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ABSTRACT: PURPOSE OF REVIEW: Liver is the major organ in mammals that possesses the capacity to release triglyceride within VLDL. VLDL assembly requires apolipoprotein (apo) B-100 with the assistance of microsomal triglyceride transfer protein (MTP), which facilitates the mobilization of triglyceride into the microsomal lumen. Recent experimental evidence has suggested that the lumenal triglyceride associated with endoplasmic reticulum (ER)/Golgi may represent an entity serving as precursors for large VLDL1. RECENT FINDINGS: Under lipid-rich conditions, discrete triglyceride-rich lipidic bodies, termed lumenal lipid droplets, are accumulated in association with ER/Golgi microsomes. Formation of the microsome-associated lumenal lipid droplets (MALD) is dependent on the activity of MTP, and the resulting apoB-free lipidic body is associated with a variety of proteins including apolipoproteins that are components of VLDL. Formation and utilization of MALD during the assembly and secretion of VLDL1 have a profound influence on hepatic cell physiology, such as ER stress responses. SUMMARY: This review summarizes current understanding of hepatic triglyceride homeostasis in general, and highlights the functional significance of triglyceride compartmentalization between cytosol and microsomes in particular. Understanding of MALD metabolism may shed new light on the prevention and treatment of liver diseases associated with abnormally elevated intracellular triglycerides.
Current opinion in lipidology 11/2012; · 6.13 Impact Factor
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ABSTRACT: Exchangeable apolipoproteins, composed mainly of amphipathic α-helices, are associated with various plasma lipoproteins and play an important role in the metabolism of those lipoproteins to which they bind. Accumulating experimental evidence suggests that exchangeable apolipoproteins, such as apoE, apoA-IV, and apoC-III, also play a role intracellularly in facilitating lipid recruitment at different stages of very low-density lipoprotein assembly and trafficking through the endoplasmic reticulum-Golgi secretory compartments. Experimental evidence also suggests that apoA-I may become lipidated intracellularly through mechanisms dependent on or independent of ATP-binding cassette transporter A1. Thus, expression of these secretory proteins may exert an impact on hepatic triglyceride and cholesterol homeostasis during their transit from the endoplasmic reticulum through the Golgi apparatus. This review summarizes findings related to the modulation of intracellular assembly of very low-density lipoprotein and high-density lipoprotein by exchangeable apolipoproteins.
Arteriosclerosis Thrombosis and Vascular Biology 05/2012; 32(5):1073-8. · 6.37 Impact Factor
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ABSTRACT: A strong positive correlation between plasma apolipoprotein (apo) C-III and triglyceride concentrations has been invariably observed in human and animal studies. The hypertriglyceridemic effect of apo C-III has been conventionally explained by its extracellular roles in inhibiting lipolysis catalysed by lipoprotein lipase and attenuating triglyceride-rich lipoprotein clearance through receptor-dependent and/or independent mechanisms. However, recent experimental evidence suggests that apo C-III may also play an intracellular role in promoting hepatic triglyceride-rich lipoprotein production.
Kinetic studies with humans and genetically modified mice have shown that apo C-III is linked with increased production of triglyceride-rich lipoproteins, such as very-low-density lipoprotein 1 (VLDL1). Mutational studies on human apo C-III variants (originally identified in humans with hypotriglyceridemia or hyperalphalipoproteinemia) provide the structure-function analysis of human apo C-III, demonstrating that loss-of-function mutations within human apo C-III impair the assembly and secretion of triglyceride-rich VLDL1 under lipid-rich conditions.
The current review summarizes recent experimental evidence for an intrahepatic role of human apo C-III in promoting mobilization and utilization of triglyceride during VLDL1 assembly/secretion. Understanding mechanisms by which hepatic apo C-III expression is regulated under insulin resistance and diabetic conditions will lead to better and more rational strategies for the prevention and treatment of diabetic hypertriglyceridemia that is closely related to premature atherosclerosis.
Current opinion in lipidology 04/2012; 23(3):206-12. · 6.13 Impact Factor
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ABSTRACT: Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.
Molecular & Cellular Proteomics 07/2011; 10(10):O111.008425. · 7.40 Impact Factor
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Wen Qin,
Meenakshi Sundaram,
Yuwei Wang,
Hu Zhou,
Shumei Zhong,
Chia-Ching Chang,
Sanjay Manhas,
Erik F Yao,
Robin J Parks,
Pamela J McFie,
Scot J Stone,
Zhenghui G Jiang,
Congrong Wang,
Daniel Figeys,
Weiping Jia, Zemin Yao
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ABSTRACT: Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.
