The liver maintains lipid homeostasis in the body by uptake, synthesis and delivery of lipid molecules. An important component is very low-density lipoprotein (VLDL) which is by hepatocytes. The significance of this molecule lies in the fact that abnormal VLDL metabolism may lead to NAFLD, fibrotic liver, or atherosclerotic heart diseases in humans. Intracellular VLDL transport tightly regulates its secretion from the liver. Our prior research studies have demonstrated that this transport process relies on ATP, GTP, and the presence of cytosolic proteins, suggesting the involvement of protein phosphorylation. To delve deeper into the mechanisms governing VLDL secretion, our laboratory has initiated a study aimed at identifying the role of specific protein kinases responsible for VLDL phosphorylation and subsequent secretion in human hepatoma cells. Utilizing various small molecule inhibitors, such as H89 dihydrochloride, Akti-1/2 and Calphostin C targeting Protein Kinase A, B, and C, respectively, we employed a pulse-chase assay supplemented with radioactive 3H-oleic acid-based Scintillation Counting and Western Blot analyses to identify the VLDL secretion. Findings from Western Blot analysis of VLDL protein-ApoB100 levels in the cell culture media, revealing a significant reduction in ApoB100 secretion from hepatocytes upon inhibition of the protein kinase C family. The distribution of intracellular VLDL molecules have also been shown to be higher in protein kinase C inhibition indicating lower secretion with the treatment. Furthermore, the pulse chase assay followed by immunocytochemistry-based imaging led to the finding that both ER to Golgi and post-Golgi trafficking is disturbed with the inhibition of protein kinase C. This study provides valuable insights into the intricate mechanisms underlying VLDL secretion from the liver, shedding light on the pivotal role of specific protein kinases in this process.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2023-1335 |
| Date | 01 January 2024 |
| Creators | Tasin, Fahim Rejanur |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Thesis and Dissertation 2023-2024 |
| Rights | In copyright |
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