Metabolism by cells in culture of low-density lipoproteins of abnormal composition from non-human primates with diet-induced hypercholesterolemia

Diet-induced hypercholesterolemia in non-human primates results in the production of a low-density lipoprotein (LDL) of abnormal size and composition. This LDL from hypercholesterolemic monkeys has been shown to be more atherogenic than the same amount of LDL from normocholesterolemic animals. Previous studies have demonstrated that hypercholesterolemic LDL is approximately twice as effective as normal LDL in stimulating cholesterol accumulation and esterification in arterial smooth muscle cells in culture. The purpose of the present study was determine whether this effect was secondary to differences in metabolism of the normal and hypercholesterolemic LDL. for this, the metabolism of 125I-labeled normal and hypercholesterolemic LDL from rhesus and cynomolgus monkeys was compared in several lines of skin fibroblasts and smooth muscle cells. Both normal and hypercholesterolemic LDL bound with high affinity to the same cell surface receptor. However, the affinity for binding of hypercholesterolemic LDL was about twice that of normal LDL (apparent dissociation constant for binding, Kd, was 2.63 micrograms protein/ml and 4.35 micrograms protein/ml, respectively). Conversely, only about 50% as many particles of hypercholesterolemic were able to bind to the receptor, compared with normal LDL. Those cells with the greatest capacity to metabolize LD generally accumulated the most cholesterol with either hypercholesterolemic or normal LDL. In all cell lines, nearly twice as much cholesterol accumulated in cells incubated with hypercholesterolemic LDL compared with normal LDL, and this differential could not be explained by differences in metabolism of the two lipoproteins, suggesting that some cholesterol entered the cells independent of the uptake of the intact LDL molecule. LDL receptors appear necessary for this to occur, since no difference in cholesterol accumulation was observed in cells genetically deficient in LDL receptors.     

Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.

Apolipoprotein B transports cholesterol in plasma as low density lipoprotein (LDL) and targets its delivery to cells by binding to a specific plasma membrane receptor. The cellular consequences of apoB binding to its receptor were investigated to determine whether it suppresses cholesterol biosynthesis and reduces the number of cellular receptors for the apoprotein. Upon preincubation of fibroblasts with lipoprotein-deficient medium alone or supplemented with either LDL or apoB complexed to BSA (apoB-BSA), LDL suppressed cholesterol biosynthesis, but apoB enhanced it. Similarly, fibroblasts preincubated in medium supplemented with LDL bound decreased amounts of either (125)I-labeled LDL or (125)I-labeled apoB-BSA to their receptors, while preincubation with apoB-BSA increased the binding relative to the controls. These latter results occurred in association with a decrease in cellular cholesterol content, indicating that apoB in the medium bound cholesterol and removed it from the cells, thus stimulating both cholesterol synthesis and cellular binding of apoB. Accordingly, fibroblast cholesterol synthesis and the number of functional LDL receptors are not suppressed by the binding of the apoprotein to the receptor, and the known role of apoB remains that of transporting cholesterol in plasma and delivering it to the cell. A possible physiologic role for apoB in depleting cells of cholesterol is presently unknown since apoB is not known to exist free in plasma; however, these findings demonstrate such a functional capability for this apoprotein.-Shireman, R. B., and W. R. Fisher. Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.     

The molecular mechanism for the genetic disorder familial defective apolipoprotein B100

