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.     

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。     

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。     

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.     

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.     

Cox-2 expression, PGE2 and cytokines production are inhibited by endogenously synthesized n-3 PUFAs in inflamed colon of fat-1 mice

There is great interest in the role of polyunsaturated fatty acids (PUFAs) in promoting (n-6 class) or inhibiting (n-3 class) inflammation. Mammalian cells are devoid of desaturase that converts n-6 to n-3 PUFAs. Consequently, essential n-3 fatty acids must be supplied with the diet. We have studied the effect of endogenously produced n-3 PUFAs on colitis development in fat-1 transgenic mice carrying the Caenorhabditis elegans fat-1 gene encoding n-3 desaturase. Colonic cell lipid profile was measured by capillary gas chromatography in fat-1 and wild-type (WT) littermates fed standard diet supplemented with 10% (w/w) safflower oil rich (76%) in n-6 polyunsaturated linoleic acid (LA). Experimental colitis was induced by administrating 3% dextran sodium sulphate (DSS). Colitis was scored by histopatological analysis. Cyclooxygenase-2 (Cox-2) expression was evaluated by real time polymerase chain reaction. Prostaglandin E₂ (PGE₂) levels and cytokine production were determined by enzyme and microsphere-based immunoassays, respectively. The n-6/n-3 PUFA ratios in colonic cells of fat-1 mice were markedly lower (9.83±2.62) compared to WT (54.5±9.24, P<.001). Results also showed an attenuation of colonic acute and chronic inflammation in fat-1 mice with significant decreases in PGE₂ production (P<.01) and Cox-2 expression (P<.01). High levels of colitis-induced proinflammatory cytokines, interleukin (IL)-18, IL-1α, IL-1β, IL-6, monocytes chemotactic proteins 1, 2 and 3 (MCP 1,2,3), matrix metalloproteinase 9 and tumor necrosis factor α (TNF-α) were down-regulated in DSS acutely and chronically treated fat-1 mice. The expression of fat-1 gene in the colon was associated with endogenous n-3 PUFAs production, decreased Cox-2 expression, increased PGE₂ and cytokine production.     

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.     

Gene transfer of the Caenorhabditis elegans n-3 fatty acid desaturase inhibits neuronal apoptosis

Previous studies have shown that n-3 polyunsaturated fatty acids (PUFAs) can exert an antiapoptotic effect on neurons. The present study was designed to investigate whether the Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase (an enzyme that converts n-6 PUFAs to corresponding n-3 PUFAs) can be expressed functionally in rat cortical neurons and whether its expression can change the ratio of n-6 : n-3 fatty acids in the cell membrane and exert an effect on neuronal apoptosis. Infection of primary rat cortical cultures with Ad-fat-1 resulted in high expression of the fat-1 gene. Lipid analysis indicated a decrease in the ratio of n-6 : n-3 PUFAs from 5.9 : 1 in control cells, to 1.45 : 1 in cells expressing the n-3 fatty acid desaturase. Accordingly, the levels of prostaglandin E2, an eicosanoid derived from n-6 PUFA, were significantly lower in cells infected with Ad-fat-1 when compared with control cells. Finally, there was a significant inhibition of growth factor withdrawal-induced apoptotic cell death in neurons expressing the fat-1 gene. These results demonstrate that expression of the fat-1 gene can inhibit apoptotic cell death in neurons and suggest that the change in the n-6 : n-3 fatty acid ratio may play a key role in this protective effect.     

Complex I-Associated Hydrogen Peroxide Production Is Decreased and Electron Transport Chain Enzyme Activities Are Altered in n-3 Enriched fat-1 Mice

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6∶n-3 ratio on liver mitochondrial H₂O₂ production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6∶n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p<0.05) in the activity of ETC complex I and increases (p<0.05) in the activities of complexes III and IV. Mitochondrial H₂O₂ production with either succinate or succinate/glutamate/malate substrates was also decreased (p<0.05) in the fat-1 mice. This change in H₂O₂ production was due to a decrease in ROS production from ETC complex I in the fat-1 animals. These results indicate that the fatty acid changes in fat-1 liver mitochondria may at least partially oppose oxidative stress by limiting ROS production from ETC complex I.     

A More Desirable Balanced Polyunsaturated Fatty Acid Composition Achieved by Heterologous Expression of Δ15/Δ4 Desaturases in Mammalian Cells

Arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the most biologically active polyunsaturated fatty acids, but their biosyntheses in mammals are very limited. The biosynthesis of DHA is the most difficult, because this undergoes the Sprecher pathway–a further elongation step from docosapentaenoic acid (DPA), a Δ6-desaturase acting on a C24 fatty acid substrate followed by a peroxisomal chain shortening step. This paper reports the successful heterologous expression of two non-mammalian genes (with modification of codon usage), coding for Euglena gracilis Δ4-desaturase and Siganus canaliculatus Δ4-desaturase respectively, in mammalian cells (HEK293 cell line). Both of the Δ4-desaturases can efficiently function, directly converting DPA into DHA. Moreover, the cooperation of the E. gracilis Δ4-desaturase with C. elegans Δ15-desaturase (able to convert a number of n-6 PUFAs to their corresponding n-3 PUFAs) in transgenic HEK293 cells made a more desirable fatty acid composition – a drastically reduced n-6/n-3 PUFAs ratio and a high level of DHA as well as EPA and ARA. Our findings provide a basis for potential applications of the gene constructs for expression of Δ15/Δ4-desaturases in transgenic livestock to produce such a fatty acid profile in the related products, which certainly will bring benefit to human health.     

