Secretion of triacylglycerol-enriched very-low-density lipoproteins (VLDLs) from the liver is vital for maintaining plasma lipid homeostasis. However, the process of VLDL assembly and lipidation is not well characterized. Here, we observed that liver of Cideb null mice had higher levels of triacylglycerols accompanied by low level of VLDL secretion. Furthermore, VLDL particles secreted from hepatocytes of Cideb null mice have low levels of triacylglycerols but normal levels of apoB. We also observed that Cideb is localized to endoplasmic reticulum and lipid droplets. Importantly, we have identified apoB as a Cideb-interacting protein. By infecting adenoviruses expressing various Cideb truncations into hepatocytes of Cideb null mice, we found that Cideb requires both its apoB-binding and lipid droplet association domains to restore the secretion of triacylglycerol-enriched VLDL particles. Our data suggest that Cideb promotes the formation of triacylglycerol-enriched VLDL particles and provides a molecular insight into VLDL lipidation and maturation in hepatocytes.

Lipid mediators participate in signal transduction pathways, proliferation, apoptosis, and membrane trafficking in the cell. Lipids are highly complex and diverse owing to the various combinations of polar headgroups, fatty acyl chains, and backbone structures. This structural diversity continues to pose a challenge for lipid analysis. Here we review the current state of the art in lipidomics research and discuss the challenges facing this field. The latest technological developments in mass spectrometry, the role of bioinformatics, and the applications of lipidomics in lipid metabolism and cellular physiology and pathology are also discussed.

To Recent studies showing that a defect in the MTP is the proximal cause of abetalipoproteinemia indicate that this protein is required for assembly and secretion of apoBcontaining lipoproteins (10, 11). Abetalipoproteinemic patients have only trace amounts of plasma apoB-containing lipoproteins, resulting in extremely low TG and cholesterol levels (12). The cause of this phenotype is a defect in the pathway responsible for assembly and secretion of apoBcontaining lipoproteins since the apoB gene (13, 14) and lipid synthesis (12) have been shown to be normal. Thus, it is clear that MTP is required for the efficient assembly and secretion of apoB-containing lipoprotein particles.Although studies of abetalipoproteinemic patients indicate that MTP is required for the production of plasma lipoproteins containing apoB, the role of MTP in this process remains unclear. Also, it is not known whether MTP is the only tissue-specific factor needed by hepatocytes and enterocytes to synthesize and secrete these particles. To address these issues, apoB-53 was expressed in either a nonlipoprotein-producing cell line (HeLa) or a derivative cell line stably expressing the large subunit ofMTP and MTP activity. These cells were evaluated for their ability to secrete apoBcontaining lipoproteins into the tissue culture medium. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 7628The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Previously, based on distinct requirement of microsomal triglyceride transfer protein (MTP) and kinetics of triglyceride (TG) utilization, we concluded that assembly of very low density lipoproteins (VLDL) containing B48 or B100 was achieved through different paths (Wang, Y., McLeod, R. S., and Yao, Z. (1997) J. Biol. Chem. 272, 12272-12278). To test if the apparent dual mechanisms were accounted for by apolipoprotein B (apoB) length, we studied VLDL assembly using transfected cells expressing various apoB forms (e.g. B64, B72, B80, and B100). For each apoB, enlargement of lipoprotein to form VLDL via bulk TG incorporation was induced by exogenous oleate, which could be blocked by MTP inhibitor BMS-197636 treatment. While particle enlargement was readily demonstrable by density ultracentrifugation for B64-and B72-VLDL, it was not obvious for B80-and B100-VLDL unless the VLDL was further resolved by cumulative rate flotation into VLDL 1 (S f > 100) and VLDL 2 (S f 20 -100). BMS-197636 diminished B100 secretion in a dose-dependent manner (0.05-0.5 M) and also blocked the particle enlargement from small to large B100-lipoproteins. These results yield a unified model that can accommodate VLDL assembly with all apoB forms, which invalidates our previous conclusion. To gain a better understanding of the MTP action, we examined the effect of BMS-197636 on lipid and apoB synthesis during VLDL assembly. While BMS-197636 (0.2 M) entirely abolished B100-VLDL 1 assembly/secretion, it did not affect B100 translation or translocation across the microsomal membrane, nor did it affect TG synthesis and cell TG mass. However, BMS-197636 drastically decreased accumulation of [ 3 H]glycerol-labeled TG and TG mass within microsomal lumen. The decreased TG accumulation was not a result of impaired B100-VLDL assembly, because in cells treated with brefeldin A (0.2 g/ml), the assembly of B100-VLDL was blocked yet lumenal TG accumulation was normal. Thus, MTP plays a role in facilitating accumulation of TG within microsomes, a prerequisite for the post-translational assembly of TG-enriched VLDL.

Excess lipid induced metabolic disorders are one of the major existing challenges for the society. Among many different causes of lipid disorders, overproduction and compromised catabolism of triacylglycerol-rich very low density lipoproteins (VLDL) have become increasingly prevalent leading to hyperlipidemia worldwide. This review provides the latest understanding in different aspects of VLDL assembly process, including structure-function relationships within apoB, mutations in APOB causing hypobetalipoproteinemia, significance of modulating microsomal triglyceride-transfer protein activity in VLDL assembly, alterations of VLDL assembly by different fatty acid species, and hepatic proteins involved in vesicular trafficking, and cytosolic lipid droplet metabolism that contribute to VLDL assembly. The role of lipoprotein receptors and exchangeable apolipoproteins that promote or diminish VLDL assembly and secretion is discussed. New understanding on dysregulated insulin signaling as a consequence of excessive triacylglycerol-rich VLDL in the plasma is also presented. It is hoped that a comprehensive view of protein and lipid factors that contribute to molecular and cellular events associated with VLDL assembly and secretion will assist in the identification of pharmaceutical targets to reduce disease complications related to hyperlipidemia.

