Effect of sex hormones on n-3 polyunsaturated fatty acid biosynthesis in HepG2 cells and in human primary hepatocytes

https://doi.org/10.1016/j.plefa.2013.12.006Get rights and content

Abstract

Female humans and rodents have been shown to have higher 22:6n-3 status and synthesis than males. It is unclear which sex hormone is involved. We investigated the specificity of the effects of physiological concentrations of sex hormones in vitro on the mRNA expression of genes involved in polyunsaturated fatty acid (PUFA) biosynthesis and on the conversion of [d5]-18:3n-3 to longer chain fatty acids. Progesterone, but not 17α-ethynylestradiol or testosterone, increased FADS2, FADS1, ELOVl 5 and ELOVl 2 mRNA expression in HepG2 cells, but only FADS2 in primary human hepatocytes. In HepG2 cells, these changes were accompanied by hypomethylation of specific CpG loci in the FADS2 promoter. Progesterone, not 17α-ethynylestradiol or testosterone, increased conversion of [d5]-18:3n-3 to 20:5n-3, 22:5n-3 and 22:6n-3. These findings show that progesterone increases n-3 PUFA biosynthesis by up-regulating the mRNA expression of genes involved in this pathway, possibly via changes in the epigenetic regulation of FADS2.

Introduction

The relative proportions of polyunsaturated fatty acids (PUFA) are an important influence on cell function as they influence the biophysical properties of the phospholipid bilayer and provide substrates for phospholipase-mediated signaling pathways. For example, the n-3 PUFA 20:5n-3 and 22:6n-3 play important roles as substrates for signaling pathways and membrane components, respectively [1]. Although these fatty acids can be obtained preformed from dietary sources such as oily fish, most mammalian species have some capacity for the synthesis of these fatty acids from the n-3 essential fatty acid 18:3n-3 which is present in specific plant oils [1]. Such molecular transformation involves desaturation at the Δ6 position by Δ6 desaturase to form 18:4n-3, elongation of the carbon chain by elongase-2 activity to form 20:4n-3, and desaturation at the Δ5 position by Δ5 desaturase to form 20:5n-3 [2]. Conversion of 20:5n-3 to 22:6n-3 involves further elongation by elongase -2 or -5 activity to form 22:5n-3 and elongation by elongase-2 activity to form 24:5n-3. 24:6n-3 is then formed by desaturation at the Δ6 position by Δ6 desaturase. 24:6n-3 is then translocated to peroxisomes where one cycle of fatty acid β-oxidation converts 24:6n-3 to 22:6n-3 which is then returned to the endoplasmic reticulum [2].

Sex appears to be an important influence on 22:6n-3 status and capacity for synthesis of 20:5n-3 and 22:6n-3 from 18:3n-3 in humans. A meta-analysis of 51 studies of the PUFA content of plasma, erythrocyte or adipose tissue in 8541 subjects has shown the proportion of 22:6n-3 to be 37% lower in men than women [3]. In addition, a meta-analysis of 2907 subjects also showed the proportion of 22:6n-3 to be 47% lower in men than in women [4]. Such sex differences in 22:6n-3 status appear to be independent of dietary background [5]. Female rodents have also been shown to have higher plasma and liver 22:6n-3 status than males [1], [6], [7], [8]. Studies in which dietary 18:3n-3 intake was increased in men have shown a dose-related increase in 20:5n-3 and 22:5n-3, but not in 22:6n-3. This implies constraint in the reactions that follow 22:5n-3 synthesis [1]. The effect of 18:3n-3 supplementation on 20:5n-3 and 22:6n-3 status in women is not known. Studies that have used 18:3n-3 labeled with stable isotopes to measure its conversion to longer chain metabolites have confirmed the findings of the observational and dietary intervention studies which showed that women were shown to convert more 18:3n-3 to 22:6n-3 than men [9], [10], [11]. In rats, the mRNA expression of Fads2, which encodes Δ6 desaturase, and Fads1, which encodes Δ5 desaturase, have been shown to be higher in the liver of females than males [6], [12] which demonstrates that at least part of the effect of sex hormones on n-3 PUFA metabolism is mediated through differences in mRNA expression. Together the findings of these studies indicate that sex hormones play a central role in the regulation of PUFA synthesis and status.

