Biomedical subjects

N Duverger

Publications and source records attributed to N Duverger.

At least 55 records · Page 3Linked to original sources

PubMed1996

Potential gene therapy for lecithin-cholesterol acyltransferase (LCAT)-deficient and hypoalphalipoproteinemic patients with adenovirus-mediated transfer of human LCAT gene.

BACKGROUND: Overexpression of human lecithin-cholesterol acyltransferase (LCAT) in transgenic mice results in an increase of the antiatherogenic HDLs. METHODS AND RESULTS: To investigate the potential use of LCAT for gene therapy, a recombinant adenovirus was constructed in which the human LCAT cDNA was expressed under the control of the human cytomegalovirus immediate/early promoter followed by a chimeric intron (AdCMV human LCAT). Human apolipoprotein (apo) A-I transgenic mice infected with AdCMV human LCAT by intravenous injection accumulated reactive LCAT in the plasma. LCAT activity was increased 201-fold in the plasma of mice infected with 1 x 10(6) pfu AdCMV human LCAT, from 45 +/- 2 to 9068 +/- 812 nmol.mL-1.h-1, in comparison with basal LCAT activity measured in control mice, 5 days after injection. Plasma HDL cholesterol levels rose from 117 +/- 12 to 797 +/- 48 mg/dL, and plasma human apo A-I concentrations increased from 247 +/- 14 to 616 +/- 17 mg/dL, in AdCMV human LCAT infected mice compared with control mice. HDL particles were larger and had a different electrophoretic mobility. Studies of cholesterol efflux by incubation of serum with cholesterol-loaded Fu5AH cells showed that serum from AdCMV human LCAT-infected mice promoted a significantly higher efflux than did that of the controls. CONCLUSIONS: These data establish the potential of this approach for treatment of subjects with LCAT gene defects as well as patients with low plasma levels of apo A-I and HDL cholesterol.

Adenoviridae↗

PubMed1996

Protection against atherogenesis in mice mediated by human apolipoprotein A-IV.

Apolipoproteins are protein constituents of plasma lipid transport particles. Human apolipoprotein A-IV (apoA-IV) was expressed in the liver of C57BL/6 mice and mice deficient in apoE, both of which are prone to atherosclerosis, to investigate whether apoA-IV protects against this disease. In transgenic C57BL/6 mice on an atherogenic diet, the serum concentration of high density lipoprotein (HDL) cholesterol increased by 35 percent, whereas the concentration of endogenous apoA-I decreased by 29 percent, relative to those in transgenic mice on a normal diet. Expression of human apoA-IV in apoE-deficient mice on a normal diet resulted in an even more severe atherogenic lipoprotein profile, without affecting the concentration of HDL cholesterol, than that in nontransgenic apoE-deficient mice. However, transgenic mice of both backgrounds showed a substantial reduction in the size of atherosclerotic lesions. Thus, apoA-IV appears to protect against atherosclerosis by a mechanism that does not involve an increase in HDL cholesterol concentration.

Animals↗

PubMed1996

Inhibition of atherosclerosis development in cholesterol-fed human apolipoprotein A-I-transgenic rabbits.

BACKGROUND: Prospective epidemiological studies support the hypothesis that high levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I limit atherosclerosis development. However, more data from studies with animal models of atherosclerosis that resemble the human disease are required to demonstrate the effect of apo A-I in the inhibition of atherogenesis. The rabbit is a good animal model for human atherosclerosis. METHODS AND RESULTS: Human apo A-I-transgenic rabbits have been produced, and we have evaluated the effect of apo A-I on the development of atherosclerosis in transgenic rabbits fed a cholesterol-rich diet for 14 weeks. Plasma cholesterol levels of atherogenic apo B-containing lipoproteins were similar for transgenic and control rabbits (> 1000 mg/dL), while plasma levels of HDL cholesterol in the transgenic group were always about twice that of the control group (68 +/- 11 versus 37 +/- 3 mg/dL at 14 weeks; P < .001). At the end of the experiment, the amount of aortic surface area covered by lesions as well as the amount of lipid accumulation in the aorta were significantly less in transgenic rabbits compared with the control group (15 +/- 12% versus 30 +/- 8%, P < .0027 for the surface area of the thoracic aorta; 116 +/- 31 versus 247 +/- 39 mumol/g aorta, P < .0068 for cholesterol content in total aorta). CONCLUSIONS: Overexpression of human apo A-I in rabbits inhibits the development of atherosclerosis in this animal model that resembles, in many respects, human atherosclerosis.

