Todd D. Porter

Policosanol Inhibits Cholesterol Synthesis in Hepatoma Cells by Activation of AMP-Kinase

Policosanol is a mixture of long-chain primary alcohols that has been shown to decrease serum cholesterol in animals and in humans. The hypocholesterolemic effect results from a decrease in cholesterol synthesis by suppression of HMG-CoA reductase activity, but the mechanism of this suppression and the active components of policosanol have not been established. In the present study, we investigated the ability of policosanol and its principal components to inhibit cholesterol synthesis in cultured rat hepatoma cells. Maximal inhibition by policosanol yielded a 30% decrease in [14C]acetate incorporation without evidence of cellular toxicity. Octacosanol (C28, the major constituent of policosanol), heptacosanol (C27), and hexacosanol (C26) yielded smaller and statistically insignificant decreases in cholesterol synthesis, whereas triacontanol (1-hydroxytriacontane; C30) replicated the inhibition obtained with policosanol. At pharmacological concentrations (<5 μg/ml), policosanol and triacontanol decreased [14C]acetate incorporation into cholesterol without affecting the incorporation of [14C]mevalonate, indicating that these compounds act at or above HMG-CoA reductase. Policosanol and triacontanol did not directly inhibit HMG-CoA reductase, and incubation of these compounds with hepatoma cells did not affect reductase enzyme levels. However, reductase activity was decreased by up to 55% in lysates prepared from these cells, suggesting that HMG-CoA reductase activity was down-regulated by policosanol treatment. Consistent with this hypothesis, a 3-fold increase in AMP-kinase phosphorylation was noted in policosanol-treated cells. Because AMP-kinase is activated by phosphorylation and is well established to suppress HMG-CoA reductase activity, these results suggest that policosanol or a metabolite decreases HMG-CoA reductase activity by activating AMP-kinase.

Green and black tea extracts inhibit HMG-CoA reductase and activate AMP kinase to decrease cholesterol synthesis in hepatoma cells.

Recent studies have demonstrated that green and black tea consumption can lower serum cholesterol in animals and in man, and suppression of hepatic cholesterol synthesis is suggested to contribute to this effect. To evaluate this hypothesis, we measured cholesterol synthesis in cultured rat hepatoma cells in the presence of green and black tea extracts and selected components. Green and black tea decreased cholesterol synthesis by up to 55% and 78%, respectively, as measured by a 3-h incorporation of radiolabeled acetate. Inhibition was much less evident when radiolabeled mevalonate was used, suggesting that the inhibition was mediated largely at or above the level of HMG-CoA reductase. Both extracts directly inhibited HMG-CoA reductase when added to microsomal preparations, although the extent of inhibition was considerably less than the decrease in cholesterol synthesis observed in whole cells. As HMG-CoA reductase activity also can be decreased by enzyme phosphorylation by AMP kinase, the phosphorylation state of HMG-CoA reductase and AMP kinase, which is activated by phosphorylation, was determined in lysates from cells treated with tea extracts. Both extracts increased AMP-kinase phosphorylation and HMG-CoA reductase phosphorylation by 2.5- to 4-fold, but with different time courses: maximal phosphorylation with green tea was evident within 30 min of treatment, whereas with black tea phosphorylation was slower to develop, with maximal phosphorylation occurring > or =3 hours after treatment. These results suggest that both green and black tea decrease cholesterol synthesis in whole cells by directly inhibiting HMG-CoA reductase and by promoting its inactivation by AMP kinase.

NADPH-cytochrome P450 oxidoreductase: roles in physiology, pharmacology, and toxicology.

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.

Modification of the Nucleotide Cofactor-binding Site of Cytochrome P-450 Reductase to Enhance Turnover with NADH in Vivo

NADPH-cytochrome P-450 reductase is the electron transfer partner for the cytochromes P-450, heme oxygenase, and squalene monooxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine has been shown to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2′-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (k cat/K m ) with NADH. Inhibition by NADP+ was reduced by up to 4 orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2–3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P-450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P-450 2E1 Ames bacterial mutagenicity assay, the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by ∼2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay.

Supernatant protein factor and tocopherol-associated protein: an unexpected link between cholesterol synthesis and vitamin E (review)

Supernatant protein factor (SPF) is a recently cloned member of a family of cytosolic lipid-binding proteins that includes Sec14p, alpha-tocopherol transfer protein, and cellular retinal-binding protein. SPF stimulates the conversion of squalene to lanosterol in the downstream pathway for cholesterol biosynthesis, and overexpression of cloned SPF in hepatoma cells increases cholesterol synthesis. The mechanism of this stimulation has yet to be defined, but SPF appears to facilitate the transfer of squalene into and between intracellular membranes. The recent identification of SPF as alpha-tocopherol-associated protein (TAP) has called into question its long-standing association with cholesterol biosynthesis. TAP binds alpha-tocopherol, but not other isomers of tocopherol, with high affinity; in the presence of alpha-tocopherol TAP translocates to the nucleus and activates reporter gene transcription. Given the ability of alpha-tocopherol to down-regulate the expression of two scavenger lipoprotein receptors, SR-A and CD36, these observations raise some interesting questions regarding the role of SPF/TAP and vitamin E in cholesterol metabolism.

