Microsomal triglyceride transfer protein expression during mouse development

Feto-maternal transfer of lipophilic nutrients is an important factor in the normal development of the fetus and may be mediated by lipoproteins as carriers of these nutrients. Two proteins that may be important in this process are apolipoprotein B (apoB, the major structural protein of secreted lipoproteins) and microsomal triglyceride transfer protein (MTP) whose normal activity is required for the secretion of apoB-containing lipoproteins. Although no abnormalities of conception and embryonic lethality are known in humans who inherit genetic deficiencies of either of these proteins, homozygous mice bearing knockouts of either apoB or MTP show early embryonic lethality. To characterize the ontogeny of MTP expression during embryonic mouse development, we have used in situ hybridization to characterize the pattern of expression. By using microwave heating of tissue sections to optimize hybridization, we show that there is robust MTP expression in the yolk sac tissues followed by expression in the primordial liver cell nests as early as day 9 post-coitum (E9.5). Intestinal expression is detected around E12.5 and attains full adult expression patterns by E14.5. No expression in any other tissues was observed, including developing heart, kidney, placenta, and maternal decidua.Thus the pattern of MTP expression is compatible with a role in the transfer of lipophilic nutrients from the yolk sac, prior to hepatic development and to the liver, once the circulatory system has been established.     

Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.     

Neonatal estrogenization leads to increased expression of cellular retinol binding protein 2 in the mouse reproductive tract

Exposure to estrogenic substances during a time window, the so-called critical period, in perinatal life causes an irregular development of the genital tract that leads to ovary-independent proliferation and cornification in the vaginal epithelium in mice. We have previously demonstrated that retinol inhibits the irreversible effects of estrogen on the vagina. Here, mice kept in a vitamin-A-deficient condition during perinatal life were shown to be more sensitive to the harmful effects of estrogen. In addition, expression of mRNA for retinol binding protein type 2 (CRBP2), a small intestine-specific cytosolic protein that captures intracellular retinal and retinol, was detected in the vaginal epithelium. Induction of increased expression of CRBP2 mRNA by estrogen was also evident in the uterus and epididymis. Both estradiol-17 and diethylstilbestrol markedly increased the tissue content of CRBP2 mRNA in the vagina and uterus during the neonatal critical period but not after 15 days of age. These results taken together imply that estrogen disrupts the local vitamin A balance by an induction of CRBP2 gene expression in the epithelium in the developing mouse genital tract, and that retinoid imbalance may contribute to the genesis of irreversible effects of estrogen on the vagina.This work was supported by Grants-in-Aid for Priority Area B (to T.M. and M.M.) and for Young Scientists (to M.M.) from the Ministry of Culture, Sports, Science and Technology, Japan     

Proteomic profile of mouse fibroblasts with a targeted disruption of the peroxisome proliferator activated receptor-beta/delta gene

The peroxisome proliferator activated receptor-beta (PPARbeta) plays an essential role in lipid metabolism, immune modulation, differentiation and cell proliferation. There is also strong evidence for a function in oncogenesis and tumor vascularization, but the underlying molecular mechanisms remain elusive. In the present study, we have used fibroblasts derived from Pparb wild-type and null mice to determine by 2-DE and PMF analysis the contribution of PPARbeta to the protein profile of fibroblasts. Thirty-one differentially expressed proteins of different functional categories were identified. For at least two proteins a role in tumorigenesis and/or tumor vascularization has previously been reported: while the calcium intracellular channel-4 (CLIC4) was expressed at lower levels in Pparb null cells, expression of the cellular retinol binding protein 1 (CRBP1) was enhanced. Clic4 and Crbp1 gene expression patterns observed in different experimental settings in vitro and in vivo confirmed the proteomics data. Our findings indicate that the expression of a defined set of proteins is altered in fibroblasts and endothelial cells from Pparb null mice, that this is due to aberrant gene regulation, and that the altered expression of these proteins is consistent with the tumor vascularization phenotype of Pparb null mice.     

微粒体甘油三脂转运蛋白MTP的结构和功能研究概况

微粒体甘油三酯转运蛋白MTP(microsomal triglyceride transfer protein,MTP)首先是从牛的肝细胞微粒体碎片中分离获得的,其作用是加速甘油三脂(triglyceride,TG)、胆固醇(cholesteryl ester,CE)和磷脂酰胆碱(phosphatidylcholine,PC)的转运和细胞或亚细胞膜的生物合成。它后来在肝细胞和小肠的微粒体膜中发现[1],由于它的位置及其转运TG可以推测与血浆脂蛋白中极低密度脂蛋白(very low density lipoprotein,VLDL)和乳糜微粒(chylomicrons,CM)的组装过程有关。     

Synthesis,DNA/protein binding and in vitro cytotoxic studies of new palladium metallothiosemicarbazones

A series of four new thiosemicarbazone complexes of palladium have been synthesized, characterized and evaluated for their DNA/protein binding with CT-DNA and BSA, respectively. The new complexes bound to CT-DNA by intercalation mode and in protein binding studies, the complexes bound to BSA binding mechanism was found as static quenching. Among them the complex 4 had a strong binding affinity with BSA. In addition, in vitro cytotoxic studies were carried out on lung cancer (A549) and liver cancer (HepG2) cell lines and found that the complexes exhibited better activity than their parent thiosemicarbazone analogues. The complex 3 exhibited better activity than other complexes and this fact supported by the increased accumulation of the complexes in to the cancer cells which are evident from inter cellular uptake studies.     

