Ellagic Acid Suppresses ApoB Secretion and Enhances ApoA-1 Secretion from Human Hepatoma Cells, HepG2

The effect of ellagic acid (EA), a naturally occurring polyphenolic compound, on the secretion of apolipoproteins from human hepatocytes, HepG2, was investigated. The levels of apoB and apoA-1 secreted in the cell culture medium were determined by sandwich ELISA. EA did not affect cell viability at the tested concentrations (up to 50 µM). EA suppressed the secretion of apoB and enhanced that of apoA-1 from HepG2 cells. However, cellular apoB levels were increased, suggesting that EA inhibited the trafficking of apoB during the process of secretion. In contrast, the increase in the cellular levels of apoA-1 was consistent with its secreted levels. These results indicate that EA inhibits the secretion of apoB from hepatocytes and increases the secretion of apoA-1. Both of these effects are beneficial for lipoprotein metabolism in the prevention of lifestyle-related diseases. The detailed mechanism underlying these effects of EA on lipoprotein metabolism should be elucidated in the future, but this naturally occurring polyphenolic compound might be antihyperlipidemic. Based on these results, EA is suggested as a candidate food-derived compound for the prevention of hyperlipidemia.

Abstract 573: Human Microrna-548p Decreases apoB Secretion and Lipid Synthesis

Objective: MicroRNAs (miRs) play important regulatory roles in lipid and lipoprotein metabolism. ApoB, as the only essential scaffolding protein in the assembly of very low density lipoproteins, is a target to treat hyperlipidemia and atherosclerosis. We aimed to find out miRs that reduce apoB expression. Approach: Bioinformatics analyses predicted that hsa-miR-548p can interact with apoB mRNA.MiR-548p mimic and control were transfected in human and mouse hepatoma cell lines to test its role in regulating apoB secretion and mRNA expression levels. Site-directed mutagenesis was used to identify the interacting site of miR-548p in human apoB 3′-untranslated region. Fatty acid oxidation and lipid syntheses were examined in miR-548p overexpressing cells to investigate its function in lipid metabolism. Results: Experimentally, we observed that miR-548p significantly reduces apoB secretion from human hepatoma cells in time and dose dependent manner. Mechanistic studies showed that miR-548p interacts with the 3′-untranslated region of human apoB mRNA to enhance posttranscriptional degradation. Bioinformatics algorithms suggested two potential binding sites of miR-548p on human apoB mRNA. Site-directed mutagenesis studies revealed that miR-548p targets site II involving both seed and supplementary sequences. MiR-548p had no effect on fatty acid oxidation but significantly decreased lipid synthesis in human hepatoma cells by reducing the expression of HMGCR and ACSL4 enzymes involved in cholesterol and fatty acid synthesis. In summary, miR-548p reduces lipoprotein production and lipid synthesis by reducing expression of different genes in human hepatoma cells. Conclusion: These studies suggest that miR-548p could be useful in treating atherosclerosis, hyperlipidemia and hepatosteatosis.

Human MicroRNA-548p Decreases Hepatic Apolipoprotein B Secretion and Lipid Synthesis

Objective— MicroRNAs (miRs) play important regulatory roles in lipid metabolism. Apolipoprotein B (ApoB), as the only essential scaffolding protein in the assembly of very-low-density lipoproteins, is a target to treat hyperlipidemia and atherosclerosis. We aimed to find out miRs that reduce apoB expression. Approach and Results— Bioinformatic analyses predicted that hsa-miR-548p can interact with apoB mRNA. MiR-548p or control miR was transfected in human and mouse liver cells to test its role in regulating apoB secretion and mRNA expression levels. Site-directed mutagenesis was used to identify the interacting site of miR-548p in human apoB 3′-untranslated region. Fatty acid oxidation and lipid syntheses were examined in miR-548p overexpressing cells to investigate its function in lipid metabolism. We observed that miR-548p significantly reduces apoB secretion from human hepatoma cells and primary hepatocytes. Mechanistic studies showed that miR-548p interacts with the 3′-untranslated region of human apoB mRNA to enhance post-transcriptional degradation. Bioinformatic algorithms suggested 2 potential binding sites of miR-548p on human apoB mRNA. Site-directed mutagenesis studies revealed that miR-548p targets site I involving both seed and supplementary sequences. MiR-548p had no effect on fatty acid oxidation but significantly decreased lipid synthesis in human hepatoma cells by reducing HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and ACSL4 (Acyl-CoA synthetase long-chain family member 4) enzymes involved in cholesterol and fatty acid synthesis. In summary, miR-548p reduces lipoprotein production and lipid synthesis by reducing expression of different genes in human liver cells. Conclusions— These studies suggest that miR-548p regulates apoB secretion by targeting mRNA. It is likely that it could be useful in treating atherosclerosis, hyperlipidemia, and hepatosteatosis.

