Absence Of Low-density Lipoprotein Research Articles

Dissociation of LPL and LDL: effects of lipoproteins and anti-apoB antibodies.

We have shown previously that the activity of lipoprotein lipase (LPL), the major enzyme responsible for hydrolysis of triglyceride contained in circulating lipoproteins, is associated with lipoproteins in postheparin plasma. In other studies, microtiter plate assays showed that LPL interaction with low density lipoprotein (LDL) and very low density lipoprotein (VLDL) was decreased by antibodies to apolipoprotein (apo)B. To test whether antibodies to apoB affected LPL-LDL association in solution, two types of assays were performed, gel filtration and coprecipitation. First we showed that LPL activity and immunoreactive mass co-eluted during gel filtration of normal postheparin plasma, approximately with the peak of low density lipoproteins. Then LPL was used for gel filtration studies in the presence and absence of LDL and anti-apoB monoclonal antibodies. LPL association with LDL was diminished by antibodies to the amino-terminal region of apoB; antibodies to the carboxyl-terminal LDL receptor binding region of apoB were less effective. LDL binding to LPL containing heparin-agarose was also disrupted by the amino-terminal antibodies to apoB. To determine the LPL-lipoprotein association in situations in which the distribution of plasma lipoproteins was altered, we studied plasma from two types of subjects with dyslipidemias. The addition of 125I-labeled LPL to type 1 postheparin plasma produced two peaks of radioactivity, one peak eluted in the void volume of the column (with the chylomicrons) and a second peak eluted just prior to the normal elution of low density lipoproteins. In postheparin plasma from an abetalipoproteinemic subject, LPL eluted with HDL. We conclude that LPL associates primarily with apoB-containing lipoproteins. The reason for this appears to be that LPL interacts with the apoB.

Progesterone stimulation of HMG-CoA reductase activity in cultured cells.

In a previous study we showed that progesterone (PG) stimulated HMG-CoA reductase (HMGR) activity in rat intestinal epithelial cells (IEC-6) incubated in the presence or absence of low-density lipoprotein (LDL) [1,2]. In the present study we examined further the mechanism of this stimulation. We observed that the stimulation of HMGR activity by PG was completely prevented by cycloheximide. Turnover studies utilizing immunoprecipitation of HMGR-labeled with [35S]methionine revealed that PG increased reductase activity by inhibiting HMGR degradation without affecting the synthesis of HMGR. The stimulation of HMGR activity by progesterone could be accounted for by a continuous synthesis of HMGR while its degradation was retarded. In the presence of LDL, the activity of HMGR in IEC-6 cells was effectively inhibited, however PG was able to stimulate HMGR in the presence of LDL. This effect was not due to an interference of normal cellular metabolism of LDL, since PG had no effect on the cellular uptake and lysosomal degradation of 125I-LDL. PG did not affect of the rate of lysosomal hydrolysis of [3H]cholesteryl linoleate-LDL. The free [3H]cholesterol derived from [3H]cholesteryl linoleate-LDL moved to the cell membrane and effluxed to HDL3 in the medium at the same rate in the presence or absence of PG. Although PG did not affect LDL metabolism, pre-treatment of cells with LDL delayed the onset of HMGR stimulation by PG. In IEC-6 cells deprived of LDL for 24 h, the HMGR activity was stimulated immediately following PG addition. In cells pre-treated with LDL for 24 h, the stimulation was delayed by 4 h. Treatment of cells with 25-hydroxycholesterol completely prevented PG stimulation of HMGR activity. We propose that the stimulation of HMGR activity in the presence or absence of LDL is related to the ability of PG to attenuate the formation and/or action of intracellular HMGR repressor molecules which accelerate the degradation of HMGR.

Regulation of intracellular cholesterol metabolism.

