Apolipoprotein C-I and Apolipoprotein E Production During Adipocyte Differentiation
Nguyen, Aivi T.
University of Delaware
Cholesterol efflux, the process in which cells transport intracellular unesterified cholesterol to extracellular cholesterol acceptors, occurs late during adipocyte differentiation and involves secreted apolipoproteins which shuttle cholesterol to extracellular cholesterol acceptors. One apolipoprotein apoE produced by adipocytes, attaches to the cell surface through proteoglycan interactions, and plays an important role in cholesterol efflux to lipid poor HDLs. The Usher lab has discovered another apolipoprotein, apoC-I, which may also play a role in cholesterol efflux in adipocytes. The aim of this study is to (1) determine whether Apoe and Apoc1 mRNA expression is dependent upon intracellular cholesterol levels; (2) detect secreted apoC-I from adipocytes; and (3) address whether apoC-I associates with heparan sulfate proteoglycans on the cell surface, like apoE. To measure Apoe and Apoc1 mRNA expression Quantitative Real-Time PCR (QPCR) was used. Mouse 3T3-L1 fibroblasts were stimulated to differentiate and either not treated or treated with LXR agonist T0901317, β-cyclodextrin, serum-free media, or Intralipid. To access human APOC-I protein production, human adipocytes were grown on glass cover slips. The cover slips were either left untreated or treated with heparinase or heparinase and cell permeabilization. Cells were then visualized with rabbit anti-human APOC-I IgG and fluorescein isothiocyanate (FITC)-conjugated goat IgG. QPCR results demonstrated that Apoe mRNA expression reaches a maximum during on Day 3 of differentiation, or intermediate-phase differentiation while maximum Apoc1mRNA expression occurs on Day 6 of late-phase differentiation.LXR agonist treatments increased expression of both Apoe and Apoc1 mRNA, while cholesterol depletion with β-cyclodextrin and HDL decreased expression. Results indicated that Apoc1 and Apoe mRNA expression is sensitive to cholesterol levels. Epifluorescence microscopy revealed that human APOC-I is produced during late-phase differentiation (Day 6) on the adipocyte surface in a distinct punctate pattern. Moreover, after heparinase treatments, little APOC-I was visualized, suggesting that APOC-I attaches to heparan sulfate proteoglycans on the cell surface. APOC-I production during adipocyte differentiation and subsequent binding to heparan sulfate proteoglycans on the adipocyte surface suggests that it may act as a cholesterol acceptor in potential lipid rafts, similar to apoE.