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© 2008 American Society for Nutrition J. Nutr. 138:476-481, March 2008

Biochemical, Molecular, and Genetic Mechanisms

Lipid Extract of Nostoc commune var. sphaeroides Kützing, a Blue-Green Alga, Inhibits the Activation of Sterol Regulatory Element Binding Proteins in HepG2 Cells1–3,

Heather E. Rasmussen4, Kara R. Blobaum4, Young-Ki Park4, Sarah J. Ehlers4, Fan Lu5 and Ji-Young Lee4,*

4 Departments of Nutrition and Health Sciences, University of Nebraska, Lincoln, NE 68583 and 5 Algaen Incorporation, Winston-Salem, NC 27101

* To whom correspondence should be addressed. E-mail: jlee8{at}unl.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Experimental Procedures
 Results
 Discussion
 LITERATURE CITED
 
Nostoc commune var. sphaeroides Kützing (N. commune), a blue-green alga, has been used as both a food ingredient and in medicine for centuries. To determine the effect of N. commune on cholesterol metabolism, N. commune lipid extract was incubated at increasing concentrations (25–100 mg/L) with HepG2 cells, a human hepatoma cell line. The addition of N. commune lipid extract markedly reduced mRNA abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and LDL receptor (LDLR) (P < 0.05), with a concomitant decrease in their protein expression (P < 0.001). Reduced HMGR activity by 90% with N. commune lipid extract confirmed the inhibitory role of N. commune in cholesterol synthesis (P < 0.006). To elucidate a molecular mechanism underlying the repression of HMGR and LDLR by N. commune lipid extract, expression of sterol regulatory element binding protein 2 (SREBP-2) was assessed. Whereas mRNA for SREBP-2 remained unchanged, SREBP-2 mature protein was reduced by N. commune (P < 0.009). In addition, N. commune lipid extract also decreased SREBP-1 mature protein by ~30% (P < 0.002) and reduced the expression of SREBP-1-responsive genes such as fatty acid synthase and stearoyl CoA desaturase 1 (SCD-1) (P < 0.05). Therefore, our results demonstrate that N. commune lipid extract inhibits the maturation process of both SREBP-1 and -2, resulting in a decrease in expression of genes involved in cholesterol and fatty acid metabolism.


   Introduction
TOP
 ABSTRACT
 Introduction
Experimental Procedures
 Results
 Discussion
 LITERATURE CITED
 
Coronary heart diseases (CHD)6 are the leading cause of death in the United States and other Westernized countries. A direct correlation exists between the concentration of plasma cholesterol and the severity of atherosclerosis (1). According to the 2001 report of the National Cholesterol Education Program, an expert panel estimated that 65 million U.S. adults should be recommended to have therapeutic lifestyle changes, including diet, and 36 million of that population also need drug therapy for treatment of elevated cholesterol levels (2). Currently, statin drugs are widely prescribed to lower plasma cholesterol concentration. Statins primarily reduce plasma cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the cholesterol biosynthetic pathway. Although statins have proven to be effective at lowering plasma cholesterol level, potential adverse effects may occur (1,3).

Increased public's interest in natural and herbal medicine has prompted the identification of natural products with heart protection. Several herbal and functional food supplements have been used for primary prevention of heart disease. Consumption of garlic (4), soy protein (5), and artichoke (6) has been reported to moderately decrease plasma cholesterol levels. In addition, soluble fibers from barley, beans, oat bran, and psyllium reduced plasma cholesterol concentration when consumed by healthy hyperlipidemic patients on a low-fat diet (7). To meet the public's demand for natural foods for the prevention of CHD, continuous efforts need to be made to discover and develop new bioactive natural products.

