The yeast acylglycerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover

Cellular phospholipids undergo deacylation and reacylation through a process known as Lands cycle. In this report, we provide evidence demonstrating that yeast YOR175c, herein designated as LCA1, encodes a key component of the Lands cycle, the acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT). Deletion of LCA1 resulted in a drastic reduction in LPCAT activity, while over expression led to a several fold increase in enzyme activity. We further show that disruption of LCA1 caused an enhanced production of glycerophosphorylcholine, a product of phosphatidylcholine (PC) deacylation and that the lysophosphatidic acid acyltransferase SLC1 was not involved in this process. Identification of LCA1 provides an essential molecular tool for further study of Lands cycle in PC turnover.     

Identification and characterization of a major liver lysophosphatidylcholine acyltransferase

Phosphatidylcholine (PC) is synthesized through the Kennedy pathway, but more than 50% of PC is remodeled through the Lands cycle, i.e. the deacylation and reacylation of PC to attain the final and proper fatty acids within PC. The reacylation step is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), and we report here the identification of a novel LPCAT, which we named LPCAT3. LPCAT3 belongs to the membrane-bound O-acyltransferase (MBOAT) family and encodes a protein of 487 amino acids with a calculated molecular mass of 56 kDa. Membranes from HEK293 cells overexpressing LPCAT3 showed significantly increased LPCAT activity as assessed by thin layer chromatography analysis with substrate preference toward unsaturated fatty acids. LPCAT3 is localized within the endoplasmic reticulum and is primarily expressed in metabolic tissues including liver, adipose, and pancreas. In a human hepatoma Huh7 cells, RNA interference-mediated knockdown of LPCAT3 resulted in virtually complete loss of membrane LPCAT activity, suggesting that LPCAT3 is primarily responsible for hepatic LPCAT activity. Furthermore, peroxisome proliferator-activated receptor alpha agonists dose-dependently regulated LPCAT3 in liver in a peroxisome proliferator-activated receptor alpha-dependent fashion, implicating a role of LPCAT3 in lipid homeostasis. Our studies identify a long-sought enzyme that plays a critical role in PC remodeling in metabolic tissues and provide an invaluable tool for future investigations on how PC remodeling may potentially impact glucose and lipid homeostasis.     

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C₁₈-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.     

Lysophosphatidylcholine acyltransferase 3 deficiency impairs 3T3L1 cell adipogenesis through activating Wnt/β-catenin pathway

Levels of polyunsaturated phosphatidylcholine (PC) influence plasma membrane structure and function. Phosphatidylcholine (PC) is synthesized de novo in the Kennedy pathway and then undergoes extensive deacylation/reacylation remodeling via Lands' cycle (non-Kennedy pathway). The reacylation is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), which adds a polyunsaturated fatty acid at the sn-2 position. Four LPCAT isoforms have been described to date, among which we found LPCAT3 to be the major isoform in adipose tissue, but its exact role in adipogenesis is unclear. In this study, we aimed to investigate whether LPCAT3 activity affects 3T3L1 cell adipogenic differentiation potential and its underline mechanism. Lentivirus-mediated LPCAT3 shRNA expression stably knocked down LPCAT3 in 3T3L1 preadipocytes and LPCAT3 deficiency dramatically reduced the levels of cellular polyunsaturated PCs. Importantly, we found that this deficiency activated the β-catenin dependent Wnt signaling pathway, which suppressed the expression of adipogenesis-related genes, thereby inhibiting 3T3L1 preadipocyte differentiation and lipid accumulation. Moreover, three different Wnt/β-catenin pathway inhibitors reversed the effect of LPCAP3 deficiency, suggesting that Wnt/β-catenin pathway activation is one of the causes for the observed phenotypes. To the best of our knowledge, we show here for the first time that PC remodeling is an important regulator of adipocyte differentiation.     

