LDL Receptor mRNA Levels Research Articles

Polyunsaturated fatty acids downregulate the low density lipoprotein receptor of human HepG2 cells

The aim of the study was to investigate the effect of different fatty acids on the low density lipoprotein (LDL) receptor of cultured human liver HepG2 cells. Previous studies investigating the effect of fatty acids on LDL expression have reported conflicting findings and are limited to measurements of LDL receptor binding activity. Therefore, this study is unique in that the relative effects of different fatty acids on the LDL receptor were investigated at three different stages of expression: 1) functional cellular LDL binding activity, 2) amount of LDL receptor protein and 3) LDL receptor mRNA level. The HepG2 cells were incubated for 24 hr with either 100 μM palmitic, oleic, linoleic or eicosapentaenoic acid (EPA). The measurement of LDL receptor binding activity was with colloidal gold-LDL conjugates, cellular LDL receptor protein was by western blotting and LDL receptor mRNA by Southern blotting of reverse-transcribed, polymerase chain reaction-amplified cDNA. The LDL receptor binding activity, protein and mRNA levels decreased as the degree of unsaturation of the fatty acids increased (palmitic acid ≥ oleic acid > linoleic acid > EPA) and the inverse relationship held whether or not cholesterol was included in the culture media. The relative differences were very similar for the three stages of expression indicating that modulation of the LDL receptor by the fatty acids occurred at the level of gene transcription. The increased susceptibility to oxidation of the polyunsaturated fatty acids was unlikely to be a factor in the effect because EPA and linoleic acid (250 μM) still downregulated the LDL receptor in the presence of the antioxidant vitamin E (50 μM). In conclusion, the polyunsaturates, linoleic acid and EPA, effectively downregulated the LDL receptor of HepG2 cells compared to palmitic acid. The effects of these fatty acids were observed at the level of LDL receptor binding activity, protein and mRNA, strongly suggesting that the fatty acid effects were at the level of gene transcription.

Cholesterol-lowering effects of maitake (Grifola frondosa) fiber, shiitake (Lentinus edodes) fiber, and enokitake (Flammulina velutipes) fiber in rats.

The effects of mushroom fibers on serum cholesterol and hepatic low-density lipoprotein (LDL) receptor mRNA in rats were investigated. Rats were fed a cholesterol-free diet with 50 g/kg cellulose powder (CP), 50 g/kg maitake (Grifola frondosa) fiber (MAF), 50 g/kg shiitake (Lentinus edodes) fiber (SF), or 50 g/kg enokitake (Flammulina velutipes) fiber (EF) for 4 weeks. There were no significant differences in the body weight, food intake, liver weight, cecum weight, and cecum pH among the groups. Cecal acetic acid, butyric acid, and total short-chain fatty acid (SCFA) concentrations in the SF and EF groups were significantly higher than those in the other groups. The serum total cholesterol concentration in the CP group was significantly higher than that in the MAF and EF groups. The very LDL (VLDL) + intermediate-density lipoprotein (IDL) + LDL-cholesterol concentration in the CP group was significantly higher than that in the MAF, SF, and EF groups, whereas the high-density lipoprotein (HDL)-cholesterol concentration in the EF group was significantly lower than that in the other groups at the end of the 4-week feeding period. The hepatic LDL receptor mRNA level in the EF group was significantly higher than that in the CP group. The fecal cholesterol excretion in the MAF, SF, and EF groups was significantly higher than that in the CP group. The results of this study demonstrate that MAF and EF lowered the serum total cholesterol level by enhancement of fecal cholesterol excretion, and in particular, by enhancement of hepatic LDL receptor mRNA in EF group.

Protective effects of Saiko-ka-ryukotsu-borei-to (Chai-Hu-Jia-Long-Gu-Mu-Li-Tang) against atherosclerosis in kurosawa and kusanagi-hypercholesterolemic (KHC) rabbits