Blue-green algae (BGA) are among the most primitive life forms on earth (8). Edible BGA, such as Spirulina and Aphanizomenon flos-aquae, are currently marketed as dietary supplements with various health claims for immune function, inflammation, and heart disease. Spirulina is known to be rich in vitamins, minerals, essential and nonessential amino acids, trace elements, and essential fatty acids (9). Currently, Spirulina is harvested from controlled ponds in dozens of countries and is being sold as an organic dietary supplement. In contrast to Spirulina, most of A. flos-aquae products are naturally harvested from the Upper Klamath Lake in southern Oregon and therefore it is called "a natural dietary supplement." Although the term "natural" could attract consumers, concerns over naturally grown BGA have been raised, because they are easily contaminated with Microcystis aeruginosa, which generates potent hepatotoxin microcystins, as well as with heavy metals (10). In response to this concern, Health Canada performed broad sampling of BGA supplements available on the Canadian market in May 1999 to test their microcystin contents. The survey revealed that only Spirulina has no detection of microcystins and many non-Spirulina BGA products harvested from natural lakes contain microcystins above levels considered acceptable by Health Canada and WHO. In addition, the quality of naturally grown BGA can vary depending on their growing environments, e.g. altitude, temperature, and sun exposure (11).

Nostoc commune var. sphaeroides Kützing (N. commune) is an edible BGA that has been used as a food delicacy as well as an herbal medicine in countries to which it is native for thousands of years. It has been historically suggested that N. commune can treat a variety of medical conditions, including inflammation, night blindness, burns, anxiety, and chronic fatigue. Studies have also demonstrated various health benefits from N. commune, including antiviral and anticancer activities (12–14). Importantly, a cholesterol-lowering effect of N. commune was reported in rats fed a high-cholesterol diet and the effect was attributed to its high-fiber content (15).

Although previous research has shown N. commune confers health benefits, the investigation into the mechanisms behind the biological effects have been limited. In this study, we investigated the ability of N. commune lipid extract to influence key regulators of both cholesterol and fatty acid metabolism in HepG2 cells, a human hepatoma cell line. Because sterol regulatory element binding proteins (SREBP) are transcriptional factors that play a central role in regulating both cholesterol and fatty acid metabolism (11,16), the effect of N. commune on the expression as well as maturation of SREBP was investigated.


   Experimental Procedures
TOP
 ABSTRACT
 Introduction
 Experimental Procedures
Results
 Discussion
 LITERATURE CITED
 
    Preparation of Nostoc lipid extract. Fresh N. commune was obtained from Algaen Incorporation. N. commune was lyophilized and ground for lipid extraction by the Bligh-Dyer method (17). Briefly, 42 mL of chloroform:methanol (1:2, v:v) was added to ~3 g of dry N. commune and vortexed. After the addition of 14 mL of chloroform, the sample was vortexed and filtered to remove solids. Subsequently, 6.3 mL of 0.05% H2SO4 was added and vortexed. To separate phases, the sample was centrifuged at 500 x g; 5 min and the lower liquid phase was recovered. Tubes containing lipid extract in chloroform were purged with N2 and tightly sealed. The lipid extract was stored at –80°C with the oxygen absorber AGELESS (Mitsubishi Gas Chemical America) to prevent oxidation until use. To incorporate the lipid extract into cell culture medium, chloroform was evaporated under nitrogen and cell culture medium was added for subsequent sonication at room temperature for 5 min.

    Cell culture. HepG2 cells (ATCC) were maintained in minimal essential medium containing 10% fetal bovine serum, 100 kU/L penicillin, 100 mg/L streptomycin, 1x vitamins, and 2 mmol/L L-glutamine in a humidified chamber at 37°C with 5% CO2. All cell culture supplies were purchased from MediaTech. Cells were plated at a density of 3 x 105 cells per well in 12-well plates. When cells reached ~90% confluence, N. commune lipid extract (0–100 mg/L) was added to culture medium for 24 h.

    Cytotoxicity measurement. Cytotoxicity of N. commune lipid extract was determined using Cell Counting Kit-8 (Dojindo Molecular Technologies) according to the manufacturer's instructions. SDS (0.5 mmol/L) was also added as a positive control for cytotoxicity. Data are expressed as cell viability (%) relative to a control that was not incubated with N. commune lipid extract.