Familial defective apolipoprotein B100 (FDB) is a genetic disorder in which low density lipoproteins (LDL) bind defectively to the LDL receptor, resulting in hypercholesterolemia and premature atherosclerosis. FDB is caused by a mutation (R3500Q) that changes the conformation of apolipoprotein (apo) B100 near the receptor-binding site. We previously showed that arginine, not simply a positive charge, at residue 3500 is essential for normal receptor binding and that the carboxyl terminus of apoB100 is necessary for mutations affecting arginine 3500 to disrupt LDL receptor binding. Thus, normal receptor binding involves an interaction between arginine 3500 and tryptophan 4369 in the carboxyl tail of apoB100. W4369Y LDL and R3500Q LDL isolated from transgenic mice had identically defective LDL binding and a higher affinity for the monoclonal antibody MB47, which has an epitope flanking residue 3500. We conclude that arginine 3500 interacts with tryptophan 4369 and facilitates the conformation of apoB100 required for normal receptor binding of LDL. From our findings, we developed a model that explains how the carboxyl terminus of apoB100 interacts with the backbone of apoB100 that enwraps the LDL particle. Our model also explains how all known ligand-defective mutations in apoB100, including a newly discovered R3480W mutation in apoB100, cause defective receptor binding.     

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatocytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptor-defective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis.     

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.     

Genetically determined hypercholesterolemia in a rhesus monkey family due to a deficiency of the LDL receptor

A family of rhesus monkeys comprising a sire, a dam, and four male offspring were fed a cholesterol-free Purina Chow diet for several months. The sire, 431-J, and two of the offspring, B-8204 and B-8806, had persistent plasma cholesterol levels in the range of 100-130 mg/dl, whereas the dam, 766-I, and the two other offspring, B-1000 and B-7643, exhibited a marked hypercholesterolemia in the 250-300 mg/dl range associated with an elevation of plasma LDL and apoB. When fed for 12 weeks a diet containing 12.5% lard and 0.25% cholesterol, sire, dam, B-1000 and B-7643 exhibited a marked hypercholesterolemia (500-800 mg/dl range), whereas B-8204 and B-8806 developed only a modest hypercholesterolemia (200-250 mg/dl). All animals were Lp[a]+. Skin fibroblasts from each animal and from control cells were grown in 10% fetal calf serum, transferred to 10% lipoprotein-deficient serum for 48 hr, and then incubated at 4 degrees C or 37 degrees C with 125I-labeled Lp[a]-free LDL. The fibroblasts from dam and offspring B-1000 and B-7643 bound and internalized 125I-labeled LDL less efficiently than control cells. Mathematical analyses of the 4 degrees C binding data indicated that there were no significant differences in LDL binding affinity between test and control cells suggesting that cells from the animals with a spontaneous hypercholesterolemia had a decreased number of LDL receptors. This conclusion was supported by the results of ligand and immunoblot analyses carried out on cell lysates separated by gradient gel electrophoresis. We conclude that a genetically determined LDL receptor deficiency was responsible, in part, for the spontaneous hypercholesterolemia observed in three out of the six family members and that this deficiency accounted for the hyperresponsiveness to a dietary fat and cholesterol challenge by the dam and the two offspring, B-1000 and B-7643. The hyperresponsiveness noted in the sire that had no evidence for LDL-receptor deficiency illustrates that factors other than the LDL receptor were responsible for the hypercholesterolemia attending the fat challenge.     

Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts.

An ultracentrifugation assay has been developed to measure low density lipoprotein (LDL) receptor activity in membranes prepared from cultured human fibroblasts. The binding site for 125I-labeled LDL in isolated membranes reflected the properties of the LDL receptor previously demonstrated in intact fibroblasts. It exhibited high affinity (Kd approximately 4 microgram of LDL protein/ml), specificity (LDL approximately 400-fold more effective than high density lipoprotein in competing with 125I-LDL for the binding site), dependence on calcium, and susceptibility to destruction by pronase. The number of LDL receptors detected in the in vitro membrane binding assay was similar to the number detected in intact cells. The number of receptors was reduced in membranes from fibroblasts that were grown in the presence of 25-hydroxycholesterol plus cholesterol and in fibroblast membranes from a subject with homozygous familial hypercholesterolemia, two situations in which the number of LDL receptors in intact fibroblasts is known to be reduced. The availability of a membrane binding assay that faithfully reflects the properties of the physiologic LDL receptor of intact cells should permit the characterization of this receptor in organs from intact humans and animals.     

