改变细胞膜的脂肪酸组成可促进乳腺癌细胞凋亡

目的: 研究n-6脂肪酸脱氢酶 fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用。方法: 构建含有fat-1 基因的重组腺病毒载体 (Ad.GFP.fat-1),通过包装细胞系（293）产生的腺病毒,感染人乳腺癌细胞MCF-7。提取细胞的总RNA,以fat-1的反义mRNA 作探针,用Northern Blot检测fat-1 基因在MCF-7细胞内的表达。MTT法分析fat-1 基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡。气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响。结果: 通过基因重组技术,得到预期的重组病毒;fat-1 基因在人乳腺癌细胞MCF-7 中能有效异源表达,2天后,可检测到fat-1 mRNA的条带。与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖（23%,p<0.05）,促进了凋亡（增加35%）;同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率。结论: 腺病毒介导的fat-1 基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖。机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率。     

Transgenic conversion of omega-6 into omega-3 fatty acids in a mouse model of Parkinson's disease

We have recently identified a neuroprotective role for omega-3 polyunsaturated fatty acids (n-3 PUFAs) in a toxin-induced mouse model of Parkinson's disease (PD). Combined with epidemiological data, these observations suggest that low n-3 PUFA intake is a modifiable environmental risk factor for PD. In order to strengthen these preclinical findings as prerequisite to clinical trials, we further investigated the neuroprotective role of n-3 PUFAs in Fat-1 mice, a transgenic model expressing an n-3 fatty acid desaturase converting n-6 PUFAs into n-3 PUFAs. Here, we report that the expression of the fat-1 transgene increased cortical n-3:n-6 PUFA ratio (+28%), but to a lesser extent than dietary supplementation (92%). Such a limited endogenous production of n-3 PUFAs in the Fat-1 mouse was insufficient to confer neuroprotection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity as assessed by dopamine levels, tyrosine hydroxylase (TH)-positive neurons and fibers, as well as nigral Nurr1 and dopamine transporter (DAT) mRNA expression. Nevertheless, higher cortical docosahexaenoic acid (DHA) concentrations were positively correlated with markers of nigral dopaminergic neurons such as the number of TH-positive cells, in addition to Nurr1 and DAT mRNA levels. These associations are consistent with the protective role of DHA in a mouse model of PD. Taken together, these data suggest that dietary intake of a preformed DHA supplement is more effective in reaching the brain and achieving neuroprotection in an animal model of PD.     

Fat-1 基因真核表达载体的构建及其对人口腔鳞癌细胞 脂肪酸含量的影响

目的:构建真核表达载体pcDNA3.1-Fat1,线性化稳定转染人口腔鳞癌细胞株Tca8113,检测其细胞内脂肪酸含量变化。方法:通过重叠延伸PCR方法人工合成利于真核表达的Fat-1基因,用基因重组技术构建真核表达载体pcDNA3.1-Fat-1,用脂质体转染真核细胞的方法转染人口腔鳞癌细胞株Tca8113,用气相色谱仪检测脂肪酸的变化情况。结果:测序及酶切鉴定成功合成真核偏好表达的Fat-1基因。与对照组相比,转染Fat-1基因的口腔癌细胞的n-3脂肪酸明显增多,n-6/n-3明显下降。结论:成功构建真核表达载体pcDNA3.1-Fat1,并对口腔鳞癌细胞内脂肪酸含量产生明显影响,为进一步研究Fat-1基因在口腔鳞癌中的生物学功能奠定了基础。     

A modified <Emphasis Type="Italic">n</Emphasis>–3 fatty acid desaturase gene from <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis> produced high proportion of DHA and DPA in transgenic mice