Animal cells synthesize cholesterol from acetyl CoA through a series of more than 20 enzymatic reactions. In addition, cells obtain cholesterol from plasma in the form of low-density lipoprotein (LDL), which is internalized via the LDL receptor and hydrolyzed to free cholesterol in lysosomes. Each cell must balance these internal and external sources while avoiding sterol shortage or overaccumulation. Both the biosynthetic and uptake pathways are well-regulated through feedback control. When cells are cultured in the presence of LDL, the activity of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase decline by more than 90% and the number of LDL receptors also decreases (1). In the absence of LDL, the cells maintain high activities of these two enzymes, which are rate-limiting enzymes of the biosynthetic pathway, and also maintain a large number of LDL receptors on their surface. In this review we assess recent progress in understanding the mechanisms involved in transcriptional and posttranscriptional regulation of intracellular cholesterol metabolism.

Differential effects of LH and PGE2 on progesterone secretion by small and large porcine luteal cells

This study examined the effects of LH and PGE2 on progesterone secretion by small and large porcine luteal cells with or without low-density lipoproteins. Corpora lutea were isolated from gilts 13-14 days after administering gonadotrophins; enzymatically dissociated and small and large cells were isolated by elutriation. Culture plates, 24-well, were then seeded with 150,000 small or 30,000 large luteal cells suspended in 1 ml M199 medium supplemented with 5 micrograms insulin ml-1, 40 ng hydrocortisone ml-1 and with or without low-density lipoproteins (50 micrograms cholesterol ml-1) or PGE2. Cells were cultured for up to 24 h in a humidified incubator at 37 degrees C under 5% CO2 in air. The low-density lipoproteins stimulated (P < 0.05) progesterone secretion by large, but not small, luteal cells. Prostaglandin E2 stimulated (P < 0.05) progesterone production by large luteal cells in a dose-dependent manner, and the stimulatory effects of PGE2 were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. Progesterone secretion by small luteal cells was not significantly affected by PGE2. Progesterone production by small luteal cells was enhanced (P < 0.05) by LH, and the stimulatory effects of LH were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. In the absence of these lipoproteins, LH had no effect on progesterone secretion by large luteal cells; however, in the presence of low-density lipoproteins, LH increased (P < 0.05) progesterone secretion by large cells, though to a lesser (P < 0.05) extent than the effect of LH on small cells. These data demonstrate that progesterone secretion by porcine luteal cells is stimulated differentially by LH and PGE2 and that small luteal cells are more responsive to LH and PGE2 acts primarily on large luteal cells.

Effects of growth hormone and triiodothyronine on insulin-induced progesterone production by granulosa cells from prepubertal gilts

Two experiments were performed using granulosa cells from medium-sized follicles (2–4 mm) derived from prepubertal gilts. Cells were cultured in a serum-free medium at a density of either 1 or 2 × 106 viable cells per well (experiments 1 and 2, respectively). For exp. 1, porcine growth hormone (pGH) (0 or 100 ng mL−1) was included in the culture medium from the time of plating, and low-density lipoprotein (LDL) (100 μg mL−1) was added at 72 h. For exp. 2, granulosa cells were plated in a culture medium containing either pGH (0 or 100 ng mL−1) or triiodothyronine (T3) (0 or 5 ng mL−1) or both pGH T3; LDL was not included. For both experiments, after 24 h of culture, bovine insulin at 0, 10, 100 or 1000 ng mL−1 was included in the medium. Hormones were replaced at 48 and 72 h, and the cultures were terminated at 96 h. Results from exp. 1 indicated that insulin increased (P &lt; 0.01) progesterone production in a dose-dependent manner, both in the presence and absence of LDL. This response was augmented (P &lt; 0.01) by co-culture with pGH. Results from exp. 2 confirmed the augmenting effect of pGH (P &lt; 0.01). It was further observed that T3 increased (P &lt; 0.01) progesterone production when cultured with insulin at 1000 ng mL−1, but at lower insulin-inclusion levels, results were equivocal. The progesterone production response was greatest (P &lt; 0.01) when cells were cultured with both pGH and T3 at insulin levels of 100 or 1000 ng mL−1. There appeared to be little relationship between the media concentrations of insulin-like growth factor 1 and progesterone. The present results suggest that relatively high levels of pGH and T3 will enhance the in vitro steroidogenic capabilities of porcine granulosa cells. Key words: Granulosa cells, GH, T3, insulin

Effects of Shosaikoto (kampo medicine) on lipid metabolism in macrophages.