A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia

Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone.     

Hypercholesterolemia and atherosclerosis in low density lipoprotein receptor mutant rats

To establish low density lipoprotein receptor (LDLR) mutant rats as a hypercholesterolemia and atherosclerosis model, we screened the rat LDLR gene for mutations using an N-ethyl-N-nitrosourea mutagenesis archive of rat gene data, and identified five mutations in its introns and one missense mutation (478T>A) in exon 4. The C160S mutation was located in the ligand binding domain of LDLR and was revealed to be equivalent to mutations (C160Y/G) identified in human familial hypercholesterolemia (FH) patients. The wild type, heterozygous, and homozygous mutant rats were fed a normal chow diet or a high fat high cholesterol (HFHC) diet from the age of 10 weeks for 16 weeks. The LDLR homozygous mutants fed the normal chow diet showed higher levels of plasma total cholesterol and LDL cholesterol than the wild type rats. When fed the HFHC diet, the homozygous mutant rats exhibited severe hyperlipidemia and significant lipid deposition from the aortic arch to the abdominal aorta as well as in the aortic valves. Furthermore, the female homozygous mutants also developed xanthomatosis in their paws. In conclusion, we suggest that LDLR mutant rats are a useful novel animal model of hypercholesterolemia and atherosclerosis.     

In vitro regulation of cholesterol metabolism by low density lipoproteins in skin fibroblasts from hypo-and hyperresponding squirrel monkeys.

When squirrel monkeys (Saimiri sciureus) are fed diets containing cholesterol, some individuals (hyperresponders) become hypercholesterolemic, while others (hyporesponders) are able to maintain nearly normal plasma cholesterol concentrations. Skin fibroblasts were grown from three hyperresponder and threehyporesponder squirrel monkeys, previously characterized on the basis of their plasma cholesterol response to two cholesterol-containing diets and the pheno-type of their parents. The rates of cholesterol synthesis and esterification were determined in the cultured fibroblasts incubated with low density lipoproteins isolated from normocholesterolemic squirrel monkeys or hypercholesterolemic rhesus monkeys. Both lipoprotein preparations influenced the metabolic parameters measured in a similar manner in cells from both hypo- and hyperresponder animals. Exposure of skin fibroblasts to low density lipoproteins resultd in a stimulation of cholesterol esterification and a suppression of cholesterol synthesis in cells from both hypo- and hyperresponder animals. When incubated with increasing concentrations of low density lipoprotein cholesterol, up to 50 microgram/ml, fibroblasts from both hypo-and hyperresponding animals responded with a similar maximum percentage suppression of sterol synthesis. Thus, hyperresponsiveness to dietary cholesterol in squirrel monkeys, although a heritable characteristic, is not associated with an inability of low density lipoprotein to suppress cholesterol synthesis or stimulate cholesterol esterification as occurs in familial hypercholesterolemia in man.     

Low density lipoprotein heterogeneity in spontaneously hypercholesterolemic pigs

We previously described a strain of spontaneously hypercholesterolemic pigs carrying an apo-B allele termed Lpb ⁵. Lpb ⁵ pigs are heterogeneous with respect to the severity of their hypercholesterolemia. We have termed Lpb ⁵ pigs with severe hypercholesterolemia Lpb 5.1 pigs, and those with moderate hypercholesterolemia Lpb 5.2, Lpb 5.1 animals have a dramatic increase in buoyant LDL relative to dense LDL, with a buoyant-to-dense LDL ratio of 2.2. In contrast, Lpb 5.2 and control pigs have buoyant-to-dense LDL ratios of 0.7 and 0.5 respectively. This ratio appears to be a stable characteristic of the Lpb 5.1 phenotype because sexually mature boars have a dramatic decrease in total plasma cholesterol concentration with no decrease in their ratio of buoyant-to-dense LDL. We have previously demonstrated a fourteen-fold overproduction of buoyant LDL in the Lpb 5.1 pigs, with very little conversion of dense LDL to buoyant LDL. In the current work, very low density lipoprotein (VLDL) turnover experiments were conducted to determine whether VLDL conversion to buoyant LDL was increased in the Lpb 5.1 pigs. VLDL conversion to buoyant LDL could not account for the increased production of buoyant LDL in Lpb 5.1 pigs. Thus, we cannot account for the increased production of buoyant LDL in the Lpb 5.1 pigs from any measurable plasma lipoprotein source. We have therefore termed this production of buoyant LDL in the Lpb 5.1 pigs direct buoyant LDL production.     

