Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells

Higashi, Yusuke; Itabe, Hiroyuki; Fukase, Hironaga; Mori, Masahiro; Fujimoto, Yasuyuki; Sato, Ryuichiro; Imanaka, Tsuneo; Takano, Tatsuya

doi:10.1016/s1388-1981(02)00157-9

Cited by 13 publications

(15 citation statements)

References 29 publications

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“…1B, calnexin expression extended over most of the 20 ER fractions. This indicated that calnexin was present in both the heavier rough endoplasmic reticulum (RER) fractions, as well as the lighter smooth endoplasmic reticulum (SER) fractions, as described previously (10,11). Based on calnexin protein expression in fractions 4 -17 (the fractions used for two-dimensional analysis), we were able to obtain a 10-fold increase in ER enrichment over the whole cell lysate positive control.…”

Section: Purity Determination Of Hepatic Er Subcellularsupporting

confidence: 76%

“…In addition, 17 abundantly expressed hepatic ER proteins were identified and are also represented in the figure. through thorough testing of the ER fractions by immunoblotting with ER marker antibodies as well as markers against other potentially contaminating organelles. We employed the ER markers calnexin and ER60, which are known to be present in ribosome-laden rough RER as well as the SER (10,11) and RER (12), respectively. We were able to obtain an enrichment of 10-and 16-fold for calnexin and ER60, respectively, over whole cell lysate with no detectable contaminating proteins.…”

Section: Discussionmentioning

confidence: 99%

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Proteomic Profiling of Hepatic Endoplasmic Reticulum-associated Proteins in an Animal Model of Insulin Resistance and Metabolic Dyslipidemia

Morand

Macri

Adeli

2005

Journal of Biological Chemistry

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Hepatic insulin resistance and lipoprotein overproduction are common features of the metabolic syndrome and insulin-resistant states. A fructose-fed, insulin-resistant hamster model was recently developed to investigate mechanisms linking the development of hepatic insulin resistance and overproduction of atherogenic lipoproteins. Here we report a systematic analysis of protein expression profiles in the endoplasmic reticulum (ER) fractions isolated from livers of fructose-fed hamsters with the intention of identifying new candidate proteins involved in hepatic complications of insulin resistance and lipoprotein dysregulation. We have profiled hepatic ER-associated proteins from chow-fed (control) and fructose-fed (insulin-resistant) hamsters using two-dimensional gel electrophoresis and mass spectrometry. A total of 26 large scale two-dimensional gels of hepatic ER were used to identify 34 differentially expressed hepatic ER protein spots observed to be at least 2-fold differentially expressed with fructose feeding and the onset of insulin resistance. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-quadrupole time of flight (MALDI-Q-TOF), MALDI-TOF-postsource decay, and database mining using ProteinProspector MS-fit and MS-tag or the PROWL ProFound search engine using a focused rodent or mammalian search. Hepatic ER proteins ER60, ERp46, ERp29, glutamate dehydrogenase, and TAP1 were shown to be more than 2-fold downregulated, whereas ␣-glucosidase, P-glycoprotein, fibrinogen, protein disulfide isomerase, GRP94, and apolipoprotein E were all found to be up-regulated in the hepatic ER of the fructose-fed hamster. Seven isoforms of ER60 in the hepatic ER were all shown to be downregulated at least 2-fold in hepatocytes from fructosefed/insulin-resistant hamsters. Implications of the differential expression of positively identified protein factors in the development of hepatic insulin resistance and lipoprotein abnormalities are discussed.

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Section: Purity Determination Of Hepatic Er Subcellularsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Proteomic Profiling of Hepatic Endoplasmic Reticulum-associated Proteins in an Animal Model of Insulin Resistance and Metabolic Dyslipidemia

Morand

Macri

Adeli

2005

Journal of Biological Chemistry

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“…Lipids were extracted from the conditioned medium with chloroform and methanol. The lipids were separated on thin-layer chromatography (TLC) as described previously (25). The radioactivities of the separated bands on TLC were quantified either using a BAS1500 bio-imaging analyzer or a liquid scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