    Total RNA isolation and quantitative real-time PCR. HepG2 cells were washed twice with cold PBS and 1 mL of TRIZOL reagent (Invitrogen) was added to each well of 12-well plates for total RNA isolation following the manufacturer's protocol. One microgram of total RNA was treated with DNase I (Promega) to remove any genomic DNA. The RNA samples were reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Promega) for subsequent real-time PCR analysis using the Sybr Green procedure and an ABI 7300 instrument (Applied Biosystems). Primers for HMGR, LDL receptor (LDLR), fatty acid synthase (FAS), stearoyl CoA desaturase 1 (SCD-1), SREBP-1c, SREBP-2, and glyceraldehyde 3-phosphate dehydrogenase were designed according to GenBank database using the Primer Express software provided by ABI. Primer sequences are given in Supplemental Table 1. Expression of mRNA values was calculated using the threshold cycle (Ct) value, i.e. the number of PCR cycles at which the fluorescent signal during the PCR reaches a fixed threshold. For each sample, {Delta}Ct, sample was calculated by subtracting the Ct value of glyceraldehyde 3-phosphate dehydrogenase, a house-keeping gene, from that of the gene of interest to normalize the data. The expression levels relative to control were estimated by calculating {Delta}{Delta}Ct ({Delta}Ct, sample – {Delta}Ct, control) and subsequently using the 2–{Delta}{Delta}Ct method (18).

    Western blot analysis. HepG2 cells were incubated with 100 mg/L N. commune lipid extract for 24 h. After removing cell culture medium, cells were washed twice with cold PBS and scraped in 1 mL of PBS. Cells were transferred into a 1.5-mL microfuge tube and centrifuged at 12,000 x g; 5 min at 4°C. The supernatant was removed and 25 µL of cell lysis buffer (150 mmol/L NaCl, 25 mmol/L Tris-HCl, pH 7.4, 1% Triton X-100) containing Protease Inhibitor Cocktail set III (Calbiochem) was added to the cell pellet. The cells were resuspended and incubated on ice for 20 min, after which the cell lysates were centrifuged at 12,000 x g; 5 min at 4°C. We collected the supernatant and measured protein concentration by BCA assay (Pierce) using bovine serum albumin as a standard.

Fifty micrograms of cell protein was subjected to 4–16% SDS-PAGE for Western blot analysis using antibodies for HMGR (Upstate), LDLR (Abcam), SREBP-1 (Santa Cruz), SREBP-2 (Santa Cruz), and β-actin (Sigma). The blots were developed using a horseradish peroxidase system (Pierce) and densitometry analysis was performed using a Chemidoc XRS (Biorad) and Quantity One software (Biorad). β-Actin was used as a loading control to normalize the data.

    HMGR activity measurement. HMGR activity was measured according to the methods by Brown et al. (19,20) with modification. Cell lysate was prepared as described above after 24 h incubation in the absence (control) or presence of N. commune lipid extract (100 mg/L). Two hundred microliters of reaction mixture was prepared as follows: 100 µg cellular protein, 0.1 mol/L potassium phosphate buffer (pH 7.5), 20 mmol/L glucose-6-phosphate, 2.5 mmol/L NADP+, 1 U glucose-6-phosphate dehydrogenase, 5 mmol/L dithiothreitol, and 6 nmol [14C]HMG-CoA. The reaction was initiated with the addition of [14C]HMG-CoA and the mixture was incubated for 2 h at 37°C. The reaction was stopped by the addition of 20 µL of 5 mol/L HCl on ice. Subsequently, 0.2 µCi of [3H]mevalonolactone was added as an internal standard for correct recovery and incubated at 37°C for 30 min to ensure lactonization of mevalonate. The mevalonolactone was extracted into 5 mL of diethyl ether twice and isolated by TLC with acetone:benzene (1:1). Mevalonolactone was identified by comigration with a mevalonolactone standard and visualized by iodine vapor. Liquid scintillation counting was performed to measure 14C and 3H counts for mevalonolactone formation and for correction of recovery, respectively. HMGR activity was expressed as pmol mevalonate synthesized·mg protein–1·min–1.

    Statistical analysis. ANOVA and Tukey's pairwise comparison or unpaired t test with Welch's correction for unequal variance when appropriate were used to identify significant differences of treatments, with P < 0.05 considered significant by GraphPad InStat 3 (GraphPad Software). Data are expressed as means ± SEM.


