Abstract
The present investigations have examined the mechanism(s) whereby Sf 60-400 very low density lipoproteins (VLDL) from Type IV hypertriglyceridemic subjects cause cholesteryl ester and triglyceride accumulation in J774 macrophages. Both apolipoprotein (apo) E-poor and apoE-rich Type IV VLDL subfractions, isolated by heparin-Sepharose chromatography, were capable of enhancing cellular cholesterol and triglyceride content. The apoE-rich fraction was significantly more effective at inducing cholesterol esterification (P < 0.05) and accumulation of esterified cholesterol (P < 0.05), whereas both subfractions caused similar increases in cellular triglyceride content. Thus, the amount of VLDL-associated apoE determined the extent to which Type IV VLDL loaded J774 cells with cholesterol but not triglyceride. Two VLDL subfractions, Sf 60-400 and Sf 20-60, isolated from Type III subjects homozygous for apoE2, caused little or no effect on cellular esterified cholesterol content, whereas both fractions induced the same degree of cellular triglyceride accumulation as Type IV VLDL. Type IV VLDL-induced cholesteryl ester accumulation was blocked by an anti-apoE monoclonal antibody, known to block the binding of apoE to the LDL receptor; however, the increase in cellular triglyceride was unaffected. Therefore, VLDL-induced triglyceride accumulation in this cell line can occur without apoE-mediated uptake of intact VLDL particles. The addition of heparin to J774 cells resulted in a 6-fold increase in lipoprotein lipase (LPL) activity in the media, and significantly enhanced the ability of Type IV VLDL to induce cellular triglyceride accumulation (P < 0.01), but significantly decreased cellular cholesteryl ester content (P < 0.025). Finally, Sf 60-400 VLDL from two subjects homozygous for apoC-II deficiency failed to increase cellular lipid content. However, the addition of exogenous apoC-II to C-II-deficient VLDL resulted in significant increases of both triglyceride and esterified cholesterol in J774 cells. In the presence of apoC-II, the anti-apoE monoclonal antibody blocked the cellular cholesteryl ester increase induced by C-II-deficient VLDL, but had no effect on the increase in cellular triglyceride. Collectively, these experiments demonstrate that extracellular lipolysis of Sf 60-400 VLDL by LPL is required for cholesteryl ester and triglyceride accumulation in J774 macrophages. After interaction with cellular LPL, VLDL triglycerides are hydrolyzed. The resulting free fatty acids are readily taken up by the macrophage, and re-esterified into triglyceride. Lipolysis proceeds until apoE epitopes are exposed, allowing the triglyceride-depleted remnant, containing all the cholesteryl ester, to be taken up via an apoE-mediated process.
Highlights
The present investigations have examined the mechanism(s) whereby St 60-400 very low density lipoproteins (VLDL) from Type IV hypertriglyceridemic subjects cause cholesteryl ester and triglyceride accumulation in 5774 macrophages
VLDL particles can be taken up as such which results in both cholesterol and triglyceride accumulation [3, 48]
Our observations provide five lines of evidence that the cholesterol and triglyceride components of Sf 60-400 VLDL particles from hypertriglyceridemic subjects are internalized by J774 cells largely by different mechanisms and that VLDL do not contribute to lipid accumulation without prior lipolysis
Summary
VLDL samples from two subjects homozygous for apoC-I1 deficiency were used in specific experiments These subjects were members of the C - 1 1 k~ind~red~ th~at ~has~ b~een described previously [31, 32]. The ratios of apoE to apoC of the re-isolated VLDL subfractions were determined by analytical isoelectric focusing gel electrophoresis as described previously [35]. For the determination of cellular esterified cholesterol and triglyceride mass, the cells were washed three times in buffer B without BSA and the lipids were extracted In situ after two 30-min incubations with 1.0 ml of hexane-isopropanol3:2 (v/v). Duplicate 2 0 0 4 aliquots of conditioned or unconditioned media and 100 pl of the substrate were added to the incubation buffer (0.15 M NaCl, 0.2 M Tris, pH 8.2, 0.05 g/ml fatty acid-free BSA, containing 12 normolipidemic human plasma (v/v) as a source of apoC-11). The data were analyzed using an unpaired Student’s t-test
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