Journal of Biological Chemistry 06/2011; 286(31):27769-80. · 4.77 Impact Factor
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ABSTRACT: Hepatic lipase (HL) plays a role in the catabolism of apolipoprotein (apo)B-containing lipoproteins through its lipolytic and ligand-binding properties. We describe a potential intracellular role of HL in the assembly and secretion of VLDL. Transient or stable expression of HL in McA-RH7777 cells resulted in decreased (by 40%) incorporation of [(3)H]glycerol into cell-associated and secreted triacylglycerol (TAG) relative to control cells. However, incorporation of [(35)S]methionine/cysteine into cell and medium apoB-100 was not decreased by HL expression. The decreased (3)H-TAG synthesis/secretion in HL expressing cells was not attributable to decreased expression of genes involved in lipogenesis. Fractionation of medium revealed that the decreased [(3)H]TAG from HL expressing cells was mainly attributable to decreased VLDL. Expression of catalytically-inactive HL (HL(SG)) (Ser-145 at the catalytic site was substituted with Gly) in the cells also resulted in decreased secretion of VLDL-[(3)H]TAG. Examination of lumenal contents of microsomes showed a 40% decrease in [(3)H]TAG associated with lumenal lipid droplets in HL or HL(SG) expressing cells as compared with control. The microsomal membrane-associated [(3)H]TAG was decreased by 50% in HL expressing cells but not in HL(SG) expressing cells. Thus, expression of HL, irrespective of its lipolytic function, impairs formation of VLDL precursor [(3)H]TAG in the form of lumenal lipid droplets. These results suggest that HL expression in McA-RH7777 cells result in secretion of [(3)H]TAG-poor VLDL.
The Journal of Lipid Research 03/2011; 52(3):540-8. · 5.56 Impact Factor
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ABSTRACT: Growing evidence links the three mammalian lipin proteins, i.e., lipin-1, lipin-2 and lipin-3, to metabolic and cardiovascular diseases such as noninsulin-dependent diabetes mellitus and atherosclerosis. Lipin proteins play a dual function in lipid metabolism by acting as phosphatidate phosphatase (PAP) enzymes and as transcriptional regulators. Genetic variants within the human LPIN1 and LPIN2 genes are associated with metabolic syndromes. The fatty liver dystrophy (fld) mice carrying mutations within the Lpin1 gene display life-long deficiency in adipogenesis, insulin resistance, neonatal hepatosteatosis and hypertriglyceridemia, as well as increased atherosclerosis susceptibility. Cell culture studies show that hepatic lipin-1 expression is selectively stimulated by glucocorticoids and repressed by insulin, and its subcellular localization governs the assembly and secretion of very low density lipoproteins (VLDL). In noninsulin-dependent diabetes, glucocorticoid signals lead to dyslipidemia characterized by overproduction of VLDL and atherogenic remnants. This puts lipin-1 as a key integrator of hormonal signals to the liver in diabetic dyslipidemia. This review summarizes the current understanding of the role that hepatic lipin-1 plays in the synthesis, storage and compartmentalization of glycerolipids, and highlights the lipid metabolic consequences associated with dysregulated lipin expression.
Biochimica et Biophysica Acta 12/2010; 1801(12):1249-59. · 4.66 Impact Factor
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ABSTRACT: 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. © 2010 Wiley Periodicals, Inc. Mass Spec Rev 29:877–929, 2010
Mass Spectrometry Reviews 10/2010; 29(6):877 - 929. · 10.46 Impact Factor
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ABSTRACT: 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.
Mass Spectrometry Reviews 10/2010; 29(6):877-929. · 10.46 Impact Factor
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Meenakshi Sundaram,
Shumei Zhong,
Maroun Bou Khalil,
Hu Zhou,
Zhenghui G Jiang,
Yang Zhao,
Jahangir Iqbal,
M Mahmood Hussain,
Daniel Figeys,
Yuwei Wang, Zemin Yao
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ABSTRACT: We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [(3)H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [(3)H]TAG from C3AT cells was markedly decreased. The lowered [(3)H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL(1). Moreover, [(3)H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [(3)H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [(3)H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL(1) maturation. Furthermore, lumenal [(35)S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL(1) maturation.
