Abstract
To determine the role of high-density lipoprotein (HDL) as an acceptor of lipolytic surface remnants of very low density lipoprotein (VLDL) in the metabolism of VLDL core remnants, we examined the effect of HDL levels in the VLDL lipolysis mixture on 1) the morphology and the apoCs to E ratio in VLDL core remnants and 2) the metabolic properties of VLDL core remnants in human hepatoma cell line HepG2 and human hepatocytes in the primary culture. Normolipidemic VLDL was lipolyzed in vitro by purified bovine milk lipoprotein lipase (LpL) in a lipolysis mixture containing a physiologic level of VLDL and albumin (30 mg VLDL-cholesterol (CH)/dl and 6% albumin) in the absence and presence of either a low HDL level (VLDL-CH:HDL-CH = 3:1) or a high HDL level (VLDL-CH:HDL-CH = 1:4). Lipolysis of VLDL in either the absence or presence of HDL resulted in the hydrolysis of >85% of VLDL-triglycerides (TG) and the conversion of VLDL into smaller and denser particles. In the absence of HDL, heterogeneous spherical particles with numerous surface vesicular materials were produced. In the presence of low or high HDL, spherical particles containing some or no detectable vesicular surface components were produced. The apoCs to apoE ratios, as determined by densitometric scanning of the SDS polyacrylamide gradient gel, were 2.89 in control VLDL and 2.27, 0.91, and 0.22 in VLDL core remnants produced in the absence and in the presence of low and high HDL levels, respectively. In vitro lipolysis of VLDL markedly increased binding to HepG2 cells at 4°C and internalization and degradation by human hepatocytes in primary culture at 37°C. However, the HDL-mediated decrease in the apoCs to apoE ratio had a minimal effect on binding, internalization, and degradation of VLDL core remnants by HepG2 cells and human hepatocytes in primary culture. In order to determine whether HepG2 bound VLDL and VLDL core remnants are deficient in apoCs, 125I-labeled VLDL and VLDL core remnants were added to HepG2 culture medium at 4°C. The bound particles were released by heparin, and the levels of 125I-labeled apoCs and apoE, relative to apoB, in the released particles were examined. When compared with those initially added to culture medium, the VLDL and VLDL core remnants released from HepG2 cells had a markedly increased (113%) level of apoE and a reduced (30–39%), but not absent, level of apoCs. We conclude that apoCs, as a minimum structural and/or functional component of VLDL and VLDL core remnants, may not have an inhibitory effect on the binding of VLDL or VLDL core remnants to hepatic apoE receptors. —Chung, B. H., and N. Dashti. Lipolytic remnants of human VLDL produced in vitro: effect of HDL levels in the lipolysis mixtures on the apoCs to apoE ratio and metabolic properties of VLDL core remnants.
Highlights
To determine the role of high-density lipoprotein (HDL) as an acceptor of lipolytic surface remnants of very low density lipoprotein (VLDL) in the metabolism of Very low density lipoprotein (VLDL) core remnants, we examined the effect of high density lipoprotein (HDL) levels in the VLDL lipolysis mixture on 1) the morphology and the apoCs to E ratio in VLDL core remnants and 2) the metabolic properties of VLDL core remnants in human hepatoma cell line HepG2 and human hepatocytes in the primary culture
The effect of increasing concentrations (2.5–40 g/ml) of unlabled VLDL core remnants produced in the absence or presence of low and high HDL levels on the binding of 125I-labeled VLDL core remnants produced in the absence of HDL to HepG2 cells at 4ЊC was determined as previously described [26, 27]
As compared to control VLDL and apoC-containing VLDL core remnants added to the culture medium, those released from HepG2 cells by heparin had a decreased apoC but increased apoE content, as evidenced by 31 – 39% decreases in the apoCs to apoB ratio and a 113% increase in the apoE to apoB ratio (Table 2); the apoCs to apoE ratios were 3.2- to 3.4-fold lower in VLDL and VLDL core remnants released from Hep G2 cells than in those added to the culture medium (Table 2)
Summary
Fresh normolipidemic plasma was obtained from the Alabama Regional Blood Bank, Birmingham, AL. LpL was isolated from fresh raw bovine milk and purified by the heparin-agarose affinity chromatographic method [21]. Fatty acid-poor bovine serum albumin was purchased from Boehringer Mannheim Biochemical Co., Indianapolis, IN, and Sigma Chemical Co., St. Louis, MO. Human hepatoma cell line HepG2 cells were obtained from the American Type Culture Collection, Rockville, MD. Cultured human primary hepatocytes in 12-well culture plates, derived from adult males, were purchased from Clonetics Corporation, San Diego, CA
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