We investigated the protective effects of the traditional Japanese herbal medicine Saiko-ka-ryukotsu-borei-to (Chai-Hu-Jia-Long-Gu-Mu-Li-Tang in Chinese) (SRBT) against hypercholesterolemia and atherosclerotic lesions. We focused on atherosclerosis using female heterozygous Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits. The total plasma cholesterol levels increased for up to 12 weeks after beginning a diet containing 0.1% cholesterol and then reached a plateau of about 600 mg dl−1. When SRBT was administered at a dose of 1.0 g kg−1per day for 24 weeks, total plasma cholesterol levels were significantly decreased after 20–24 weeks. On the other hand, pravastatin at a dose of 10 mg kg−1per day produced a significant decrease in total plasma cholesterol levels from 4 to 24 weeks (about 105–130 mg dl−1). Moreover, 1.0 g kg−1per day of SRBT significantly decreased plasma low density lipoprotein (LDL) cholesterol levels but did not change either very low density lipoprotein (VLDL), or high density lipoprotein (HDL) cholesterol levels. Animals that received pravastatin had significantly decreased LDL cholesterol levels and VLDL cholesterol levels after 8 weeks and at 24 weeks. We also examined the expression of apoB, E and LDL receptor mRNA levels in the liver at 24 weeks after beginning the administration of 1.0 g kg−1per day of SRBT. Both apoE and LDL receptor mRNA levels were significantly increased compared with those in rabbits receiving the 0.1% cholesterol diet. SRBT at a dose of 1.0 g kg−1per day significantly depressed the intimal surface area of the thoracic aortae involved with atheromatous plaques. The present results suggest that SRBT may protect against hypercholesterolemia and atheromatous lesions by affecting apoE and LDL receptor mRNA gene expression in the liver.

Regulation of Sterol Regulatory Element-binding Proteins in Hamster Intestine by Changes in Cholesterol Flux

A control chow diet or diets containing 1% cholesterol (cholesterol-enriched) or 4% cholestyramine and 0.15% lovastatin (cholesterol-depletion) were fed to hamsters for 2 weeks. Sterol regulatory element-binding protein (SREBP)-1a, SREBP-1c, SREBP-2, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, and LDL receptor mRNA levels and SREBP-1 and -2 protein expression were estimated in villus cell populations from duodenum, jejunum, and ileum. SREBP-1a was a minor transcript in hamster intestine, and its gene expression was not altered by changes in dietary cholesterol flux. In contrast, SREBP-1c gene expression was increased by dietary cholesterol and decreased by cholesterol depletion. mRNA levels for SREBP-2 and the other sterol-responsive genes were increased in intestines of animals on the cholesterol depletion diet but minimally suppressed if at all, by the diet enriched in cholesterol. In general, the amount of the precursor form of SREBP-1 was higher in cells of the upper villus and lower in cells of the lower villus. SREBP-2 precursor was higher in cells of the lower villus and lower in cells of the upper villus. Protein expression of precursor correlated with the location of gene expression for SREBPs. The amount of precursor mass of SREBP-2 was not altered by cholesterol feeding but was increased by cholesterol depletion. The mature form of SREBP-2 was in very low abundance and difficult to detect in intestines of animals fed control chow or cholesterol. It was readily detectable and increased in intestines of animals on the cholesterol-depletion diet. The diets did not significantly alter the amount of precursor or mature forms of SREBP-1. Cholesterol feeding had no effect on cholesterol or fatty acid synthesis, whereas synthesis of these lipids was increased in intestines of hamsters on the cholesterol-depleted diet. These results suggest that SREBP-1a has little or no role in regulating intestinal cholesterol synthesis. It is postulated that under basal conditions, SREBP-1c regulates intestinal fatty acid synthesis and SREBP-2 regulates cholesterol synthesis. Following marked changes in cholesterol flux across the intestine, SREBP-2 assumes the role of SREBP-1 and regulates both cholesterol and fatty acid synthesis in intestine.

Effects of diets enriched in n-6 or n-3 fatty acids on cholesterol metabolism in older rats chronically fed a cholesterol-enriched diet.

