that lipoprotein-mediated sphingomyelin transport helps to maintain plasma membrane integrity of other tissues and ceramide transport protects the liver and intestine from ceramide toxicity.
In the previous funding cycle, we illustrated mechanisms involved in the absorption and transport of free and esterified cholesterol. In this grant, we propose to delineate mechanisms involved and roles played by MTP and ABCA1 in the absorption and transport of ceramide and sphingomyelin with B-lp and HDL. These studies will provide novel information about the role of hepatic and intestinal MTP and ABCA1 in the regulation of plasma and tissue sphingolipids.
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