HMG-CoA Reductase Gene Expression Research Articles

Artemisinic acid inhibits melanogenesis through downregulation of C/EBP α-dependent expression of HMG-CoA reductase gene

Cholesterol is associated with the regulation of melanogenesis which is the major physiological defense against solar irradiation. The present study was designed to determine the effects of artemisinic acid on melanogenesis and its mechanisms of action in human epidermal melanocytes. In this study, we found that artemisinic acid inhibited melanin content. The mRNA levels of microphthalmia-associated transcription factor (MITF) and its downstream genes tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 were reduced by artemisinic acid treatment. Additionally, the mRNA levels of melanogenesis-related genes (c-KIT, stem cell factor (SCF), and macrophage migration inhibitory factor (MIF)) were down-regulated by artemisinic acid. Furthermore, cAMP production and protein kinase A (PKA) activity were suppressed by artemisinic acid. Moreover, attempts to elucidate a possible mechanism underlying the artemisinic acid-mediated effects revealed that artemisinic acid regulated melanogenesis by inhibiting cholesterol synthesis through downregulation of the hydroxymethylglutaryl CoA (HMG CoA) reductase gene, which was mediated through reduced expression of the CCAAT/enhancer-binding protein (C/EBP) α gene.Taken together, these findings indicate that the inhibition of melanogenesis by artemisinic acid occurs through reduced expression of the HMG CoA reductase gene, which is mediated by C/EBP α inhibition and suggest that artemisinic acid may be useful as a hyperpigmentation inhibitor.

Brain Isoprenoids Farnesyl Pyrophosphate and Geranylgeranyl Pyrophosphate are Increased in Aged Mice

The mevalonate/isoprenoids/cholesterol pathway has a fundamental role in the brain. Increasing age could be associated with specific changes in mevalonate downstream products. Other than age differences in brain cholesterol and dolichol levels, there has been little if any evidence on the short-chain isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), as well as downstream lipid products. The purpose of the present study was to determine whether brain levels of FPP, GGPP and sterol precursors and metabolites would be altered in aged mice (23 months) as compared to middle-aged mice (12 months) and young mice (3 months). FPP and GGPP levels were found to be significantly higher in brain homogenates of 23-months-old mice. The ratio of FPP to GGPP did not differ among the three age groups suggesting that increasing age does not alter the relative distribution of the two isoprenoids. Gene expression of FPP synthase and GGPP synthase did not differ among the three age groups. Gene expression of HMG-CoA reductase was significantly increased with age but in contrast gene expression of squalene synthase was reduced with increasing age. Levels of squalene, lanosterol and lathosterol did not differ among the three age groups. Desmosterol and 7-dehydroxycholesterol, which are direct precursors in the final step of cholesterol biosynthesis were significantly lower in brains of aged mice. Levels of cholesterol and its metabolites 24S- and 25S-hydroxycholesterol were similar in all three age groups. Our novel find ings on increased FPP and GGPP levels in brains of aged mice may impact on protein prenylation and contribute to neuronal dysfunction observed in aging and certain neurodegenerative diseases.

Blueberry anthocyanins at doses of 0.5 and 1 % lowered plasma cholesterol by increasing fecal excretion of acidic and neutral sterols in hamsters fed a cholesterol-enriched diet

The present study investigated the underlying mechanism associated with the hypocholesterolemic activity of blueberry anthocyanins by examining its effect on fecal sterol excretion and gene expression of major receptors, enzymes, and transporters involved in cholesterol metabolism. Hamsters were divided into three groups and fed a 0.1 % cholesterol diet containing 0 % (CTL), 0.5 % (BL), and 1.0 % (BH) blueberry anthocyanins, respectively, for six weeks. Plasma total cholesterol (TC), triacylglycerols (TAG), and non-high-density lipoproteins cholesterol (non-HDL-C) were measured using the enzymatic kits, and the gene expression of transporters, enzymes, and receptors involved in cholesterol absorption and metabolism was quantified using the quantitative PCR. GC analysis was used to quantify hepatic cholesterol and fecal acidic and neutral sterols. Dietary supplementation of 0.5 and 1.0 % blueberry anthocyanins for 6 weeks decreased plasma TC concentration by 6-12 % in a dose-dependent manner. This was accompanied by increasing the excretion of fecal neutral and acidic sterols by 22-29 % and 41-74 %, respectively. Real-time PCR analyses demonstrated that incorporation of blueberry anthocyanins into diet down-regulated the genes of NPC1L1, ACAT-2, MTP, and ABCG 8. In addition, blueberry anthocyanins were also able to down-regulate the gene expression of hepatic HMG-CoA reductase. The cholesterol-lowering activity of blueberry anthocyanins was most likely mediated by enhancing the excretion of sterols accompanied with down-regulation on gene expression of intestinal NPC1L1, ACAT-2, MTP, and ABCG 8.