Detection and quantitation of low density lipoprotein (LDL) receptors in human liver by ligand blotting, immunoblotting, and radioimmunoassay. LDL receptor protein content is correlated with plasma LDL cholesterol concentration

Low density lipoprotein (LDL) receptor activity has been detected and identified in human liver samples by ligand blotting with biotinylated lipoproteins and by immunoblotting with a monoclonal antibody raised against the bovine adrenal LDL receptor. The molecular weight of the human liver LDL receptor, approximately 132,000 on nonreduced polyacrylamide gels, is identical to that of LDL receptors detected in normal human skin fibroblasts by the same methods. LDL receptor-dependent binding activity in human liver samples has been semi-quantitated by integrating the areas under the peaks after scanning photographs of ligand blots, and receptor protein determined by radioimmunoassay with purified bovine adrenal LDL receptor protein as the standard. There was a highly significant correlation between the values obtained by each method for seven different liver samples (r = 0.948). The LDL receptor protein content of liver membranes from 10 subjects as determined by radioimmunoassay was inversely related to the plasma LDL cholesterol concentration (r = 0.663, p = 0.05) but not to other plasma lipid values, including total plasma cholesterol, high density lipoprotein cholesterol, or plasma triglyceride concentrations.     

Defective receptor binding of low density lipoprotein from pigs possessing mutant apolipoprotein B alleles

We previously identified a defect in the in vivo catabolism of low density lipoprotein (LDL) from hypercholesterolemic pigs carrying a mutant apolipoprotein B allele. In the present studies, we examined the in vitro metabolism of mutant LDL in cultured pig fibroblasts. A 3-fold higher concentration of mutant LDL (compared to control) was needed to displace 50% of control 125I-LDL binding. Mutant LDL had a 6-fold higher dissociation constant than control LDL. Scatchard plots of the binding data were concave upward, suggesting multiple classes of binding sites or negative cooperativity. The mutant LDL degradation rate was reduced by 40%; this decrease could be attributed to a dense LDL subspecies. Mutant and control buoyant LDL subspecies were degraded more slowly than the corresponding dense LDL subspecies. Together, these studies show that diminished LDL receptor binding can result from mutations in apolipoprotein B and from changes in the lipid composition of LDL particles.     

Molecular modeling of D151Y and M391T mutations in the LDL receptor

The low-density lipoprotein receptor (LDLR) is a key regulator of cholesterol homeostasis, and defects in the function of LDLR result in familial hypercholesterolemia (FH). In the present study, we performed structural analyses of two novel LDLR mutations, D151Y and M391T. Both mutations occurred in conserved residues of LDLR. The D151Y mutation, in the ligand binding domain, caused an elimination of a hydrogen bond in the calcium binding site, higher solvent accessibility and a loss of negative charge in the Y151 residue. On the other hand, the M391T mutation, in the β-propeller of the epidermal growth factor (EGF) precursor homology domain, caused an additional hydrogen bond to form, higher solvent accessibility and a distortion of the β-strand. These data suggest that the irregular structures of the mutated LDLRs are likely to cause the functional defect that contributes to the pathology of FH.     