Analysis of Variance↗

PubMed1996

Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice.

The regulation of liver apolipoprotein (apo) A-I gene expression by fibrates was studied in human apo A-I transgenic mice containing a human genomic DNA fragment driving apo A-I expression in liver. Treatment with fenofibrate (0.5% wt/wt) for 7 d increased plasma human apo A-I levels up to 750% and HDL-cholesterol levels up to 200% with a shift to larger particles. The increase in human apo A-I plasma levels was time and dose dependent and was already evident after 3 d at the highest dose (0.5% wt/wt) of fenofibrate. In contrast, plasma mouse apo A-I concentration was decreased after fenofibrate in nontransgenic mice. The increase in plasma human apo A-I levels after fenofibrate treatment was associated with a 97% increase in hepatic human apo A-I mRNA, whereas mouse apo A-I mRNA levels decreased to 51%. In nontransgenic mice, a similar down-regulation of hepatic apo A-I mRNA levels was observed. Nuclear run-on experiments demonstrated that the increase in human apo A-I and the decrease in mouse apo A-I gene expression after fenofibrate occurred at the transcriptional level. Since part of the effects of fibrates are mediated through the nuclear receptor PPAR (peroxisome proliferator-activated receptor), the expression of the acyl CoA oxidase (ACO) gene was measured as a control of PPAR activation. Both in transgenic and nontransgenic mice, fenofibrate induced ACO mRNA levels up to sixfold. When transgenic mice were treated with gemfibrozil (0.5% wt/wt) plasma human apo A-I and HDL-cholesterol levels increased 32 and 73%, respectively, above control levels. The weaker effect of this compound on human apo A-I and HDL-cholesterol levels correlated with a less pronounced impact on ACO mRNA levels (a threefold increase) suggesting that the level of induction of human apo A-I gene is related to the PPAR activating potency of the fibrate used. Treatment of human primary hepatocytes with fenofibric acid (500 microM) provoked an 83 and 50% increase in apo A-I secretion and mRNA levels, respectively, supporting that a direct action of fibrates on liver human apo A-I production leads to the observed increase in plasma apo A4 and HDL-cholesterol.

Analysis of Variance↗

PubMed1996

Transgenic rabbits expressing human apolipoprotein A-I in the liver.

Human apolipoprotein A-I (apo A-I) transgenic rabbits were created by use of an 11-kb genomic human apo A-I construct containing a liver-specific promoter. Five independent transgenic lines were obtained in which human apo A-I gene had integrated and was expressed. Plasma levels of human apo A-I ranged from 8 to 100 mg/dL for the founder and up to 175 mg/dL for the progeny. Rabbit apo A-I levels were substantially decreased in the transgenic rabbits. HDL cholesterol (HDL-C) levels were higher in two of the five transgenic rabbit lines than in controls (line 20 versus nontransgenic littermate, HDL-C = 80 +/- 7 versus 37 +/- 6 mg/dL; line 8 versus nontransgenic littermate, HDL-C = 54 +/- 16 versus 35 +/- 6 mg/dL). This resulted in less atherogenic lipoprotein profiles, with very low (VLDL + LDL-C)/HDL-C ratios. HDL size and protein and lipid compositions were similar between transgenic and littermate nontransgenic rabbits. However, a large amount of pre-beta apo A-I-containing lipoproteins was observed in the plasma of the highest human apo A-I expressor. Cell cholesterol efflux was evaluated with the incubation of whole serum from transgenic and control rabbits. Cell cholesterol efflux was highly correlated with HDL cholesterol, with apo A-I, and with the presence of pre-beta apo A-I-containing lipoproteins. These rabbits will be an extremely useful model for the evaluation of the effect of increased hepatic apo A-I expression on atherosclerosis.