Objective-Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes and therefore plays an important role in controlling circulating levels of LDL-cholesterol. To date, the relationship between PCSK9 and metabolism of apolipoprotein B (apoB), the structural protein of LDL, has been controversial and remains to be clarified. Methods and Results-We assessed the impact of PCSK9 overexpression (≈400-fold above baseline) on apoB synthesis and secretion in 3 mouse models: wild-type C57BL/6 mice and LDLR-null mice (Ldlr −/− and Ldlr). Irrespective of LDLR expression, mice transduced with the PCSK9 gene invariably exhibited increased levels of plasma cholesterol, triacylglycerol, and apoB. Consistent with these findings, the levels of very-low-density lipoprotein and LDL were also increased whereas high-density lipoprotein levels were unchanged. Importantly, we demonstrated that endogenous PCSK9 interacted with apoB in hepatocytes. The PCSK9/apoB interaction resulted in increased production of apoB, possibly through the inhibition of intracellular apoB degradation via the autophagosome/lysosome pathway. Conclusion-We propose a new role for PCSK9 that involves shuttling between apoB and LDLR. The present study thus provides new insights into the action of PCSK9 in regulating apoB metabolism.

Hyperlipidemia, one of the most important risk factors for coronary heart disease, is often associated with inflammation. We identified lymphotoxin (LT) and LIGHT, tumor necrosis factor cytokine family members that are primarily expressed on lymphocytes, as critical regulators of key enzymes that control lipid metabolism. Dysregulation of LIGHT expression on T cells resulted in hypertriglyceridemia and hypercholesterolemia. In low-density lipoprotein receptor-deficient mice, which lack the ability to control lipid levels in the blood, inhibition of LT and LIGHT signaling with a soluble lymphotoxin beta receptor decoy protein attenuated the dyslipidemia. These results suggest that the immune system directly influences lipid metabolism and that LT modulating agents may represent a novel therapeutic route for the treatment of dyslipidemia.

This article is available online at http://www.jlr.org secretion under lipid-rich conditions. J. Lipid Res. 2010. 51: 150-161. Supplementary key words triacylglycerol • hypotriglyceridemia • hypertriglyceridemia • microsomal triglyceride transfer protein • hepatocyteApolipoprotein (apo) C-III is a small exchangeable apolipoprotein (79 amino acids) that is synthesized mainly in the liver and intestine from the APOA5-APOA4-APOC3-APOA1 gene locus and is secreted into the plasma as a component of VLDL and HDL ( 1 ). Elevated plasma apoC-III concentration is commonly observed in human subjects with insulin resistance and central obesity and is positively correlated with plasma triacylglycerol (TAG) concentrations in hypertriglyceridemia subjects ( 2, 3 ). Early studies have shown that mutations within the APOA4-APOC3-APOA1 gene locus were associated with patients of premature atherosclerosis ( 4, 5 ). However, the close proximity of apoA-IV, apoC-III, and apoA-I encoded within this gene cluster makes it diffi cult to ascertain the contribution of apoC-III defi ciency per se to the development of atherosclerosis. Recently, a genome-wide association study has discovered an apoC-III null allele (R19×) in Lancaster Amish population and shown that individuals heterozygous of the R19× allele have a favorable plasma lipid proAbstract Apolipoprotein (apo) C-III plays a regulatory role in VLDL lipolysis and clearance. In this study, we determined a potential intracellular role of apoC-III in hepatic VLDL assembly and secretion. Stable expression of recombinant apoC-III in McA-RH7777 cells resulted in increased secretion effi ciency of VLDL-associated triacylglycerol (TAG) and apoB-100 in a gene-dosage-dependent manner. The stimulatory effect of apoC-III on TAG secretion was manifested only when cells were cultured under lipid-rich (i.e., media supplemented with exogenous oleate) but not lipid-poor conditions. The stimulated TAG secretion was accompanied by increased secretion of apoB-100 and apoB-48 as VLDL 1 . Expression of apoC-III also increased mRNA and activity of microsomal triglyceride transfer protein (MTP). Pulse-chase experiments showed that apoC-III expression promoted VLDL 1 secretion even under conditions where the MTP activity was inhibited immediately after the formation of lipid-poor apoB-100 particles, suggesting an involvement of apoC-III in the second-step VLDL assembly process. Consistent with this notion, the newly synthesized apoC-III was predominantly associated with TAG within the microsomal lumen that resembled lipid precursors of VLDL. Introducing an Ala23-to-Thr mutation into apoC-III, a naturally occurring mutation originally identifi ed in two Mayan Indian subjects with hypotriglyceridemia, abolished the ability of apoC-III to stimulate VLDL secretion from transfected cells. Thus, expression of apoC-III in McA-RH7777 cells enhances hepatic TAG-rich VLDL assembly and secretion under lipid-rich conditions. -Sundaram, M.,