The functions of polyunsaturated fatty acids (PUFAs) have been widely investigated. In mammals, levels of n-3 PUFAs are relatively low compared to those of n-6 PUFAs. Either a lack of n-3 PUFAs or an excess of n-6 PUFAs could potentially cause health problems in humans. Hence, methods to increase the amount of n-3 PUFAs in diet have been intensely sought. In this study, we demonstrated that the n-3 fatty acid desaturase gene (sFat-1) synthesized from revised and optimized codons based on roundworm Caenorhabditis briggsae genomic gene for enhanced expression in mammals was successfully expressed in Chinese hamster ovary (CHO) cells and significantly elevated cellular n-3 PUFA contents. We generated sFat-1 transgenic mice by introducing mammal expression vector DNAs containing the sFat-1 gene into regular mice through the method of microinjection. Fatty acid compositions were then altered and the levels of docosahexaenoic acid (DHA, 22:6n-3) and docosapentaenoic acid (DPA, 22:5n-3) were greatly increased in these transgenic mice. Various types of tissues in the transgenic mice produced many types of n-3 PUFAs, such as alpha-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), DPA, and DHA, for example, muscle tissues of the transgenic mice contained 12.2% DHA, 2.0% DPA, and 23.1% total n-3 PUFAs. These research results demonstrated that the synthesized sFat-1 gene with modified and optimized codons from C. briggsae possess functional activity and greater capability of producing n-3 PUFAs, especially DHA and DPA, in transgenic mice.     

Fat-1基因在n-3多不饱和脂肪酸功能研究中的应用

n-3多不饱和脂肪酸是机体脂肪酸成分之一,维持体内合理的n-3多不饱和脂肪酸比例具有重要的生理意义。但n-3多不饱和脂肪酸在大多数动物体内不能合成,只能从食物中补充。fat-1基因的出现改变了这一现状,它可以将多不饱和脂肪酸从n-6形式转化为n-3形式。文章综述了n-3多不饱和脂肪酸的功能,并介绍了fat-1基因的功能及转fat-1基因动物在n-3多不饱和脂肪酸功能研究上的应用。     

ω-6脂肪酸脱氢酶基因在乳腺癌细胞内的表达和作用

为探讨ω- 6脂肪酸脱氢酶基因fat -1在人类乳腺癌细胞MCF- 7中表达和对其生长的作用,将fat -1基因插入到腺病毒载体中,构建腺病毒重组载体(Ad·GFP·fat1) .通过包装细胞系(2 93)产生重组腺病毒,感染MCF 7细胞.用核糖核酸酶保护性分析技术,检测fat -1基因在MCF- 7细胞内的表达,细胞增殖试剂盒(MTT)和凋亡染色试剂盒染色分析fat 1基因对MCF- 7细胞增殖和凋亡的影响,用酶联免疫分析花生酸类(eicosanoids)前列腺素E2 (prostaglandinE2 )的含量.结果显示,腺病毒介导的fat- 1基因能在MCF- 7细胞内有效异源表达,抑制MCF -7细胞的增殖且导致凋亡,前列腺素的含量也明显地减少.结果说明,fat- 1基因在乳腺癌的基因治疗中具有良好利用价值.     

过表达ω-3多不饱和脂肪酸脱氢酶基因fat-1保护小鼠胚胎成纤维细胞避免凋亡

由于膳食原因,人体摄入ω-6PUFAs/ω-3PUFAs比例过高,脂类代谢严重失衡.鉴于ω-3PUFAs获取困难而且人体无催化ω-6PUFAs向ω-3PUFAs转化的ω-3多不饱和脂肪酸脱氢酶,本研究体外扩增来源于秀丽线虫(Caenorhabditis elegans)的ω-3多不饱和脂肪酸脱氢酶基因（fat-l）cDNA,构建了真核表达载体pEGFPC1-fat-1,将该基因转染入小鼠胚胎成纤维细胞;激发荧光与RT-PCR方法检测转染pEGFPC1-fat-1细胞表达该基因,气相色谱分析显示该转染细胞中ω-6PUFAs/ω-3PUFAs的比例降低;细胞抑制率实验显示转染细胞的MTT吸光值升高（P <0.05）;双染法流式细胞仪分析转染细胞凋亡降低,结果表明fat-1基因即使在高浓度ω-6PUFAs的细胞毒作用下,依然能够发挥将ω-6PUFAs脱氢转化为与ω-3PUFAs的生理功能,抑制3T3细胞凋亡,促进细胞生长增殖,实现了较强的细胞保护功能.     