We investigated effects of Shosaikoto treatment on cholesterol metabolism in macrophages. Although macrophages, harvested from mice treated with Shosaikoto, took up a small amount of control low density lipoprotein (LDL) (thiobarbituric acid-reactive substance (TBA-RS) value was 0.27 pmol/mg of protein) as control macrophages, they took up more LDL modified with CuSO4 (TBA-RS value was 6.12 pmol/mg of protein) than control macrophages. Degradation of both control LDL and oxidized LDL was enhanced in Shosaikoto treated macrophages. In the presence of control LDL or in the absence of LDL, incorporation of [3H]oleic acid into chlesteryl oleate was significantly reduced in Shosaikoto treated macrophages. This suggests that acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in macrophages was partly inhibited by Shosaikoto treatment. On the other hand, in the present of oxidized LDL, cholesteryl ester accumulated in Shosaikoto treated macrophages as much as in controls. However, cholesteryl oleate efflux from macrophages in the presence of high density lipoprotein (HDL) was enhanced in Shosaikoto treated macrophages. These result indicate that Shosaikoto facilitates oxidized LDL catabolism in macrophages, resulting in the augmentation of oxidized LDL uptake and the elimination of cholesterol from macrophages by HDL. These Shosaikoto effects may prevent foam cell formation and the progression of atherosclerotic lesions.

Evidence for sterol-independent regulation of low-density lipoprotein receptor activity in Hep-G2 cells

The relationship between the serum factor(s)-mediated induction of low-density lipoprotein (LDL) receptor activity and changes in cellular cholesterol metabolism was examined in the human hepatoma cell line Hep-G2. Relative to incubation with serum-free media [Eagle's minimal essential medium (MEM) control], short-term (less than 8 h) incubation with medium containing 15% of either calf serum (MEM + serum) or the d greater than 1.25 fraction of calf serum (MEM + d greater than 1.25) produced a time- and concentration-dependent increase in the uptake of 125I-LDL. Immunoblotting with anti-(LDL receptor) antibodies demonstrated that this was correlated with a 2-fold increase in the amount of the mature 136,000 Da LDL receptor protein in detergent-solubilized Hep-G2 cell membranes. Incubation with MEM + serum, but not MEM + d greater than 1.25, increased the efflux of radiolabelled cholesterol from Hep-G2 cells. However, the induction of 125I-LDL uptake by MEM + d greater than 1.25 (2.3-fold) and MEM + serum (2.2-fold) was virtually identical. Addition of the d less than 1.063 lipoproteins of calf serum to MEM + d greater than 1.25 at their original or three times their serum concentration decreased the induction of 125I-LDL uptake by MEM + d greater than 1.25 by only 20-30%. Together, these results suggest that the stimulation of 125I-LDL uptake was not due to the presence of high-density lipoprotein, the absence of LDL or the stimulation of cholesterol efflux. MEM + serum stimulated 125I-LDL uptake in cells cholesterol-loaded by incubation with rat very-low-density lipoprotein with beta electrophoretic mobility (beta-VLDL). Compared to incubation with the MEM control, either MEM + serum or MEM + d greater than 1.25 produced time-dependent increases in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase which also occurred in cholesterol-loaded cells. However, cholesterol biosynthesis, whether measured from 3H2O, [14C]acetate or [3H]mevalonic acid, was not increased. Incubation with MEM + serum or MEM + d greater than 1.25 did not affect [3H]oleate incorporation into cellular cholesteryl esters, hydrolysis of intracellular [3H]cholesteryl esters or the cellular mass of unesterified or esterified cholesterol. Incubation with MEM + serum or MEM + d greater than 1.25 produced a transient increase in the level of LDL receptor mRNA, reaching a maximum of 5-10-fold by 2 h and decreasing to near baseline levels by 4 h. Actinomycin D blocked the serum-factor-mediated induction of LDL receptor mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Transformations of reconstituted high-density lipoprotein subclasses as a function of temperature or LDL concentration

