LDL Receptor Related Protein-1 in Metabolic and Cardiovascular Disease

描述(申请人提供)：几项大规模的人群研究已经确定了低密度脂蛋白受体相关蛋白-1(LRP1)基因的多态与广泛的代谢性疾病之间的关联，包括动脉粥样硬化、肥胖/糖尿病、非酒精性脂肪性肝病(NAFLD)和肝细胞癌(HCC)的不良预后。后者的观察表明，肝脏中的LRP1功能障碍可能会加速NAFLD进展为脂肪性肝炎和肝硬变，并增加肝细胞癌的风险。该项目过去资助期间的研究表明，肝细胞特异性LRP1基因的失活或突变改变了小鼠的LRP1内吞作用和细胞表面循环，加剧了脂肪诱导的脂肪变性和细胞死亡。由于LRP1基因的全部缺失和突变影响LRP1细胞表面循环的研究以前在人类中还没有报道，我们启动了研究，以探讨LRP1在肝细胞表面的表达是否以及如何受到生理调节。我们的初步结果表明，脂肪酸和高脂饮食抑制LRP1转位到肝细胞表面，而胰岛素则相反，刺激LRP1细胞表面转位。这一更新应用的目的是破译LRP1循环和转位到细胞表面的调控机制。我们将验证这一假设，即肝细胞表面LRP1表达的减少直接导致脂肪诱导的肝炎和肝硬变，并直接导致与高脂肪/胆固醇饮食和脂肪肝疾病相关的肝细胞癌进展加速。此外，我们认为增加LRP1对细胞表面的循环是缓解这些代谢异常的可行策略。具体目标1将确定LRP1转位到质膜的脂质抑制机制，验证脂质积累改变LRP1细胞质结构域的磷酸化，从而抑制其与转位到细胞表面所需的接头蛋白的相互作用的假设。我们还将评估两种增加LRP1循环的策略，并测试它们在改善肝细胞功能和预防脂质诱导的肝功能障碍和肝硬变方面的有效性。具体目标2将描述LRP1转位到细胞表面的胰岛素激活的机制，验证胰岛素诱导脂蛋白激酶激活产生磷脂酰肌醇(Poly)磷酸盐的假设，以增加LRP1-适配器复合体到循环内体的募集，从而转位到细胞表面。后续实验将探索透性肌醇多磷酸作为一种策略来克服胰岛素抵抗以改善肝脏代谢健康的潜力。特指目标3将验证肝细胞LRP1功能障碍促进脂质诱导性肝硬变的假设，从而提供一个理想的局部环境来加速肝脏肿瘤的进展。这些研究的完成将确定调节LRP1转位到细胞表面的机制，并提供新的策略来抑制NAFLD进展为NASH、肝硬变和肝细胞癌。

DESCRIPTION (provided by applicant): Several large-scale population studies have identified the association between polymorphisms of the LDL receptor-related protein-1 (LRP1) gene with a wide spectrum of metabolic diseases, including atherosclerosis, obesity/diabetes, nonalcoholic fatty liver diseases (NAFLD), and poor prognosis of hepatocellular carcinoma (HCC). The latter observations imply that LRP1 dysfunction in the liver may accelerate the progression of NAFLD to steatohepatitis and cirrhosis with increased HCC risk. Studies from the past funding period of this project have shown that hepatocyte-specific Lrp1 gene inactivation or mutations that alter LRP1 endocytosis and cell surface recycling in mice potentiates fat-induced steatosis and cell death. Since total LRP1 gene deletion and mutations that affect LRP1 cell surface recycling have not been reported previously in humans, we initiated studies to examine if and how LRP1 expression at the cell surface of hepatocytes may be regulated physiologically. Our preliminary results showed that fatty acid and high fat diet suppress LRP1 translocation to the cell surface of hepatocytes, whereas insulin has the opposite effect and stimulates LRP1 cell surface translocation. The objective of this renewal application is to decipher the mechanisms underlying the regulation of LRP1 recycling and translocation to the cell surface. We will test the hypothesis that reduction of cell surface expression of LRP1 in hepatocytes directly contributes to fat-induced liver inflammation and hepatic cirrhosis, and is directly responsible for the accelerated HCC progression associated with high fat/cholesterol diet and fatty liver disease. Moreover, we propose that increasing LRP1 recycling to the cell surface is a viable strategy to alleviate these metabolic abnormalities. Specific Aim 1 will identify the mechanism underlying lipid suppression of LRP1 translocation to plasma membrane, testing the hypothesis that lipid accumulation alters phosphorylation of the cytoplasmic domain of LRP1, thereby suppressing its interaction with adaptor proteins required for its translocation to the cel surface. We will also evaluate two strategies to increase LRP1 recycling and test their effectiveness to improve hepatocyte functions and prevent lipid-induced liver dysfunction and cirrhosis. Specific Aim 2 will delineate the mechanism underlying insulin activation of LRP1 translocation to the cell surface, testing the hypothesis that insulin induces lipid kinase activit to generate phosphatidylinositol (poly)phosphates to increase the recruitment of LRP1-adaptor complex to the recycling endosomes for translocation to the cell surface. Follow up experiments will explore the potential of permeable inositide polyphosphates as a strategy to overcome insulin resistance to improve metabolic health of the liver. Specific Aim 3 will test the hypothesi that LRP1 dysfunction in hepatocytes promotes lipid-induced liver cirrhosis, thereby providing an ideal local environment to accelerate liver tumor progression. The completion of these studies will identify mechanisms regulating LRP1 translocation to the cell surface, and offers novel strategies to suppress NAFLD progression to NASH, liver cirrhosis, and HCC.