Fat-1基因真核表达载体的构建及其对人口腔鳞癌细胞脂肪酸含量的影响

目的：构建真核表达载体pcDNA3．1-Fat1,线性化稳定转染人口腔鳞癌细胞株Tca8113,检测其细胞内脂肪酸含量变化。方法：通过重叠延伸PCR方法人工合成利于真核表达的Fat-1基因,用基因重组技术构建真核表达载体pcDNA3．1-Fat—1,用脂质体转染真核细胞的方法转染人1：／腔鳞癌细胞株Tca8113,用气相色谱仪检测脂肪酸的变化情况。结果：测序及酶切鉴定成功合成真核偏好表达的Fat-1基因。与对照组相比,转染Fat—1基N的口腔癌细胞的n-3脂肪酸明显增多,n-6／n-3明显下降。结论：成功构建真核表达载体pcDNA3．1-Fat1,并对口腔鳞癌细胞内脂肪酸含量产生明显影响,为进一步研究Fat-1基因在口腔鳞癌中的生物学功能奠定了基础。     

Fat-1 transgenic mice: a new model for omega-3 research

An appropriate animal model that can eliminate confounding factors of diet would be very helpful for evaluation of the health effects of nutrients such as n-3 fatty acids. We recently generated a fat-1 transgenic mouse expressing the Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase that converts n-6 to n-3 fatty acids (which is absent in mammals). The fat-1 transgenic mice are capable of producing n-3 fatty acids from the n-6 type, leading to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in their organs and tissues, without the need of a dietary n-3 supply. Feeding an identical diet (high in n-6) to the transgenic and wild-type littermates can produce different fatty acid profiles in these animals. Thus, this model allows well-controlled studies to be performed, without the interference of the potential confounding factors of diet. The transgenic mice are now being used widely and are emerging as a new tool for studying the benefits of n-3 fatty acids and the molecular mechanisms of their action.     

Activation of Stat5 and induction of a pregnancy-like mammary gland differentiation by eicosapentaenoic and docosapentaenoic omega-3 fatty acids

The protective effect of early pregnancy against breast cancer can be attributed to the transition from undifferentiated cells in the nulliparous to the differentiated mature cells during pregnancy. Considerable evidence suggests strongly that the n-3 polyunsaturated fatty acid (PUFA) content of adipose breast tissue is inversely associated with an increased risk of breast cancer. Here, we report that there was a decrease in the n-6/n-3 PUFA ratio and a significant increase in concentration of n-3 PUFA docosapentaenoic acid and eicosapentaenoic acid in the pregnant gland. The functional role of n-3 PUFAs on differentiation was supported by the studies in the fat-1 transgenic mouse, which converts endogenous n-6 to n-3 PUFAs. Alternation of the n-6/n-3 ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid, in the mammary gland of fat-1 mouse resulted in development of lobulo-alveolar-like structure and milk protein beta-casein expression, mimicking the differentiated state of the pregnant gland. Docosapentaenoic acid and eicosapentaenoic acid activated the Jak2/Stat5 signaling pathway and induced a functional differentiation with production of beta-casein. Expression of brain type fatty acid binding protein brain type fatty acid binding protein in virgin transgenic mice also resulted in a reduced ratio of n-6/n-3 PUFA, a robust increase in docosapentaenoic acid accumulation, and mammary differentiation. These data indicate a role of mammary derived growth inhibitor related gene for preferential accumulation of n-3 docosapentaenoic acid and eicosapentaenoic acid in the differentiated gland during pregnancy. Thus, alternation of n-6/n-3 fatty acid compositional ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid and eicosapentaenoic acid, is one of the underlying mechanisms of pregnancy-induced mammary differentiation.     

Gene and protein expression profiling of the fat-1 mouse brain

Polyunsaturated fatty acids (PUFAs) are essential structural components of all cell membranes and, more so, of the central nervous system. Several studies revealed that n-3 PUFAs possess anti-inflammatory actions and are useful in the treatment of dyslipidemia. These actions explain the beneficial actions of n-3 PUFAs in the management of cardiovascular diseases, inflammatory conditions, neuronal dysfunction, and cancer. But, the exact molecular targets of these beneficial actions of n-3 PUFAs are not known. Mice engineered to carry a fat-1 gene from Caenorhabditis elegans add a double bond into an unsaturated fatty acid hydrocarbon chain and convert n-6 to n-3 fatty acids. This results in an abundance of n-3 eicosapentaenoic acid and docosapentaenoic acid specifically in the brain and a reduction in n-6 fatty acids of these mice that can be used to evaluate the actions of n-3 PUFAs. Gene expression profile, RT-PCR and protein microarray studies in the hippocampus and whole brain of wild-type and fat-1 transgenic mice revealed that genes and proteins concerned with inflammation, apoptosis, neurotransmission, and neuronal growth and synapse formation are specifically modulated in fat-1 mice. These results may explain as to why n-3 PUFAs are of benefit in the prevention and treatment of diseases such as Alzheimer's disease, schizophrenia and other diseases associated with neuronal dysfunction, low-grade systemic inflammatory conditions, and bronchial asthma. Based on these data, it is evident that n-3 PUFAs act to modulate specific genes and formation of their protein products and thus, bring about their various beneficial actions.     