Characterisation of the LDL receptor in Epstein-Barr virus transformed lymphocytes

Non-dividing human lymphocytes were transformed upon infection with the Epstein-Barr virus (EBV) into lymphoblasts which are capable of continuous growth in culture. We studied the properties of the LDL receptor in EBV-transformed human lymphocytes (EBV-L) by binding experiments and by ligand blotting. EBV-L show a high affinity binding of LDL in the same order of magnitude as found with fibroblasts; EBV-L obtained from a homozygous familial hypercholesterolemic (FH) patient fail to express LDL receptor activity. Similar to that of fibroblasts, the LDL receptor activity in EBV-L is Ca2(+)-dependent and is down-regulated by the presence of an exogenous source of cholesterol in the medium. The LDL receptor protein of EBV-L has an apparent molecular weight of 130,000. Since our results show that EBV-L display a LDL receptor protein similar to the LDL receptor present in fibroblasts, we conclude that in comparison with other cell types the EBV-L offer a suitable model system to investigate LDL receptor protein abnormalities in FH patients.     

Studies on the expression of genes encoding apolipoproteins B100 and B48 and the low density lipoprotein receptor in nonhuman primates. Comparison of dietary fat and cholesterol

African green monkeys were fed diets containing low and moderate cholesterol concentrations with either polyunsaturated or unsaturated fat as 40% of calories. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apoB concentrations generally were higher in animals fed (a) the higher dietary cholesterol concentration and (b) saturated fat. At necropsy, liver and intestine were removed, and measurement of mRNAs for LDL receptors (liver) and for apolipoprotein B (liver and intestine) was done. Monkey small intestine mucosa made exclusively apoB48 while the liver made only apoB100, although apoB mRNA in both tissues was the same size (14 kilobases). No dietary cholesterol or fat effects were found for apoB mRNA abundance in the liver, while the animals fed the higher dietary cholesterol level had 50% lower levels of hepatic LDL receptor mRNA. In a separate group of animals, livers were perfused and the rate of apoB secretion was measured. No dietary fat effect on apoB secretion rate was found, and no relationship between plasma LDL cholesterol concentration and the rate of hepatic apoB production existed. These findings support the idea that the dietary factors that increase LDL concentrations act by reducing clearance of apoB-containing particles rather than by increasing production of these lipoproteins. Hepatic LDL receptor mRNA was similar in abundance in polyunsaturated fat and saturated fat-fed animals, suggesting that the difference in plasma cholesterol concentration between these groups is not mediated via effects on LDL receptor mRNA abundance. The level of intestinal apoB mRNA was about 30% higher in animals fed the moderate dietary cholesterol concentration. Earlier studies have shown that more cholesterol is transported in chylomicrons from the intestine when dietary cholesterol levels are higher, and the increased intestinal apoB mRNA abundance may reflect increased intestinal cholesterol transport and chylomicron apoB48 production.     

Differential effects of saturated fatty acids on low density lipoprotein metabolism in the guinea pig.

Studies have shown that dietary fat saturation affects guinea pig plasma low density lipoprotein (LDL) levels by altering both LDL receptor-mediated catabolism and flux rates of LDL (Fernandez et al. 1992. J. Lipid Res. 33: 97-109). The present studies investigated whether saturated fatty acids of varying chain lengths have differential effects on LDL metabolism. Guinea pigs were fed 15% (w/w, 35% calories) fat diets containing either palm kernel oil (PK), 52% lauric acid/18% myristic acid; palm oil (PO), 43% palmitic acid/4% stearic acid; or beef tallow (BT), 23% palmitic acid/14% stearic acid. Plasma LDL cholesterol levels were significantly higher for animals fed the PK diet (P < 0.001) with values of 83 +/- 19 (n = 12), 53 +/- 8 (n = 12) and 44 +/- 16 (n = 10) mg/dl for PK, PO, and BT diets, respectively. The relative percentage composition of LDL was modified by fat type; however, LDL diameters and peak densities were not different between diets, indicating no effect of saturated fatty acid composition on LDL size. ApoB/E receptor-mediated LDL fractional catabolic rates (FCR) were significantly lower in animals fed the PK diet (P < 0.01) and LDL apoB flux rates were reduced (P < 0.01) in animals fed the BT diet. A correlation was found between plasma LDL levels and receptor-mediated LDL catabolism (r = -0.66, P < 0.01). A higher apoB/E receptor number (Bmax), determined by in vitro LDL binding to guinea pig hepatic membranes, was observed for animals fed BT versus PK or PO diets and Bmax values were significantly correlated with plasma LDL levels (r = -0.776, P < 0.001). These results indicate that saturated fatty acids of varying chain length have differential effects on hepatic apoB/E receptor expression and on LDL apoB flux rates which in part account for differences in plasma LDL cholesterol levels of guinea pigs fed these saturated fats.     