Animals↗

PubMed1995

Tissue-specific expression of the human gene for lecithin: cholesterol acyltransferase in transgenic mice alters blood lipids, lipoproteins and lipases towards a less atherogenic profile.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the reverse cholesterol pathway but its role in lipid metabolism is still unclear. We have generated mice transgenic for a 7-kb genomic DNA fragment comprising the 6 exons and 5 introns of the LCAT gene with 1932 bp of 5' flanking and 908 bp of 3' flanking sequences. One line had integrated about 30 copies and expressed about 40-fold increased LCAT activity in a human test system. The expression showed correct tissue specificity of the human LCAT gene. Increased LCAT activity resulted in a decrease of plasma triacylglycerols below 50% of fasting controls. This reduction was seen in all lipoprotein fractions. Lipoprotein lipase activity did not change significantly, whereas hepatic triacylglycerol lipase increased markedly. Plasma total cholesterol was similar in fasting transgenic and control mice, but low-density lipoprotein and very low-density lipoprotein cholesterol were reduced to about 50%. High-density lipoprotein cholesterol increased about 20%, accompanied by a correspondingly increased size and a higher cholesterol efflux-stimulating activity of transgenic LCAT high-density lipoprotein. Both apolipoprotein A-I and A-II plasma concentrations increased in transgenic mice. Plasma triacylglycerol and cholesteryl ester fatty acid distribution showed an increased proportion of palmitic acid, whereas oleic, linoleic and arachidonic acid decreased, thus resembling more closely the human situation. Overexpression of the human LCAT gene provokes major changes in plasma lipoprotein and apolipoprotein concentrations, resulting in a less atherogenic plasma lipoprotein profile through a reduction in atherogenic and an increase in anti-atherogenic lipoproteins.

Animals↗

PubMed1995

In vitro expression of structural defects in the lecithin-cholesterol acyltransferase gene.

Classic LCAT deficiency (CLD) and fish eye disease (FED) are two clinically distinct syndromes, associated with defects in the lecithin-cholesterol acyltransferase (LCAT) gene resulting in total (CLD) or partial (FED) enzyme deficiency. In order to investigate the underlying molecular mechanisms that lead to different phenotypic expression in CLD and FED, LCAT mutants associated with either CLD (LCAT147, LCAT156, and LCAT228) or FED (LCAT10, LCAT123, LCAT158, LCAT293, LCAT300, and LCAT347) were expressed in vitro in human embryonic kidney 293 cells and characterized with respect to LCAT expression and enzyme activity. Evaluation of mutant LCAT gene transcription by Northern blot analysis demonstrated LCAT mRNA of normal size and concentration. Although all constructs gave rise to similar intracellular LCAT mass, the amount of enzyme present in the media for LCAT147, LCAT156, and LCAT300 was reduced to less than 10% of normal, suggesting that these mutations disrupted LCAT secretion. Western blot analysis of cell culture media containing wild type or mutant LCAT demonstrated the presence of a single normal-sized band of 67 kDa. The ability of the different enzymes to esterify free cholesterol in high density lipoprotein-like proteoliposomes (alpha-LCAT-specific activity) was reduced to less than 5% of normal for CLD mutants LCAT147 and LCAT228 and FED mutants LCAT10, LCAT123, LCAT293, and LCAT347, whereas that of LCAT156, LCAT158, and LCAT300 ranged from 45 to 110% of control. Although most FED mutant LCAT enzymes retained the ability to esterify free cholesterol present in alpha- and beta-lipoproteins of heat-inactivated plasma, esterification was undetectable in all CLD mutants (LCAT147, LCAT156, and LCAT228). In contrast, all mutant enzymes retained the ability to hydrolyze the water soluble, short-chained fatty acid substrate p-nitrophenolbutyrate. In summary, our studies establish the functional significance of nine LCAT gene defects associated with either FED or CLD. Characterization of the expressed LCAT mutants identified multiple, overlapping functional abnormalities that include defects in secretion and/or disruption of enzymic activity. All nine LCAT mutants retained the ability to hydrolyze the water-soluble PNPB substrate, indicating intact hydrolytic function. Based on these studies we propose that mutations in LCAT residues 147, 156, 228 (CLD) and 10, 123, 158, 293, 300, and 347 (FED) do not disrupt the functional domain mediating LCAT phospholipase activity, but alter structural domains involved in lipid binding or transesterification.

Blotting, Northern↗

PubMed1995

Cholesterol efflux from human monocyte-derived macrophages in the presence of LpA-I:A-II.