Effect of dietary fatty acids on expression of lipogenic enzymes and fatty acid profile in tissues of bulls

This study investigated the effects of dietary linolenic acid (C18:3n-3) v. linoleic acid (C18:2n-6) on fatty acid composition and protein expression of key lipogenic enzymes, acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD) and delta 6 desaturase (Δ6d) in longissimus muscle and subcutaneous adipose tissue of bulls. Supplementation of the diet with C18:3n-3 was accompanied by an increased level of n-3 fatty acids in muscle which resulted in decrease of n-6/n-3 ratio. The diet enriched with n-3 polyunsaturated fatty acids (PUFAs) significantly inhibited SCD protein expression in muscle and subcutaneous adipose tissue, and reduced the Δ6d expression in muscle. There was no significant effect of the diet on ACC protein expression. Inhibition of the Δ6d expression was associated with a decrease in n-6 PUFA level in muscles, whereas repression of SCD protein was related to a lower oleic acid (C18:1 cis-9) content in the adipose tissue. Expression of ACC, SCD and Δ6d proteins was found to be relatively higher in subcutaneous adipose tissue when compared with longissimus muscle. It is suggested that dietary manipulation of fatty acid composition in ruminants is mediated, at least partially, through the regulation of lipogenic enzymes expression and that regulation of the bovine lipogenic enzymes expression is tissue specific.     

Expression of an ovine growth hormone transgene in mice increases archidonic acid in cellular membranes

The degree of unsaturation of membrane lipids has been implicated in a number of physiological disorders, yet its regulation remains poorly understood, especially the regulation of the synthesis and distribution of arachidonic acid levels, the most abundant long chain polyunsaturated fatty acid in membranes. Transgenic mice expressing the ovine metallothionein 1a — ovine growth hormone (oMt1a-oGH) fusion gene exhibited significantly elevated levels of a number of long chain polyusaturated fatty acids in serum, including arachidonic acid. In oMt1a-oGH transgenic mice the products of all three desaturation pathways are affected by the expression of the ovine growth hormone trangene. The essential precursors of membrane long chain polyunsturated fatty acids, 18:2n-6 and 18:3n-3, were reduced in transgenic relative to controls, and their desaturation and elongation products, arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3), were elevated. As rare intermediate long chain polyunsaturated fatty acids such as eicosatrienoic acid (20:3n-9) were also signficantly elevated, we conclude that these observations reflect increased activity of the Δ-5 and Δ-6 desaturase enzymes. In contranst, the products of the stearoyl CoA or Δ-9 desaturase, were significantly reduced in oMt1a-oGH expressing transgenics relative to their levels in control mice.     

Generation of cloned transgenic pigs rich in omega-3 fatty acids

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001).     

A genome-wide association study of n-3 and n-6 plasma fatty acids in a Singaporean Chinese population

Polyunsaturated fatty acids (PUFAs) have a major impact on human health. Recent genome-wide association studies (GWAS) have identified several genetic loci that are associated with plasma levels of n-3 and n-6 PUFAs in primarily subjects of European ancestry. However, the relevance of these findings has not been evaluated extensively in other ethnic groups. The primary aim of this study was to evaluate for genetic loci associated with n-3 and n-6 PUFAs and to validate the role of recently identified index loci using data from a Singaporean Chinese population. Using a GWAS approach, we evaluated associations with plasma concentrations of three n-3 PUFAs [alphalinolenic acid (ALA), eicosapentaenoic acid and docosahexaenoic acid], four n-6 PUFAs [linoleic acid (LA), gammalinolenic acid, dihomogammalinolenic acid (DGLA) and arachidonic acid], and estimates of delta-5 desaturase and delta-6 desaturase activities among the participants (N = 1361) of the Singaporean Chinese Health Study. Our results reveal robust genome-wide associations (p value <5 × 10⁻⁸) with ALA, all four n-6 PUFAs, and delta-6 desaturase activity at the FADS1/FADS2 locus. We further replicated the associations between common index variants at the NTAN1/PDXDC1 locus and n-6 PUFAs LA and DGLA, and between the JMJD1C locus and n-6 PUFA LA (p value between 0.0490 and 9.88 × 10⁻⁴). These associations were independent of dietary intake of PUFAs. In aggregate, we show that genetic loci that influence plasma concentrations of n-3 and n-6 PUFAs are shared across different ethnic groups.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0502-2) contains supplementary material, which is available to authorized users.     

