Influence of resveratrol and resveratol enriched extracts on endogenous synthesis longchained omega-3-fatty acids - Studies in cell culture in vitro and in rainbow trout and seabass in aquaculture

二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)属于长链多不饱和脂肪酸家族(LCPUFA)，对人体有显著的健康益处。这两种脂肪酸都主要参与调节甘油三酯水平、炎症过程和神经元发育。鱼是人类饮食中LCPUFA的主要来源。在水产养殖中生长的鱼必须添加饲料中的LCPUFA，以维持鱼的健康和产品质量，因为饲料中的α-亚麻酸到EPA和DHA的转化率在淡水鱼物种中都很低，特别是在海水鱼物种中。因此，需要创新的战略来提高水产养殖鱼类内源LCPUFA的合成。Delta6-去饱和似乎是LCPUFA合成的限速步骤。Delta6-去饱和酶将α-亚麻酸转化为硬脂酸，硬脂酸进一步加工成EPA和DHA。我们已经确定二苯乙烯类白藜芦醇是培养肝细胞中Delta6-去饱和度的有效诱导剂。白藜芦醇可提高体外培养肝细胞的Delta6-去饱和酶基因和蛋白水平以及EPA/DHA浓度。在这项资助计划中，我们的目标是研究白藜芦醇对体内LCPUFA代谢的影响。为此，将在不同的水温下添加白藜芦醇来补充虹鲑鱼(淡水)和海鱼(海水)。我们将测量鱼的Delta6-去饱和酶、EPA/DHA水平以及血浆、肝脏和鱼片中的脂肪酸组成。此外，白藜芦醇影响EPA和DHA合成的潜在分子机制将在体外D11鲑鱼肝细胞中进行检测。D11-细胞将用非细胞毒性浓度的白藜芦醇处理，以测定脂肪酸光谱和Delta6-去饱和酶活性。此外，RNAi和电迁移率改变分析将被用来确定参与白藜芦醇介导的Delta6-去饱和酶诱导的可能的转录因子(PPARpha，STAT，HNF1，RXR，ER)。

Eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) belong to the long-chain polyunsaturated fatty acid family (LCPUFA) and exhibit significant health benefits in humans. Both fatty acids are centrally involved in the regulation of triglyceride levels, inflammatory processes and neuronal development. Fish is the major source for LCPUFA in human diets. Fish grown in aquaculture has to be supplemented with dietary LCPUFA to maintain fish health and product quality as conversion rates of dietary alpha-linolenic acid to EPA and DHA are poor both in freshwater fish species and especially in seawater fish species. Thus innovative strategies are needed to improve endogenous LCPUFA synthesis in aquaculture fish species. Delta6-desaturation seems to be the rate-limiting step in LCPUFA-synthesis. Delta6-desaturase converts alpha-linolenic acid into stearidonic acid which is further processed to EPA and DHA. We have identified the stilbene resveratrol as a potent inducer of delta6-desaturation in cultured hepatocytes. Resveratrol enhanced delta6-desaturase mRNA and protein levels as well EPA/DHA concentrations in hepatocytes in vitro. Within this grant proposal we aim to investigate the impact of resveratrol on LCPUFA metabolism in vivo. To this end rainbow trouts (fresh water) and sea bass (seawater) will be supplemented with resveratrol at varying water temperatures. We will measure delta6-desaturase, EPA/DHA levels and fatty acid composition in plasma, liver and filet in fish. Furthermore underlying molecular mechanisms by which resveratrol affects EPA and DHA synthesis will be examined in vitro in D11-trout hepatocytes. D11-cells will be treated with non-cytotoxic concentrations of resveratrol for the determination of fatty acid spectra and delta6-desaturase activities. Furthermore RNAi and electro mobility shift assays will be used to identify putative transcription factors (PPARalpha, STAT, HNF1, RXR, ER) involved in the resveratrol mediated induction of delta6-desaturase.