The objectives of this study were to determine the structural changes in defined, reconstituted high density lipoproteins (rHDL) resulting from spontaneous phospholipid depletion in the presence or absence of low-density lipoproteins (LDL), to establish the precursor-product relationships among the rHDL particles and to assess the differences in behavior of rHDL particles containing apo A-I or apo A-II. The rHDL particles were prepared by the sodium cholate dialysis method, and were incubated in buffer at 50 degrees C, or in buffer containing different concentrations of LDL at 37 degrees C, for up to 24 h. The changes in the rHDL particle distributions with time were followed by non-denaturing gradient gel electrophoresis, and the rHDL were isolated at various time points for chemical analysis. We found that rHDL particles containing apo A-I or apo A-II lose phospholipid and gain cholesterol when incubated with LDL. Increasing LDL concentrations remove increasingly larger amounts of phospholipid. With phospholipid loss the apo A-I containing particles undergo major structural rearrangements that give rise to 78 A and 106 A particles from 86 A and 94 A precursors. The 78 A products appear to be the most stable, lipid-poor species. Reconstituted HDL particles prepared with apo A-II (94 and 101 A in diameter) are more resistant to structural rearrangements than the apo A-I counterparts under similar reaction conditions.

The human asialoglycoprotein receptor is a possible binding site for low-density lipoproteins and chylomicron remnants.

Binding and internalization of chylomicron remnants from rat mesenteric lymph by HepG2 cells was inhibited by both excess remnants and low-density lipoprotein (LDL) to the same extent. Ligand blots revealed binding of remnants and LDL to the LDL receptor. Measures regulating LDL receptor activity greatly influenced the binding of remnants: ethinyloestradiol, the hydroxymethylglutaryl-CoA reductase inhibitor pravastatin and the absence of LDL all increased binding, whereas high cell density or the presence of LDL decreased binding. Also, asialofetuin, asialomucin, the neoglycoprotein galactosyl-albumin and an antibody against the asialoglycoprotein receptor all decreased substantially the binding of remnants. At high cell density, binding internalization and degradation of chylomicron remnants was inhibited by up to 70-80%, yet binding of LDL was inhibited by no more than 20-30%. In cross-competition studies, the binding of 125I-asialofetuin was efficiently competed for by asialofetuin itself or by the antibody, and also by LDL and remnants, yet remnants displayed an approx. 100-fold higher affinity than LDL. Likewise, remnants of human triacylglycerol-rich lipoproteins and asialofetuin interfered with each others' binding to HepG2 cells or human liver membranes. It is concluded that the LDL receptor mediates the internalization of chylomicron remnants into hepatocytes depending on its activity, according to demand for cholesterol. Additionally, the asialoglycoprotein receptor may contribute to the endocytosis of LDL, but predominantly of chylomicron remnants.

Enhancement of Thyroxine Entry into Low Density Lipoprotein (LDL) Receptor-Competent Fibroblasts by LDL: An Additional Mode of Entry of Thyroxine into Cells