   Results
TOP
 ABSTRACT
 Introduction
 Experimental Procedures
 Results
Discussion
 LITERATURE CITED
 
    Cytotoxicity of N. commune lipid extract in HepG2 cells. Cytotoxicity of N. commune lipid extract was assessed by incubating HepG2 cells with increasing concentrations of lipid extract for 24 h and subsequently measuring cell viability. Cells incubated with 25–100 mg/L of N. commune lipid extract showed no reduction in cell viability compared with control (data not shown). Although the percentage of viable cells was lowered at 200 mg/L of N. commune lipid extract (P < 0.05) compared with control, >80% of cells were still viable. Because there were no decreases in cell viability in incubations with N. commune lipid extract concentrations of up to 100 mg/L, these concentrations were used for the cell culture experiments.

    Effect of N. commune lipid extract on the expression of HMGR and LDLR. Quantitative real-time PCR analysis demonstrated that N. commune lipid extract significantly reduced mRNA abundance of HMGR and LDLR in HepG2 cells (Fig. 1A). Protein levels of HMGR and LDLR were measured after incubation with 100 mg/L of N. commune lipid extract for 24 h. Western blot analysis revealed that both HMGR and LDLR protein levels were significantly diminished by the addition of N. commune lipid extract by ~70% and ~60%, respectively (Fig. 1B). The inhibitory effect of N. commune lipid extract on HMGR was further confirmed by reduced HMGR activity by ~90% compared with control (Fig. 2). This result indicates a potential reduction in cholesterol biosynthesis by N. commune lipid extract.


Figure 1
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  		FIGURE 1  N. commune lipid extract reduces mRNA and protein expression of genes involved in cholesterol metabolism in HepG2 cells. (A) Real-time PCR analysis for mRNA expression of HMGR and LDLR in HepG2 cells incubated with N. commune lipid extract (0–100 mg/L) for 24 h. Data are expressed as percentage of control that was not treated with N. commune lipid extract (0 mg/L). Values are means ± SEM, n = 6. Means without a common letter differ, P < 0.05. (B) Western blot analysis for HMGR and LDLR protein levels in HepG2 cells incubated with 100 mg/L N. commune lipid extract for 24 h. Data are expressed as arbitrary units. Values are means ± SEM, n = 6. *Different from control, P < 0.005 for HMGR, P = 0.0014 for LDLR.

 

Figure 2
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  		FIGURE 2  N. commune lipid extract reduces HMGR activity in HepG2 cells. HepG2 cells were incubated in the absence (control) or presence of N. commune lipid extract (100 mg/L) for 24 h and cell lysates were used for HMGR activity. Data are expressed as pmol mevalonate synthesized·mg protein–1·min–1. Values are means ± SEM, n = 6. *Different from control, P = 0.02.

 
    Inhibition of SREBP maturation by N. commune lipid extract. To determine the molecular mechanisms underlying the inhibition of HMGR and LDLR expression by N. commune lipid extract, we evaluated the modulation of SREBP-2 by N. commune. At 25–100 mg/L, N. commune lipid extract did not affect SREBP-2 mRNA abundance (data not shown). Because transcriptional activity of SREBP is largely regulated by the maturation process of precursor proteins (21), Western blot analysis was performed to determine the level of mature SREBP-2 protein. At 100 mg/L, N. commune lipid extract reduced SREBP-2 mature protein levels by 35% (P = 0.02) (Fig. 3). Our next question was whether N. commune lipid extract specifically targets the SREBP-2 pathway or if SREBP-1 could also be inhibited by N. commune lipid extract. To address this question, expression and maturation of SREBP-1 as well as mRNA levels of SREBP-1-responsive genes were measured. Similar to SREBP-2, mRNA level of SREBP-1c was not significantly changed by incubation with N. commune lipid extract (data not shown). However, the SREBP-1 mature protein level was significantly reduced by ~30% (Fig. 4A). From 25–100 mg/L of N. commune lipid extract, both FAS and SCD-1 mRNA levels were significantly decreased (Fig. 4B), indicating that N. commune lipid extract represses the expression of genes involved in fatty acid biosynthetic pathways through the inhibition of SREBP-1 maturation. These data combined indicate that N. commune lipid extract can widely affect the expression of genes involved in cholesterol and fatty acid metabolism by inhibiting the maturation process of both SREBP-1 and SREBP-2.