Hypocholesterolemic effects in older animals after long-term feeding are unknown. Therefore, aged rats (24 wk of age) fed a conventional diet were shifted to diets containing 10% perilla oil [PEO; oleic acid + linoleic acid + alpha-linolenic acid; n-6/n-3, 0.3; polyunsaturated fatty acid/saturated fatty acid (P/S), 9.6], borage oil [oleic acid + linoleic acid + alpha-linolenic acid; n-6/n-3, 15.1; P/S, 5.3], evening primrose oil (EPO; linoleic acid + gamma-linolenic acid; P/S, 10.5), mixed oil (MIO; oleic acid + linoleic acid + gamma-linolenic acid + alpha-linolenic acid; n-6/n-3, 1.7; P/S, 6.7), or palm oil (PLO; palmitic acid + oleic acid + linoleic acid; n-6/n-3, 25.3; P/S, 0.2) with 0.5% cholesterol for 15 wk in this experiment. There were no significant differences in the food intake and body weight gain among the groups. The liver weight in the PEO (n-6/n-3, 0.3) group was significantly higher than those of other groups in aged rats. The serum total cholesterol and very low density lipoprotein (VLDL) + intermediate density lipoprotein (IDL) + low density lipoprotein (LDL)-cholesterol concentrations of the PLO (25.3) group were consistently higher than those in the other groups. The serum high density lipoprotein cholesterol concentrations of the PEO (0.3) and EPO groups were significantly lower than in the other groups at the end of the 15-wk feeding period. The liver cholesterol concentration of the PLO (25.3) group was significantly higher than those of other groups. There were no significant differences in the hepatic LDL receptor mRNA level among the groups. Hepatic apolipoprotein (apo) B mRNA levels were not affected by the experimental conditions. The fecal neutral steroid excretion of the PLO (25.3) group tended to be low compared to the other groups. The results of this study demonstrate that both n-6 fatty acid and n-3 fatty acids such as gamma-linolenic acid and alpha-linolenic acid inhibit the increase of serum total cholesterol and VLDL + IDL + LDL-cholesterol concentrations of aged rats in the presence of excess cholesterol in the diet compared with dietary saturated fatty acid.

Low density lipoprotein receptor mRNA in rat liver is affected by resistant starch of beans

The effects of resistant starches of beans on serum cholesterol and hepatic low density lipoprotein (LDL) receptor mRNA in rats were investigated. Rats were fed a cholesterol-free diet with 150 g/kg corn starch (CS), 150 g/kg adzuki (Vigna angularis) starch (AS), 150 g/kg kintoki (Phaseolus vulgaris, variety) starch (KS), or 150 g/kg tebou (P. vulgaris, variety) starch (TS) for 4 wk. There were no significant differences in body weight among groups through the experimental period. The liver weight in the CS group was 1.1-1.2 times higher than that in the AS, KS, and TS groups. The cecum weight in the TS was 1.4 times higher than that in the CS group, and the cecal pH in the CS group was significantly higher than in the other groups. The serum total cholesterol, very low density lipoprotein + intermediate density lipoprotein + LDL-cholesterol and high density lipoprotein (HDL)-cholesterol concentrations in the bean starch groups were significantly lower than those in the CS group through the feeding period. The total cholesterol/HDL-cholesterol ratio in the bean starch groups was also significantly lower than that in the CS group at the end of the 4-wk feeding period. The hepatic cholesterol concentration in the TS group was significantly higher than in the CS group at the end of the 4-wk feeding period. The relative quantity of hepatic apo B mRNA in the AS group was 1.2 times higher than that in the CS group, and the hepatic LDL receptor mRNA levels in the AS and TS groups were 1.8-2.0 times higher than that in the CS group. The results of this study demonstrate that AS, KS, and TS lowered the serum total cholesterol level by enhancing the hepatic LDL receptor mRNA level.

Gene expression of sterol regulatory element-binding proteins in hamster small intestine

Gene expression of sterol regulatory element-binding proteins 1a, 1c, and 2 (SREBP-1a, -1c, and -2) and of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low density lipoprotein (LDL) receptor was examined in hamster small intestine. SREBP-1c transcript predominated over SREBP-1a. mRNA levels for SREBP-1a, -1c, and -2, LDL receptor, and HMG-CoA synthase were highest in jejunum and ileum. Expression of SREBP-1a and SREBP-1c was highest in cells of the upper villus and decreased in cells of the lower villus. Gene expression of SREBP-2 was lowest in cells of the upper villus and increased in cells of the lower villus. Ileal SREBP-2 gene expression was highest in cells of the midvillus. mRNA levels for HMG-CoA synthase and the LDL receptor followed a pattern similar to that of SREBP-2. A positive correlation existed between SREBP-2 gene expression and rates of cholesterol synthesis. Fatty acid synthesis was highest in jejunum and ileum, correlating positively with the expression of SREBP-1c. Sterol influx into intestinal cells decreased mRNA levels of SREBP-2, HMG-CoA reductase, HMG-CoA synthase, and LDL receptor. In ileum, sterol influx decreased gene expression of SREBP-1a and increased expression of SREBP-1c. The results suggest that SREBP-2 regulates cholesterol synthesis in the small intestine. SREBP-1a is a minor transcript and its expression does not correlate with cholesterol-synthesizing activity. SREBP-1c is a major transcript in small intestine and its expression along the length of the gut correlates with fatty acid synthesis. Sterols regulate gene expression of sterol-responsive genes, including SREBP-2, in small intestine.—Field, F. J., E. Born, S. Murthy, and S. N. Mathur. Gene expression of sterol regulatory element-binding proteins in hamster small intestine. J. Lipid Res. 2001. 42: 1–8.