Having demonstrated that plasma low density lipoproteins (LDL) bind T4 through a specific interaction with their sole apolipoprotein, apoB-100, we tested the hypothesis that cells could internalize the LDL-T4 complex via cell surface LDL receptors. These receptors are down-regulated by cholesterol loading and up-regulated by cholesterol deficiency. We, therefore, studied the uptake of [125I]T4 or [125I]T3 by human skin fibroblasts grown in 10% lipoprotein-deficient serum in the absence or presence of LDL. At concentrations of LDL (12.5 and 22 micrograms protein/ml) that gave significant binding of T4 but did not exceed LDL receptor capacity, both the initial rate of saturable T4 uptake and the uptake at equilibrium increased by 27-63%. No significant increase occurred at a LDL concentration of 1.6 micrograms protein/ml (less than 3% occupancy), whereas there was a 20 to 31% reduction at 125 micrograms/ml (approximately 5 times the saturation dose for the LDL receptor). These changes were confirmed with several different LDL preparations and were mimicked by isolated apoB-100 and apoE, the sole ligands for the LDL receptors (apoB/E receptors). T4 uptake did not increase in normal fibroblasts with down-regulated LDL receptors or in LDL receptor-deficient fibroblasts from a patient with familial hypercholesterolemia. In the latter cell line the uptake of T4 (and T3) in the absence of LDL was indistinguishable from that of normal fibroblasts. T3 uptake in normal fibroblasts was not enhanced by LDL. The specificity of the LDL effect was shown by the finding that T4-binding globulin, prealbumin, or serum albumin, at concentrations giving 10-90% T4 bound, failed to increase T4 uptake. Instead, each of these major thyroid hormone-binding plasma proteins caused a dose-dependent decrease in T4 entry. It is concluded that at least two modes of entry into fibroblasts are available for T4. The first is the cell surface thyroid hormone-binding sites, which recognize the free hormone and are present in both normal and LDL receptor-negative fibroblasts. The second, and additional, mode of entry is via the LDL receptors, which recognize the T4-LDL complex, are absent in LDL receptor-negative fibroblasts, are reduced in down-regulated fibroblasts, and are unavailable for T3, owing to the low affinity of T3 for LDL.

Unesterified Fatty Acids Inhibit the Binding of Low Density Lipoproteins to the Human Fibroblast Low Density Lipoprotein Receptor

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.

Regulation of Low Density Lipoprotein Receptor Gene Expression in Human Lymphocytes

Cholesterol homeostasis is maintained by coordinate regulation of endogenous synthesis and exogenous uptake of lipoprotein cholesterol by low density lipoprotein (LDL) receptors. In the lymphocyte, limiting the availability of exogenous cholesterol is known to increase the rate of endogenous sterol biosynthesis. However, the effect of cholesterol deprivation on the expression and regulation of the LDL receptor gene has not been delineated in lymphocytes. Here, LDL receptor mRNA was detected in freshly isolated human peripheral mononuclear cells. LDL receptor mRNA levels increased by 3-fold during a one-h in vitro culture in lipoprotein-deficient medium and by 6-fold during a 2-h incubation. Actinomycin D blocked the synthesis of LDL receptor mRNA in these cultures. However, neither cycloheximide nor LDL or oxygenated sterols suppressed the increase in LDL receptor mRNA levels observed after a 2-h incubation. The increase in LDL receptor mRNA was maintained for 24 h of culture in the absence of LDL. Ongoing gene transcription and not mRNA stabilization accounted for this expression. Inhibition of protein synthesis with cycloheximide completely prevented the sustained increase in LDL receptor mRNA levels measured after 24 h. Low concentrations of LDL (5 micrograms of cholesterol/ml) and oxygenated sterols also suppressed the level of LDL receptor mRNA measured after a 24-h incubation. These data show that the initial upregulation of LDL receptor gene expression is independent of protein synthesis and not suppressed by either LDL or oxygenated sterols. In contrast, the continued transcription necessary for the maintenance of steady-state levels of LDL receptor mRNA requires synthesis of new protein and is regulated by LDL and oxygenated sterols.

The antidiabetic drug Metformin decreases cholesterol metabolism in cultured human fibroblasts

The effect of the hypoglycemic biguanide drug Metformin was investigated after a 72 h pretreatment of human cultured fibroblasts. Metformin induced a moderate increase in low density lipoprotein binding, uptake and internalization (25% increase after treatment with 5 × 10 −4 M of drug). A decrease in sterol, fatty acid and triacylglycerol synthesis from sodium acetate was observed after pretreatment with the drug, with a dose-dependent effect in the range of 5 × 10 −5 to 5 × 10 −4 M (50% reduction of sterol synthesis after treatment with Metformin 5 × 10 −4 M). This effect was also observed in fibroblasts from a patient with homozygous familial hypercholesterolemia. Cholesterol esterification studied by incorporation of radiolabeled oleic acid was reduced by Metformin (40% of control after treatment with Metformin 5 × 10 −4 M) whereas incorporation into triacylglycerols was less impaired. These effects of Metformin on cholesterol metabolism were observed either in the presence or in the absence of low density lipoproteins. Moreover, Metformin also reduced cholesterol esterification in J774 monocyte-macrophage cells. Metformin also induced a decrease of hydroxymethylglutaryl coenzyme A reductase activity in cultured fibroblasts and a reduction of acyl-coenzyme A :cholesterol- O-acyltransferase activity in cultured fibroblasts and J774 cells.