Figure 3
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  		FIGURE 3  N. commune lipid extract reduces mature protein levels of SREBP-2 in HepG2 cells. HepG2 cells were incubated with 100 mg/L N. commune lipid extract for 24 h, after which Western blot analysis for mature SREBP-2 was performed. A representative blot of 2 separate experiments is shown.

 

Figure 4
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  		FIGURE 4  N. commune lipid extract reduces mature protein levels of SREBP-1 and the expression of SREBP-1-responsive genes in HepG2 cells. (A) Western blot analysis for SREBP-1 in HepG2 cells incubated with 100 mg/L N. commune lipid extract for 24 h. The upper panel shows a representative blot of 3 separate experiments and the lower panel is densitometry analysis of the blots. Data are express as arbitrary units. Values are means ± SEM, n = 9. *Different from control, P = 0.003. (B) Real-time PCR analysis of SREBP-1-responsive genes in HepG2 cells incubated with 0–100 mg/L N. commune lipid extract for 24 h. Data are expressed as percentage of control that was not treated with N. commune lipid extract. Bars without a common letter differ, P < 0.05. Values are means ± SEM, n = 6. Means without a common letter differ, P < 0.05.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Experimental Procedures
 Results
 Discussion
LITERATURE CITED
 
Consumption of foods with bioactive compounds that may confer a beneficial effect on health is increasingly popular. Although N. commune has been consumed for centuries for its purported health benefits, not all claims have been substantiated. In this study, we investigated the effect of N. commune lipid extract on the expression and function of key regulatory proteins involved in cholesterol and fatty acid metabolism in HepG2 cells. Our results demonstrate for the first time, to our knowledge, that N. commune lipid extract represses the expression of both cholesterol and fatty acid biosynthetic genes by inhibiting the maturation process of SREBP-1 and -2.

SREBP are key transcriptional regulators in cholesterol and fatty acid metabolism. They were first discovered as specific transcription factors that bind to the 10-base pair sterol regulatory element within the promoter of the gene encoding LDLR (22,23). When cellular cholesterol is depleted, SREBP are activated to increase the transcription of genes involved in the cholesterol biosynthetic pathway, including HMGR, HMG-CoA synthase, farnesyl diphosphate synthase, and squalene synthase (24–26). Enzymes involved in fatty acid synthesis and uptake, such as acetyl-CoA carboxylase, FAS, SCD-1, and lipoprotein lipase, are also under the transcriptional regulation of SREBP (27–31). Later studies using cell lines and transgenic animals revealed that 2 isoforms of SREBP, i.e. SREBP-1 and SREBP-2, have different functions. SREBP-1 plays an important role in fatty acid metabolism, whereas SREBP-2 plays a greater regulatory role in cholesterol metabolism than fatty acid metabolism (32–35). SREBP are primarily regulated at the posttranscriptional level and 2 major regulatory proteins are involved in the activation of SREBP, i.e. insulin-induced genes (INSIG) and SREBP cleavage-activating protein (SCAP). SCAP/SREBP precursor complex is retained in the endoplasmic reticulum membrane via sterol-induced interaction of SCAP with INSIG (36,37). Upon the signal for low cellular cholesterol level, SCAP is released from INSIG and guides SREBP to the Golgi where a mature form of SREBP is released through 2-step proteolytic cleavages by site-1-protease (S1P) and site-2-protease (S2P) (38). The mature SREBP enters the nucleus and enhances the expression of genes involved in cholesterol and fatty acid metabolism. Therefore, alterations in the SREBP maturation process could have a wide-range of effects on body lipid metabolism. It needs to be further determined which regulatory proteins participating in the maturation process of SREBP are targeted by N. commune lipid extract. Nonetheless, our observation that N. commune lipid extract did inhibit SREBP maturation implicates its potential as a functional food to reduce lipogenesis as well as cholesterol biosynthesis.

