Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Cholesterol metabolism in pigeon aortic smooth muscle cells lacking a functional low density lipoprotein receptor pathway

Cholesterol metabolism was examined in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons that have been shown to lack a functional LDL receptor pathway. In cells incubated in the presence of whole serum or low density lipoprotein (LDL) the rate of cholesterol synthesis from [1-14C]acetate or of HMG-CoA reductase activity was 20-100 times greater than for mammalian cells incubated under the same conditions. Unexpectedly, cholesterol synthesis decreased by nearly 50% after preincubation for 24 hr with lipoprotein-deficient serum (LPDS). This occurred without a change in cellular cholesterol content. Neither the high rate of cholesterol synthesis nor the effect of LPDS could be accounted for by differences in cell turnover or state of growth. Cholesterol added in ethanol was ineffective in altering cellular cholesterol synthesis or esterification even though a near doubling in cellular free cholesterol content occurred. Cholesterol synthesis and esterification were, however, able to be regulated with 25-OH cholesterol and mevalonolactone, as indicated by their ability to suppress cholesterol synthesis and to stimulate cholesterol esterification. In spite of the high rate of endogenous cholesterol synthesis, cellular cholesterol content was maintained at a constant level by the efficient efflux of the newly synthesized cholesterol from the cell. Unlike mammalian cells that require a cholesterol acceptor in the medium for efflux to occur, cholesterol efflux from pigeon cells occurred in the absence of a cholesterol acceptor. This suggests either that pigeon cells utilize a different mechanism for cholesterol efflux or that they produce their own cholesterol acceptor. As a result of a lack of a functional LDL receptor pathway, pigeon smooth muscle cells do not maintain cholesterol homeostasis through the controlled uptake of exogenous LDL cholesterol, as do mammalian cells. Rather, pigeon smooth muscle cells would appear to regulate cholesterol concentrations at the level of either cholesterol synthesis or efflux.     

Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.     

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

Expression of low density lipoprotein receptor gene in human placenta during pregnancy

Mammalian cells require cholesterol as a structural component of plasma membranes. It is also required for placental steroid synthesis. De novo synthesis of cholesterol is limited in human placenta and cholesterol is obtained mainly from plasma low density lipoprotein (LDL). Cholesterol delivery from LDL is mediated by receptor-mediated uptake and the receptor amount is the most important factor for cellular delivery. Thus, the regulation of receptor synthesis is important for placental development and function. Since the regulation of LDL receptor gene expression has not been studied in human placenta, LDL receptor mRNA was measured in placentae of 5-40 weeks of gestation by hybridization of RNA with 32P-labeled cDNA for human LDL receptor. Two mRNA species for LDL receptor were demonstrated by Northern blot analysis. The longer mRNA [5.3 kilobases (kb)] was much more abundant than the shorter mRNA (3.7 kb). The amount of 5.3 kb mRNA was highest early in gestation and decreased during pregnancy. However, the amount of 3.7 kb mRNA did not change appreciably during gestation. Dot blot analysis of 26 placental mRNAs obtained from various stages of gestation revealed a negative correlation between LDL receptor mRNA and gestation (r = -0.76, P less than 0.001). Considering the rapid growth of the trophoblast during gestation, especially in the first and the second trimester, increased expression of the LDL receptor gene and subsequent translation are expected for efficient cholesterol uptake to provide a sufficient substrate for cell growth. Possible mechanisms for the appearance of two mRNA species for LDL receptor are also discussed.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

beta-Hydroxyaspartic acid or beta-hydroxyasparagine in bovine low density lipoprotein receptor and in bovine thrombomodulin

All of the vitamin K-dependent plasma proteins with domains that are homologous to the epidermal growth factor (EGF) precursor have 1 hydroxylated aspartic acid residue in the NH2-terminal EGF-homology region. In addition, protein S has 1 hydroxylated asparagine residue in each of the three COOH-terminal EGF-homology regions. All of these proteins have been found to have the amino acid sequence, CX(D or N)XXXX(F or Y)XCXC (corresponding to residues 20 to 33 in EGF), where the Asp or Asn residue is hydroxylated. This sequence also appears in two of the three EGF-homology regions of the human low density lipoprotein receptor and in two of the six EGF-homology regions of bovine thrombomodulin so far identified, suggesting that they may have the modified amino acid. We have now identified beta-hydroxyaspartic acid in acid hydrolysates of both these proteins.     