Lipopolysaccharide-binding protein: localization in secretory granules of Paneth cells in the mouse small intestine

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host’s response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58–60 kDa protein of LBP distinctly labeled a small population of cells located deep into the crypts. This cell population was also positive for lysozyme and α-defensin 4, identifying Paneth cells as the main intestinal LBP-producing cells. By immunogold electron microscopy, intense labeling was observed in the secretory granules of these cells. We conclude that Paneth cells express LBP together with other proteins acting in the innate immune response of the gut, such as lysozyme, defensins and intelectin.     

Lead influences translational or posttranslational regulation of ia expression and increases invariant chain expression in mouse B cells

The molecular mechanisms governing the increased cell surface expression of major histocompatibility complex (MHC) class II molecules (Ia) on lead-treated mouse B cells was investigated. Lead has been shown to directly cause a selective, two-fold increase in the B cell's surface density of both products of the I region of the mouse MHC, I-A and I-E. In the present study, Western blot analysis showed that Pb increases the total cellular pool of I-A β-chain by twofold. The increase in cellular I-A was not found to be due to increased messenger RNA (mRNA) for either the α- or the β-chain of I-A. Biosynthetic labeling studies showed that Pb increases the translation or the stability of the Ia-associated invariant chain (Ii or γ) and possibly the β-chain of Ia. Collectively these results suggest that Pb increases the B cell's surface Ia by influencing translational or posttranslational regulation of Ia and/or Ia-associated chains.     

Immunohistochemical analysis of paraoxonases-1, 2, and 3 expression in normal mouse tissues

The paraoxonase (PON) enzyme family, comprising PON1, PON2, and PON3, are antioxidant enzymes that degrade oxidised phospholipids. We describe the immunohistochemical localisation of the PON proteins in the normal mouse. Antibodies were obtained by inoculating rabbits with peptides derived from specific sequences of mature PONs. PON1 and PON3 were detected in the skin external epithelium, acini of the sebaceous glands, tongue epithelium, acini of the submandibular gland, surface epithelia of the stomach and the intestine, hepatocytes, exocrine pancreas acini, fibre tracts of the encephalon and the spinal cord, skeletal and cardiac muscle, eye lens epithelium and retinal layers, adipocytes, chondrocytes, epithelial cells of the trachea and bronchiole, ovary follicular fluid, seminiferous tubules, spermatozoa, and kidney proximal tubules. PON2 expression was weaker than that of PON1 and PON3, and was absent in some of the tissues studied, such as submandibular gland, nerve cells, and adipocytes. In muscle cells, PON2 expression was restricted to the endomysium. Apolipoprotein A-I did not colocalise with PONs, suggesting local synthesis. This study provides an experimental model to investigate the role played by these enzymes as antioxidants and their relationship with the development of a variety of diseases.     

Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

A role for scavenger receptor B-I in selective transfer of rhodamine-PE from liposomes to cells

We investigated the potential role of scavenger receptor B-I (SR-BI) in the selective removal of liposomal markers from blood by hepatocytes. Liposomes were labeled with [(3)H]cholesteryloleyl-ether ([(3)H]COE), 1,2-di[1-(14)C]palmitoyl-phosphatidylcholine ([(14)C]PC), and N-(lissamine rhodamine-B sulfonyl)-phosphatidylethanolamine (N-Rh-PE). The radiolabels were eliminated at identical rates from plasma, while N-Rh-PE was cleared twice as fast. Involvement of SR-BI in the selective removal of N-Rh-PE from liposomes was studied in transfected Chinese hamster ovary cells over-expressing SR-BI. Uptake of N-Rh-PE from liposomes containing phosphatidylserine was higher than [(3)H]COE, and was further enhanced by apolipoprotein A-I, confirming involvement of SR-BI in the selective uptake of liposomal N-Rh-PE by cells.     

