The liver is tasked with managing the concentration of various metabolites in the blood, and of particular importance is the uptake of free fatty-acid (FFA), as elevated concentrations of FFA are toxic to cells. FFAs are transported across the cell membrane by CD36 and distributed by LFABP to the endoplasmic reticulum (ER), where they are esterified to glycerol, yielding more chemically inert triglyceride (TAG), which is essential to the process of VLDL assembly. VLDL secretion distributes energy rich TAG to peripheral tissues, and its dysfunction leads to hepatic steatosis, which may progress into hepatocellular carcinoma. The present study examined the role of cathepsin B (CatB) in regulating very-low density lipoprotein (VLDL) secretion through liver fatty-acid binding protein (LFABP) cleavage as well as CD36 expression in response to 0.5 mM oleic acid:BSA treatment, which has been reported to redistribute CatB from the lysosome to the cytosol, where the majority of cellular LFABP is localized. Genetic knock-down of CatB in McA-RH7777 cells resulted in increased VLDL secretion as measured by 3H TAG DPM counting and immunoblot for ApoB in cell culture media, due to increased expression of LFABP and CD36 and increased FFA uptake. Knock-down of CatB also resulted in decreased cellular TAG as measured by 3H DPM counting due to increased VLDL secretion. CatB over-expression in McA-RH7777 cells resulted in decreased FFA uptake leading to decreased VLDL secretion, which was due to increased cleavage of LFABP. Co-localization of LFABP and CatB was observed exclusively under conditions of 0.5 mM oleic acid:BSA treatment. Based on these results, we can conclude that CatB plays a distinct physiological role in the turnover of LFABP and CD36 protein, which leads to suppressed uptake of FFA, and thus, reduced TAG synthesis and VLDL secretion.
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Master of Science (M.S.)
College of Medicine
Molecular Biology and Microbiology
Length of Campus-only Access
Masters Thesis (Campus-only Access)
Thibeaux, Simeon, "Cathepsin B Regulates VLDL Secretion Through LFABP Cleavage" (2017). Electronic Theses and Dissertations, 2004-2019. 5495.