Previous epidemiological studies have suggested that the LpA-I subfraction of HDL is more protective than the LpA-I:A-II subfraction against the development of cardiovascular disease. A possible basis for a specific anti-atherogenic function of LpA-I emerged from studies of cholesterol efflux from cultured mouse adipocytes. LpA-I efficiently removed excess cholesterol from the mouse adipocytes, while LpA-I:A-II was ineffective. On the other hand, LpA-I:A-II was able to stimulate cholesterol efflux from a number of other cell types including rodent macrophages. Because of previously reported differences in HDL stimulation of cholesterol clearance from macrophages of different origins, we determined whether LpA-I:A-II could induce cholesterol efflux from cultured human monocyte-macrophages. Our findings showed that LpA-I:A-II and HDL3 effectively stimulated cholesterol efflux from human monocyte-macrophages enriched with cholesterol by incubation with AcLDL. LpA-I:A-II also decreased by one-half the amount of cholesterol accumulated when macrophages were incubated with AcLDL and LpA-I:A-II together. Thus, it would appear that the differential anti-atherogenic effects of LpA-I:A-II and LpA-I do not derive from their effects on macrophage cholesterol efflux. Possibly these HDL subfractions differentially affect other biologic processes that modulate the development of cardiovascular disease.

Cardiovascular Diseases↗

PubMed1995

Characterization of high-density apolipoprotein particles A-I and A-I:A-II isolated from humans with cholesteryl ester transfer protein deficiency.

The cholesteryl ester transfer protein (CETP) plays an important role in metabolism of high-density lipoprotein and reverse cholesterol transport in humans. The two major classes of high-density lipoprotein particles are those containing apolipoprotein A-I (LpA-I) and those containing both apoA-I and apoA-II (LpA-I:A-II). We isolated and characterized the apoA-I-containing lipoprotein particles from three subjects with homozygous CETP deficiency (CETP-D) and compared the results with those from normolipidemic control subjects. Plasma concentrations of apoA-I in both LpA-I and LpA-I:A-II were significantly elevated in CETP-D subjects. Both LpA-I and LpA-I:A-II from these subjects were larger and contained more cholesteryl ester per particle than control particles. In CETP-D, subpopulations of LpA-I and LpA-I:A-II with an unusually large size (Stokes diameters 13.8 nm and 12.6 nm, respectively) not detected in normal subjects were isolated. The molar ratio of apoA-I to apoA-II in LpA-I:A-II isolated from CETP-D subjects was higher (mean 2.4) than those of controls (mean 1.4). ApoE was primarily associated with LpA-I:A-II in CETP-D subjects. A subclass of LpA-I with pre-beta migration on agarose electrophoresis was increased in CETP-D subjects. Both LpA-I and LpA-I:A-II from CETP-D subjects bound with higher affinity but less capacity to HepG2 cells compared with control particles, and were internalized to a lesser extent than control particles. These data suggest that the absence of CETP in humans significantly affects the plasma concentration, size, composition, and cellular interaction of both major classes of apoA-I-containing lipoprotein particles.

Adult↗

PubMed1994

Lipoproteins containing apolipoprotein A-IV: composition and relation to cholesterol esterification.

In order to investigate the relationship of lipid and apolipoprotein composition to cholesterol esterification in lipoproteins containing apolipoprotein (apo) A-IV, apo A-containing lipoprotein particles were isolated from fresh human plasma using a system of sequential immunoaffinity chromatography. Plasma was first depleted of apo B- and apo E-containing lipoproteins. Four major subpopulations of apo A-containing lipoprotein particles were separated: Lp A-I, Lp A-I: A-II, Lp A-IV and Lp A-I: A-IV: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained less total lipid, less cholesterol and more triacylglycerol than Lp A-I and Lp A-I: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained more sphingomyelin and less phosphatidylcholine than Lp A-I and Lp A-I: A-II and were richer in (16:0 + 18:0) saturated fatty acids. Among these isolated lipoprotein particles, Lp A-IV contained the highest lecithin: cholesterol acyltransferase (LCAT) activity per micrograms of protein. Cholesterol esterification rates were 2.6 +/- 0.5, 5.3 +/- 0.4 and 0.8 +/- 0.2 mumol of cholesterol per hour per mg of lipoproteins for Lp A-IV, Lp A-I and Lp A-I: A-II, respectively. The apolipoprotein and lipid composition and LCAT activity of Lp A-IV suggest that this lipoprotein may be a source of cholesterol esterification in plasma.