Familial hypercholesterolaemia : effect of low density lipoproteins on esterification of cholesterol in lymphocytes from homozygous and heterozygous subjects

The effect of low density lipoproteins on esterification of cholesterol was studied in lymphocytes from patients with familial hypercholesterolaemia; results were compared with those obtained using cells from normal individuals. Freshly isolated lymphocytes were maintained in lipoprotein-deficient medium for 48 h and the rate of formation of [3H] cholesteryl oleate from [3H] oleate was then determined in the presence or absence of low density lipoproteins. In the absence of low density lipoproteins, incorporation of [3H] oleate was higher in heterozygote and homozygote cells than in normal lymphocytes. Incorporation in the presence of low density lipoproteins was increased relative to that measured in their absence for all of the subjects studied; heterozygotes and homozygotes showed marked changes in some cases but not in others.

Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

Activity and regulation of low density lipoprotein receptors in a human hepatoblastoma cell line.

The responsiveness of low density lipoprotein (LDL) binding and uptake was measured in a human hepatoblastoma cell line, Hep G2. These cells exhibited both saturable, high-affinity and nonsaturable low-affinity components similar to that described for LDL binding in other mammalian cells. In addition, receptor-mediated uptake of [125I]LDL was dependent on the presence of Ca++ and required intact lysine residues in LDL as evidenced by abolition of binding following reductive methylation of LDL. The receptor activity was rapidly responsive to changes made in LDL concentrations in the medium, up-regulating by 200% in the absence of LDL, and down-regulating by 56% in the presence of LDL, both changes occurring within 2 hr. Cholesterol synthesis was estimated by measuring 3-hydroxymethylglutaryl coenzyme A reductase activity. The activity of this enzyme was also found to increase rapidly in the absence of LDL and decrease rapidly in the presence of LDL in the media. The human cell line Hep G2 possesses an active and responsive receptor for LDL and provides a useful model for the study of lipoprotein uptake and metabolism in intact human liver.

Regulation of low density lipoprotein receptors by plasma lipoproteins from patients with abetalipoproteinemia.

Despite an absence of low density lipoproteins (LDLs) and chylomicron remnants from plasma, the rates of cholesterol synthesis or the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased. These observations suggest that other lipoprotein particles present in the plasma of patients with abetalipoproteinemia may regulate LDL receptor activity and the rates of cellular cholesterol synthesis in this disorder. In the present report we have studied the effects of lipoprotein fractions from the plasma of normal subjects, patients with abetalipoproteinemia, and a patient with dysbetalipoproteinemia on the binding, internalization, and degradation of 125I-labeled LDL (125I-LDL) by cultured human fibroblasts. LDL from normal subjects or the high density lipoprotein fraction HDL2 from the plasma of patients with abetalipoproteinemia effectively down-regulated LDL receptor activity (greater than 50% inhibition at 20 micrograms of protein per ml). HDL2 from the plasma of patients with abetalipoproteinemia also effectively reduced the binding, internalization, and degradation of 125I-LDL by cultured human fibroblasts. 125I-HDL2 from the plasma of patients with abetalipoproteinemia was bound, internalized, and degraded by cultured human fibroblasts; this process was competitively inhibited by unlabeled normal LDL or HDL2 from abetalipoproteinemic plasma and was 1/6th to 1/8th times as high when 125I-HDL2 was incubated with fibroblasts from a patient with receptor-negative homozygous familial hypercholesterolemia. We conclude that lipoproteins present in the HDL2 fraction of plasma from patients with abetalipoproteinemia (which are relatively rich in apoprotein E) are effective regulators of LDL receptor activity in normal human fibroblasts. These in vitro findings may explain why the in vivo rates of cholesterol synthesis and the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased.