Identification of Brassica napus lysophosphatidylcholine acyltransferase genes through yeast functional screening

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which acylates lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), is a key enzyme in the Lands cycle. There is evidence that acyl exchange involving LPCAT is a prevailing metabolic process during triacylglycerol (TAG) synthesis in seeds. In this study, by complementing the yeast lca1Δ mutant deficient in LPCAT activity with an Arabidopsis seedling cDNA library, it was found that the previously reported lysophospholipid acyltransferases (LPLATs), At1g12640 and At1g63050, were the only two acyltransferase genes that restored hyposensitivity of the lca1Δ mutant to lyso-platelet-activating factor (lyso-PAF). A developing seed cDNA library from Brassica napus L. cv Hero was constructed to further explore the heterologous yeast complementation approach. Three B. napus LPCAT homologs were identified, of which BnLPCAT1-1 and BnLPCAT1-2 are orthologous to Arabidopsis AtLPLAT1 (At1g12640) while BnLPCAT2 is an ortholog of AtLPLAT2 (At1g63050). The proteins encoded by BnLPCAT1-1 and BnLPCAT2 were chosen for further study. Enzymatic assays demonstrated that both proteins exhibited a substrate preference for LPCs and unsaturated fatty acyl-CoAs. In addition to the enzymatic properties of plant lysophosphatidylcholine acyltransferases uncovered in this study, this report describes a useful technique that facilitates subsequent analyses into the role of LPCATs in PC turnover and seed oil biosynthesis.     

Deciphering the roles of Arabidopsis LPCAT and PAH in phosphatidylcholine homeostasis and pathway coordination for chloroplast lipid synthesis

Phosphatidylcholine (PC) is a key intermediate in the metabolic network of glycerolipid biosynthesis. Lysophosphatidylcholine acyltransferase (LPCAT) and phosphatidic acid phosphatase (PAH) are two key enzymes of PC homeostasis. We report that LPCAT activity is markedly induced in the Arabidopsis pah mutant. The quadruple pah lpcat mutant, with dual defects in PAH and LPCAT, had a level of lysophosphatidylcholine (LPC) that was much higher than that in the lpcat mutants and a PC content that was higher than that in the pah mutant. Comparative molecular profile analysis of monogalactosyldiacylglycerol and digalactosyldiacylglycerol revealed that both the pah and pah lpcat mutants had increased proportions of 34:6 from the prokaryotic pathway despite differing levels of LPCAT activity. We show that a decreased representation of the C_16:0C_18:2 diacylglycerol moiety in PC was a shared feature of pah and pah lpcat, and that this change in PC metabolic profile correlated with the increased prokaryotic contribution to chloroplast lipid synthesis. We detected increased PC deacylation in the pah lpcat mutant that was attributable at least in part to the induced phospholipases. Increased LPC generation was also evident in the pah mutant, but the phospholipases were not induced, raising the possibility that PC deacylation is mediated by the reverse reaction of LPCAT. We discuss possible roles of LPCAT and PAH in PC turnover that impacts lipid pathway coordination for chloroplast lipid synthesis.     

Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region

Lipid droplets (LDs), the major intracellular storage sites for neutral lipids, consist of a neutral lipid core surrounded by a phospholipid monolayer membrane. In addition to their function in lipid storage, LDs participate in lipid biosynthesis and recently were implicated in proteasomal protein degradation and autophagy. To identify components of the protein degradation machinery on LDs, we studied several candidates identified in previous LD proteome analyses. Here, we demonstrate that the highly conserved and broadly expressed ancient ubiquitous protein 1 (AUP1) localizes to LDs, where it integrates into the LD surface in a monotopic fashion with both termini facing the cytosol. AUP1 contains a C-terminal domain with strong homology to a domain known as G2BR, which binds E2 ubiquitin conjugases. We show that AUP1, by means of its G2BR domain, binds to Ube2g2. This binding is abolished by deletion or mutation of the G2BR domain, although the LD localization of AUP1 is not affected. The presence of the AUP1-Ube2g2 complex at LDs provides a direct molecular link between LDs and the cellular ubiquitination machinery.     

