The role of amino acids in ApoB100 synthesis and catabolism in human HepG2 cells.

Abstract

These studies were initiated to determine the effects of extracellular amino acids on the hepatic synthesis, secretion, and metabolism of apolipoprotein B100 (ApoB100), the major protein component of low‐density lipoproteins (LDLs). As a model, the authors used a human hepatoma cell line, HepG2, which retains many liver‐specific functions including the biosynthesis of lipoprotein particles. The cells were cultured in minimum essential medium in which amino acids are present at levels that reflect average fasting plasma concentrations in humans (0.5 mg/mL) or in minimum essential medium supplemented with a concentrated stock of essential amino acids to a final concentration of 5.5 mg/mL. The authors' initial results demonstrated that after 24 hours of culture ApoB100 accumulation was reduced by 87%, in spite of a fivefold increase in the rate of total protein secretion under the same conditions. The extent of ApoB100 inhibition was dependent on the concentration of essential amino acids in the medium. In contrast, the synthesis of apolipoprotein A1, the major protein component of high‐density lipoprotein, was unaffected by amino acid levels. The remainder of the studies addressed the mechanism by which high amino acid levels inhibit the production of ApoB100.Because ApoB100 is secreted as a component of a lipoprotein particle (very low density lipoprotein), it must first be synthesized in the rough endoplasmic reticulum, packaged with cholesterol, cholesterol esters, triglycerides, and phospholipids in the Golgi, then either degraded or finally secreted via the exocytotic pathway. The multistep nature of this biosynthetic process allows the rate of production to be regulated at any one of the steps (protein synthesis, lipid or cholesterol synthesis, rate of secretion, and intracellular degradation). To determine the effect of amino acids on lipid metabolism, the authors incubated the cells for 24 hours in the presence of normal or high amino acid levels in the presence of 14C‐acetate to measure cholesterol synthesis and 3H‐glucose to determine triglyceride production. The results indicated that elevated levels of essential amino acids in the culture medium had no significant influence on lipid metabolism, which suggested that the amino acid‐dependent inhibition of ApoB100 production must be attributable to changes in the secretion efficiency and/or synthetic or degradative rates of this protein.Pulse‐label experiments indicated that high amino acid levels stimulated the secretion of total labeled protein into the medium fivefold but failed to enhance the rate of incorporation of radiolabeled leucine into protein intracellularly. An alternative explanation for the inhibition of ApoB100 production by elevated amino acid levels in the medium is that the secretion efficiency of this protein is reduced under these conditions, even though total protein secretion is enhanced. To address this possibility, the authors pulse‐labeled cells for 10 minutes with labeled leucine followed by a chase with unlabeled leucine for 0 to 180 minutes. Both albumin and ApoB100 were immunoprecipitated from cell extracts and the medium at specific time points, and the amount of radioactivity was quantitated and compared with that at the maximal incorporation time point (10 to 15 minutes after pulse). The resulting ratio of counts per minute secreted to initial counts per minute yields an index of the secretion efficiency. The data indicate that the secretion efficiency for each protein in low and high amino acid media were 36 ± 6% and 46 ± 8%, respectively, for ApoB100 and 91 ± 14% and 71 ± 7%, respectively, for albumin. However, the initial ApoB100 synthesized was 84% higher in low amino acid medium, whereas the initial amount of albumin synthesized was twofold greater in high amino acid medium. These experiments demonstrated that the translational rate of each of these proteins is altered by extracellular amino acid levels but that the secretory process (posttranslational events) is relatively unaltered. For ApoB100, the authors demonstrated that the 1.8‐fold induction in translational rate in the absence of high amino acids correlates with a 1.6‐fold to 2.1‐fold increase in its messenger RNA levels.Finally, the LDL receptor‐mediated pathway for ApoB100 degradation was shown to be enhanced more than twofold in low amino acid medium, which suggested that the extent of inhibition of ApoB100 production by high levels of essential amino acids was actually underestimated.