Nomenclature for alleles of the cytochrome P450 oxidoreductase gene

Recent focus on the cytochrome P450 oxidoreductase (POR) gene has resulted in the discovery of numerous new polymorphic alleles. Many of these were found [1–6] because of their association with steroidogenic disorders and congenital skeletal malformations resembling the phenotype of Antley-Bixler syndrome [7], whereas other alleles have been found as a consequence of sequencing the POR gene in normal unrelated individuals [8,9]. The association of POR variants with clinical phenotypes is the result of POR serving as the major electron donor for cytochrome P450 (CYP) enzymes with important endogenous functions in hormone biosynthesis. Consequently, defective POR alleles can be the cause of abnormal glucocorticoid, mineralocorticoid, and sex steroid synthesis [10], thus leading to a form of congenital adrenal hyperplasia. In addition, POR deficiency can cause skeletal defects, the mechanism of which is yet unknown but has been suggested to result from impaired sterol synthesis [11] because of decreased electron flow from POR to lanosterol 14-alpha-demethylase (CYP51A1) and squalene monooxygenase (SQLE). In addition, as POR is equally important as an electron donor to CYP enzymes involved in the metabolism of drugs, POR variants may affect drug bioavailability. The effect of POR mutations on the activity of some drug-metabolizing CYP enzymes has been documented in vitro [12–14], but not yet in vivo. In addition, POR is an electron donor for heme oxygenase, cytochrome b5, and several additional small molecules that can be directly metabolized by POR without CYP enzymes. Thus, an increasing focus on the importance of POR in drug response and adverse drug reactions is to be expected. Until now, no systematic guidelines have been proposed for the naming of POR alleles. To standardize POR allelic nomenclature, the Human CYP Allele Nomenclature Chair and Committee have taken the initiative to devise a system for the designation of POR alleles that follows the guidelines for CYP allelic star (CYP*) nomenclature (http://www.cypalleles.ki.se/criteria.htm). The POR allele nomenclature web page (http://www.cypalleles.ki.se/por.htm) was launched in September 2008, listing 35 different alleles. On this POR web page, the alleles are presented together with their corresponding nucleotide and amino acid changes, and the phenotypic consequences observed by in vitro and in vivo studies. Among the more important POR variants are POR*2 and *5 (Arg457His and Ala287Pro, respectively), the former being the most frequent mutation in Japanese and Chinese POR-deficient patients [5,15], whereas the latter is the POR mutation most frequently found in Caucasians. Alleles with frameshift mutations (POR*9, *10, and *20–24), deletions, insertions, and several of the alleles that result in amino acid substitutions are also associated with in vivo phenotypes, as is a splice defect in the POR*3 allele. To maintain a common nomenclature system within the field, fellow scientists investigating POR polymorphisms are highly recommended to submit novel POR allelic variants to the Human CYP Allele Nomenclature Committee (http://www.cypalleles.ki.se/criteria.htm) by contacting the Webmaster for designation and reservation of novel POR allele names. The authors of this Letter, a number of whom have identified the novel POR alleles, are supportive of this new nomenclature system, and will use this system in their future work.

Correlation between codon usage, regional genomic nucleotide composition, and amino acid composition in the cytochrome P-450 gene superfamily

The codon usage bias of 110 mammalian cytochrome P-450 genes has been determined and analyzed in relation to a variety of genetic, biochemical, and physiological parameters. In those P-450 genes exhibiting biased usage the preferred codons generally do not differ among the four species examined (rat, rabbit, man, and mouse) or from the predominantly used codons identified for all sequenced genes in a recent data base analysis (Wada et al. (1992) Nucleic Acids Res. 20 (Suppl.), 2111-2118). Codon usage bias does not correlate with evolutionary relationships, evolutionary age, or with the extent of evolutionary conservation of orthologous proteins; there is no obvious correlation with the level of expression of a given P-450, with its inducibility, nor with its physiologic role; and neither the preferred codons nor the degree of bias differ for P-450s expressed in different tissues. Codon usage bias does correlate with the C+G content at the codon third position, and thus preferred codons usually end in C or G; for those P-450s for which gene sequences are available this bias also correlates with the C + G content of the intronic and flanking regions of these genes. Moreover, a lesser increase in the C + G content at the codon first and second positions is also evident in genes located in regions of high C + G content; this leads to predictable differences in the amino acid compositions of P-450 enzymes that correlate with genomic nucleotide composition and the degree of bias in codon usage.

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.

Mutagenicity of nitrosamines in methyltransferase-deficient strains of Salmonella typhimurium coexpressing human cytochrome P450 2E1 and reductase.

Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds is problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. We have constructed two mutagenicity tester strains that co-express full-length human cytochrome P450 2E1 and P450 reductase in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt- and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar to or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. These strains were also sensitive to nitrosamines with longer alkyl side chains including diethylnitrosamine, dipropylnitrosamine and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples.

Resveratrol inhibits human squalene monooxygenase

Abstract Epidemiological studies have suggested that moderate consumption of red wine may protect against the development of cardiovascular diseases. Resveratrol (trans-3,4′,5-trihydroxystilbene) is a naturally occurring polyphenolic compound found in a variety of sources, including red wine, that has been shown to have a number of beneficial effects on cardiovascular health, including prevention of oxidative damage, promotion of vasodilation, and prevention of platelet aggregation. Herein we report that resveratrol specifically inhibits purified human squalene monooxygenase, a rate-limiting enzyme in cholesterol biosynthesis, in a noncompetitive manner with respect to both squalene (K i = 35 μM) and FAD (K i = 69 μM). These data raise the possibility that the protective effect of resveratrol on the development of cardiovascular disease may be explained in part by the inhibition of endogenous cholesterol biosynthesis.