HMGR is the key rate-limiting enzyme in cholesterol synthesis. Repression of HMGR by competitive inhibitors such as statins in humans and by inhibition of the regulatory transcription factor SREBP-2 in rats has been shown to decrease the plasma cholesterol level (39,40). This study shows that N. commune lipid extract reduces HMGR mRNA, protein, and activity by inhibiting the maturation process of SREBP-2. N. commune lipid extract also significantly reduced LDLR mRNA and protein levels. LDLR is responsible for the uptake of LDL from circulation and decreased LDLR expression is associated with increased plasma LDL cholesterol concentration (41). Therefore, N. commune lipid extract may not induce a favorable change in plasma total cholesterol concentration due to reduced LDLR expression despite its effect on decreased cholesterol biosynthesis. Alternatively, depending on the body's cholesterol status, inhibition of cholesterol biosynthesis by N. commune lipid extract could be enough to lower the plasma total cholesterol level. However, evidence exists showing that inhibition of SREBP-2 pathway with diminished expression of both HMGR and LDLR decreases plasma total cholesterol level. When the abundance of nuclear SREBP-2 was reduced in rats, which resulted in diminished mRNA abundance of HMGR and LDLR, plasma total cholesterol concentration as well as liver cholesterol content decreased (40). In addition, inhibition of SREBP maturation by knocking down S1P in mice induced reductions in SREBP expression, cholesterol, and fatty acid biosynthesis as well as plasma cholesterol concentration (42). These studies suggest that the SREBP-2 maturation pathway can be a therapeutic target to lower plasma cholesterol level. In addition to its role in regulating cholesterol metabolism through SREBP-2, N. commune lipid extract inhibited key enzymes in fatty acid biosynthetic pathways, such as FAS and SCD-1, through repressed maturation of SREBP-1 in HepG2 cells. Therefore, our results strongly suggest that N. commune lipid extract contains bioactive compounds that can inhibit the SREBP maturation process. A study is underway in our laboratory to determine bioactive compounds responsible for the repressed SREBP pathway in N. commune lipid extract.

Atherosclerosis is a progressive and continuous process throughout life and its progression can be accelerated by dyslipidemia, including high plasma total cholesterol and triglyceride concentrations. Despite extensive efforts to reduce the risk of CHD for the last several decades, CHD remain as the leading cause of death in the US. Given that the public's interest in the prevention and/or therapy for CHD has shifted from the use of drugs to that of natural products, discovery and development of natural sources bearing atheroprotective properties are needed. Our current study indicates that N. commune lipid extract contains bioactive compounds that can decrease the expression and function of key regulatory genes involved in cholesterol and fatty acid biosynthetic pathways through the repression of the SREBP maturation process. This property of N. commune lipid extract could contribute to lower plasma total cholesterol as well as triglyceride concentrations. Although further studies are required to ensure the biological functions of N. commune, particularly in vivo, this study suggests a strong potential of N. commune to be developed as a new natural food to reduce the risk of CHD.


   FOOTNOTES
 
1 Supported by the National Science Foundation-EPSCoR grant EPS-0346476, the Hatch Act and University of Nebraska-Lincoln Research Council Interdisciplinary grant to J-Y. Lee, by the University of Nebraska-Lincoln Agricultural Research Division Honors Student Undergraduate Research to K. R. Blobaum, and by the USDA SBIR grant 2003-00102 to F. Lu. This study is a contribution of the University of Nebraska Agricultural Research Division. Back

2 Author disclosures: H. E. Rasmussen, K. R. Blobaum, Y.-K. Park, S. J. Ehlers, F. Lu, and J.-Y. Lee, no conflicts of interest. Back

3 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org. Back

6 Abbreviations used: CHD, coronary heart disease; BGA, blue-green algae; Ct, threshold cycle; HMGR, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase; LDLR, LDL receptor; SREBP, sterol regulatory element binding protein; FAS, fatty acid synthase; SCD-1, stearoyl CoA desaturase 1; INSIG, insulin-induced gene; SCAP, SREBP cleavage-activating protein; S1P, site-1-protease. Back

Manuscript received 30 October 2007. Initial review completed 14 November 2007. Revision accepted 21 December 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Experimental Procedures
 Results
 Discussion
 LITERATURE CITED
 

1. Carr TP, Parks JS, Rudel LL. Hepatic ACAT activity in African green monkeys is highly correlated to plasma LDL cholesteryl ester enrichment and coronary artery atherosclerosis. Arterioscler Thromb. 1992;12:1274–83.[Abstract/Free Full Text]