Investigation of Gene Expressions Related to Cholesterol Metabolism in Rats Fed Diets Enriched in n-6 or n-3 Fatty Acid with a Cholesterol after Long-term Feeding Using Quantitative-competitive RT-PCR Analysis.

We have developed a method to quantitate hepatic apolipoprotein (apo) B, LDL receptor, 3-hydroxy-3-methylglutary coenzyme A reductase (HMG-CoA reductase) and cholesterol 7alpha-hydroxylase mRNA expression in rats fed a cholesterol-enriched diet after long-term feeding using competitive RT-RCR. Rats (8 wk of age) fed a conventional diet were shifted to diets containing 10% perilla oil (PEO, oleic acid+linoleic acid+alpha-linolenic acid), borage oil (BRO, oleic acid+linoleic acid+gamma-linolenic acid), evening primrose oil (EPO, linoleic acid+gamma-linolenic acid), mixed oil (MIO, oleic acid+linoleic acid+gamma-linolenic acid+alpha-linolenic acid), or palm oil (PLO, palmitic acid+oleic acid+linoleic acid) with 0.5% cholesterol for 15 wk. There were no significant differences in the food intake and body weight gain among the groups. The liver weight in the PEO and PLO groups was significantly higher than other groups. The serum total cholesterol and very low density lipoprotein (VLDL)+intermediate density lipoprotein (IDL)+low density lipoprotein (LDL)-cholesterol concentrations were consistently higher in PLO group than in the other groups. The serum high density lipoprotein cholesterol concentration was significantly lower in the PEO group than in the other groups. The liver cholesterol concentration group was significantly higher in the PEO than in the other groups. There were no significant differences in the hepatic LDL receptor mRNA level among the groups. Hepatic apo B, HMG-CoA reductase and cholesterol 7alpha-hydroxylase mRNA levels were not affected by the experimental conditions. However, hepatic cholesterol 7alpha-hydroxylase mRNA level in the PEO and MIO groups tended to be higher than in the other groups. The fecal cholesterol extraction was significantly higher in the MIO and PLO groups than in the PEO and EPO groups and the total bile acid extraction was significantly higher in the PEO and MIO groups than in the PLO group. The results of this study demonstrated that both n-6 fatty acid and n-3 fatty acids such as gamma-linolenic acid and alpha-linolenic acid lowered serum total cholesterol and VLDL+IDL+LDL-cholesterol concentrations of rats in the presence of excess cholesterol in the diet compared with dietary saturated fatty acid.

Hepatic LDL Receptor mRNA in Rats Is Increased by Dietary Mushroom (Agaricus bisporus) Fiber and Sugar Beet Fiber

Plasma cholesterol concentration is reduced by feeding some dietary fibers and mushroom fruit body, but the mechanism is not fully understood. We examined the effects of mushroom (Agaricus bisporus) fiber and sugar beet fiber on serum cholesterol and hepatic LDL receptor mRNA in rats. Rats were fed a cholesterol-free diet with 50 g/kg cellulose powder (CP), 50 g/kg mushroom (Agaricus bisporus) fiber (MSF) or 50 g/kg sugar beet fiber (BF) for 4 wk. There were no significant differences in the body weight, food intake and cecum weight among the groups. The relative liver weight in the CP group was significantly greater than that in the MSF and BF groups. The cecal pH in the CP and MSF groups was significantly higher than that in the BF group. Cecal acetic acid, butyric acid and total short-chain fatty acid (SCFA) concentrations in the BF group were significantly higher than those in the other groups. The serum total cholesterol, VLDL + intermediate density lipoprotein (IDL) + LDL cholesterol concentrations in the CP group were significantly greater than those in the MSF and BF groups. The HDL cholesterol concentration in the MSF group was significantly lower than that in the CP group. The hepatic LDL receptor mRNA level in the MSF and BF groups was significantly higher than that in the CP group. The results of this study demonstrate that mushroom fiber and sugar beet fiber lowered the serum total cholesterol level by enhancement of the hepatic LDL receptor mRNA.