Adult↗

PubMed1994

Distribution of subclasses of HDL containing apoA-I without apoA-II (LpA-I) in normolipidemic men and women.

Women have significantly higher plasma concentrations of high-density lipoprotein (HDL) and apolipoprotein (apo) A-I than men. Human HDL consists of two major species of apoA-I-containing lipoproteins: LpA-I (lipoprotein containing apoA-I but not apoA-II) and LpA-I:A-II (lipoprotein containing both apoA-I and apoA-II). LpA-I is itself heterogeneous and contains several subclasses of different size and composition. We analyzed LpA-I subclasses in 12 male and 12 female healthy normolipidemic adults. LpA-I concentrations were significantly higher in women (72.4 +/- 5.6 mg/dL) than in men (50.2 +/- 2.2 mg/dL) (P < .05). LpA-I was preparatively isolated from fasting plasma by immunoaffinity chromatography. Gel filtration chromatography was then used to isolate LpA-I subclasses based on size. Three major subclasses were eluted: large, medium, and small LpA-I. No differences between men and women in the size or composition of individual LpA-I subclasses were observed. In contrast, the distribution and plasma concentration of LpA-I subclasses were significantly different between men and women. As a fraction of total LpA-I, the large LpA-I was significantly higher (68.0% to 48.4%) and the medium LpA-I was significantly lower (26.4% to 44.9%) in women than in men. The fraction of small LpA-I was not significantly different. Plasma concentrations of large LpA-I in women (49.2 mg/dL) were twice that in men (24.3 mg/dL), whereas plasma concentrations of medium LpA-I (19.1 mg/dL versus 22.5 mg/dL) and small LpA-I (4.0 mg/dL versus 3.0 mg/dL) were similar in women and men.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Markedly accelerated catabolism of apolipoprotein A-II (ApoA-II) and high density lipoproteins containing ApoA-II in classic lecithin: cholesterol acyltransferase deficiency and fish-eye disease.

Classic (complete) lecithin:cholesterol acyltransferase (LCAT) deficiency and Fish-eye disease (partial LCAT deficiency) are genetic syndromes associated with markedly decreased plasma levels of high density lipoprotein (HDL) cholesterol but not with an increased risk of atherosclerotic cardiovascular disease. We investigated the metabolism of the HDL apolipoproteins (apo) apoA-I and apoA-II in a total of five patients with LCAT deficiency, one with classic LCAT deficiency and four with Fish-eye disease. Plasma levels of apoA-II were decreased to a proportionately greater extent (23% of normal) than apoA-I (30% of normal). In addition, plasma concentrations of HDL particles containing both apoA-I and apoA-II (LpA-I:A-II) were much lower (18% of normal) than those of particles containing only apoA-I (LpA-I) (51% of normal). The metabolic basis for the low levels of apoA-II and LpA-I:A-II was investigated in all five patients using both exogenous radiotracer and endogenous stable isotope labeling techniques. The mean plasma residence time of apoA-I was decreased at 2.08 +/- 0.27 d (controls 4.74 +/- 0.65 days); however, the residence time of apoA-II was even shorter at 1.66 +/- 0.24 d (controls 5.25 +/- 0.61 d). In addition, the catabolism of apoA-I in LpA-I:A-II was substantially faster than that of apoA-I in LpA-I. In summary, genetic syndromes of either complete or partial LCAT deficiency result in low levels of HDL through preferential hypercatabolism of apoA-II and HDL particles containing apoA-II. Because LpA-I has been proposed to be more protective than LpA-I:A-II against atherosclerosis, this selective effect on the metabolism of LpA-I:A-II may provide a potential explanation why patients with classic LCAT deficiency and Fish-eye disease are not at increased risk for premature atherosclerosis despite markedly decreased levels of HDL cholesterol and apoA-I.

Adult↗

PubMed1993

Very low high-density lipoproteins without coronary atherosclerosis.

Epidemiological studies have established that concentrations of plasma high-density lipoproteins (HDL) are inversely associated with premature atherosclerosis, but the physiological basis of this relationship remains unknown. We investigated 5 probands with very low plasma HDL. None had clinical or biochemical findings typical of the known genetic disorders with low HDL nor had evidence of premature coronary atherosclerosis by sensitive diagnostic methods. All 5 probands and the son of 1 of them had rapid catabolism of the HDL apolipoproteins A-I and A-II. These results indicate that not all people with low HDL are necessarily at risk of premature coronary heart disease and that further investigation is required before decisions can be made about their management.