Metabolic heterogeneity in the formation of low density lipoprotein from very low density lipoprotein in the rat: evidence for the independent production of a low density lipoprotein subfraction.

The formation of low density lipoprotein (LDL) from very low density lipoprotein (VLDL) was studied after injecting 14C-radiomethylated or 125I-radioiodinated VLDL into rats. VLDL and LDL B apoprotein specific radioactivity time curves were obtained after tetramethylurea extraction of the lipoproteins. In all experiments, the specific activity of LDL B apoprotein did not intercept the VLDL curve at maximal heights, suggesting that not all LDL B apoprotein is derived from VLDL B apoprotein. Further subfractionation of LDL into the Sf 12-20, 5-12, and 0-5 ranges showed that most (65%) LDL B apoprotein was present in the Sf 0-5 fraction and that only a small proportion (6-15%) of this fraction was derived from VLDL. However, the curves obtained for the Sf 12-20 and 5-12 subfractions were consistent with a precursor-product relationship in which all of these fractions were derived entirely from VLDL catabolism. These results contrasted strikingly with similar data obtained for normal humans in which all LDL is derived from VLDL. In the rat, it appears that most of the B apoprotein in the Sf 0-5 range, which contains 65% of the total LDL B apoprotein, enters the plasma independently of VLDL secretion.     

Impaired trafficking of the very low density lipoprotein receptor caused by missense mutations associated with dysequilibrium syndrome

Dysequilibrium syndrome (DES, OMIM 224050) is a genetically heterogeneous condition that combines autosomal recessive non-progressive cerebellar ataxia with mental retardation. The subclass dysequilibrium syndrome type 1 (CAMRQ1) has been attributed to mutations in the VLDLR gene encoding the very low density lipoprotein receptor (VLDLR). This receptor is involved in the Reelin signaling pathway that guides neuronal migration in the cerebral cortex and cerebellum. Three missense mutations (c.1459G > T; p.D487Y, c.1561G > C; p.D521H and c.2117G > T; p.C706F) have been previously identified in VLDLR gene in patients with DES. However, the functional implications of those mutations are not known and therefore we undertook detailed functional analysis to elucidate the cellular mechanisms underlying their pathogenicity. The mutations have been generated by site-directed mutagenesis and then expressed in cultured cell lines. Confocal microscopy and biochemical analysis have been employed to examine the subcellular localization and functional activities of the mutated proteins relative to wild type. Our results indicate that the three missense mutations lead to defective intracellular trafficking and ER retention of the mutant VLDLR protein. This trafficking impairment prevents the mutants from reaching the plasma membrane and binding exogenous Reelin, the initiating event in Reelin signaling. Collectively, our results provide evidence that ER quality control is involved in the functional inactivation and underlying pathogenicity of these DES-associated mutations in the VLDLR.     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

A direct role for the macrophage low density lipoprotein receptor in atherosclerotic lesion formation.

To evaluate the contribution of the macrophage low density lipoprotein receptor (LDLR) to atherosclerotic lesion formation, we performed bone marrow transplantation studies in different mouse strains. First, LDLR(-/-) mice were transplanted with either LDLR(+/+) marrow or LDLR(-/-) marrow and were challenged with an atherogenic Western type diet. The diet caused severe hypercholesterolemia of a similar degree in the two groups, and no differences in the aortic lesion area were detected. Thus, macrophage LDLR expression does not influence foam cell lesion formation in the setting of extreme LDL accumulation. To determine whether macrophage LDLR expression affects foam cell formation under conditions of moderate, non-LDL hyperlipidemia, we transplanted C57BL/6 mice with either LDLR(-/-) marrow (experimental group) or LDLR(+/+) marrow (controls). Cholesterol levels were not significantly different between the two groups at baseline or after 6 weeks on a butterfat diet, but were 40% higher in the experimental mice after 13 weeks, mostly due to accumulation of beta-very low density lipoprotein (beta-VLDL). Despite the increase in cholesterol levels, mice receiving LDLR(-/-) marrow developed 63% smaller lesions than controls, demonstrating that macrophage LDLR affects the rate of foam cell formation when the atherogenic stimulus is beta-VLDL. We conclude that the macrophage LDLR is responsible for a significant portion of lipid accumulation in foam cells under conditions of dietary stress.     