Interleukin-6 stimulates LDL receptor gene expression via activation of sterol-responsive and Sp1 binding elements.

Inflammatory or malignant diseases are associated with elevated levels of cytokines and abnormal low density lipoprotein (LDL) cholesterol metabolism. In the acute-phase response to myocardial injury or other trauma or surgery, total and LDL cholesterol levels are markedly decreased. We investigated the effects of the proinflammatory cytokine interleukin (IL)-6 on LDL receptor (LDL-R) function and gene expression in HepG2 cells. IL-6 dose-dependently increased the binding, internalization, and degradation of (125)I-LDL. IL-6-stimulated HepG2 cells revealed increased steady-state levels of LDL-R mRNA. In HepG2 cells transiently transfected with reporter gene constructs harboring the sequence of the LDL-R promoter extending from nucleotide -1563 (or from nucleotide -234) through -58 relative to the translation start site, IL-6 dose-dependently increased promoter activity. In the presence of LDL, a similar relative stimulatory effect of IL-6 was observed. Studies using a reporter plasmid with a functionally disrupted sterol-responsive element (SRE)-1 revealed a reduced stimulatory response to IL-6. In gel-shift assays, nuclear extracts of IL-6-treated HepG2 cells showed an induced binding of SRE binding protein (SREBP)-1a and SRE binding protein(SREBP)-2 to the SRE-1 that was independent of the cellular sterol content and an induced binding of Sp1 and Sp3 to repeat 3 of the LDL-R promoter. Our data indicate that IL-6 induces stimulation of the LDL-R gene, resulting in enhanced gene transcription and LDL-R activity. This effect is sterol independent and involves, on the molecular level, activation of nuclear factors binding to SRE-1 and the Sp1 binding site in repeat 2 and repeat 3 of the LDL-R promoter, respectively.

Hormonal regulation of steroidogenic enzyme expression in granulosa cells during the peri-ovulatory interval in monkeys.

Although progesterone plays an essential role in ovulation and the luteiniziation of the primate follicle, the expression of cellular components required for progesterone synthesis and their control is not well defined. This study was designed to determine the time course and gonadotrophin versus steroid regulation of the transcription of genes involved in progesterone synthesis in peri-ovulatory follicles. Granulosa cells or whole ovaries were obtained from macaques undergoing controlled ovarian stimulation either before (0 h) or up to 36 h following the administration of an ovulatory human chorionic gonadotrophin (HCG) bolus with or without a 3beta-hydroxysteroid dehydrogenase (3beta-HSD) inhibitor, with or without a non-metabolizable progestin. Granulosa cell concentrations of low density lipoprotein receptor (LDL-R) and steroidogenic acute regulatory protein (StAR) mRNA increased transiently 12 h following HCG administration (P < 0.05) at which time steroid depletion tended to reduce StAR mRNA (P = 0.06). At 36 h post-HCG progesterone suppressed the LDL-R mRNA levels (P < 0.05). P450 side-chain cleavage (P450scc) mRNA decreased in a time-dependent fashion up to 24 h, whereas 3beta-HSD mRNA increased within 12 h of HCG administration (P < 0.05) in a steroid-independent manner. Whole ovarian 17alpha-hydroxylase (P450c17) and granulosa cell P450 aromatase (P450arom) mRNA declined in a time-dependent fashion; by 36 h after HCG administration, steroid depletion increased P450arom mRNA, although progestin replacement did not return aromatase to control values (P < 0.05). These data demonstrate diverse patterns of steroidogenic enzyme expression that generally reflect the conversion of the macaque peri-ovulatory follicle from an oestrogen to progesterone producing gland. Although mRNAs associated with progesterone synthesis and metabolism are primarily regulated by gonadotrophins, cholesterol uptake and utilization may be modulated locally by steroids in luteinizing granulosa cells.

Analysis of expression of genes involved in apolipoprotein E-based lipoprotein metabolism in pregnant mice deficient in the receptor-associated protein, the low density lipoprotein receptor, or apolipoprotein E.

