Δ5 desaturase mRNA levels are increased by simvastatin via SREBP-1 at early stages, not via PPARα, in THP-1 cells

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Abstract

In addition to inhibiting cholesterol biosynthesis, statins increase the conversion of linoleic acid to its derivatives, in particular to arachidonic acid, both in vivo and in vitro. Desaturases are the rate-limiting enzymes in this metabolic process and statins markedly enhance Δ5 desaturase activity. To evaluate the Δ5 desaturase gene expression and the transcription factors involved, THP-1 cells (a monocytic cell line) were incubated with 5 μM simvastatin for different time periods. The activity of the enzyme, evaluated as product/precursor ratio in the metabolic pathway (starting from [1-14C] linoleic acid), increased in treated cells with respect to controls after 24 h, whereas, mRNA levels of the Δ5 desaturase increased after 12 h of incubation with simvastatin. Fatty acid desaturase genes are regulated by both sterol regulatory element binding proteins (SREBPs) and peroxisome proliferators activated receptors (PPARs). Both PPARα (WY 14643 and fenofibrate) and PPARγ (ciglitazone) agonists did not affect linoleic acid conversion and the Δ5 desaturase activity at any time considered (8–48 h), but they increased the Δ5 desaturase mRNA levels, after 48 h; only fenofibrate showed a synergistic effect with simvastatin at this time, with a concomitantly increase in PPARα expression and β-oxidation. Simvastatin alone increased SREBP-1 levels with respect to controls, starting from 8 h of incubation, whereas PPARα and linoleic acid β-oxidation (a PPARα mediated process) were not affected after 48 h of incubation. These results taken together suggest that SREBP-1 is involved in the early regulation of Δ5 desaturase gene by simvastatin, in THP-1 cells.

Introduction

Statins, in addition to reducing cholesterol synthesis by inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, have some pleiotropic effects (Corsini et al., 1993, Hochgraf et al., 1994, Colli et al., 1997). The effects on lipid metabolism involve also the production of the long chain polyunsaturated fatty acids; in fact, statins induce elevation of arachidonic acid levels, as a consequence of increased linoleic acid conversion, in vitro and in vivo (Risé et al., 1997, Hrboticky et al., 1994, Van Doormal et al., 1989, Risé et al., 2001, Nakamura et al., 1998). Linoleic acid (18:2 n-6) is converted to arachidonic acid (20:4 n-6), its most relevant metabolite, by elongation and desaturation reactions: Δ6 desaturase catalyzes the step from 18:2 to 18:3 (γ-linolenic acid) and the Δ5 desaturase the step from 20:3 (eicosatrienoic acid) to 20:4.

Human Δ6 and Δ5 desaturase genes have been cloned (Cho et al., 1999a, Cho et al., 1999b): as to the relative abundance of Δ6 and Δ5 desaturase mRNA, in different tissues, Δ6 mRNA levels are higher than those of Δ5 mRNA, the highest levels being found in the liver (Cho et al., 1999b). In addition, in mice fed diets rich in polyunsaturated fatty acids, the decreased hepatic Δ6 and Δ5 desaturase activities reflected a reduction in their mRNA levels (Nakamura et al., 2000). The genes of human Δ6 and Δ5 desaturases are located on chromosome 11 where they are positioned in a reverse sequence orientation to each other and, between the start sites of translation for these genes, there is a sequence of about 11 kb. The proximity of the promoters suggests a coordinated and similar regulation of desaturase transcription (Leonard et al., 2000).

Fatty acid desaturase genes are regulated by peroxisome proliferator activated receptors (PPARs) and sterol regulatory element binding proteins (SREBPs), two different classes of nuclear receptors (Nakamura and Nara, 2002). Cellular cholesterol and fatty acid homeostasis are regulated by SREBPs; in mammalian cells three isoforms have been identified and characterized, and their distribution and abundance differ in cultured cells and in various animal tissues. Of the three isoforms, SREBP-1a, expressed at very low levels, is responsible for maintaining basal cholesterol levels and fatty acid synthesis; SREBP-1c regulates genes involved in lipogenesis and SREBP-2 is more specific in the control of cholesterol homeostasis. Recently, Matsuzaka et al. (Matsuzaka et al., 2002) reported that Δ6 and Δ5 desaturase mRNA levels are elevated in mice overexpressing SREBP-1. The SRE (sterol-regulatory element) sequence, that binds SREBP-1, was identified in human Δ9 and Δ6 desaturase promoters (Bené et al., 2001, Nara et al., 2002).

