LepR-PI3K-AKT通过Nedd4-2调节孕前超重/肥胖孕妇胎盘氨基酸转运的机制研究

Pre-pregnancy overweight/obesity increased the risk of having low/high birth weight neonates, which was closely associated with placental amino acid transport capacity. Leptin regulates placental amino acid transport, however, the mechanisms of leptin signaling affects amino acid transport remain to be clarified. Placental amino acid transport capacity is determined by the nutrient exchange area of the syncytiotrophoblast and the expression of transporters in microvillous plasma membranes (MVM). Leptin bands to its receptors and activates its downstream signaling, which may affect the membrane trafficking of amino acid transporters. Recent studies found that expression levels of placental leptin receptors (LepR) was associated with fetal birth weight. We propose that in pre-pregnancy overweight/obesity women, the expression of LepRs (LepRa and LepRb) and the transduction of downstream signaling might be disrupted, which affects placental amino acid transport capacity, results in abnormal fetal growth. In addition, ubiquitination of amino acid transporters in MVM by Nedd4-2 decreased the transporter expression in the plasma membrane. There might be interaction between Nedd4-2 and PI3K-AKT signaling.. The present investigation aims to study the placental leptin signaling and amino acid transport in pre-pregnancy overweight/obesity women, using Western blotting and placenta fragment culture technique to investigate the relationship among birth weight and maternal leptin, cord blood leptin, placental leptin, LepRa and LepRb expression levels, and analyze the differences of placental leptin signaling and amino acid transport capacity in different birth outcomes. Next, we use human trophoblast cell line JEG-3 to study the mechanisms of leptin signaling in the regulation of placental amino acid transporters expression. We use pharmacology, lentiviral transfection, Co-Immunoprecipitation, in situ proximity ligation assay, site-directed mutagenesis methods to investigate the role of Nedd4-2 in mediating LepR-PI3K-AKT signaling in the regulation of System A amino acid transporters expression in MVM. Then, further analyze the mechanisms of PI3K-AKT signaling in the regulation of Nedd4-2 activity. This study aims to demonstrate the molecular mechanisms of the regulation of placental amino acid transport by LepR-PI3K-AKT signaling via Nedd4-2 in pre-pregnancy overweight/obesity women (from post translational level), and may help to identify novel therapeutic strategies to improve birth outcomes and prevent the occurrence of obesity and related metabolic diseases in adolescents.

孕前超重/肥胖是引起新生儿出生体重过高或过低的高危因素，其不同妊娠结局的产生与胎盘氨基酸转运能力密切相关。研究发现胎盘氨基酸转运受瘦素影响，但其调控机制尚待阐明。本研究以孕前超重/肥胖孕妇为研究对象，收集正常、高、低出生体重儿胎盘，通过蛋白印记法与绒毛膜片段培养技术，观察不同出生结局胎盘中瘦素信号通路的激活情况、氨基酸转运体表达及转运能力，分析胎盘瘦素敏感性、氨基酸转运能力与胎儿出生体重的关系；以人滋养层细胞系JEG-3为细胞模型，采用慢病毒转染、免疫共沉淀、邻位连接、定点突变等技术，研究泛素连接酶Nedd4-2在LepR-PI3K-AKT信号通路调节微绒毛膜氨基酸转运体表达过程中的作用，并进一步探讨PI3K-AKT调控Nedd4-2活性的机制，从翻译后水平揭示胎盘瘦素信号通路调控氨基酸转运体表达的分子机制。本研究为临床干预改善不良出生结局及青少年代谢疾病的预防提供有价值的研究资料。

孕前超重/肥胖增加了妊娠糖尿病（GDM）、高血压、先兆子痫、早产及其他并发症如巨大儿的发生风险。胎盘连接母胎循环，在母胎营养交换中发挥重要作用。然而孕前超重/肥胖引起胎盘氨基酸及脂肪酸转运异常的机制尚不明确。本研究以孕前超重及GDM孕妇为研究对象，分析不同组孕妇生产巨大儿及大于胎龄儿的危险因素，并进一步探究胎盘氨基酸及脂肪酸转运体的表达、瘦素信号通路的激活及其与胎儿出生体重的关系；建立母体妊娠期高脂饮食动物模型，检测胎盘形态学改变，氨基酸转运体表达变化及瘦素信号通路激活情况及其与Nedd4-2活性之间的关系，探讨PI3K-AKT、ERK1/2及mTORC1信号通路调节营养转运体表达的分子机制。. 研究发现：1、孕前超重是生产大于胎龄儿的独立风险因素；2、相较GDM，孕前超重是影响胎盘营养转运的主要因素，且孕前超重孕妇胎盘氨基酸转运体SNAT2及脂肪酸转运体表达明显升高，瘦素受体表达显著升高，mTORC1，ERK1/2通路激活程度较正常孕妇显著增高，且ERK1/2的激活水平与胎儿出生体重正相关，但胎盘AKT信号通路激活水平未见显著改变；3、母体妊娠期高脂饮食增加母体孕期脂肪含量、血浆瘦素水平、降低胎盘滋养层厚度、并增加胎盘SNAT2的基因及蛋白表达，胎盘mTORC1信号通路激活程度增强，但AKT信号通路激活水平减弱，且Nedd4-2的表达降低，Nedd4-2的表达水平与AKT的磷酸化程度正相关。以上结果提示孕前超重是影响母体代谢表型及胎盘营养物质转运的主要因素，而动物模型中高脂饮食母鼠胎盘SNAT2表达增加与PI3K-AKT通路调节Nedd4-2的表达有关，但在人类胎盘ERK1/2信号通路可能存在更为显著的调节作用。该研究结果为临床早期干预调节胎盘营养转运提供了基础研究数据。