PPARα, PPARγ and SREBP-1 pathways mediated waterborne iron (Fe)-induced reduction in hepatic lipid deposition of javelin goby Synechogobius hasta

Abstract

The 42-day experiment was conducted to investigate the effects and mechanism of waterborne Fe exposure influencing hepatic lipid deposition in Synechogobius hasta. For that purpose, S. hasta were exposed to four Fe concentrations (0 (control), 0.36, 0.72 and 1.07 μM Fe) for 42 days. On days 21 and 42, morphological parameters, hepatic lipid deposition and Fe contents, and activities and mRNA levels of enzymes and genes related to lipid metabolism, including lipogenic enzymes (6PGD, G6PD, ME, ICDH, FAS and ACC) and lipolytic enzymes (CPTI, HSL), were analyzed. With the increase of Fe concentration, hepatic Fe content tended to increase but HSI and lipid content tended to decrease. On day 21, Fe exposure down-regulated the lipogenic activities of 6PGD, G6PD, ICDH and FAS as well as the mRNA levels of G6PD, ACCa, FAS, SREBP-1 and PPARγ, but up-regulated CPT I, HSLa and PPARα mRNA levels. On day 42, Fe exposure down-regulated the lipogenic activities of 6PGD, G6PD, ICDH and FAS as well as the mRNA levels of 6PGD, ACCa, FAS and SREBP-1, but up-regulated CPT I, HSLa, PPARα and PPARγ mRNA levels. Using primary S. hasta hepatocytes, specific pathway inhibitors (GW6471 for PPARα and fatostatin for SREBP-1) and activator (troglitazone for PPARγ) were used to explore the signaling pathways of Fe reducing lipid deposition. The GW6471 attenuated the Fe-induced down-regulation of mRNA levels of 6PGD, G6PD, ME, FAS and ACCa, and attenuated the Fe-induced up-regulation of mRNA levels of CPT I, HSLa and PPARα. Compared with single Fe-incubated group, the mRNA levels of G6PD, ME, FAS, ACCa, ACCb and PPARγ were up-regulated while the CPT I mRNA levels were down-regulated after troglitazone pre-treatment; fatostatin pre-treatment down-regulated the mRNA levels of 6PGD, ME, FAS, ACCa, ACCb and SREBP-1, and increased the CPT I and HSLa mRNA levels. Based on these results above, our study indicated that Fe exposure reduced hepatic lipid deposition by down-regulating lipogenesis and up-regulating lipolysis, and PPARα, PPARγ and SREBP-1 pathways mediated the Fe-induced reduction of hepatic lipid deposition in S. hasta.

Introduction

Iron (Fe) is an essential micronutrient for vertebrates because it plays important roles in multiple metabolic processes, including oxygen transport, detoxification, electron transport, DNA synthesis and protein synthesis (Kwong and Niyogi, 2009). However, excessive Fe in the aquatic environments can be toxic, which has a devastating effects on fish species, affecting growth performance, Fe accumulation and absorption, and physiological response (Peuranen and Hollender, 1994, Dalzell and Macfarlane, 1999, Lappivaara and Marttinen, 2005, Debnath et al., 2012, Glover et al., 2016).

The liver is the main site for Fe storage and also an important site for lipid metabolism. In mammals, studies indicated that variations in hepatic Fe stores modified lipid metabolism (Silva et al., 2008, Ahmed et al., 2012, Wlazlo and van Greevenbroek, 2012). However, in fish, the studies on waterborne Fe exposure influencing lipid metabolism were poorly described. Fish use lipids as main energy reserves and lipids serve a vast array of functions in the life histories of fish (Sheridan, 1988). Recently, in our laboratory, Chen et al. (2016) pointed out that 1.128 μM of waterborne Fe exposure influenced Cu-induced changes of hepatic lipid deposition in Synechogobius hasta, a carnivorous and euryhaline fish species. However, in the study by Chen et al. (2016), only one Fe concentration (relatively high Fe concentration) was selected. At present, to our best knowledge, no other attempts have been made to demonstrate the relationship between different Fe exposure concentrations in water and lipid metabolism in fish.

Lipid accumulation results from the balance between synthesis of fatty acids (lipogenesis) and fat catabolism via β-oxidation (lipolysis), and many key enzymes and transcriptional factors are involved in these metabolic processes. These enzymes include lipogenic enzymes, such as glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS, and lipolytic enzymes, such as hormone-sensitive lipase (HSL) and carnitine palmitoyltransferase I (CPT I) (Elliott and Elliott, 2009). On the other hand, several transcription factors, such as peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) and sterol-regulator element-binding protein (SREBP-1), play an intermediary role in lipid homeostasis, by orchestrating the gene transcription of the enzymes involved in these pathways (Spiegelman and Flier, 2001). SREBP-1 and PPARγ are key transcriptional factors involved in lipogenesis whereas PPARα plays key roles in the catabolism of fatty acids (Spiegelman and Flier, 2001). Studies suggested that effects of metal elements (such as Cu and Zn) on enzymatic activities and expression of genes mentioned above were concentration-dependent in fish (Zheng et al., 2013, Zheng et al., 2014, Chen et al., 2013a, Chen et al., 2013b, Chen et al., 2015, Huang et al., 2014, Huang et al., 2016, Wu et al., 2016a). In addition, investigation into toxicity of metal elements has focused on high-doses exposure, which is a very different situation from the low-doses exposure encountered in the environment by fish. Usually, under natural environment, fish often face the challenge from low waterborne concentration of metal elements, which poses the potential long-term influences on their physiological parameters. Theoretically, the effects of lower doses of Fe cannot be extrapolated from high doses and accordingly studies into the possible impacts of low-dose Fe exposure are needed. Thus, it is very meaningful and necessary to explore the effects of lower Fe concentration on lipid metabolism. Using the in vivo and in vitro experiments, our study was conducted to investigate the potential mechanism of low-dose Fe exposure influencing hepatic lipid deposition in S. hasta.