Mice deficient in receptor-associated protein (RAP) were phenotypically normal, but in contrast to results previously reported in RAP(-/-) mice, nearly 50% of the offspring died at or shortly after birth. To attempt to determine the reason for this, we analyzed the regulation of expression of genes involved in apolipoprotein E (apoE)-based mechanisms in RAP-deficient mice and compared this to results in mice deficient in low density lipoprotein receptor (LDLR) or apoE. The major finding concerned a large increase in hepatic lipoprotein receptor-related protein (LRP) mRNA and LDLR mRNA levels in pregnant RAP knockout mice. This is in contrast to the down-regulation of LRP mRNA and LDLR mRNA, which is normally seen in wild-type mice. Also in LDLR knockout mice, a significant up-regulation in expression of LRP mRNA was demonstrated. In apoE knockout mice, hepatic LRP mRNA did not change significantly, while hepatic LDLR mRNA expression was increased. In placenta and uterus, the deficiency of RAP did not markedly affect the expression of LRP and LDLR. Lipoprotein lipase mRNA and apoE mRNA increased during pregnancy in all mice, independent of their genetic status. The current study does not directly explain the increased mortality of RAP(-/-) pups. The data demonstrate, however, important relative changes in expression of the genes analyzed, an indication that LRP and LDLR play an important role in lipid metabolism during pregnancy.

Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARα activation

3-Thia fatty acids are modified fatty acids that promote hepatic peroxisome proliferation and decrease serum triacylglycerol, cholesterol and free fatty acid levels in rats. In vivo administration of tetradecylthioacetic acid (TTA) to rats led to a significant decrease in liver apolipoproteins apoA-I, A-II, A-IV, and C-III mRNA levels, and to an increase of liver acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-II, and 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-CoA synthase) mRNA levels and activities. By contrast, no significant changes of lipoprotein lipase (LPL) mRNA levels were detected in rat epididymal adipose tissue. Liver carnitine palmitoyltransferase-I, apoB, apoE, and LDL receptor mRNA levels were not significantly affected. When tested in vitro, TTA increased rat ACO and carnitine palmitoyltransferase-I mRNA levels in primary rat hepatocytes and also LPL mRNA levels in 3T3-L1 preadipocytes. TTA also enhanced the transcriptional activity of chimeras containing the DNA binding domain of the yeast transcription factor Gal4 fused to the ligand binding domain of either human PPARalpha or human PPARgamma. The effect depended on the concentration tested and the cell type. In conclusion, our data suggest that in vitro, TTA activates both PPARalpha and PPARgamma, but the latter with much lower affinity. TTA affects serum lipid levels in vivo in rats by acting mainly on the liver via PPARalpha where it decreases the liver expression of genes involved in vascular lipid transport and increases the expression of genes involved in intracellular fatty acid metabolism. -Raspé, E., L. Madsen, A-M. Lefebvre, I. Leitersdorf, L. Gelman, J. Peinado-Onsurbe, J. Dallongeville, J-C. Fruchart, R. Berge, and B. Staels. Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation.

MDR1 Gene Expression in Normal and Atherosclerotic Human Arteries1

Recent studies have shown that a membrane p-glycoprotein, encoded by MDR1 gene, is involved in the transport of free cholesterol from the plasma membrane to endoplasmic reticulum, the site of cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT). Moreover, results deriving from our previous studies have shown that the rate of cell proliferation was positively correlated with cholesteryl ester levels as well as with ACAT and MDR1 gene expression. In this study, lipid content and the expression of the genes involved in cholesterol metabolism such as hydroxy-methylglutaryl coenzyme A reductase (HMGCoA-R), low-density lipoprotein receptor (LDL-R), ACAT and MDR1 have been investigated in control and atherosclerotic arteries. The results have shown that the levels of cholesteryl ester increase with the age of cadaveric donors in arteries prone to atherosclerosis (abdominal aorta, superficial femoral artery) and become predominant in advanced atherosclerotic lesions. The mRNA levels of ACAT and MDR1 showed the same age correlation, reaching the highest values in atherosclerotic specimens. These results suggest that MDR1 may be involved in the accumulation of intracellular cholesterol ester levels found in atherosclerotic lesions. Moreover, the levels of HMGCoA-R, LDL-R and ACAT gene expressions progressively increased with the age of cadaveric donors; conversely, in atherosclerotic specimens, the mRNA levels of HMGCoA-R and LDL-R drastically decreased while ACAT gene expression reached its maximum. These findings suggest a reactivation of normal homeostatic regulation of cholesterol in advanced and complicated lesions.