Linalool reduces the expression of 3-hydroxy-3-methylglutaryl CoA reductase via sterol regulatory element binding protein-2- and ubiquitin-dependent mechanisms

We investigated hypocholesterolemic mechanisms of linalool, an aromatic anti-oxidative monoterpene, which is abundant in teas and essential oils. Oral administration of linalool to mice for 6weeks significantly lowered total and low-density lipoprotein cholesterol concentrations, and HMG-CoA reductase protein expression (−46%; P<0.05) by both transcriptional and posttranscriptional mechanisms. Linalool suppressed the gene expression of HMG-CoA reductase by reducing the binding of SREBP-2 to its promoter, as assessed by qPCR and chromatin immunoprecipitation, and by inducing ubiquitin-dependent proteolysis of the HMG-CoA reductase. These findings suggest that food molecules with a pleasant scent could exert beneficial metabolic effects through multiple mechanisms.

Study of directed insilico mutations on polyphenol oxidase to improve enzyme–substrate binding efficiency

7S protein is one of four major storage proteins in soybean. The present study compared the relative cholesterol-lowering activity of the 7S protein with that of total soybean protein isolate (SPI). Forty-eight hamsters were divided into six groups fed non-cholesterol diet or one of five high-cholesterol diets containing 24.2% casein, 12.1–24.2% SPI, 12.1–24.2% 7S protein, respectively. Results showed the 7S protein reduced plasma total cholesterol (TC) by 20–41% while SPI reduced it by 9–16%. 7S protein increased the fecal excretion of neutral sterols by 43–116%, while SPI only had 18–68% increase. 7S protein increased the excretion of total bile acids by 208–367%, while SPI only increased it by 41–86%. 7S protein could not only down regulate gene expression of HMG-CoA reductase but also up regulate gene expression of CYP7A1. It was concluded that dietary 7S protein was more hypocholesterolemic than SPI, mediated by increasing the sterol excretion and decreasing the cholesterol synthesis.

Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cell steroidogenesis

We have previously reported that simvastatin (SIM), a competitive inhibitor of HMG-CoA reductase, a rate-limiting step of the mevalonate pathway, inhibits rat theca-interstitial cell steroidogenesis by decreasing CYP17A1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol (RES), a bioflavonoid abundant in red grapes, decreases HMG-CoA reductase gene expression in rat theca-interstitial cells and may enhance the inhibitory effect of simvastatin on the mevalonate pathway. The present study evaluated the effect of combining RES and SIM treatments on rat theca-interstitial cell steroidogenesis. In-vitro study. Rat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 hrs in the absence (control) or in the presence of SIM (1 μM) and/or RES (10-30 μM). Gene expression of steroidogenic enzymes (CYP11A1, CYP17A1) was evaluated by qrt-PCR. Androstenedione and androsterone levels in culture media were determined using liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test. SIM decreased androstenedione levels in culture media by 92% (P<0.001) and androsterone production by 60% (P<0.001), whereas the addition of RES to SIM-treated cultures decreased androstenedione levels by 79-88% (P<0.001) and androsterone production by 82-91% (P<0.001), respectively. SIM inhibited both CYP11A1 and CYP17A1 gene expression, respectively, by 24% (P<0.001) and 87% (P<0.001). However, RES in combination with SIM decreased both CYP11A1 and CYP17A1 gene expression, respectively, by 55-72% (P<0.001) and 95-99% (P<0.001). SIM inhibits androgen production in theca-interstitial cells, and this SIM-induced decrease in steroidogenesis is enhanced by the addition of RES. The present findings may lead to the development of novel treatments of PCOS involving a combination of SIM and RES.