The Journal of Lipid Research 06/2010; 51(6):1524-34. · 5.56 Impact Factor
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Shumei Zhong,
Antonia Lucia Magnolo,
Meenakshi Sundaram,
Hu Zhou,
Erik F Yao,
Enza Di Leo,
Paola Loria,
Shuai Wang,
Michelle Bamji-Mirza,
Lisheng Wang,
C Jamie McKnight,
Daniel Figeys,
Yuwei Wang,
Patrizia Tarugi, Zemin Yao
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ABSTRACT: Five nontruncating missense APOB mutations, namely A31P, G275S, L324M, G912D, and G945S, were identified in heterozygous carriers of familial hypobetalipoproteinemia (FHBL) in the Italian population. To test that the FHBL phenotype was a result of impaired hepatic secretion of mutant apoB proteins, we performed transfection studies using McA-RH7777 cells stably expressing wild type or mutant forms of human apolipoprotein B-48 (apoB-48). All mutant proteins displayed varied impairment in secretion, with G912D the least affected and A31P barely secreted. Although some A31P was degraded by proteasomes, a significant proportion of it (although inappropriately glycosylated) escaped endoplasmic reticulum (ER) quality control and presented in the Golgi compartment. Degradation of the post-ER A31P was achieved by autophagy. Expression of A31P also decreased secretion of endogenous apoB and triglycerides, yet the impaired lipoprotein secretion did not lead to lipid accumulation in the cells or ER stress. Rather, expression of genes involved in lipogenesis was down-regulated, including liver X receptor alpha, sterol regulator element-binding protein 1c, fatty acid synthase, acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1, and lipin-1. These results suggest that feedback inhibition of hepatic lipogenesis in conjunction with post-ER degradation of misfolded apoB proteins can contribute to reduce fat accumulation in the FHBL liver.
Journal of Biological Chemistry 02/2010; 285(9):6453-64. · 4.77 Impact Factor
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Shumei Zhong,
Antonia Lucia Magnolo,
Meenakshi Sundaram,
Hu Zhou,
Erik F. Yao,
Enza Di Leo,
Paola Loria,
Shuai Wang,
Michelle Bamji-Mirza,
Lisheng Wang,
C. Jamie McKnight,
Daniel Figeys,
Yuwei Wang,
Patrizia Tarugi, Zemin Yao
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ABSTRACT: Five nontruncating missense APOB mutations, namely A31P, G275S, L324M, G912D, and G945S, were identified in heterozygous carriers of familial hypobetalipoproteinemia
(FHBL) in the Italian population. To test that the FHBL phenotype was a result of impaired hepatic secretion of mutant apoB
proteins, we performed transfection studies using McA-RH7777 cells stably expressing wild type or mutant forms of human apolipoprotein
B-48 (apoB-48). All mutant proteins displayed varied impairment in secretion, with G912D the least affected and A31P barely
secreted. Although some A31P was degraded by proteasomes, a significant proportion of it (although inappropriately glycosylated)
escaped endoplasmic reticulum (ER) quality control and presented in the Golgi compartment. Degradation of the post-ER A31P
was achieved by autophagy. Expression of A31P also decreased secretion of endogenous apoB and triglycerides, yet the impaired
lipoprotein secretion did not lead to lipid accumulation in the cells or ER stress. Rather, expression of genes involved in
lipogenesis was down-regulated, including liver X receptor α, sterol regulator element-binding protein 1c, fatty acid synthase,
acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1, and lipin-1. These results suggest that feedback inhibition of hepatic
lipogenesis in conjunction with post-ER degradation of misfolded apoB proteins can contribute to reduce fat accumulation in
the FHBL liver.
Journal of Biological Chemistry 02/2010; 285(9):6453-6464. · 4.77 Impact Factor
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ABSTRACT: 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.
Nutrition & Metabolism 01/2010; 7:35. · 2.88 Impact Factor
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ABSTRACT: 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 efficiency 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 identified 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.
The Journal of Lipid Research 08/2009; 51(1):150-61. · 5.56 Impact Factor
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ABSTRACT: Phosphatidylcholine (PC) is the predominant phospholipid associated with high density lipoproteins (HDL). Although the hepatic uptake of cholesteryl esters from HDL is well characterized, much less is known about the fate of PC associated with HDL. Thus, we investigated the uptake and subsequent metabolism of HDL-PC in primary mouse hepatocytes.
The absence of scavenger receptor-BI resulted in a 30% decrease in cellular incorporation of [(3)H]PC whereas [(3)H]cholesteryl ether uptake was almost completely abolished. Although endocytosis is not involved in the uptake of cholesteryl esters from HDL, we demonstrate that HDL internalization accounts for 40% of HDL-PC uptake. Extracellular remodeling of HDL by secretory phospholipase A(2) significantly enhances HDL lipid uptake. HDL-PC taken up by hepatocytes is partially converted to triacylglycerols via PC-phospholipase C-mediated hydrolysis of PC and incorporation of diacylglycerol into triacylglycerol. The formation of triacylglycerol is independent of scavenger receptor-BI and occurs in extralysosomal compartments.