“…After 4-h incubation with [ 14 C]acetate, cells recovered from culture dishes by trypsin treatment were washed extensively with ice-cold PBS. Cell fractionation was carried out as described previously (25). Briefly, cells were homogenized in cell-homogenization buffer (250 mM sucrose, 0.1% ethanol, and 20 mM Tricine, pH 7.8) using a Teflon homogenizer.…”

Section: Measurement Of [ 14 C]acetic Acid Incorporation Intomentioning

confidence: 99%

“…HuH-7 is well differentiated and is considered to be relevant to hepatic cells physiologically (23). Although HepG-2, one of the widely used hepatic cell lines, secretes only high density lipoprotein-sized, lipid-poor particles (24), low density lipoprotein-sized lipoprotein particles containing apoB-100 are secreted from HuH-7 cells (25). Thus we used the cell line as a good model to investigate human lipoprotein metabolism.…”

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confidence: 99%

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Transmembrane Lipid Transfer Is Crucial for Providing Neutral Lipids during Very Low Density Lipoprotein Assembly in Endoplasmic Reticulum

Higashi

Itabe

Fukase

et al. 2003

Journal of Biological Chemistry

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Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [ 14 C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [ 14 C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.The liver plays a central role in lipoprotein metabolism through secretion of very low density lipoprotein (VLDL) 1 , which carries neutral lipids to all peripheral tissues. VLDL, a large particle containing apolipoprotein B (apoB) and various lipids, including triglyceride (TG), free cholesterol (FC), and cholesteryl ester (CE), is assembled in endoplasmic reticulum (ER) in hepatic cells. Mechanisms of VLDL particle formation are likely to be rigorously regulated, because association of lipids to apoB needs to occur cotranslationally, and poorly lipidated apoB-containing lipoprotein is degraded by proteasome or ER-resident proteases such as ER-60 prior to secretion (1-3). Thus it is thought that the association of lipids to apoB is a rate-limiting step for VLDL secretion, with regulation of this process controlled by as yet unknown mechanisms. One of the factors for this lipid transfer is microsomal triglyceride transfer protein (MTP), a lack of which was identified as the cause of abetalipoproteinemia, an autosomal recessive disease with total loss of apoB-containing lipoprotein in plasma (4, 5). MTP locates in the luminal space of ER by forming heterodimers with protein disulfide isom...

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Proteomic profiling of the prechylomicron transport vesicle involved in the assembly and secretion of apoB‐48‐containing chylomicrons in the intestinal enterocytes

et al. 2009

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Intracellular assembly of chylomicrons (CM) occurs in intestinal enterocytes through a series of complex vesicular interactions. CM are transported from the ER to the Golgi using a specialized vesicular compartment called the prechylomicron transport vesicle (PCTV). In this study, PCTVs were isolated from the enteric ER of the Syrian Golden hamster, and characterized using 2-DE and MS. Proteomic profiles of PCTV-associated proteins were developed with the intention of identifying proteins involved in the formation, transport, lipidation, and assembly of CM particles. Positively identified proteins included those involved in lipoprotein assembly, namely microsomal triglyceride transfer protein and apolipoprotein B-48, as well as proteins involved in vesicular transport, such as Sar1 and vesicle-associated membrane protein 7. Other groups of proteins found were chaperones, intracellular vesicular trafficking proteins, fatty acid-binding proteins, and lipid-related proteins. These findings have increased our understanding of the transport vesicle involved in the intracellular assembly and transport of CM and can provide insight into potential cellular factors responsible for dysregulation of intestinal CM production.

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Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells

Cited by 13 publications

References 29 publications

Proteomic Profiling of Hepatic Endoplasmic Reticulum-associated Proteins in an Animal Model of Insulin Resistance and Metabolic Dyslipidemia

Proteomic Profiling of Hepatic Endoplasmic Reticulum-associated Proteins in an Animal Model of Insulin Resistance and Metabolic Dyslipidemia

Transmembrane Lipid Transfer Is Crucial for Providing Neutral Lipids during Very Low Density Lipoprotein Assembly in Endoplasmic Reticulum

Proteomic profiling of the prechylomicron transport vesicle involved in the assembly and secretion of apoB‐48‐containing chylomicrons in the intestinal enterocytes

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