Design, synthesis and biological study of pinacolyl boronate-substituted stilbenes as novel lipogenic inhibitors

A pilot library of novel 4,4,5,5-tetramethyl-2-(4-substitutedstyrylphenyl)-1,3,2 dioxaborolane derivatives has been synthesized. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaboratophenyl)-methyl triphenylphosphonium bromide 3 was treated with various aldehydes in the presence of 3 equiv of (t)BuONa in DMF, and stirred at room temperature for 4-6h to yield the corresponding boron-containing stilbene derivatives in 71-94% yields. Several of them, including BF102 and BF175, have the lipogenesis inhibitory effect by suppressing lipogenic gene expression in mammalian hepatocytes. Further, BF102 also inhibits cholesterol biosynthesis by suppressing HMG-CoA reductase gene expression in hepatocytes. Interestingly, our preliminary in vivo data suggests that BF102 has no significant toxicity in mice at the highest possible dose we can administered. Thus, BF102 is a potential lead for the next generation of lipid-lowering drugs.

A new TCM formula FTZ lowers serum cholesterol by regulating HMG-CoA reductase and CYP7A1 in hyperlipidemic rats

Based on a theory of Chinese Medicine, Regulating Gan (liver) to lower lipids that is in brief to regulate the lipid metabolic related factors in the liver will improve serum lipid profile, we have developed Fufang Zhenzhu Tiao Zhi (FTZ) which includes eight herbs that are quality assured. FTZ has been developed with the potential to correct abnormal lipid metabolism. This Chinese herbal medicine has been prescribed for 20 years, which has been issued patent and clinically proven for use in the treatment of dyslipidemia. To investigate the cholesterol-lowering effect and the mode of action of FTZ extract on high lipid diet induced hyperlipidemic rats. The FTZ was prepared by alcohol and water extraction of eight herbs that have been quality-controlled according to the protocol. The cholesterol-lowering effect of FTZ was evaluated on SD rats fed with high-lipid diet. RT-PCR and western blot were used to analyze the gene expression of cholesterol metabolism-related enzymes including HMG-CoA reductase and cholesterol 7α-hydroxylase (CYP7A1) in the livers of the rats. The activity of HMG-CoA reductase and CYP7A1 were assessed by colorimetrical method and by quantification of the cholesterol metabolite of CYP7A1 using HPLC analysis respectively. FTZ significantly decreased the levels of serum total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C), whilst elevated the serum high-density lipoprotein cholesterol (HDL-C) and decreased serum atherogenic index (A.I.) values in high lipid diet induced hyperlipidemic rats. Furthermore, FTZ showed significant antihyperlipidemic effect by at least three pathways in the high lipid diet induced hyperlipidemic rats: (1) upregulating the gene expression and activity of CYP7A1 which promotes the conversion of cholesterol into bile acid; (2) downregulating the gene expression and activity of HMG-CoA reductase to reduce de novo synthesis of cholesterol; (3) increasing the cholesterol excretion from feces. In these three pathways, HMG-CoA reductase and CYP7A1 are two pivotal enzymes in lipid cholesterol metabolism and are expressed mainly in hepatic cells, which support our new TCM treatment strategy: Modulating Liver to Treat Hyperlipemia.

Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5′-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure.

Synergic effect of Eicosapentaenoic acid and Lovastatin on gene expression of HMGCoA reductase and LDL receptor in cultured HepG2 cells

BackgroundPUFAs are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an enzyme catalyzing the conversion of HMGCoA to mevalonate, the rate limiting step in cholesterol biosynthesis. Statins represent a class of drugs that are widely used to treat hypercholesterolemia for their ability to inhibit cholesterol biosynthesis and to up-regulate the synthesis of Low Density Lipoprotein (LDL) receptors in the liver. PUFAs mediate many, if not all, actions of statins and this could be one mechanism by which they lower cholesterol levels. The purpose of this study was to investigate whether combined treatment with Eicosapentaenoic acid (EPA) and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line.ResultsThe combined treatment with EPA and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line. Moreover, we detected a synergistic effect on the inhibition of cancer cell proliferation obtained by combination of EPA and Lovastatin.ConclusionsThe use of EPA, in combination with low doses of Lovastatin may have potential value in treatment of neoplastic diseases.