高内涵脂滴三维影像定量分析研究细胞内的脂质储存

摘要 目的：研究细胞内脂滴含量的变化对肥胖、糖尿病等代谢性疾病发生发展的影响。方法：建立高内涵脂滴三维成像和定量分析系统,获得脂滴三维动态表型参数,例如细胞内脂滴的总体积量、脂滴平均体积、单一细胞内脂滴平均数量等指标。选择HeLa、AML-12、COS-7和3T3-L1四种细胞系进行油酸、基因沉默、酶活性抑制剂的处理,量化处理后四种细胞内的脂滴数量与大小的表型差异。结果：在加入油酸情况下,细胞随油酸浓度增加而生成更多、更大的脂滴,但AML-12细胞只有展现增加脂滴数量的变化表型;在HeLa细胞中进行19种中性脂合成通路上关键基因的转录表达沉默,发现需要同时双敲降两种甘油三酯合成酶DGAT1和DGAT2才能显着降低细胞内脂滴总体积储存量,但在COS-7细胞中只需要单敲降DGAT1即可降低脂滴存量;进一步使用了DGAT1/2抑制剂处理四种细胞后,发现对抑制剂响应可区分为两类细胞分组（HeLa、AML-12与COS-7、3T3-L1）的脂滴存量表型差异,其原因是DGAT1和DGAT2的转录表达谱在这两类细胞分组中的不同。结论：建立了高内涵脂滴三维成像和定量分析系统,量化了四种细胞系的脂滴数量与大小的表型差异,揭示了细胞的脂滴脂储存方式与蛋白酶表达谱的关系。     

Hepatitis C virus core protein decreases lipid droplet turnover: a mechanism for core-induced steatosis

Steatosis is a frequent complication of hepatitis C virus infection. In mice, this condition is recapitulated by the expression of a single viral protein, the nucleocapsid core. Core localizes to the surface of lipid droplets (LDs) in infected liver cells through a process dependent on host diacylglycerol acyltransferase 1 (DGAT1), an enzyme that synthesizes triglycerides in the endoplasmic reticulum. Whether DGAT1 also plays a role in core-induced steatosis is uncertain. Here, we show that mouse embryonic fibroblasts isolated from DGAT1(-/-) mice are protected from core-induced steatosis, as are livers of DGAT1(-/-) mice expressing core, demonstrating that the steatosis is DGAT1-dependent. Surprisingly, core expression did not increase DGAT1 activity or triglyceride synthesis, thus excluding the possibility that core activates DGAT1 to cause steatosis. Instead, we find that DGAT1-dependent localization of core to LDs is a prerequisite for the steatogenic properties of the core. Using biochemical and immunofluorescence microscopy techniques, we show that the turnover of lipids in core-coated droplets is decreased, providing a physiological mechanism for core-induced steatosis. Our results support a bipartite model in which core first requires DGAT1 to gain access to LDs, and then LD-localized core interferes with triglyceride turnover, thus stabilizing lipid droplets and leading to steatosis.     

Fat Storage-inducing Transmembrane Protein 2 Is Required for Normal Fat Storage in Adipose Tissue

Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.     

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation

Accurate protein inventories are essential for understanding an organelle’s functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.     

Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) Specifically Interacts with Phospholipid Transfer Protein StarD10 to Facilitate Surfactant Phospholipid Trafficking in Alveolar Type II Cells

Pulmonary surfactant, a mixture of proteins and phospholipids, plays an important role in facilitating gas exchange by maintaining alveolar stability. Saturated phosphatidylcholine (SatPC), the major component of surfactant, is synthesized both de novo and by the remodeling of unsaturated phosphatidylcholine (PC) by lyso-PC acyltransferase 1 (LPCAT1). After synthesis in the endoplasmic reticulum, SatPC is routed to lamellar bodies (LBs) for storage prior to secretion. The mechanism by which SatPC is transported to LB is not understood. The specificity of LPCAT1 for lyso-PC as an acyl acceptor suggests that formation of SatPC via LPCAT1 reacylation is a final step in SatPC synthesis prior to transport. We hypothesized that LPCAT1 forms a transient complex with SatPC and specific phospholipid transport protein(s) to initiate trafficking of SatPC from the endoplasmic reticulum to the LB. Herein we have assessed the ability of different StarD proteins to interact with LPCAT1. We found that LPCAT1 interacts with StarD10, that this interaction is direct, and that amino acids 79–271 of LPCAT1 and the steroidogenic acute regulatory protein-related lipid transfer (START) domain of START domain-containing protein 10 (StarD10) are sufficient for this interaction. The role of StarD10 in trafficking of phospholipid to LB was confirmed by the observation that knockdown of StarD10 significantly reduced transport of phospholipid to LB. LPCAT1 also interacted with one isoform of StarD7 but showed no interaction with StarD2/PC transfer protein.     

Targeting lipid droplet lysophosphatidylcholine for cisplatin chemotherapy

This study aims to explore lipidic mechanism towards low‐density lipoprotein receptor (LDLR)‐mediated platinum chemotherapy resistance. By using the lipid profiling technology, LDLR knockdown was found to increase lysosomal lipids and decrease membranous lipid levels in EOC cells. LDLR knockdown also down‐regulated ether‐linked phosphatidylethanolamine (PE‐O, lysosomes or peroxisomes) and up‐regulated lysophosphatidylcholine [LPC, lipid droplet (LD)]. This implies that the manner of using Lands cycle (conversion of lysophospholipids) for LDs might affect cisplatin sensitivity. The bioinformatics analyses illustrated that LDLR‐related lipid entry into LD, rather than an endogenous lipid resource (eg Kennedy pathway), controls the EOC prognosis of platinum chemotherapy patients. Moreover, LDLR knockdown increased the number of platinum‐DNA adducts and reduced the LD platinum amount. By using a manufactured LPC‐liposome‐cisplatin (LLC) drug, the number of platinum‐DNA adducts increased significantly in LLC‐treated insensitive cells. Moreover, the cisplatin content in LDs increased upon LLC treatment. Furthermore, lipid profiles of 22 carcinoma cells with differential cisplatin sensitivity (9 sensitive vs 13 insensitive) were acquired. These profiles revealed low storage lipid levels in insensitive cells. This result recommends that LD lipidome might be a common pathway in multiple cancers for platinum sensitivity in EOC. Finally, LLC suppressed both cisplatin‐insensitive human carcinoma cell training and testing sets. Thus, LDLR‐platinum insensitivity can be due to a defective Lands cycle that hinders LPC production in LDs. Using lipidome assessment with the newly formulated LLC can be a promising cancer chemotherapy method.     

DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes

The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types.     

The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide induces large lipid droplet accumulation and highlights the effect of LAMP-2 deficiency on lipid droplet degradation

Excess accumulation of intracellular lipids leads to various diseases. Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage. LDs are hydrolyzed via cytosolic lipases (lipolysis) and also degraded in lysosomes through autophagy; namely, lipophagy. A recent study has shown the size-dependent selection of LDs by the two major catabolic pathways (lipolysis and lipophagy), and thus experimental systems that can manipulate the size of LDs are now needed. The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide (PDMP) affects the structures and functions of lysosomes/late endosomes and the endoplasmic reticulum (ER), and alters cholesterol homeostasis. We previously reported that PDMP induces autophagy via the inhibition of mTORC1. In the present study, we found that PDMP induced the accumulation of LDs, especially that of large LDs, in mouse fibroblast (L cells). Surprisingly, the LD accumulation was relieved by PDMP in L cells deficient in lysosome-associated membrane protein-2 (LAMP-2), which is reportedly important for lipophagy. An electron microscopy analysis demonstrated that the LAMP-2 deficiency caused enlarged autophagosomes/autolysosomes in L cells, which may promote the sequestration and degradation of the PDMP-dependent large LDs. Accordingly, PDMP will be useful to explore the mechanism of LD degradation, by inducing large LDs.     

