Role of Lipoprotein Assembly in Maternal-Fetal Transport of Beta-Carotene

脂蛋白组装在 β-胡萝卜素母胎转运中的作用

• 批准号: 10390463
• 负责人: M Mahmood Hussain
• 金额: $ 37.02万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-12 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10390463
• 关键词: Adult; Affect; Anabolism; Animal Model; Apolipoproteins B; Beta Carotene; Biological Assay; Biological Process; Blood Circulation; Carotenoids; Cells; Congenital Abnormality; Data; Developed Countries; Developing Countries; Development; Dietary Component; Dietary Practices; Dose; Elements; Embryo; Embryonic Development; Event; Fetal health; Fetus; Generations; Genetic Transcription; HNF4A gene; Health; Human; In Vitro; Incidence; Intake; Intervention; Intraperitoneal Injections; Knowledge; Link; Lipoproteins; Mediating; Metabolism; Micronutrients; Molecular; Mothers; Mus; Nutrient; Oxides; Oxygenases; Placenta; Pregnancy; Pregnant Women; Premature Birth; Production; Public Health; Publishing; Regulation; Reporter; Retinaldehyde; Retinoids; Risk; Role; Small Interfering RNA; Source; Time; Tretinoin; Vitamin A; Vitamin A Deficiency; Vitamins; Work; base; chromatin immunoprecipitation; dietary; embryo tissue; enhancing factor; fetal; human model; in vivo; inhibitor; insight; intervention program; knock-down; microsomal triglyceride transfer protein; mouse model; novel; promoter; transcription factor; trophoblast

Embryonic vitamin A deficiency or excess have been linked to congenital defects in humans and animal models, owing to the defective transcriptional action of retinoic acid (RA), the active form of vitamin A, during embryogenesis. β-carotene (BC) is the most abundant dietary vitamin A precursor. In the embryo, BCO1 cleaves intact BC, from the maternal circulation, to generate retinaldehyde, which is then oxidized to RA. Asymmetric cleavage of BC by BCO2, generates β-apo-10'-carotenal (apo10AL) which can serve as a precursor of retinoids (vitamin A and its derivatives), but also antagonize RA action. Our data suggest that lipoproteins synthesized within placenta mediate the transfer of BC towards the fetus. We hypothesize that apo10AL, generated in placenta by BCO2 from BC, upregulates microsomal triglyceride transfer protein (Mttp; MTP) through HNF4α and/or COUP-TFs, by antagonizing the action of RA. We propose three Specific Aims to understand the molecular mechanisms of BC placental-fetal transfer under a normal maternal vitamin A status. Aim 1. To investigate in vivo the regulation of placental lipoprotein biosynthesis by apo10AL and RA. We will assess placental carotenoids and retinoids levels and metabolism, and lipoprotein biosynthesis in WT, Bco1-/- and Bco2-/- mice. Aim 1A: To define the molecular events related to carotenoid metabolism and lipoprotein production that occur in placenta after a single maternal administration of BC. Dams will be sacrificed at various time points post-BC administration (at 13.5 dpc, by IP injection). Aim 1B: To establish the effects of a single maternal BC administration at various times during gestation on the molecular events related to carotenoid metabolism and lipoprotein production. Dams will be administered BC at various times during gestation and sacrificed after 4 and 24 h. Aim 2. To establish that placenta MTP deficiency abrogates accumulation of embryonic BC after its administration to the mother. A mouse model with placental-specific inactivation of Mttp will be used. Dams will be sacrificed 4 and 24 h after BC administration at 13.5 dpc, and placentas analyzed as in Aim 1. Aim 3. To investigate in vitro the regulation of human placental lipoprotein biosynthesis by apo10AL and RA. In this Aim, we will expand our studies to human models. Aim 3A: To study the apo10AL-mediated regulation of MTP function in human placenta explants and Bewo cells. We will: (i) determine the time course and dose-dependent effect of BC, apo10AL and RA on carotenoid and retinoid metabolism and lipoprotein biosynthesis; (ii) inactivate MTP by siRNA or an MTP inhibitor and (iii) Bco1 and Bco2 by siRNA; (iv) study the competing effects of apo10AL and RA on MTP expression. Aim 3B: To identify the cis-elements in the Mttp promoter and the transcription factors required for apo10AL-mediated increase in Mttp transcription. Promoter reporter assays, transcription factors knockdown, and ChIP assays will establish how placental Mttp expression is regulated by apo10AL through HNF4a and/or COUP-TFs.

胚胎维生素 A 缺乏或过量与人类和动物模型的先天性缺陷有关，这是由于视黄酸 (RA)（维生素 A 的活性形式）在胚胎发生过程中的转录作用有缺陷。 β-胡萝卜素 (BC) 是最丰富的膳食维生素 A 前体。在胚胎中，BCO1 从母体循环中裂解完整的 BC，生成视黄醛，然后氧化为 RA。 BCO2 对 BC 进行不对称裂解，生成 β-apo-10'-胡萝卜素 (apo10AL)，它可以作为类视黄醇（维生素 A 及其衍生物）的前体，同时还能拮抗 RA 作用。我们的数据表明胎盘内合成的脂蛋白介导 BC 向胎儿的转移。我们假设 apo10AL 由 BC 中的 BCO2 在胎盘中产生，通过 HNF4α 和/或 COUP-TF 拮抗 RA 的作用，上调微粒体甘油三酯转移蛋白（Mttp；MTP）。我们提出了三个具体目标来了解正常母体维生素 A 状态下 BC 胎盘-胎儿转移的分子机制。目的 1. 体内研究apo10AL和RA对胎盘脂蛋白生物合成的调节作用。我们将评估 WT、Bco1-/- 和 Bco2-/- 小鼠的胎盘类胡萝卜素和类视黄醇水平、代谢以及脂蛋白生物合成。目标 1A：确定母体单次施用 BC 后胎盘中发生的与类胡萝卜素代谢和脂蛋白产生相关的分子事件。母鼠将在 BC 给药后的不同时间点被牺牲（在 13.5 dpc，通过 IP 注射）。目标 1B：确定妊娠期间不同时间单次母体 BC 给药对与类胡萝卜素代谢和脂蛋白产生相关的分子事件的影响。母鼠将在妊娠期间的不同时间施用BC并在4小时和24小时后处死。目标 2. 确定胎盘 MTP 缺陷会消除给予母亲后胚胎 BC 的积累。将使用 Mttp 胎盘特异性失活的小鼠模型。母鼠将在 BC 给药后 13.5 dpc 后 4 和 24 小时处死，并按目标 1 中的方法对胎盘进行分析。目标 3. 体外研究 apo10AL 和 RA 对人胎盘脂蛋白生物合成的调节。在这个目标中，我们将把我们的研究扩展到人体模型。目标 3A：研究 apo10AL 介导的人胎盘外植体和 Bewo 细胞中 MTP 功能的调节。我们将： (i) 确定 BC、apo10AL 和 RA 对类胡萝卜素和类视黄醇代谢以及脂蛋白生物合成的时程和剂量依赖性影响； (ii) 通过 siRNA 或 MTP 抑制剂灭活 MTP，以及 (iii) 通过 siRNA 灭活 Bco1 和 Bco2； (iv) 研究 apo10AL 和 RA 对 MTP 表达的竞争效应。目标 3B：鉴定 Mttp 启动子中的顺式元件以及 apo10AL 介导的 Mttp 转录增加所需的转录因子。启动子报告基因检测、转录因子敲低和 ChIP 检测将确定 apo10AL 如何通过 HNF4a 和/或 COUP-TF 调节胎盘 Mttp 表达。