Rapamycin down-regulates LDL-receptor expression independently of SREBP-2

As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffected by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2.

Early induction of LDL receptor gene expression by genistein in DLD-1 colon cancer cell line

The phytoestrogen genistein has been demonstrated to possess anti-tumor properties by mechanisms not yet clearly established. The present study was designed to investigate the effects of isoflavone genistein exposure at concentrations ranging from 0.01 microM to 50 microM on the LDL receptor and HMGCoA reductase gene expression in the estrogen receptor positive DLD-1 human colon cancer cell line. LDL receptor and HMGCoA reductase gene expressions were evaluated by reverse transcription followed by real-time PCR. Genistein induced an early increase of LDL receptor gene expression and later decreased HMGCoA reductase mRNA expression in DLD-1 cells. These findings provide direct evidence on the role of genistein in regulating LDL receptor and HMGCoA reductase gene expression in colon cancer.

Effect of genistein on cholesterol metabolism-related genes in a colon cancer cell line

The major soy-derived isoflavones such as genistein has been demonstrated to possess anticarcinogenic activity in animal model systems. The present study was designed to investigate the effects of isoflavone genistein exposure at concentrations ranging from 0.01 to 50 muM on the LDL receptor and HMG-CoA reductase gene expression in the estrogen receptor positive DLD-1 human colon cancer cell line. LDL receptor and HMG-CoA reductase gene expressions were evaluated by reverse transcription followed by real-time PCR. Genistein induced an increase of LDL receptor gene expression and later decrease of HMG-CoA reductase mRNA expression in DLD-1 cells. These findings provide direct evidence on the role of genistein in regulating LDL receptor and HMG-CoA reductase gene expression in colon cancer.

Policosanol fails to lower HMG‐CoA reductase activity in HepG2 cells

The cholesterol‐lowering properties of policosanol are controversial. One research group in Cuba have reported that policosanol intake of 5–10 mg/day in humans can lower plasma LDL cholesterol up to 25%, although other laboratories have failed to repeat these findings. Moreover, the cellular/molecular effects of policosanol are uncertain. Using HepG2 cells, we assessed the regulation of HMG‐CoA reductase (HMGR) activity and gene expression by incubating cells for 1.5, 3.0 or 4.5 hrs with bile salt micelles (taurocholate, lecithin) with and without policosanol. Policosanol uptake into HepG2 cells was confirmed by gas chromatography, demonstrating the efficacy of our micelle delivery system. Our results indicated no significant differences at any time point between control and policosanol treated cells; however, after 4.5 hrs of incubation, HMGR activity was significantly increased in both control and policosanol treated cells, suggesting that prolonged exposure to micelles may promote cholesterol depletion in HepG2 cells. We also quantified mRNA gene expression of HMGR and its transcription factor SREBP2. HMGR mRNA abundance was determined by real‐time PCR and indicated no difference in gene expression after 3 hrs. We conclude that policosanol has little or no effect on HMGR activity or gene expression in HepG2 cells. [Supported by USDA‐NRI Competitive Grant No. 2004‐35503‐14824]

Endogenous 24(S),25-Epoxycholesterol Fine-tunes Acute Control of Cellular Cholesterol Homeostasis

Certain oxysterols, when added to cultured cells, are potent regulators of cholesterol homeostasis, decreasing cholesterol synthesis and uptake and increasing cholesterol efflux. However, very little is known about whether or not endogenous oxysterol(s) plays a significant role in cholesterol homeostasis. 24(S),25-Epoxycholesterol (24,25EC) is unique among oxysterols in that it is produced in a shunt of the mevalonate pathway which also produces cholesterol. We investigated the role of endogenously produced 24,25EC using a novel strategy of overexpressing the enzyme 2,3-oxidosqualene cyclase in Chinese hamster ovary cells to selectively inhibit the synthesis of this oxysterol. First, loss of 24,25EC decreased expression of the LXR target gene, ABCA1, substantiating its role as an endogenous ligand for LXR. Second, loss of 24,25EC increased acute cholesterol synthesis, which was rationalized by a concomitant increase in HMG-CoA reductase gene expression at the level of SREBP-2 processing. Therefore, in the absence of 24,25EC, fine-tuning of the acute regulation of cholesterol homeostasis is lost, supporting the hypothesis that 24,25EC functions to protect the cell against the accumulation of newly synthesized cholesterol.