HMG CoA Reductase and LDL Receptor Genes Are Regulated Differently by 15-Ketosterols in Hep G2 Cells

The ability of two 15-ketosubstituted sterols, 5α-cholest-8(14)-en-3β-ol-15-one and 3β-(2-hydroxyethoxy)-5α-cholest-8(14)-en-15-one, to alter the mRNA levels of 3-hydroxy-3-methylglutaryl-CoA reductase, low density lipoprotein receptor, and oxysterol binding protein was studied and compared with the effects of 25-hydroxycholesterol in Hep G2 cells. All three oxysterols decreased the level of HMG CoA reductase mRNA at concentrations of 10–30 μM, although 25-hydroxycholesterol was effective at concentrations of 1–3 μM. 25-Hydroxycholesterol lowered the level of LDL receptor mRNA more efficiently after 8 hours than after 24 hours of incubation, whereas 15-ketosterols did not decrease the mRNA level of the LDL receptor. The transcriptions of HMG CoA reductase and LDL receptor genes are therefore independently regulated by 15-ketosterols in Hep G2 cells. In addition, the level of the oxysterol binding protein mRNA is not affected by oxysterols in Hep G2 cells.

Effects of fibrillar C-terminal fragment of cleaved alpha1-antitrypsin on cholesterol homeostasis in HepG2 cells.

Amyloid fibrils of diverse origin are known to disturb vital cellular functions and induce cell death. In this study, the effects of amyloid fibrils from the C-terminal fragment (C-36) of cleaved alpha1-antitrypsin (AAT) on low-density lipoprotein (LDL) metabolism were investigated in HepG2 cells. Treatment of the cells with C-36 fibrils (10 micromol/L) enhanced 125I-LDL binding and uptake 10 to 15 times, and highly up-regulated levels of LDL receptor mRNA, as compared with control cells. Competition experiments using excess of unlabeled LDL and blockage experiments with a monoclonal LDL receptor antibody diminished or completely abolished the stimulatory effects of fibrils on LDL binding and LDL receptor mRNA levels, suggesting that fibrils act via the LDL receptor pathway. However, C-36 fibrils had no significant effect on [2-14C]acetate incorporation into cholesterol biosynthesis and cholesterol ester formation, but inhibited 125I-LDL degradation by 20% and reduced bile acid biosynthesis up to 48% in a dose-dependent manner. Preincubation of the cells with fibrils before the addition of LDL totally abolished the LDL inhibitory effect on unesterified cholesterol synthesis, further confirming the LDL receptors to be the target for C-36 fibrils. Moreover, the expression of sterol regulatory element binding protein-1 (SREBP-1) was found to increase twofold and more after 24 hours of incubation of the cells with several concentrations of C-36 fibrils. Our study suggests that the cytotoxicity of C-36 fibrils on HepG2 cells is associated with perturbed intracellular cholesterol homeostasis, induced through fibril-stimulated expression of the LDL receptors via the sterol-responsive element.

Effect of dietary cholesterol on low density lipoprotein-receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and low density lipoprotein receptor-related protein mRNA expression in healthy humans.

We investigated the possibility that dietary cholesterol downregulates the expression of low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase genes of circulating mononuclear cells in vivo in healthy humans. We also studied the variations of the LDL receptor-related protein (LRP) gene in the same conditions. Dieters (n = 5) were submitted to a 4-d fat restriction (mean cholesterol intake: 6+/-4 mg/d), followed by a 7-d cholesterol (a mean of 791+/-150 mg/d) supplementation. Controls (n = 3) did not change their diet. During fat restriction, serum total and LDL cholesterol decreased significantly (P < 0.05), and LDL receptor and HMG-CoA reductase mRNA copy numbers in mononuclear cells increased by 57 and 147%, respectively (P < 0.05). After reintroducing cholesterol, serum cholesterol was stable whereas LDL receptor and HMG-CoA reductase mRNA decreased by 46 and 72% (P < 0.05) and LRP mRNA increased by 59% (P < 0.005). The changes in LDL receptor and HMG-CoA reductase mRNA abundance were correlated (r = +0.79, P = 0.02) during cholesterol reintroduction as were LDL receptor and LRP mRNA levels, but negatively (r = -0.70, P = 0.05). Also, 70% of the variability in LRP mRNA (P < 0.005) was explained by dietary cholesterol. Thus, the basic mechanisms regulating cellular cholesterol content, the coordinate feedback repression of genes governing the synthesis and uptake of cholesterol, are operating in vivo in humans. However, serum cholesterol did not increase in response to dietary cholesterol, suggesting that these mechanisms may not play as predominant a role as previously believed in the short-term control of serum cholesterol in vivo in humans. A new finding is that LRP gene is also sensitive to dietary cholesterol, suggesting that it may participate in the control of serum cholesterol. Further in vivo studies in humans are warranted to explore the molecular mechanisms of the physiological response to dietary cholesterol in humans.