Improving lipoprotein profiles by liver-directed gene transfer of low density lipoprotein receptor gene in hypercholesterolaemia mice

The defect of low density lipoprotein receptor disturbs cholesterol metabolism and causes familial hypercholesterolaemia (FH). In this study, we directly delivered exogenous Ldlr gene into the liver of FH model mice (Ldlr^?/?) by lentiviral gene transfer system. The results showed that the Ldlr gene controlled by hepatocyte-specific human thyroxine-binding globulin (TBG) promoter successfully and exclusively expressed in livers. We found that, although, the content of high density lipoprotein in serum was not significantly affected by the Ldlr gene expression, the serum low density lipoprotein level was reduced by 46%, associated with a 30% and 28% decrease in triglyceride and total cholesterol, respectively, compared to uninjected Ldlr^?/? mice. Moreover, the TBG directed expression of Ldlr significantly decreased the lipid accumulation in liver and reduced plaque burden in aorta (32%). Our results indicated that the hepatocyte-specific expression of Ldlr gene strikingly lowered serum lipid levels and resulted in amelioration of hypercholesterolaemia.     

Modulation of low density lipoprotein receptor activity in squamous carcinoma cells by variation in cell density

Morphological and biochemical studies on low density lipoprotein (LDL) receptor metabolism were performed in squamous carcinoma cells (SCC-15 and SCC-12F2). Modulation of terminal differentiation was achieved by culturing these cells at different cell densities. Studies on these cells cultured at low density (hardly any terminal differentiation) showed the following results: High affinity binding of LDL was excessive; LDL binding to SCC-15 cells was twice as high as that in SCC-12F2 cells and in fibroblasts. The distribution of the LDL binding visualized by LDL receptor antibodies was non-linear. There was no contact inhibition of LDL binding. LDL-gold particles were mainly bound to the plasma membrane outside coated pits. LDL-gold particles were internalized and delivered to dense bodies (= lysosomes). Degradation of LDL took place after a lag period of 10 min. Dissociation of LDL from the plasma membrane was substantial (more than 40% after a 120 min chase period). The same experiments on the cells cultured at high density (terminal differentiation present) showed several differences: A sharp decrease in high affinity LDL binding in both cell types. The internalization of surface bound LDL was defective in most of the squamous carcinoma cells. Dissociation of LDL from the plasma membrane was substantial, and after a chase period of 120 min at 37 degrees C still more than 20% of LDL remained intracellular and was not degraded. We postulate that LDL receptor-mediated endocytosis and degradation take place in squamous carcinoma cells but that during the process of terminal differentiation modulation of LDL-receptor metabolism occurs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peroxisome proliferator-activated receptor beta/delta regulates very low density lipoprotein production and catabolism in mice on a Western diet

The results of recent studies using selective agonists for peroxisome proliferator-activated receptor beta (PPARbeta) suggest that this receptor may have a role in regulating levels of serum lipids in animal models of obesity and insulin resistance. To further examine this possibility, serum lipid profiles of mice lacking a functional PPARbeta receptor were determined. PPARbeta-null mice maintained on either normal chow or a 10-week high fat (HF) diet, a condition that has been shown to induce insulin resistance and obesity in mice, have elevated levels of serum triglycerides primarily associated with very low density lipoprotein (VLDL) with no difference in either total cholesterol or phospholipids. Consistent with this finding, PPARbeta-null mice on a HF-diet were shown to have an increased rate of hepatic VLDL production as well as lowered lipoprotein lipase activity in serum compared with wild-type controls. The latter parallels an increase in the hepatic expression of the genes encoding angiopoietin-like proteins 3 and 4 in PPARbeta-null mice on a HF diet, both proteins of which have recently been shown to inhibit lipoprotein lipase (LPL) activity in vivo. Consistent with elevated VLDL production, a marked increase in plasma VLDL apoB48, -E, -AI, and -AII, as well as a sharp depletion of the hepatic lipid stores was also found in PPARbeta-null mice. In addition, PPARbeta-null mice on a HF diet were shown to have increased adiposity, despite lower total body weight. Together, these results indicate a clear role for PPARbeta in regulating levels of serum triglycerides in mice on a high fat Western diet by modulating both VLDL production and LPL-mediated catabolism of VLDL-triglycerides and also suggest a potential therapeutic role for PPARbeta in the improvement of serum lipids in the setting of metabolic syndrome.     

Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knockout mice

Scavenger receptors, which include various classes, play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins, resulting in the massive accumulation of cholesteryl esters. Because macrophage-derived foam cells are considered to be an important feature in early atherogenesis, we investigated the role of scavenger receptor class A (SR-A) overexpression, especially on macrophages in lipoprotein metabolism and atherosclerosis. Bone marrow from human SR-A (MSR1)-overexpressing mice was transplanted into irradiated low density lipoprotein receptor knockout [LDLR(-/-)] mice. The transplantation resulted in an increase in total serum cholesterol (approximately 15 to 25%), especially in the VLDL fraction, when compared with LDLR(-/-) mice that were transplanted with bone marrow of wild-type littermates. Quantification of atherosclerotic lesions in the mice that were fed a "Western-type" diet for 3 months revealed that there were no differences in mean lesion area between LDLR(-/-) mice transplanted with MSR1 overexpressing and wild-type littermate bone marrow, despite increased scavenger receptor activity in vitro. The presence or absence of the LDLR in the transplanted bone marrow did not influence these results.In conclusion, introduction of MSR1-overexpressing bone marrow in LDLR(-/-) mice via bone marrow transplantation resulted in a slight increase in lipoprotein levels, but had no effect on the atherosclerotic lesion area, despite increased scavenger receptor activity in vitro.     

Analysis of a recycling-impaired mutant of low density lipoprotein receptor in familial hypercholesterolemia

A mutant low density lipoprotein (LDL) receptor with abnormal ligand binding and recycling abilities was found in a patient with familial hypercholesterolemia. The molecular weights of the precursor and the mature form of the receptor were 72,000 and 115,000, respectively, which were about 45,000 smaller than those of the normal receptor. The mutant receptor was concluded to be present on the cell surface because the mature form was susceptible to Pronase digestion, and specific monoclonal antibody against the LDL receptor (IgG-C7) could bind to the cell surface. This mutant receptor could not bind LDL, but could bind other ligands for the LDL receptor, beta-migrating very low density lipoprotein, and the apolipoprotein E-lipid complex. After the receptor bound to the ligand, it disappeared from the cell surface of the mutant cells faster than that of normal cells, showing that, in the mutant cells, the receptor was not efficiently recycled back to the cell surface. Southern blotting of the genomic DNA from the patient showed a large deletion of about 12 kilobases around the epidermal growth factor precursor homology domain. For further characterization of the mutant, we cloned a 9.4-kilobase EcoRI/XbaI fragment, which was expected to contain the deletion joint. Mapping and sequencing analyses of the receptor gene showed that exons 7-14 were deleted. The nucleotide sequence suggested that this mutation may have occurred by recombination between repetitive Alu sequences in introns 6 and 14 of the receptor gene. The recombination brought about a complete deletion of the gene coding the epidermal growth factor precursor homology domain. The characteristics of the receptor protein produced by this mutation were similar to those of an artificial mutation constructed by Davis et al. (Davis, C. G., Goldstein, J. L., Südhof, T. C., Anderson, R. G. W., Russell, D. W., and Brown, M. S. (1987) Nature 326, 760-765) in which the whole gene coding this domain was deleted. The clinical phenotype of the patient having this mutation was similar to that of so-called "receptor-defective" type familial hypercholesterolemia, in which cells show detectable, but markedly reduced activity of the LDL receptor.     

Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor

Studies of low density lipoprotein (LDL) metabolism in nonhuman model systems have indicated that the mammalian liver has dual mechanisms for the uptake and regulation of the concentration of plasma LDL. Heretofore, direct evaluation of lipoprotein uptake mechanisms in human hepatocytes has not been possible. In order to compare hepatocyte LDL uptake with fibroblast LDL metabolism, human hepatocytes were isolated and cultured from small biopsy specimens obtained from normolipidemic and homozygous familial hypercholesterolemic patients. Cells cultured in serum-free culture medium retained the morphological and biochemical characteristics of hepatocytes for at least 7 days. The uptake and degradation of LDL by hepatocytes was compared to that of the cultured human fibroblasts. Like fibroblasts, hepatocytes bound, internalized, and degraded LDL. In both cell types, uptake approached saturation at a concentration of 50 micrograms of LDL protein/ml. Competition for LDL binding by LDL, high density lipoprotein, and modified LD revealed that the hepatocyte binding was specific for LDL. Cellular cholesterol loading by incubation in LDL-enriched culture medium resulted in diminished LDL uptake in both cell types. Chemical modification of LDL by acetoacetylation, acetylation, and reductive methylation abolished LDL uptake and degradation by fibroblasts. However, hepatocytes bound and degraded the modified LDL at 30-50% the level of native LDL. Homozygous familial hypercholesterolemic hepatocytes were devoid of the LDL receptor pathway but metabolized native LDL to the extent observed with modified LDL uptake by normal hepatocytes. In contrast to the classic LDL receptor pathway, the second or alternate pathway does not lead to regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity. These findings indicate the presence of two separate pathways of LDL uptake in human hepatocytes which have different effects on hepatocytic cholesterol metabolism.     