Carbohydrate restriction and dietary cholesterol modulate the expression of HMG-CoA reductase and the LDL receptor in mononuclear cells from adult men

The liver is responsible for controlling cholesterol homeostasis in the body. HMG-CoA reductase and the LDL receptor (LDL-r) are involved in this regulation and are also ubiquitously expressed in all major tissues. We have previously shown in guinea pigs that there is a correlation in gene expression of HMG-CoA reductase and the LDL-r between liver and mononuclear cells. The present study evaluated human mononuclear cells as a surrogate for hepatic expression of these genes. The purpose was to evaluate the effect of dietary carbohydrate restriction with low and high cholesterol content on HMG-CoA reductase and LDL-r mRNA expression in mononuclear cells. All subjects were counseled to consume a carbohydrate restricted diet with 10–15% energy from carbohydrate, 30–35% energy from protein and 55–60% energy from fat. Subjects were randomly assigned to either EGG (640 mg/d additional dietary cholesterol) or SUB groups [equivalent amount of egg substitute (0 dietary cholesterol contributions) per day] for 12 weeks. At the end of the intervention, there were no changes in plasma total or LDL cholesterol (LDL-C) compared to baseline (P > 0.10) or differences in plasma total or LDL-C between groups. The mRNA abundance for HMG-CoA reductase and LDL-r were measured in mononuclear cells using real time PCR. The EGG group showed a significant decrease in HMG-CoA reductase mRNA (1.98 ± 1.26 to 1.32 ± 0.92 arbitrary units P < 0.05) while an increase was observed for the SUB group (1.13 ± 0.52 to 1.69 ± 1.61 arbitrary units P < 0.05). Additionally, the LDL-r mRNA abundance was decreased in the EGG group (1.72 ± 0.69 to 1.24 ± 0.55 arbitrary units P < 0.05) and significantly increased in the SUB group (1.00 ± 0.60 to 1.67 ± 1.94 arbitrary units P < 0.05). The findings indicate that dietary cholesterol during a weight loss intervention alters the expression of genes regulating cholesterol homeostasis.

Effects of ezetimibe and/or simvastatin on LDL receptor protein expression and on LDL receptor and HMG-CoA reductase gene expression: A randomized trial in healthy men

Objective The combination of simvastatin, an HMG-CoA reductase inhibitor, and ezetimibe, an inhibitor of Niemann-Pick C1-like 1 protein, decreases cholesterol synthesis and absorption and reduces circulating LDL-cholesterol concentrations. The molecular mechanisms underlying the pronounced lipid-lowering effects of this combination have not been fully elucidated in humans. Methods and results One center, prospective, randomized, parallel three-group study in 72 healthy men (mean age 32 ± 9 years, mean body mass index 25.7 ± 3.2 kg/m 2). Each group of twenty-four subjects received a 14-day treatment with either ezetimibe (10 mg/day), simvastatin (40 mg/day) or their combination. Lipid levels, the ratio of non-cholesterol sterols to cholesterol concentrations (used as markers of cholesterol synthesis and absorption), cell surface LDL receptor (LDLR) protein as well as LDLR and HMG-CoA reductase gene expression in mononuclear blood cells were measured at baseline and at the end of the study. LDL-C decreased in all groups. Simvastatin decreased, ezetimibe increased and their combination had no effect on HMG-CoA reductase activity. Simvastatin and the combination of ezetimibe and simvastatin increased the HMG-CoA reductase and LDLR gene expression while ezetimibe had no effect. The cell surface LDLR protein expression remained unchanged in all groups. The combination of ezetimibe and simvastatin increased the expression of the serine protease proprotein convertase subtilisin/kexin 9 (PCSK9), an enzyme shown to down-regulate LDLR protein levels. Conclusions The co-administration of ezetimibe and simvastatin abrogates the ezetimibe-induced increase in cholesterol synthesis and up-regulates the LDLR gene but not protein expression, an effect possibly mediated through a parallel upregulation of PCSK9 expression.