N-3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure

In cardiac cells the effects of n-3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n-3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and cardiac matrix remodelling, calcium movements and ROS generation. Our data allow to speculate that the cardioprotective effect of n-3 PUFAs is related to a direct modulation of genes in cardiac cells.     

Drosophila lacks C20 and C22 PUFAs

Drosophila melanogaster has been considered a model organism for investigating human diseases and genetic pathways. Whether Drosophila is an ideal model for nutrigenomics, especially for FA metabolism, however, remains to be illustrated. The aim of this study was to examine the metabolism of C20 and C22 PUFAs in Drosophila. Analysis of FA composition revealed a complete lack of C20 and C22 PUFAs in the body tissue of larvae, pupae, and adult flies fed either a base or supplemented diet abundant in the PUFA precursors linoleic acid and α-linolenic acid. PUFA with >C20 could only be found in flies supplemented with specific FAs. Interestingly, the supplemented C22 PUFAs docosahexaenoic acid (22:6n-3) and docosatetraenoic acid (22:4n-6) were largely converted to the shorter chain C20 PUFAs eicosapentaenoic acid (20:5n-3) and arachidonic acid (20:4n-6), respectively. Furthermore, a genome sequence scan indicated that no gene encoding Δ-6/ Δ-5 desaturases, the key enzymes for the synthesis of C20/C22 PUFA, was present in Drosophila. These findings demonstrate that Drosophila lacks the capability to synthesize the biologically important C20 and C22 PUFAs, and thereby argue that Drosophila is not a valid model for the study of lipid metabolism and related diseases.     

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.     

Fatty-acid metabolism is involved in stress-resistance mechanisms of Caenorhabditis elegans

Fatty acids are the major components of the phospholipid bilayer and are involved in several functions of cell membrane. We previously reported that fatty-acid metabolism is involved in the regulation of DAF-2/insulin signal in Caenorhabditis elegans. In this study, we investigate the role of fatty-acid metabolism in stress resistance with respect to daf-16 in nematode. We found that fatty-acid metabolism regulates heat, osmotic, and oxidative-stress resistance in C. elegans. RNA interference (RNAi) of fat-6, fat-7, and elo-2 enhanced heat resistance but decreased oxidative-stress tolerance. RNAi of fat-2 strongly increased osmotic-stress resistance, whereas nhr-49-RNAi remarkably reduced osmotic and oxidative-stress tolerance. In daf-16 mutants (mgDf50), RNAi of fat-2 and fat-7 increased viability under osmotic stress, while RNAi of fat-6, fat-7, and elo-2 enhanced heat resistance. Exposure of saturated fatty acids to RNAi worms of fat-1-, fat-7-, and nhr-49 increased osmotic resistance. On the other hand, polyunsaturated fatty acids (PUFAs) reduced osmotic-stress tolerance in fat-2-RNAi worms, whereas PUFAs enhanced it in nhr-49-RNAi worms. Heat-stress resistance in fat-6- and fat-7-RNAi worms was suppressed by oleic acid.These results suggest that stress-resistance mechanisms are regulated by fatty-acid metabolism with or without DAF-16 activity.     

The opposing effects of n-3 and n-6 fatty acids

Polyunsaturated fatty acids (PUFAs) can be classified in n-3 fatty acids and n-6 fatty acids, and in westernized diet the predominant dietary PUFAs are n-6 fatty acids. Both types of fatty acids are precursors of signaling molecules with opposing effects, that modulate membrane microdomain composition, receptor signaling and gene expression. The predominant n-6 fatty acid is arachidonic acid, which is converted to prostaglandins, leukotrienes and other lipoxygenase or cyclooxygenase products. These products are important regulators of cellular functions with inflammatory, atherogenic and prothrombotic effects. Typical n-3 fatty acids are docosahexaenoic acid and eicosapentaenoic acid, which are competitive substrates for the enzymes and products of arachidonic acid metabolism. Docosahexaenoic acid- and eicosapentaenoic acid-derived eicosanoids antagonize the pro-inflammatory effects of n-6 fatty acids. n-3 and n-6 fatty acids are ligands/modulators for the nuclear receptors NFkappaB, PPAR and SREBP-1c, which control various genes of inflammatory signaling and lipid metabolism. n-3 Fatty acids down-regulate inflammatory genes and lipid synthesis, and stimulate fatty acid degradation. In addition, the n-3/n-6 PUFA content of cell and organelle membranes, as well as membrane microdomains strongly influences membrane function and numerous cellular processes such as cell death and survival.     

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.