Rationale: ANGPTL3 has been identified as an important regulator of lipoprotein metabolism as well as a determinant of plasma levels of apolipoprotein B-containing lipoproteins. It has been reported that the genetically determined ANGPTL3 absence or deficit is linked to lower plasma levels of apoB-containing lipoproteins. Therefore, a way to address the action of ANGPTL3 is to evaluate the in vivo lipoprotein metabolism in subjects carrying genetic mutations lowering ANGPTL3 as compared to normal controls. PCSK9 is also a known regulator of plasma cholesterol through the modulation of LDL-R expression in the cell surface. In a previous study we demonstrated that the two proteins show a direct intracellular interaction and a co-regulated secretion in conditions of feeding and fasting. Hence, it is possible that they both exert an intracellular role in controlling cholesterol and triglycerides metabolism through the modulation of metabolic signals carried by the MAPK pathways.
Overall Goal: To establish how ANGPTL3 and PCSK9 differentially regulate HepG2 lipid metabolism in feeding and fasting conditions through the regulation of intracellular pathways.
Methods: In the first part of the project HepG2 cells overexpressing ANGPTL3, PCSK9, or both will be analyzed through a proteome profiler array in order to identify the most deregulated pathways in intracellular MAPKs signaling.
In the second part of the project, the specific intracellular kinases identified will be either inhibited or induced in order to simulate ANGPTL3 and/or PCSK9 overexpression state. Intracellular and secreted levels of ANGPTL3 and PCSK9 will be associated with the apoB containing lipoprotein secretion rates.
ANGPTL3 and PCSK9 have an extremely important role in the regulation of circulating lipids, particularly apolipoprotein-B-containing particles. Several inhibitors of both proteins are under intense research in phase III clinical trials. Although their extracellular function has been extensively investigated as circulating plasma proteins, their role in the intracellular environment is not yet clear. The proposed research might unveil the intracellular role of ANGPTL3 and PCSK9 that are still neglected in lipidology. A specific intracellular role of the two proteins might be translated into specific indications or contra-indications to the use of antisense oligonucleotides against ANGPTL3 (Vupanorsen) or PCSK9 (Inclisiran). Moreover, this study might set a cornerstone in the under-investigated area of lipid metabolism regulation mediated by specific intracellular signals (potentially MAPKs).