Hepatic low density lipoprotein receptor binding and lipid profile in Mastomys natalensis during Plasmodium berghei infection

Plasmodium berghei infection to Mastomys natalensis showed hyper beta-lipoproteinemia. The increase in serum cholesterol is associated with decreased uptake of low density lipoprotein (LDL) by the liver through receptor mediated endocytosis. The membranes prepared from infected M. natalensis exhibit up to 50% decline in high affinity binding sites for human 125I-LDL. Significant increases in serum lipids, cholesterol, triglyceride and lipid peroxide (LPO) contents of liver membrane were observed. Effects of lipid constituents and LPO content of liver membrane in relation to LDL catabolism and other possible mechanisms have been explained.     

Cholesterol delivered to macrophages by oxidized low density lipoprotein is sequestered in lysosomes and fails to efflux normally

Oxidized low density lipoprotein (LDL) has been found to exhibit numerous potentially atherogenic properties, including transformation of macrophages to foam cells. It is believed that high density lipoprotein (HDL) protects against atherosclerosis by removing excess cholesterol from cells of the artery wall, thereby retarding lipid accumulation by macrophages. In the present study, the relative rates of HDL-mediated cholesterol efflux were measured in murine resident peritoneal macrophages that had been loaded with acetylated LDL or oxidized LDL. Total cholesterol content of macrophages incubated for 24 h with either oxidized LDL or acetylated LDL was increased by 3-fold. However, there was no release of cholesterol to HDL from cells loaded with oxidized LDL under conditions in which cells loaded with acetylated LDL released about one-third of their total cholesterol to HDL. Even mild degrees of oxidation were associated with impairment of cholesterol efflux. Macrophages incubated with vortex-aggregated LDL also displayed impaired cholesterol efflux, but aggregation could not account for the entire effect of oxidized LDL. Resistance of apolipoprotein B (apoB) in oxidized LDL to lysosomal hydrolases and inactivation of hydrolases by aldehydes in oxidized LDL were also implicated. The subcellular distribution of cholesterol in oxidized LDL-loaded cells and acetylated LDL-loaded cells was investigated by density gradient fractionation, and this indicated that cholesterol derived from oxidized LDL accumulates within lysosomes. Thus impairment of cholesterol efflux in oxidized LDL-loaded macrophages appears to be due to lysosomal accumulation of oxidized LDL rather than to impaired transport of cholesterol from a cytosolic compartment to the plasma membrane.     

Identification of a splice-site mutation in the low density lipoprotein receptor gene by denaturing gradient gel electrophoresis

We have applied the denaturing gradient gel electrophoresis (DGGE) technique to detect sequence variations in exon 9 of the low density lipoprotein receptor (LDLR) gene in individuals with heterozygous familial hypercholesterolemia (FH). A fragment containing exon 9 and 25 base pairs (bp) of the intron boundary sequence at either side was amplified. To this fragment a 40-bp GC-clamp was attached by the polymerase chain reaction (PCR). We have analyzed a total of 165 DNA samples of FH patients and have detected a mutation in three cases. Two patients were found to have the previously described South African G to A transition in codon 408. In a third patient, we observed a different banding pattern of the DNA fragments on DGGE indicating a different mutation. The mutant homoduplex band of this sample was purified from the gel, cloned in an AT-vector and sequenced. Sequence analysis demonstrated a G to A transition of the consensus G-nucleotide at the intron 9 splice donor site. Cosegregation between this mutation and elevated plasma cholesterol levels was observed in family members of this FH patient. This mutation probably prevents normal splicing of the mRNA and represents the first identified splice-site mutation in the LDLR gene. We conclude that the use of DGGE of GC-clamped PCR-amplified exon sequences offers a general strategy for the detection of disease-producing mutations in the LDLR gene.