There have been relatively few studies of the effects of sex hormones on 22:6n-3 status or synthesis. Women who use an oral contraceptive pill have higher 22:6n-3 status [13] and greater conversion of 18:3n-3 to 22:6n-3 than those who do not. Male transsexuals undergoing hormone therapy treated with synthetic estrogens combined showed increased 22:6n-3 levels in blood compared to untreated men, while those treated with cyproterone acetate, and anti-androgen and weak progesterone mimetic, showed no difference to untreated men [13]. Treatment of postmenopausal women with conjugated estrogens and medroxyprogesterone acetate also increased 20:4n-6 and 22:6n-3 levels in blood compared to untreated individuals [14]. Furthermore, female transsexuals who were treated with testosterone showed a reduction in 22:6n-3 status [13]. Such findings point to a specific positive effect of females sex hormones estrogens on 22:6n-3 synthesis. However, it is not clear which hormone is involved. One recent study in rats has shown that the increase in 22:6n-3 status associated with pregnancy was associated positively with progesterone concentration [6]. Such effects were associated with increased mRNA expression of Fads2. These findings imply that the regulation of PUFA synthesis by sex hormones involves effects on the transcription of genes that encode key genes in this pathway.

In order to investigate the specificity of the effects of hormones on the regulation of n-3 PUFA synthesis, we treated the human hepatocarcinoma cell line HepG2 with a physiologically relevant estrogen (17α-ethynylestradiol (EE2) which has similar potency to naturally occurring 7β-estradiol [15]), progesterone and testosterone. Because sex hormones have been shown previously to alter Fads2 mRNA expression in rat liver, we measured the effect of treatment with these hormones on the mRNA expression of FADS and ELOVL genes. We then determined whether any changes in mRNA levels were associated with altered synthesis of 20:5n-3 and 22:6n-3. In addition, we investigated whether any effects of sex hormones on genes involved in n-3 PUFA metabolism in HepG2 cells were also induced in primary human hepatocytes.

Section snippets

Materials

HepG2 cells and primary hepatocytes were obtained from ECACC and Invitrogen. [17,17,18,18,18-d5]-18:3n-3 (98%) was purchased from Cambridge Isotope Laboratories. Primers for real time PCR were from Biomers (FADS2) and Qiagen (FADS1, ELOVL2, ELOVL5 and cyclophilin). Pyrosequencing and PCR and sequencing primers were synthesized by Biomers. All other reagents were obtained from Sigma and PAA, with noted exceptions.

Cell culture

Cells were maintained at 37 °C under 5% CO2. HepG2 cells were grown in DMEM-high

mRNA expression

Treatment of HepG2 cells with progesterone for 48 h induced a linear (r=0.67, P<0.0001) dose-related increase in FADS2 mRNA expression which reached statistical significance compared to control at 25 nM (2.1-fold) and 50 nM (Fig. 1). However, maximum mRNA levels of FADS1, ELOVL 5 and ELOVL 2 were induced at the lowest concentration of progesterone tested (10 nmole/l); 2.1-fold, 2.5-fold and 3.9-fold, respectively (Fig. 1). There was no significant effect of treatment with EE2 or testosterone on the

Discussion

Although there is compelling evidence from studies of humans and of rodents that males and females differ in n-3 PUFA status, in particular 22:6n-3, and in capacity for conversion of 18:3n-3 to 22:6n-3, there remains uncertainty in nature of the endocrine regulation of the PUFA biosynthesis. While some studies have implicated estrogen as an agonist [9], [13], there is also evidence for positive regulation by progesterone [6]. In contrast, testosterone appears to antagonize conversion of 18:3n-3

Source of funding

The work reported in this article was supported by a grant from the Nutricia Research Foundation (project code 2011-03).

Author contributions

GCB and KAL designed the study. CMS, SPH, R C-H, PL and JTB carried out the experiments and analyzed the data. GCB wrote the first draft of the manuscript with subsequent input form all authors. The authors declare no conflict of interest.

Acknowledgments

GC/MS analysis was supported in part by NIH Grant R01 AT007003 from the National Center for Complementary and Alternative Medicine (NCCAM) and the Office of Dietary Supplements (ODS). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCCAM, ODS, or the National Institutes of Health.

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