Stearic acid reduces gene expression in FHs 74 Int and HepG2 cells

Dietary stearic acid (18:0), in contrast to other saturated fatty acids, has cholesterol-lowering or neutral effects on plasma LDL and liver cholesterol concentration. Several mechanisms of action have been suggested, including reduced cholesterol absorption, increased bile acid excretion, and increased endogenous cholesterol excretion. We therefore hypothesized that several regulatory genes in liver and small intestine may be responsive to 18:0. Using human intestinal cell line FHs 74 Int and liver-derived HepG2 cells, we measured gene expression of Niemann-Pick C1-Like 1 (NPC1L1), HMG-CoA reductase (HMGR), acyl-CoA:cholesterol acyltransferase (ACAT2), ABCG5 sterol transporter (ABCG5), and LDL receptor (LDLR). Cells were incubated 24 h with 100 μM of 18:0 or palmitic acid (16:0) complexed to BSA, and mRNA was quantified by real time PCR. Results indicate: these genes are responsive to fatty acids in both FHs 74 Int and HepG2 cells, FHs 74 Int cells treated with 18:0 relative to 16:0 had significantly lower NPC1L1, HMGR, ABCG5 and ACAT2 gene expression, HepG2 cells treated with 18:0 relative to 16:0 had significantly lower HMGR gene expression. We conclude that the hypocholesterolemic effects of 18:0 are due to its ability to down-regulate genes involved in cholesterol transport and synthesis. (Supported by NSF EPSCoR grant EPS-0346476)

Policosanol reduces HMG‐CoA reductase mRNA in HepG2 cells

Policosanol is a natural food component that is believed to lower plasma LDL cholesterol by inhibiting HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. The cellular and molecular mechanisms of regulation by policosanol have not been delineated. Moreover, virtually all published studies supporting the beneficial effects of policosanol have been published by a single research group in Cuba, whereas others have failed to reproduce these findings. We hypothesize that inconsistencies are related to policosanol insolubility and the choice of appropriate delivery systems to cells. Therefore, we tested the following methods of solubilizing policosanol for delivery to HepG2 cells: dissolved in ethanol, associated with cyclodextrin, dissolved in intralipid, or incorporated in micelles. Of the methods tested, micelles proved most successful. Cells were incubated 24 h with 0.24 mM policosanol in micelles (bile salts, lecithin, and cholesterol), resulting in decreased expression of HMGR and SREBP2 mRNA. We conclude that, with an appropriate delivery system policosanol inhibits cholesterol synthesis by decreasing gene expression of HMGR. (Supported by USDA-NRI Competitive Grant No. 2004-35503-14824)

Cyclic fluctuations of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase in aortic smooth muscle cell cultures

The cyclic fluctuations of HMG-CoA reductase activity and mRNA are reportedly related to feeding the cells in culture or to variations in food consumption by the animals over a 24-h cycle. In this work, we demonstrate cyclic increments in HMG-CoA reductase activity in smooth muscle cells (SMC) not associated with the culture feeding. Since reductase activity also shows a marked rise preceding the S phase, one of the major goals of the present work was to evaluate this dual role of reductase activity and mRNA fluctuations related to the cell cycle and to food intake in the SMC-C/SMC-Ch cultures derived from control-fed (SMC-C) and cholesterol-fed (SMC-Ch) chicks. The period and amplitude oscillations in HMG-CoA reductase activity varied depending on culture conditions: lipoprotein-deficient serum vs. FBS, young vs. senescent cells, or confluent vs. nonconfluent cultures. The HMG-CoA reductase mRNA concentration showed a marked rise after feeding not correlated to the fluctuation activity, suggesting posttranscriptional modulation. Reductase activity and mRNA were down-regulated in SMC-Ch. Since the nutritional culture conditions were the same in both cell lines, these findings indicate that consumption of a high-cholesterol diet by the animals prior to the establishment of the SMC cultures induced changes in the HMG-CoA reductase gene expression in-aortic SMC.