The high-level accumulation of n-3 polyunsaturated fatty acids in transgenic pigs harboring the n-3 fatty acid desaturase gene from Caenorhabditis briggsae

Livestock meat is generally low in n-3 polyunsaturated fatty acids (PUFAs), which are beneficial to human health. An alternative approach to increasing the levels of n-3 PUFAs in meat is to generate transgenic livestock animals. In this study, we describe the generation of cloned pigs that express the cbr-fat-1 gene from Caenorhabditis briggsae, encoding an n-3 fatty acid desaturase. Analysis of fatty acids demonstrated that the cbr-fat-1 transgenic pigs produced high levels of n-3 fatty acids from n-6 analogs; consequently, a significantly reduced ratio of n-6/n-3 fatty acids was observed. We demonstrated that the n-3 desaturase gene from C. briggsae was functionally expressed, and had a significant effect on the fatty acid composition of the transgenic pigs, which may allow the production of pork enriched in n-3 PUFAs.     

Neurite outgrowth stimulation by n-3 and n-6 PUFAs of phospholipids in apoE-containing lipoproteins secreted from glial cells

PUFAs, which account for 25–30% of the total fatty acids in the human brain, are important for normal brain development and cognitive function. However, it remains unclear how PUFAs are delivered to neurons and exert their effects. In this study, we demonstrated that n-3 and n-6 PUFAs added to the medium are incorporated into membrane phospholipids of primary glial cells from rat cortices, and then secreted as the fatty acid moiety of phospholipids in apoE-containing lipoproteins (LpEs). Tandem mass spectrometry analysis further showed that LpEs secreted from glial cells contain a variety of metabolites of PUFAs produced in glial cells by elongation and unsaturation. LpEs are absorbed by endocytosis into neurons via LDL receptor-related protein 1. LpE-containing n-3 and n-6 PUFAs exhibit a strong effect on neurite outgrowth of hippocampal neurons by increasing the number of branches. This study sheds light on the novel role of LpEs in the central nervous system and also a novel pathway in which PUFAs act on neurons.     

Fatty acid desaturation in red blood cell membranes of patients with type 2 diabetes is improved by zinc supplementation

Background/ObjectiveMembrane flexibility can be a determining factor in pathophysiological mechanisms of type 2 diabetes (T2D). As a cofactor of delta-5 desaturase (D5D) and delta-6 desaturase (D6D), and gene expression regulator, zinc may play a role modulating membrane flexibility by increasing membrane polyunsaturated fatty acids (PUFA) abundance. The objective of this study was to evaluate the effect of a 24-month zinc supplementation (30 mg elemental zinc) on membrane fatty acid composition in patients with T2D.Subjects/methodsSixty patients with T2D were evaluated. Thirty were randomly assigned to the zinc supplemented group and thirty to the placebo group. Fatty acid composition in red blood cell (RBC) membranes was determined by gas chromatography. Expression of gene encoding for D5D (FADS1), and D6D (FADS2) were evaluated in peripheral blood mononuclear cells by real-time polymerase chain reaction.ResultsAfter 24 months of supplementation, a greater abundance of docosapentaenoic acid (C22:5 n-3), arachidonic acid (C20:4 n-6), adrenic acid (C22:4 n-6), and total n-6 PUFA was found (p = 0.001, p = 0.007, p = 0.033, p = 0.048, respectively). The unsaturated fatty acids/saturated fatty acids ratio, and unsaturation index was increased in the zinc supplemented group at month 24 (p = 0.003 and p = 0.000, respectively). FADS1 gene was upregulated in the zinc group in relation to placebo at month 12 (p = 0.020).ConclusionsSupplementation with 30 mg/d elemental zinc during 24 months in patients with T2D had an effect on the composition of RBC membranes increasing PUFA abundance and in turn, improving membrane flexibility. This effect may be mediated by induction of D5D gene expression.