Abstract
BackgroundNAFLD as a result of inappropriate diet and obesity, may progress to sever conditions such as: type 2 diabetes mellitus or steatohepatitis, and has recently become a prevalent topic of numerous investigations. Due to its dangerous aftermaths, finding new substances, such as polyphenols and their derivatives, which might reduce liver steatosis is the main target of research into NAFLD treatment. Hence, the aim of the present study was to evaluate the effect(s) of enterolactone (ENL), a metabolite of secoisolariciresinol (SECO), on lipid metabolism together with changes in the expression of fatty acid transporters in fatty liver.MethodsThe experiments were conducted on HepG2 cells incubated with either ENL and/or palmitic acid during 16 h exposure. The expression of selected fatty acid transport proteins: FATP2, FATP5, CD36, FABPpm, ABCA1, MTP, ACBP and L-FABP, as well as the proteins directly involved in lipogenesis (FAS), oxidation pathway (CPT 1), and lipid metabolism (PPARα, LXR, SREBP1c, pAMPK) was estimated by Western Blot. Intra and extracellular lipid contents were assessed by Gas-Liquid Chromatography. The data was analyzed with two-way analysis of variance (ANOVA), and results were considered to be statistically significant at p ≤ 0.05.ResultsENL stimulated extracellular efflux of free fatty acids (FFA) and triacylglicerols (TAG) to the medium, while, it had no influence on FATP-family mediated intracellular fatty acid uptake. Moreover, ENL decreased the expression of CPT 1, pAMPK, PPARα, increased SREBP1c and had no effect on LXR, and FAS content.ConclusionsThe findings of our study demonstrate that ENL had opposite effect on liver steatosis in comparison with other polyphenols what suggests that it may be an inactive metabolite. ENL did not affect significantly the intracellular accumulation of FFA, DAG and TAG, yet it promoted their extracellular efflux. Furthermore, it inhibited ß-oxydation and intracellular lipid metabolism what may contribute to the progression of NAFLD.
Highlights
Non-alcoholic fatty liver disease (NAFLD) as a result of inappropriate diet and obesity, may progress to sever conditions such as: type 2 diabetes mellitus or steatohepatitis, and has recently become a prevalent topic of numerous investigations
Immunoblotting analyses Routine Western Blotting procedures were used to detect the total expression of fatty acid transport proteins: FATP2, FATP5 (Santa Cruz Biotechnology, USA), FAT/ CD36, Fatty acid binding protein (FABPpm) (Abcam, UK), ATP-binding cassette transporter 1 (ABCA1) (Thermo Scientific, USA), Mitochondrial trifunctional protein (MTP) (Santa Cruz Biotechnology, USA), Acyl binding protein (ACBP) and liver-type fatty acid binding protein (L-FABP) (Abcam, UK) as well as the proteins directly involved in lipogenesis (FAS; Cell Signaling, USA), oxidation pathway (CPT 1; Santa Cruz Biotechnology, USA) and lipid metabolism (PPARα, liver X receptor (LXR), Sterol regulatory element-binding protein 1c (SREBP1c), phosphorylated 5′ AMP-activated protein kinase (pAMPK); Cell Signaling, USA) as previously described in details by Konstantynowicz-Nowicka et al [17]
Effects of HepG2 cells incubation with PA and/or ENL on total expression of proteins involved in extra- and intracellular lipid transport Among all the examined proteins involved in fatty acid uptake (FAT/CD36, FATP2, FATP5, FABPpm), we detected a significant increase only in FATP5 expression in PA-treated group exposed to enterolactone (ENL + PA: +14.6%, P < 0.05) (Fig. 3)
Summary
NAFLD as a result of inappropriate diet and obesity, may progress to sever conditions such as: type 2 diabetes mellitus or steatohepatitis, and has recently become a prevalent topic of numerous investigations. Taking into consideration all the above-mentioned aftermath, it is clear that NAFLD is the first step in the development of many diseases caused by a disruption of cell metabolic pathways. As it was shown previously, many polyphenols or phytoestrogens are investigated nowadays as a potential medicine for NAFLD treatment and some of them, such as resveratrol, have reached the level of clinical trials, providing promising outcomes [10]. ENL has not been examined before in the context of NAFLD development in connection with fatty acid transporters activity and lipid overload related impairment of the lipid metabolism [16, 15]
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