2. Expert Panel on Detection EaToHBCiA. Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001;285:2486–97.[Free Full Text]

3. McKenney JM, Davidson MH, Jacobson TA, Guyton JR. Final conclusions and recommendations of the National Lipid Association Statin Safety Assessment Task Force. Am J Cardiol. 2006;97:C89–94.[Medline]

4. Stevinson C, Pittler MH, Ernst E. Garlic for treating hypercholesterolemia. A meta-analysis of randomized clinical trials. Ann Intern Med. 2000;133:420–9.[Abstract/Free Full Text]

5. Anderson JW, Johnstone BM, Cook-Newell ME. Meta-analysis of the effects of soy protein intake on serum lipids. N Engl J Med. 1995;333:276–82.[Abstract/Free Full Text]

6. Pittler MH, Thompson CO, Ernst E. Artichoke leaf extract for treating hypercholesterolaemia. Cochrane Database Syst Rev. 2002;CD003335.

7. Jenkins DJ, Wolever TM, Rao AV, Hegele RA, Mitchell SJ, Ransom TP, Boctor DL, Spadafora PJ, Jenkins AL, et al. Effect on blood lipids of very high intakes of fiber in diets low in saturated fat and cholesterol. N Engl J Med. 1993;329:21–6.[Abstract/Free Full Text]

8. Stanier RY, Cohen-Bazire G. Phototrophic prokaryotes: the cyanobacteria. Annu Rev Microbiol. 1977;31:225–74.[Medline]

9. Iwata K, Inayama T, Kato T. Effects of Spirulina platensis on plasma lipoprotein lipase activity in fructose-induced hyperlipidemic rats. J Nutr Sci Vitaminol (Tokyo). 1990;36:165–71.[Medline]

10. Gilroy DJ, Kauffman KW, Hall RA, Huang X, Chu FS. Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements. Environ Health Perspect. 2000;108:435–9.[Medline]

11. Potts M, Olie JJ, Nickels JS, Parsons J, White DC. Variation in phospholipid ester-linked fatty acids and carotenoids of desiccated Nostoc commune (Cyanobacteria) from different geographic locations. Appl Environ Microbiol. 1987;53:4–9.[Abstract/Free Full Text]

12. Esser MT, Mori T, Mondor I, Sattentau QJ, Dey B, Berger EA, Boyd MR, Lifson JD. Cyanovirin-N binds to gp120 to interfere with CD4-dependent human immunodeficiency virus type 1 virion binding, fusion, and infectivity but does not affect the CD4 binding site on gp120 or soluble CD4-induced conformational changes in gp120. J Virol. 1999;73:4360–71.[Abstract/Free Full Text]

13. Knubel G, Larsen LK, Moore RE, Levine IA, Patterson GM. Cytotoxic, antiviral indolocarbazoles from a blue-green alga belonging to the Nostocaceae. J Antibiot (Tokyo). 1990;43:1236–9.[Medline]

14. Smith CD, Zhang X, Mooberry SL, Patterson GM, Moore RE. Cryptophycin: a new antimicrotubule agent active against drug-resistant cells. Cancer Res. 1994;54:3779–84.[Abstract/Free Full Text]

15. Hori K, Ishibashi G, Okita T. Hypocholesterolemic effect of blue-green alga, ishikurage (Nostoc commune) in rats fed atherogenic diet. Plant Foods Hum Nutr. 1994;45:63–70.[Medline]

16. Horton JD, Shimomura I. Sterol regulatory element-binding proteins: activators of cholesterol and fatty acid biosynthesis. Curr Opin Lipidol. 1999;10:143–50.[Medline]

17. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Med Sci. 1959;37:911–7.[Medline]

18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods. 2001;25:402–8.[Medline]

19. Brown MS, Dana SE, Goldstein JL. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem. 1974;249:789–96.[Abstract/Free Full Text]

20. Brown MS, Dana SE, Dietschy JM, Siperstein MD. 3-Hydroxy-3-methylglutaryl coenzyme A reductase. Solubilization and purification of a cold-sensitive microsomal enzyme. J Biol Chem. 1973;248:4731–8.[Abstract/Free Full Text]

21. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997;89:331–40.[Medline]

22. Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, Brown MS. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J Biol Chem. 1993;268:14497–504.[Abstract/Free Full Text]

23. Briggs MR, Yokoyama C, Wang X, Brown MS, Goldstein JL. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence. J Biol Chem. 1993;268:14490–6.[Abstract/Free Full Text]

24. Osborne TF. Transcriptional control mechanisms in the regulation of cholesterol balance. Crit Rev Eukaryot Gene Expr. 1995;5:317–35.[Medline]

25. Ericsson J, Jackson SM, Lee BC, Edwards PA. Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene. Proc Natl Acad Sci USA. 1996;93:945–50.[Abstract/Free Full Text]

26. Guan G, Jiang G, Koch RL, Shechter I. Molecular cloning and functional analysis of the promoter of the human squalene synthase gene. J Biol Chem. 1995;270:21958–65.[Abstract/Free Full Text]

27. Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS, Goldstein JL. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest. 1996;98:1575–84.[Medline]

28. Magana MM, Osborne TF. Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter. J Biol Chem. 1996;271:32689–94.[Abstract/Free Full Text]

29. Lopez JM, Bennett MK, Sanchez HB, Rosenfeld JM, Osborne TE. Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid. Proc Natl Acad Sci USA. 1996;93:1049–53.[Abstract/Free Full Text]

30. Kim JB, Spiegelman BM. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev. 1996;10:1096–107.[Abstract/Free Full Text]

31. Tontonoz P, Kim JB, Graves RA, Spiegelman BM. ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation. Mol Cell Biol. 1993;13:4753–9.[Abstract/Free Full Text]

32. Kawabe Y, Suzuki T, Hayashi M, Hamakubo T, Sato R, Kodama T. The physiological role of sterol regulatory element-binding protein-2 in cultured human cells. Biochim Biophys Acta. 1999;1436:307–18.[Medline]

33. Shimano H, Yahagi N, Amemiya-Kudo M, Hasty AH, Osuga J, Tamura Y, Shionoiri F, Iizuka Y, Ohashi K, et al. Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes. J Biol Chem. 1999;274:35832–9.[Abstract/Free Full Text]

34. Horton JD, Shimomura I, Brown MS, Hammer RE, Goldstein JL, Shimano H. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2. J Clin Invest. 1998;101:2331–9.[Medline]

35. Pai JT, Guryev O, Brown MS, Goldstein JL. Differential stimulation of cholesterol and unsaturated fatty acid biosynthesis in cells expressing individual nuclear sterol regulatory element-binding proteins. J Biol Chem. 1998;273:26138–48.[Abstract/Free Full Text]

36. Yabe D, Brown MS, Goldstein JL. Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins. Proc Natl Acad Sci USA. 2002;99:12753–8.[Abstract/Free Full Text]

37. Yang T, Espenshade PJ, Wright ME, Yabe D, Gong Y, Aebersold R, Goldstein JL, Brown MS. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell. 2002;110:489–500.[Medline]

38. Goldstein JL, Rawson RB, Brown MS. Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis. Arch Biochem Biophys. 2002;397:139–48.[Medline]

39. Olsson AG, Pears J, McKellar J, Mizan J, Raza A. Effect of rosuvastatin on low-density lipoprotein cholesterol in patients with hypercholesterolemia. Am J Cardiol. 2001;88:504–8.[Medline]

40. Konig B, Koch A, Spielmann J, Hilgenfeld C, Stangl GI, Eder K. Activation of PPARalpha lowers synthesis and concentration of cholesterol by reduction of nuclear SREBP-2. Biochem Pharmacol. 2007;73:574–85.[Medline]

41. Stein CS, Martins I, Davidson BL. Long-term reversal of hypercholesterolemia in low density lipoprotein receptor (LDLR)-deficient mice by adenovirus-mediated LDLR gene transfer combined with CD154 blockade. J Gene Med. 2000;2:41–51.[Medline]

42. Yang J, Goldstein JL, Hammer RE, Moon YA, Brown MS, Horton JD. Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene. Proc Natl Acad Sci USA. 2001;98:13607–12.[Abstract/Free Full Text]





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