PPARs are transcription factors that bind to specific DNA sequences (PPREs) on the promoter of target genes. All the three isoforms, α, β/δ and γ, are important modulators of lipid metabolism. PPARα is highly expressed in liver, muscle, heart, kidney and stimulates fatty acid β-oxidation; PPARγ is expressed mainly in the adipose tissue and promotes lipid storage and cell differentiation; PPARβ/δ is ubiquitous and appears to control adipogenesis (Barbier et al., 2002, Vosper et al., 2002). Peroxisome proliferators, such as fibrates and 4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14643), ligands of PPARα, also induce Δ9 and Δ6 desaturase mRNA and it was found that mouse Δ9 desaturase and human Δ6 desaturase promoters have a consensus sequence similar to PPRE (Miller and Ntambi, 1996, Tang et al., 2003).

We have previously shown that, in THP-1 cells (a human monocytic cell line), 5 μM simvastatin significantly increases the conversion of [1-14C] 18:2 n-6 to its long chain polyunsaturated fatty acid derivatives. The increment of 18:2 n-6 conversion is related to increased desaturase activity, Δ6 and Δ5 desaturase activities being enhanced versus controls respectively of about 100% and 380%, in treated cells (Risé et al., 1997). Δ5 desaturation step is more affected than Δ6 after statin, oxysterol and cigarette smoke treatments, in different types of cells (Risé et al., 2004, Risé et al., 1997; Marangoni et al., 2004). Δ5 desaturase is an enzymatic system more specific than that of Δ6 desaturase in humans and animals. In fact, a restricted range of higher plants synthesize 18:3 n-6 from 18:2 n-6, due to the presence of Δ6 desaturase, but not arachidonic acid or eicosapentaenoic and docosahexaenoic acids (Tucker, 2003, Napier et al., 1999) that are direct products of Δ5 desaturase.

Based on these considerations, the purpose of the present study is to investigate in detail, in THP-1 (the cell line used in our previous experiments), the effects of simvastatin on the expression of Δ5 desaturase and also to study the possible transcription factors involved in the action of simvastatin.

Section snippets

Chemicals

RPMI 1640 medium, penicillin, streptomycin, glutamine and foetal calf serum were from Sigma-Aldrich (St. Louis, MO, USA). All the solvents used were from E. Merck (Darmstadt, Germany); [1-14C] 18:2 n-6 (specific activity 55 mCi/mmol) and [1-14C] acetic acid sodium salt (specific activity 57 mCi/mmol) were from Amersham. All the reagents for the reverse transcription-polymerase chain reaction (RT-PCR) were from Invitrogen, Life Technologies (Carlsbad, CA, USA), whereas the TaqMan probes were

Linoleic acid total conversion and cholesterol synthesis

Based on our previous findings (Risé et al., 1997), we performed different time-course experiments for [1-14C] 18:2 n-6 total conversion (expressed as the sum of labelled 18:2 n-6 metabolites, i.e. 18:3, 20:3, 20:4, 22:4 and 22:5 n-6 fatty acids) in control and simvastatin treated cells. As reported in Fig. 1, 5 μM simvastatin increased significantly 18:2 n-6 total conversion at 24 and 48 h versus control, of about 30 and 60% respectively, whereas at 8 and 16 h simvastatin had no effect. The

Discussion

Statins affect not only cholesterol biosynthesis but also polyunsaturated fatty acid production, as shown by the increased levels of linoleic acid (18:2 n-6) conversion products, i.e. arachidonic acid, in vitro and in vivo (Risé et al., 1997, Risé et al., 2001). These effects were reported for simvastatin, fluvastatin and atorvastatin at different concentrations ranging from 0.1 to 5 μM and the increased linoleic acid conversion is concomitant with the inhibition of cholesterol biosynthesis (

Acknowledgments

The authors thank Dr. Cristina Banfi for the suggestion and comments concerning PPARα agonists, and Dr. Maria Grazia Levati for the contribution on primer design. A special thank to Dr. Cristina Manzoni for the technical aid.

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