Raphael Cheung

Simvastatin, an HMG-CoA reductase inhibitor, induces the synthesis and secretion of apolipoprotein AI in HepG2 cells and primary hamster hepatocytes

Clinical studies have recently suggested that statin treatment may beneficially elevate plasma concentrations of high density lipoprotein (HDL)-cholesterol in patients with hyperlipidemia. Here, we have investigated the effect of a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase on the synthesis and secretion of apolipoprotein AI (apoAI) in two model systems, HepG2 cells and primary hamster hepatocytes. Cultured cells were incubated with different doses of simvastatin (0.1-10 microM) for a period of 18 h. A dose-dependent increase in synthesis and secretion of apoAI was observed in both cell types. There was a significant increase in the synthesis of apoAI in HepG2 cells (44.3+/-12.1%), and hamster hepatocytes (212+/-2%) after treatment with 10 microM of the statin. The increase in apoAI synthesis appeared to result in a higher level of apoAI secreted into the culture media in both cell types (49.2+/-7.8% in HepG2, 197+/-0.2% in hamster hepatocytes). ApoAI mRNA levels were also significantly increased in both cell types in response to statin treatment. Control experiments with transferrin confirmed specificity of the effect on apoAI secretion. Analysis of a density fraction containing HDL particles in culture media revealed an increase in HDL-associated apoAI of 94.3+/-2.1% in HepG2 cells and 27.0+/-0.03% in hamster hepatocytes following 10 microM simvastatin-treatment. Comparative studies of simvastatin and lovastatin indicated a differential ability to induce apoAI synthesis and secretion, with simvastatin having a more significant effect. Thus, acute statin treatment of cultured hepatocytes (transformed as well as primary) resulted in a significant upregulation of apoAI mRNA and apoAI synthesis, causing oversecretion of apoAI and HDL extracellularly. The stimulatory effect on apoAI synthesis and secretion may thus explain the clinical observation of an elevated plasma HDL-cholesterol level in hyperlipidemic patients treated with certain statins.

A simple micromethod for rapid assessment of the distribution of apolipoprotein C isoforms in very-low-density lipoprotein

A simple and rapid isoelectric focusing method for quantifying Apo C isoforms of triglyceride-rich lipoprotein was developed. The very-low-density lipoprotein (VLDL) was isolated from 100 microL of EDTA plasma using a Beckman Airfuge ultracentrifuge. The delipidated VLDL was applied to an ultrathin flat acrylamide gel, and focused using a Bio-Rad Mini IEF Cell, for 1.5 h at a maximum of 500 V. Apo CII and Apo CIII in VLDL were resolved into four major bands, CIII0 (PI 4.91), CII (PI 4.78), CIII1 (PI 4.72), and CIII2 (PI 4.53). The method demonstrated within-run and between-run CVs of 2.7% to 11.9% and 4.4% to 12.2%, respectively. The relative percentage of C apoproteins and the ratio of CII to CIII found in VLDL from plasma of normal, chronic renal failure, and hyperlipidemic subjects agreed with previously published data.

A simple micromethod for rapid phenotyping of apolipoprotein E

Abstract Polymorphysim of apolipoprotein E (Apo E) accounts for a substantial amount of the genetically determined variance of cholesterol levels in the population, and is an important factor in the development of type III hyperlipidemia. In this report, we describe a simple and rapid isoelectric focusing method for phenotyping apolipoprotein E of triglyceride-rich lipoprotein. The very-low-density lipoprotein (VLDL) was isolated within 3 h from 100 μl of EDTA plasma using a Beckman Airfuge ultracentrifuge. The delipidated VLDL was applied to an ultrathin flat acrylamide gel, and focused using a Bio-Rad Mini IEF Cell, for 1.5 h at a maximum of 500 V. Apo E was resolved into three major bands, Apo E4 (pI 5.98 ± 0.02), Apo E3 (pI 5.83 ± 0.03), and Apo E2 (pI 5.71 ± 0.02). These bands were identified by cysteamine treatment and by immunofixation isoelectric focusing using goat antibody to Apo E. The present method can be used in routine clinical laboratories and can be performed in 1 day.