Comparative effects of short- and long-term feeding of safflower oil and perilla oil on lipid metabolism in rats

Diets high in linoleic acid (20% safflower oil contained 77.3% linoleic acid, SO-diet) and α-linolenic acid (20% perilla oil contained 58.4% α-linolenic acid, PO-diet) were fed to rats for 3, 7, 20, and 50 days, and effects of the diets on lipid metabolism were compared. Levels of serum total cholesterol and phospholipids in the rats fed the PO-diet were markedly lower than those fed the SO-diet after the seventh day. In serum and hepatic phosphatidylcholine and phosphatidylethanolamine, the proportion of n-3 fatty acids showed a greater increase in the PO group than in the SO group in the respective feeding-term. At the third and seventh days after the commencement of feeding the experimental diets, expressions of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA were significantly higher in the SO group than those in the PO group, although the difference was not observed in the longer term. There were no significant differences in the LDL receptor mRNA levels between the two groups through the experimental term, except 3-days feeding. These results indicate that α-linolenic acid has a more potent serum cholesterol-lowering ability than linoleic acid both in short and long feeding-terms.

Sterol-independent, sterol response element-dependent, regulation of low density lipoprotein receptor gene expression

Stimulation with phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin increased native low density lipoprotein (LDL) receptor gene expression in the human leukemic T cell line Jurkat when cells were cultured in the absence of sterols and also increased nuclear accumulation of sterol regulatory element binding protein (SREBP)-1. PMA and ionomycin likewise increased LDL receptor mRNA levels when cells were cultured in the presence of suppressive concentrations of sterols, when neither SREBP-1 nor SREBP-2 was detectable in the nucleus. These findings indicated that mitogen-induced up-regulation of the LDL receptor gene could be independent of sterol-regulated transcription factors. The involvement of sterol regulatory element (SRE)-1 was analyzed by transfection of LDL receptor promoter constructs. Promoter fragments of either the 5′ 1472 or 142 base pairs induced reporter gene expression after mitogenic stimulation when cells were cultured in the absence or presence of sterols. Mutation of the SRE-1 sequence in either construct abolished sterol-mediated regulation of transcription. However, mutation of the SRE-1 sequence in the 1472 base pair promoter fragment did not alter mitogenic induction of transcription, whereas mutation of SRE-1 in the 142 base pair promoter fragment completely prevented up-regulation of transcription.▪ Taken together, these results demonstrate that the LDL receptor promoter contains at least one 5′ SRE-independent as well as an SRE-dependent response element. Furthermore, the data suggest that the SRE-dependent response may not involve the action of either SREBP-1 or –2. Thus, mitogen-induced transcription of the LDL receptor promoter is regulated by diverse sterol-independent mechanisms.—Makar, R. S. J., P. E. Lipsky, and J. A. Cuthbert. Sterol-independent, sterol response element-dependent, regulation of low density lipoprotein receptor gene expression.

Gemfibrozil reduces non-high-density lipoprotein cholesterol in exogenously hypercholesterolemic (ExHC) rats fed a high-cholesterol diet

Gemfibrozil is a widely used drug prescribed to elevate serum high-density lipoprotein (HDL) cholesterol levels and lower triacylglycerols. The present study was done to determine if the drug would alleviate hypercholesterolemia in exogenously hypercholesterolemic (ExHC) rats. In the drug-treated ExHC rats, the serum non-HDL cholesterol levels were lowered and the ratio of the non HDL cholesterol to serum triacylglycerols was decreased to the extent seen in the drug-treated SD rats. Liver cholesterol and triacylglycerols were lowered in the drug-treated rats. There was also an increase in fecal excretion of neutral sterols and bile acids, particularly chenodeoxycholic and β-muricholic acids. The drug elevated cholesterol 7 α-hydroxylase activity and mRNA abundance and acyl-CoA cholesterol acyltransferase activity in the liver, but did not influence low-density lipoprotein receptor mRNA level in the liver. Thus, gemfibrozil is effective in alleviating hypercholesterolemia in exogenously hypercholesterolemic rats, by partitioning hepatic cholesterol into biliary excretion.