Abstract 192: Characterization of Microsomal Triglyceride Transfer Protein Missense Mutations Found in Abetalipoproteinemia and Hybobetalipoproteinemia Subjects

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels and lipid malabsorption with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their effects on function. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. Similarly, H297Q, D384A, and G661A mutants supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP’s three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, there probably exist novel mutations in other genes that cause severe hypolipidemia and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.

Abstract 387: Regulation of Apolipoprotein B Secretion by Hepatic Sortilin is Dependent on Secretory Stress

Sortilin is a multi-ligand sorting receptor involved in trafficking of proteins from the Golgi apparatus to the lysosome and has been widely shown to be associated with plasma lipid traits and coronary artery disease. While over expression of sortilin in the liver reduces VLDL production, the reported effects of the genetic loss of sortilin on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing; loss of sortilin has been shown in different studies to result in both increased and decreased apoB/VLDL secretion. These conflicting studies were carried out in a variety of different models and used different methods of knocking down sortilin expression. To attempt to further clarify the role of sortilin, we explored the role of sortilin deficiency on apoB secretion in the hepatocyte by utilizing 2 in vitro models of sortilin knockdown; primary hepatocytes from Sort1-/- mice and siRNA -treated McA-RH7777 (McA) cells. In both primary hepatocytes and McA cells, loss of sortilin alone was not associated with any change in apoB secretion. The previously reported increases in VLDL secretion occurred on either the background of apoB over expression or in livers of mice on a high fat diet, suggesting the requirement for a metabolic stress. We found that apoB secretion was increased with Sort1 knockdown as compared to control in isolated primary hepatocytes from Apobec1-/-; hAPOB Tg mice and McA cells stably over expressing apoB. We then sought to increase apoB secretion by lipid loading with oleic acid (OA). While OA increased apoB secretion in all cells, there was no effect of Sort1 knockdown in this context. However, when the cells were further treated with either palmitic acid, proteasomal inhibitors, or tunicamycin (an ER stress inducer), there was an observed increase in apoB secretion with Sort1 knockdown, suggesting that sortilin regulates apoB secretion only when both apoB secretion is increased and the cell is stressed. Based on this data, we propose that hepatic sortilin regulates the post-ER fate of apoB for degradation and export and acts to coordinate intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi and the level of post-ER pre-secretory proteolysis activity.

Abstract 232: A Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of the N-Terminal ß-Sheet in the Lipid Transfer and ApoB Secretion Activities of Microsomal Triglyceride Transfer Protein

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. Mutations in the MTTP gene cause abetalipoproteinemia (ABL). Missense mutations in ABL have revealed that the central α-helical and C-terminal β-sheet domains are important for the lipid transfer activity of MTP and for the assembly and secretion of apoB-containing lipoproteins. The N-terminal domain, on the other hand, has mainly been implicated in apoB- and membrane-binding. Here, we describe a novel ABL missense mutation (D169V) in the N-terminal β-sheet of MTP. Although this mutant MTP (MTPD169V) is expressed and localized to the endoplasmic reticulum, it is unable to transfer triglycerides and phospholipids. Further, MTPD169V does not support the assembly and secretion of apoB-containing lipoproteins. Computational molecular modeling suggests that D169 could form an internal salt bridge with K187 and K189. Indeed, mutagenesis of these lysine residues to leucine abolishes triglyceride transfer and apoB secretion activities of MTP. Furthermore, conservative mutagenesis that preserves charges on these residues partially restores triglyceride transfer and apoB secretion activities of MTP. Therefore, D169 is probably involved in an internal salt bridge with K187 and K189. Disruption of this internal salt bridge in the N-terminal region affects the lipid transfer activity present in the C-terminal end of the MTP molecule. We speculate that this salt bridge, although away from the speculated lipid transfer site, might be important in providing structural integrity necessary for the lipid transfer activity of MTP.