Extrinsic effectors regulating genes for plasmalogen biosynthetic enzymes in HepG2 cells

Plasma plasmalogens (Pls) may serve as potential biomarkers not only for rare peroxisomal diseases but also for general disorders related to oxidative stress and aging. Recent clinical observational studies demonstrated that low levels of plasma Pls are risk factors for atherosclerosis and dementia. Serum levels of Pls showed a strong positive correlation with high-density lipoprotein (HDL) cholesterol concentration, suggesting that Pls may be involved in metabolism or the function of HDL. Increasing the levels of plasma Pls may serve as a novel therapeutic strategy for preventing diseases associated with oxidative stress and aging. Therefore, we and other groups elevated plasma Pl levels in laboratory animals or humans through administration of myo-inositol, monounsaturated long-chain fatty acids, and the hypolipidemic agent, statin. However, their effects on the gene expression of Pl biosynthetic enzymes remain unknown. To gain insight into the manipulation of Pl biosynthesis and the relationship between Pl biosynthesis and HDL metabolism, we examined target gene expression by real time reverse transcription polymerase chain reaction (RT-PCR) in hepatoma HepG2 cells treated with various test substances. Monounsaturated long-chain fatty acids such as oleic acid and erucic acid, myo-inositol, and the Pl precursor alkylglycerol, all of which supply materials or coenzymes for Pl biosynthesis, unexpectedly reduced the expression of the genes for Pl biosynthetic enzymes. These results suggest the presence of strict regulation of Pl homeostasis. In contrast, pitavastatin induced peroxisome biogenesis and promoted the expression of peroxisomal Pl biosynthetic enzymes and HDL metabolism-associated proteins such as apoprotein A1 and ATP-binding cassette transporter A1. This was likely through enhancement of peroxisome proliferator-activated receptor (PPAR) expression. These findings suggest that there may be a physiological relationship between Pl biosynthesis and HDL metabolism via peroxisomal status. Correspondence to: Ryouta Maeba, Ph.D., Department of Biochemistry, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan, Tel: +81-3-3964-1211; E-mail: maeba@med.teikyo-u.ac.jp


Introduction
Age-related diseases, such as atherosclerosis and dementia, are associated with oxidative stress and chronic inflammation [1]. Peroxisomal as well as mitochondrial dysfunction may be related to aging and age-related pathologies, possibly through the derangement of redox homeostasis [2,3]. Plasmalogens (PIs), a subclass of glycerophospholipids possessing a vinyl-ether bond at the sn-1 position, are biosynthesized and regulated in peroxisomes [4][5][6]. Therefore, plasma Pls may reflect the systemic functional state of peroxisomes, and serve as potential biomarkers for diseases related to oxidative stress and aging [7][8][9]. Human plasma Pls are synthesized mainly in the liver and secreted into the blood as lipoprotein components. To investigate the clinical significance of plasma Pls, we developed three promising analytical methods [10][11][12][13]. Our research lab and other investigators have demonstrated in clinical observational studies that low levels of plasma Pl are a risk factor for atherosclerosis and dementia [14][15][16][17][18]. Serum levels of Pl showed a strong positive correlation with high-density lipoprotein (HDL) cholesterol concentration [14,15], suggesting that Pls may be involved in metabolism or HDL functions. Accordingly, we attempted to increase the levels of plasma Pls as a preventative strategy for diseases associated with oxidative stress and aging. This was achieved in laboratory animals as well as humans through administration of the Pl precursor alkylglycerol [19], myo-inositol (MI) [20,21], monounsaturated long-chain fatty acids [22], and the hypolipidemic agent, statin [23]. However, their effects on the gene expression of Pl biosynthetic enzymes remain unknown. To gain insight into the mechanisms mediating the enhancement of Pl biosynthesis, and its relationship with HDL metabolism, we examined target gene expression in HepG2 cells treated with various test substances.

Cell culture and treatment with test substances
HepG2 cells (RIKEN BioResource Center, Tukuba, Ibaragi, Japan) were cultured in Dulbecco's Modified Eagle Medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/mL streptomycin sodium, and 100 U/mL penicillin G sodium (Meiji Seika Pharma Co., Ltd. Tokyo, Japan) at 37°C and 5% CO 2 . Test substances were dissolved in distilled water, ethanol, or dimethyl sulfoxide and then passed through a membrane filter (0.45 µm) for sterilization. The test substance solution was added to the cell culture medium at a desired concentration, adjusted to a vehicle concentration of less than 0.1%. Cells were incubated with test substances at 37°C for 24 h.