Gut triglyceride production

Our knowledge of how the body absorbs triacylglycerols (TAG) from the diet and how this process is regulated has increased at a rapid rate in recent years. Dietary TAG are hydrolyzed in the intestinal lumen to free fatty acids (FFA) and monoacylglycerols (MAG), which are taken up by enterocytes from their apical side, transported to the endoplasmic reticulum (ER) and resynthesized into TAG. TAG are assembled into chylomicrons (CM) in the ER, transported to the Golgi via pre-chylomicron transport vesicles and secreted towards the basolateral side. In this review, we mainly focus on the roles of key proteins involved in uptake and intracellular transport of fatty acids, their conversion to TAG and packaging into CM. We will also discuss intracellular transport and secretion of CM. Moreover, we will bring to light few factors that regulate gut triglyceride production. Furthermore, we briefly summarize pathways involved in cholesterol absorption. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Plasma lecithin:cholesterol acyltransferase activity modifies the inverse relationship of C-reactive protein with HDL cholesterol in nondiabetic men

Lecithin:cholesterol acyltransferase (LCAT) is instrumental in high-density lipoprotein (HDL) maturation, but high LCAT levels do not predict low cardiovascular risk. LCAT may affect antioxidative or anti-inflammatory properties of HDL. We determined the relationship of plasma high-sensitivity C-reactive protein (CRP) with LCAT activity and evaluated whether LCAT activity modifies the decreasing effect of HDL cholesterol (HDL-C) on CRP, as an estimate of its anti-inflammatory properties. Plasma HDL-C, apolipoprotein (apo) A-I and LCAT activity (exogenous substrate method) were measured in 260 nondiabetic men without cardiovascular disease. CRP was correlated inversely with HDL-C and apo A-I, and positively with LCAT activity (P < 0.01 to 0.001). Multivariate regression analysis demonstrated that age- and smoking-adjusted plasma CRP levels were associated negatively with HDL-C (β = − 0.224, P < 0.001) and positively with LCAT activity (β = 0.119, P = 0.034), as well as with the interaction between HDL-C and LCAT activity (β = 0.123, P = 0.026). There was also an interaction between apo A-I and LCAT activity on CRP (β = 0.159, P = 0.005). These relationships remained similar after adjustment for apo B-containing lipoproteins. In conclusion, the inverse relationship of HDL-C with CRP is attenuated by LCAT activity at higher HDL-C levels. It is hypothesized that LCAT could mitigate HDL's anti-inflammatory or antioxidative properties at higher HDL-C concentrations.     

Gene expression profiling related to the enhanced erythropoiesis in mouse bone marrow cells

Peroxiredoxin II knockout (Prdx II(-/-)) mice had a spontaneous phenotype of hemolytic anemia. In this study, we found that Ter-119(+)CD71(+) cells increased in Prdx II(-/-) mice bone marrow (BM) at 8 weeks of age. We examined the differential expression profiles to bone marrow cells (BMCs) between Prdx II(+/+) and Prdx II(-/-) mice using a cDNA microarray. We identified the 136 candidates were differentially expressed a greater twofold increase or decrease than EPO receptor. In this study, we focused on the up-regulated NBPs during erythropoietic differentiation. According to cDNA microarray results, six NBPs except zfp-127 were up-regulated during erythropoiesis in Prdx II(-/-) mice. Among the six candidates, eIF3-p44, hnRNPH1, G3bp, and Zfpm-1 were dramatically increased at day 7 of the in vitro erythropoietic differentiation of human CD34(+) cells. However, DJ-1 and Rbm3 were slightly increased only at day 12. Our results suggest that up-regulated NBPs might be involved during erythropoietic differentiation.     

Intestinal fatty acid binding protein and microsomal triglyceride transfer protein polymorphisms in French-Canadian youth

Growing evidence suggests an association between lipid abnormalities and fatty acid binding protein (FABP) and microsomal triglyceride transfer protein (MTP) gene variants. Our objectives were to determine whether Ala54Thr FABP2 and G-493T MTP polymorphisms are associated with increased risks of insulin resistance syndrome (IRS) in youth and/or modify the expression of accompanying dyslipidemia. Our study of 1,742 French-Canadians aged 9, 13, and 16 years did not provide evidence of a potential predisposition to IRS related to either FABP2 or MTP genotypes. However, we observed a heterogeneity of the FABP2 effect by IRS status on total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), and apolipoprotein B (apoB) concentrations (P for interaction=0.045, 0.018, and 0.017, respectively). Among the metabolic components of IRS, only triglyceride (TG) displayed an interaction with FABP2 polymorphism: compared with Thr/Ala and Ala/Ala, the Thr/Thr genotype was associated with a steeper increase in TC, LDL-C, and apoB parallel to TG concentrations (P <0.001). IRS did not modify the associations between the MTP polymorphism and any of the biochemical parameters. Our study suggests that the effects of FABP2 allelic variations on lipid traits are context dependent, indicating that this variant may play an important role in cardiovascular pathogenesis in the presence of IRS or hypertriglyceridemia.