The beta very low density lipoprotein present in hepatic lipase deficiency competitively inhibits low density lipoprotein binding to fibroblasts and stimulates fibroblast acyl-CoA:cholesterol acyltransferase

Beta very low density lipoprotein (VLDL) was isolated from a patient with hepatic lipase deficiency. The particles were found to contain apolipoprotein B-100 (apoB) and apolipoprotein E (apoE) and were rich in cholesterol and cholesteryl ester relative to VLDL with pre beta electrophoretic mobility. These particles were active in displacing human low density lipoprotein (LDL) from the fibroblast apoB,E receptor and produced a marked stimulation of acyl-CoA:cholesterol acyltransferase. Treatment of intact beta-VLDL with trypsin abolished its ability to displace LDL from fibroblasts. Incubation of trypsin treated beta-VLDL with fibroblasts resulted in a significant stimulation of acyl-CoA:cholesterol acyltransferase activity. beta-VLDL isolated from a patient with Type III hyperlipoproteinemia and an apoE2/E2 phenotype had a higher cholesteryl ester/triglyceride ratio than the beta-VLDL of hepatic lipase deficiency and contained apoB48. It displaced LDL from fibroblasts to a small but significant extent. The Type III beta-VLDL stimulated acyl-CoA:cholesterol acyltransferase to a level similar to that of trypsin-treated beta-VLDL isolated from the hepatic lipase-deficient patient. These results demonstrate that the cholesterol-rich beta-VLDL particles present in patients with hepatic lipase deficiency are capable of interacting with fibroblasts via the apoB,E receptor and that this interaction is completely due to trypsin-sensitive components of the beta-VLDL. These particles were very effective in stimulating fibroblast acyl-CoA:cholesterol acyltransferase. This stimulation was due to both trypsin-sensitive and trypsin-insensitive components.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Thyroid hormone regulation and cholesterol metabolism are connected through Sterol Regulatory Element-Binding Protein-2 (SREBP-2)

High affinity uptake of serum-derived low density lipoprotein (LDL) cholesterol is accomplished through the LDL receptor in the liver. In mammals, thyroid hormone depletion leads to decreased LDL receptor expression and elevated serum cholesterol. The clinical association in humans has been known since the 1920s; however, a molecular explanation has been lacking. LDL receptor levels are subject to negative feedback regulation by cellular cholesterol through sterol regulatory element-binding protein-2 (SREBP-2). Here we demonstrate that the SREBP-2 gene is regulated by thyroid hormone and that increased SREBP-2 nuclear protein levels in hypothyroid animals results in thyroid hormone-independent activation of LDL receptor gene expression and reversal of the associated hypercholesterolemia. This occurs without effects on other thyroid hormone-regulated genes. Thus, we propose that the decreased LDL receptor and increased serum cholesterol associated with hypothyroidism are secondary to the thyroid hormone effects on SREBP-2. These results suggest that hypercholesterolemia associated with hypothyroidism can be reversed by agents that directly increase SREBP-2. Additionally, these results indicate that mutations or drugs that lower nuclear SREBP-2 would cause hypercholesterolemia.