Adult↗

PubMed1993

Biochemical characterization of the three major subclasses of lipoprotein A-I preparatively isolated from human plasma.

Apolipoprotein (apo) A-I is the major protein constituent of plasma high-density lipoproteins (HDL). HDL consist of two major classes of apoA-I-containing lipoproteins: LpA-I and LpA-I:A-II. LpA-I includes heterogeneous lipoprotein particles that differ in size and hydrated density. LpA-I was isolated by immunoaffinity chromatography from the fasting plasma of 24 normal human subjects and separated by gel filtration chromatography. Three major subclasses of LpA-I were eluted: large (Lg-LpA-I), medium (Md-LpA-I), and small LpA-I (Sm-LpA-I). By nondenaturing gradient PAGE, Lg-LpA-I, Md-LpA-I, and Sm-LpA-I had mean Strokes diameters of 10.8 +/- 0.5, 8.9 +/- 0.5, and 7.5 +/- 0.3 nm, respectively. The lipid/protein ratios were 1.25 +/- 0.12 for Lg-LpA-I, 0.75 +/- 0.10 for Md-LpA-I, and 0.38 +/- 0.08 for Sm-LpA-I. Lg-LpA-I was relatively lipid and cholesteryl ester rich compared with Md-LpA-I and Sm-LpA-I. Sm-LpA-I contained phospholipids as the major lipid component. ApoA-I was the major apolipoprotein in all LpA-I subfractions, whereas apoE was present only in Lg-LpA-I and apoA-IV was associated with both Md-LpA-I and Sm-LpA-I. All three LpA-I subclasses exhibited predominantly alpha mobility on agarose electrophoresis. Lg-LpA-I migrated as a diffuse band in the fast alpha position, whereas Md-LpA-I and Sm-LpA-I migrated to the slow alpha position.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Tangier disease: isolation and characterization of LpA-I, LpA-II, LpA-I: A-II and LpA-IV particles from plasma.

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 +/- 0.14 and 0.14 +/- 0.04 mumol of cholesterol esterified/h/micrograms of protein, and 7 +/- 2.5 and 1.4 +/- 0.3 mumol of cholesteryl ester transferred/h/micrograms of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2-1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 +/- 0.16 and 0.05 +/- 0.01 mumol of cholesterol esterified/h/micrograms of protein and, 41 +/- 3.7 and 1 +/- 0.4 mumol of cholesteryl ester transferred/h/micrograms of protein). The LpA-II particle in TD represented in fact an LpA-II:A-IV complex (75% mol apoA-II and 22% mol apoA-IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1993

Interaction of reconstituted high density lipoprotein discs containing human apolipoprotein A-I (ApoA-I) variants with murine adipocytes and macrophages. Evidence for reduced cholesterol efflux promotion by apoA-I(Pro165-->Arg).

Interaction of cells with both native and reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I mediates efflux of cellular cholesterol. To characterize structural requirements for this activity in apoA-I, six different genetic apoA-I variants and the corresponding normal allele products were isolated from heterozygous carriers, reconstituted with dimyristoylphosphatidylcholine (DMPC) into discoidal HDL and compared with regard to their ability to release intracellular cholesterol from murine adipocytes and peritoneal macrophages. In previous studies we determined the amino acid changes of these variants: Pro3-->Arg, Pro4-->Arg, Lys107-->0, Lys107-->Met, Pro165-->Arg, and Glu198-->Lys (von Eckardstein, A., Funke, H., Walter, M., Altland, K., Benninghoven, A., and Assmann, G. (1990) J. Biol. Chem. 265, 8610-8617) and demonstrated that all apoA-I variants except apoA-I(Lys107-->0) form discoidal HDL particles with phosphatidylcholine analogues indistinguishable from normal apoA-I (Jonas, A., von Eckardstein, A., Kezdy, K. E., Steinmetz, A., and Assmann, G. (1991) J. Lipid Res. 32, 95-106). In the present study, all apoA-I.DMPC complexes except those containing apoA-I(Pro165-->Arg) promoted cholesterol efflux as effectively as those containing normal apoA-I. Cholesterol efflux from adipocytes obtained after 180 min of incubation with apoA-I(Pro165-->Arg).DMPC complexes was decreased by 30% although this variant was bound to adipocytes with normal affinity. By contrast, apoA-I(Lys107-->Met).DMPC complexes were decreased in their binding affinity compared to normal apoA-I.DMPC complexes but normally promoted cholesterol efflux. Incubation of mouse peritoneal macrophages with apoA-I(Pro165-->Arg).DMPC complexes did also result in a 30% decreased efflux of radiolabeled cholesterol if compared to rHDL with the normal allele product from the same donor. Together the observations suggest that the substitution of proline at residue 165 interferes with the formation of a structural domain in apoA-I that is crucial for cellular cholesterol efflux stimulation. Furthermore, our results suggest that binding of HDL to adipocytes and cholesterol efflux promotion by HDL requires different structural domains.