Stimulation of phospholipid and cholesterol ester synthesis by platelet-derived growth factor in normal and homozygous familial hypercholesterolemia human skin fibroblasts

Pure human platelet-derived growth factor at nanogram levels stimulates cholesterol ester, phospholipid and DNA synthesis in normal and familial hypercholesterolemia mutant human skin fibroblasts. Stimulation of DNA synthesis did not begin until 15–24 h after addition of platelet-derived growth factor to quiescent normal and FH mutant fibroblasts. In contrast, stimulation of [ 3H]oleic acid incorporation into cholesterol ester and phospholipid was evident 3–6 h after the addition of platelet-derived growth factor. In the normal cells, the rate of cholesterol ester synthesis was maximal at 24 h, then rapidly declined. Compared to the normal cells, cholesterol esterification was much lower in the FH cells; however, platelet-derived growth factor stimulated the rate of [ 3H]oleic acid incorporation into cholesterol ester by 5-fold, 31 h after addition of the growth factor. The stimulation of [ 3H]oleic acid incorporation into cholesterol ester by platelet-derived growth factor was inhibited in both normal and FH mutant skin fibroblasts by progesterone, an inhibitor of acyl-CoA: cholesterol acyltransferase. The rate of cholesterol ester synthesis in the normal cells increased as the concentration of platelet-poor plasma or low density lipoprotein (LDL) was increased, especially in the presence of platelet-derived growth factor. Linearization of the LDL dose-response curve indicated that platelet-derived growth factor increased the rate rather than the affinity of the overall cholesterol esterification system. The rate of cholesterol esterification in the FH mutant cells was highest in the absence of LDL or at low levels of platelet-poor plasma. Consequently, platelet-derived growth factor can stimulate cholesterol ester synthesis by LDL- and non-LDL-mediated processes.

Regulation of low-density lipoprotein receptors in cultured bovine adrenocortical cells

Bovine adrenocortical cells in monolayer culture produce cortisol and respond to corticotropin (ACTH) by an increase in cortisol secretion. Several lines of evidence are indicative that much of the cholesterol that serves as precursor for steroid hormone biosynthesis by these cells is derived from low-density lipoprotein (LDL) cholesterol that is taken up endocytotically by means of specific receptors localized in bovine adrenocortical plasma membranes. ACTH stimulated this process concomitant with an increase in steroid production. In the absence of LDL, ACTH had no effect on steroid biosynthesis. ACTH action in bovine adrenocortical cells resulted in an increase in the number of LDL receptor sites in the membrane fractions, whereas the dissociation constant for LDL binding was not changed. Chloroquine and NH 4Cl, considered to be inhibitors of lysosomal degradative activity, caused an increase in the number of [ 125I]iodoLDL binding sites in the plasma membrane but the effect of ACTH was still apparent in the presence of these agents. These results are suggestive that the lifetime of the LDL receptor is increased when lysosomal activity is inhibited. When aminoglutethimide was added to block cholesterol side-chain cleavage activity and inhibit steroid production, the number of [ 125I]iodoLDL binding sites in the membrane fractions prepared from bovine adrenocortical cells cultured in the presence of ACTH was reduced to 50% of that in cells maintained in aminoglutethimide-free medium. However, under these conditions the number of binding sites was still significantly greater than in cells maintained in the absence of ACTH. The effects of aminoglutethimide on uptake and degradation of [ 125I]iodoLDL were similar to the effects on the number of [ 125I]iodoLDL binding sites. Based on these results, we conclude that the action of ACTH to stimulate LDL metabolism in bovine adrenocortical cells results from an increase in the number of LDL binding sites in the plasma membranes. This action of ACTH appears to be, at least in part, independent of cholesterol utilization for cortisol biosynthesis. However, the effect of aminoglutethimide is indicative that changes in the intracellular cholesterol concentration might modulate the action of ACTH to increase the number of LDL binding sites and therefore to stimulate LDL degradation.