Re-patterning of skeletal muscle energy metabolism by fat storage-inducing transmembrane protein 2

Triacylglyceride stored in cytosolic lipid droplets (LDs) constitutes a major energy reservoir in most eukaryotes. The regulated turnover of triacylglyceride in LDs provides fatty acids for mitochondrial β-oxidation and ATP generation in physiological states of high demand for energy. The mechanisms for the formation of LDs in conditions of energy excess are not entirely understood. Fat storage-inducing transmembrane protein 2 (FIT2/FITM2) is the anciently conserved member of the fat storage-inducing transmembrane family of proteins implicated to be important in the formation of LDs, but its role in energy metabolism has not been tested. Here, we report that expression of FIT2 in mouse skeletal muscle had profound effects on muscle energy metabolism. Mice with skeletal muscle-specific overexpression of FIT2 (CKF2) had significantly increased intramyocellular triacylglyceride and complete protection from high fat diet-induced weight gain due to increased energy expenditure. Mass spectrometry-based metabolite profiling suggested that CKF2 skeletal muscle had increased oxidation of branched chain amino acids but decreased oxidation of fatty acids. Glucose was primarily utilized in CKF2 muscle for synthesis of the glycerol backbone of triacylglyceride and not for glycogen production. CKF2 muscle was ATP-deficient and had activated AMP kinase. Together, these studies indicate that FIT2 expression in skeletal muscle plays an unexpected function in regulating muscle energy metabolism and indicates an important role for lipid droplet formation in this process.     

Phospholipid Flippase Activities and Substrate Specificities of Human Type IV P-type ATPases Localized to the Plasma Membrane

Type IV P-type ATPases (P4-ATPases) are believed to translocate aminophospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. The yeast P4-ATPases, Drs2p and Dnf1p/Dnf2p, flip nitrobenzoxadiazole-labeled phosphatidylserine at the Golgi complex and nitrobenzoxadiazole-labeled phosphatidylcholine (PC) at the plasma membrane, respectively. However, the flippase activities and substrate specificities of mammalian P4-ATPases remain incompletely characterized. In this study, we established an assay for phospholipid flippase activities of plasma membrane-localized P4-ATPases using human cell lines stably expressing ATP8B1, ATP8B2, ATP11A, and ATP11C. We found that ATP11A and ATP11C have flippase activities toward phosphatidylserine and phosphatidylethanolamine but not PC or sphingomyelin. By contrast, ATPase-deficient mutants of ATP11A and ATP11C did not exhibit any flippase activity, indicating that these enzymes catalyze flipping in an ATPase-dependent manner. Furthermore, ATP8B1 and ATP8B2 exhibited preferential flippase activities toward PC. Some ATP8B1 mutants found in patients of progressive familial intrahepatic cholestasis type 1 (PFIC1), a severe liver disease caused by impaired bile flow, failed to translocate PC despite their delivery to the plasma membrane. Moreover, incorporation of PC mediated by ATP8B1 can be reversed by simultaneous expression of ABCB4, a PC floppase mutated in PFIC3 patients. Our findings elucidate the flippase activities and substrate specificities of plasma membrane-localized human P4-ATPases and suggest that phenotypes of some PFIC1 patients result from impairment of the PC flippase activity of ATP8B1.     

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.     

Lysophosphatidylcholine acyltransferase 3 knockdown-mediated liver lysophosphatidylcholine accumulation promotes very low density lipoprotein production by enhancing microsomal triglyceride transfer protein expression

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.