These findings indicate that HDL-associated PC is incorporated into primary hepatocytes via a pathway that differs significantly from that of HDL-cholesteryl ester, and shows that HDL-PC is more than a framework molecule, as evidenced by its partial conversion to hepatic triacylglycerol.
Biochimica et Biophysica Acta 03/2009; 1790(6):538-51. · 4.66 Impact Factor
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Maroun Bou Khalil,
Meenakshi Sundaram,
Hong-Yu Zhang,
Philip H Links,
Jennifer F Raven,
Boripont Manmontri,
Meltem Sariahmetoglu,
Khai Tran,
Karen Reue,
David N Brindley, Zemin Yao
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ABSTRACT: Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.
The Journal of Lipid Research 09/2008; 50(1):47-58. · 5.56 Impact Factor
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ABSTRACT: Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for beta-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.
The Journal of Lipid Research 06/2008; 49(5):1056-67. · 5.56 Impact Factor
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ABSTRACT: Hepatic secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins, is regulated through posttranslational
degradation. We reported a degradation pathway, post-ER pre secretory proteolysis (PERPP), that is increased by reactive oxygen
species (ROS) generated within hepatocytes from dietary polyunsaturated fatty acids (PUFA). We now report the molecular processes
by which PUFA-derived ROS regulate PERPP of apoB. ApoB exits the ER; undergoes limited oxidant-dependent aggregation; and
then, upon exit from the Golgi, becomes extensively oxidized and converted into large aggregates. The aggregates slowly degrade
by an autophagic process. None of the oxidized, aggregated material leaves cells, thereby preventing export of apoB-lipoproteins
containing potentially toxic lipid peroxides. In summary, apoB secretory control via PERPP/autophagosomes is likely a key
component of normal and pathologic regulation of plasma apoB levels, as well as a means for remarkably late-stage quality
control of a secreted protein.
Proceedings of the National Academy of Sciences 04/2008; 105(15):5862-5867. · 9.68 Impact Factor
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ABSTRACT: LRP1 [LDL (low-density lipoprotein) receptor-related protein 1]-null CHO cells (Chinese-hamster ovary cells) (13-5-1 cells) exhibited accelerated cell growth and severe tumour progression after they were xenografted into nude mice. Reconstitution of LRP1 expression in these cells, either with the full-length protein or with a minireceptor, reduced growth rate as well as suppressed tumour development. We tested the role of the tyrosine residue in the FXNPXY63 motif within the LRP1 cytoplasmic domain in signal transduction and cell growth inhibition by site-specific mutagenesis. The LRP1 minireceptors harbouring Tyr63 to alanine or Tyr63 to phenylalanine substitution had diametrically opposite effects on cell growth, cell morphology and tumour development in mice. The Y63F-expressing cells showed suppressed cell growth and tumour development, which were associated with decreased beta-catenin and cadherin concentrations in the cells. On the other hand, the Y63A-expressing cells lacked inhibition on cell growth and tumour development, which were associated with hyperactivation of ERKs (extracellular-signal-regulated kinases), FAK (focal adhesion kinase) and cyclin D1 in the cells. The mutant Y63A minireceptor also exhibited reduced capacity in binding to the Dab2 (disabled 2) adaptor protein. In addition, the Y63A mutant showed increased caveolar localization, and cells expressing Y63A had altered caveolae architecture. However, tyrosine to alanine substitution at the other NPXY29 motif had no effect on cell growth or tumorigenesis. These results suggest that the FXNPXY63 motif of LRP1 not only governs cellular localization of the receptor but also exerts multiple functional effects on signalling pathways involved in cell growth regulation.
Biochemical Journal 02/2008; 409(1):53-64. · 4.90 Impact Factor
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John R Burnett,
Shumei Zhong,
Zhenghui G Jiang,
Amanda J Hooper,
Eric A Fisher,
Roger S McLeod,
Yang Zhao,
P Hugh R Barrett,
Robert A Hegele,
Frank M van Bockxmeer,
Hongyu Zhang,
Dennis E Vance,
C James McKnight, Zemin Yao
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ABSTRACT: Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the alpha-helical domain within the N terminus of apoB. Thus, proper folding of the alpha-helical domain of apoB-100 is essential for efficient secretion.
Journal of Biological Chemistry 09/2007; 282(33):24270-83. · 4.77 Impact Factor