Real-time RT-PCR
Total RNA was prepared using Trizol (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized from 1.0 μg RNA with GeneAmp™ RNA PCR (Applied Biosystems, Branchburg, NJ, USA) using random hexamers. Real-time RT-PCR was performed using LightCycler-FastStart DNA Master SYBR-Green 1 (Roche, Tokyo, Japan), according to the manufacturer's instructions. The reaction mixture (20 μL) contained LightCycler-FastStart DNA Master SYBR-Green 1, 4 mM MgCl 2 , 0.5 μM of the upstream and downstream PCR primers, and 2 mL of the first-strand cDNA as a template. The target genes and their primers are shown in Table 1. To control variations in the reactions, all PCR reactions were normalized against GAPDH or β-actin expression. The results of pitavastatin are shown as the mean ± SEM (Figure 1). Statistical analyses were performed using Stat Flex ver.6 (Artech Co. Ltd., Osaka, Japan).

Discussion
Monounsaturated long-chain fatty acids such as oleic acid (C18:1) and erucic acid (C22:1), myo-inositol, and Pl precursors, alkylglycerol and HG, have been reported to increase Pl levels in laboratory animals and humans [19][20][21][22][23]. However, they unexpectedly reduced the gene expression of Pl biosynthetic enzymes in HepG2 cells (Table 2). Monounsaturated long-chain fatty acids are preferred substrates for peroxisomal β-oxidation, and the resulting acetyl CoA is preferentially utilized for the synthesis of ether phospholipids including Pls [29,30]. The decreased expression of FAR1 in HepG2 cells treated with C18:1 and C22:1 may have resulted from the negative feedback from the overproduction of Pls. MI is presumed to enhance Pl biosynthesis through NADPH generation during MI catabolism [31], since Far 1 is activated via NADPH binding [32]. Therefore, the suppressed expression of Pl biosynthetic enzymes in cells treated with MI could also be caused by the negative feedback from overproduction of Pl. Similarly, the reduced expression of Pl biosynthetic enzymes in cells treated with HG was thought to be attributed to overload of Pls in the cells.
However, DHA and pitavastatin increased the gene expression of Pl biosynthetic enzymes in HepG2 cells (Table 2, Figure 1). Because DHA is preferentially incorporated into Pls at the sn-2 position and Pls may function as reservoirs for these biologically active lipid mediators [33], DHA supplementation was considered to potentiate  [4,34]. In addition, the rate-limiting enzyme of Pl biosynthesis, is also peroxisomal [5]. Pitavastatin further enhanced the expression of peroxisomal PPARA and TYSND1, as well as β-oxidation enzymes (Table 2, Figure 1). This suggests that pitavastatin may increase Pl biosynthesis by facilitating peroxisome biogenesis. In addition, pitavastatin increased the expression of PEMT, which may be involved in the conversion of PlsEtn to PlsCho. Our clinical observational studies indicated that serum levels of Pls, particularly PlsCho were significantly but negatively associated with diverse risk factors for metabolic syndrome and/or atherosclerosis. Furthermore, PlsCho showed the stronger positive correlation with HDL cholesterol concentration than PlsEtn [14,15]. Pitavastatin is a strong HMG-CoA reductase inhibitor and is more potent than other statins in lowering serum total cholesterol, low-density lipoprotein cholesterol, and  triglycerides with modest elevation of HDL cholesterol [35]. Recently, pitavastatin was reported to increase Pl content in HDL particles in relation to improving HDL functionality [36]. Moreover, pitavastatin promoted the expression of HDL metabolism-associated proteins such as APOA1. It is proposed that this is probably via enhancement of PPAR expression, since APOA1 and ABCA1 expression are upregulated by PPAR agonists [37].
In conclusion, the supply of materials or coenzymes for Pl biosynthesis such as acetyl CoA derived from peroxisomal β-oxidation of monounsaturated long-chain fatty acids, the Pl precursor alkylglycerol, and NADPH from MI catabolism, suppressed the expression of Pl biosynthetic enzymes ( Table 2). These results suggest that Pl homeostasis is strictly regulated, and the supplementation of these materials may be effective in restoring normal levels of Pls in Pl-deficient individuals. Since peroxisome biogenesis induced by treatment with pitavastatin promoted both the gene expression of Pl biosynthetic enzymes and HDL metabolism-associated proteins, there may be a close relationship between them as a result of their peroxisomal status. Our findings of the strong association between the serum levels of Pls, especially PlsCho and HDL-cholesterol