Adipose Tissue↗

PubMed1993

Characterization of apoA-IV-containing lipoprotein particles isolated from human plasma and interstitial fluid.

Apolipoprotein (apo) A-IV has been proposed to play a role in reverse cholesterol transport. ApoA-IV-containing lipoprotein particles (A-IVLp) were isolated from human plasma and interstitial fluid and characterized by immunoaffinity chromatography. Two major A-IVLp subpopulations, lipoprotein particles containing apoA-IV with apoA-I (LpA-I:A-IV) and lipoprotein particles containing apoA-IV without apoA-I (LpA-IV), were identified. The larger subpopulation of A-IVLp is the LpA-IV that represents 70% (protein mass) of the initial particles. Only 5.8% of apoA-IV was recovered in the retained fraction after affinity chromatography with an anti-apoA-I immunosorbent. ApoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoE, apoH, lecithin: cholesterol acyltransferase (LCAT), cholesteryl ester transfer (CET) protein, proline-rich protein, and a protein of Mr 59,000 were detected in the A-IVLp. These particles contain more than 20% triglycerides (lipid mass). ApoA-IV-containing particles that were isolated from plasma are heterogeneous in size, consisting of two major populations with Stokes' diameters of 10.3 nm and 9.3 nm. Both subpopulations of A-IVLp contain LCAT and CET activities and promote cholesterol efflux from cholesterol-preloaded adipose cells. These data support the hypothesis that A-IVLp particles may be involved in reverse cholesterol transport.

Adipose Tissue↗

PubMed1993

Fish eye syndrome: a molecular defect in the lecithin-cholesterol acyltransferase (LCAT) gene associated with normal alpha-LCAT-specific activity. Implications for classification and prognosis.

We have identified the molecular defect in two siblings presenting with classical clinical and biochemical features of Fish Eye disease (FED), including corneal opacities, HDL cholesterol < 10 mg/dl, normal plasma cholesteryl esters, and elevated triglycerides. In contrast to previously reported patients with FED who are unable to esterify HDL-associated cholesterol, our patients' plasma lecithin-cholesterol acetyltransferase (alpha-LCAT)-specific activities assayed using an HDL-like proteoliposome substrate were 12.7-25.7 nmol/micrograms (19.5 +/- 1.8 in controls). In addition, significant residual cholesterol esterification was present in VLDL/LDL-depleted plasma, confirming the presence of HDL-associated alpha-LCAT activity. DNA sequence analysis of the proband's LCAT gene identified deletion of the triplet coding for leu300, which resulted in the loss of a restriction site for MlnI. Digestion of PCR-amplified DNA using MlnI established that both siblings are homozygous for this defect. Expression of LCAT300-del. in human embryonic kidney-293 cells revealed normal mRNA and intracellular LCAT concentrations. However, reduced amounts of LCAT300-del., which had a normal specific alpha-LCAT activity, were present in the media. In summary, we report the first case of FED associated with a mutant enzyme that has a normal alpha-LCAT-specific activity. The functional significance of this LCAT gene defect has been established in an in vitro expression system, which demonstrates that very small amounts of this functional LCAT mutant enzyme accumulate in the media. Characterization of LCAT300-del. established that selective alpha-LCAT deficiency is not a prerequisite for the development of FED. On the basis of our combined results, we propose that the residual amounts of total plasma LCAT activity and not its distribution on lipoproteins primarily determines the heterogeneity in phenotypic expression observed in familial LCAT deficiency syndromes.

Base Sequence↗