Modulation of gene expression in eicosapentaenoic acid and docosahexaenoic acid treated human colon adenoma cells

Epidemiological studies suggest that high fish intake is associated with a decreased risk of colorectal cancer which has been linked to the high content of the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acids (EPA) and docosahexaenoic acid (DHA) in some fish. The aim of the study was to compare the modulation of gene expression in LT97 colon adenoma cells in response to EPA and DHA treatment. Therefore, we used custom-designed cDNA arrays containing probes for 306 genes related to stress response, apoptosis and carcinogenesis and hybridised them with cDNA from LT97 cells which were treated for 10 or 24 h with 50 μM EPA or DHA. There was a marked influence of n-3 PUFA on the expression of several gene types, such as detoxification, cell cycle control, signaling pathways, apoptosis and inflammation. DHA and EPA generally modulated different sets of genes, although a few common effects were noted. In our approach, we used preneoplastic adenoma cells which are a relevant model for target cells of chemoprevention. If verified with real time PCR, these results identify genes and targets for chemoprevention of colon cancer.     

Antarctic microorganisms as source of the omega-3 polyunsaturated fatty acids

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are long-chain polyunsaturated fatty acids (PUFAs) that belong to the omega-3 group. They are essential fatty acids found in phospholipid of cell membranes. There is strong evidence that these nutrients may also favorably modulate many diseases. Primary sources of omega-3 PUFAs in the human diet are fish and fish-derived products. The fishing industry worldwide, however, is becoming unable to satisfy the growing demand for these PUFAs. A promising cost-effective alternative source of PUFAs is bacterial production. We identified 40 Antarctic marine bacterial isolates by 16S rRNA gene sequence analysis. Fifteen genera in three phyla were represented in the collection. Isolates were tested for ability to produce EPA using a method in which their ability to reduce 2,3,5-triphenyltetrazolium chloride (TTC) is determined and by gas chromatography coupled to mass spectrometry (GC–MS). All isolates could reduce TTC, and GC–MS analysis showed that four produced EPA and that six produced DHA. We show for the first time that isolates identified as Cellulophaga, Pibocella and Polaribacter can produce EPA and DHA, only DHA or only EPA, respectively. One isolate, Shewanella sp. (strain 8-5), is indicated to be a good candidate for further study to optimize growth and EPA production. In conclusion, a rapid method was tested for identification of new EPA producing strains from marine environments. New EPA and DHA producing strains were found as well as a potentially useful PUFA production strain.     

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.     

Induction of apoptosis and lipogenesis in human preadipocyte cell line by n −3 PUFAs

We examined the effect of n ?3 PUFAs (polyunsaturated fatty acids) on the growth and maturation of human preadipocyte cell line AML‐I. On day 3 of the culture, n ?3 fatty acids such as DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), but not n ?6 fatty acid LA (linoleic acid), induced growth arrest accompanied by the appearance of characteristics of apoptosis in AML‐I cells at concentrations between 250 and 500 μM by Annexin V‐FITC staining. In Western blotting analysis, the loss of NF‐κB, Bcl‐2 and p‐Akt and the accumulation of Bad and Akt were observed in the cytoplasmic protein from the EPA‐treated cells. Exposure of AML‐I to EPA or DHA increased the cytoplasmic lipid accumulation compared with the vehicle‐treated cells in a time‐dependent manner during 4 and 6 days culture period by Oil Red O staining. The expression of FAS (fatty acid synthase) and PPAR‐γ (peroxisome proliferator‐activated receptor‐γ) were increased in EPA‐treated cells. These results suggest that EPA and DHA promote differentiation, inhibit proliferation and induce apoptosis in preadipocyte cell line AML‐I.     

Growth Inhibitory Effect of Polyunsaturated Fatty Acids (PUFAs) on Colon Cancer Cells via Their Growth Inhibitory Metabolites and Fatty Acid Composition Changes

Background

Colorectal cancer is common. Polyunsaturated fatty acids (PUFAs) exert growth-inhibitory and pro-apoptotic effects on colon cancer cells. Metabolites of PUFAs such as prostaglandins (PGs), leukotrienes (LTs) and lipoxins (LXs) play a significant role in colon cancer.

Methods

Human colon cancer LoVo and RKO cells were cultured with different concentration of PUFAs and 5-fluorouracil (5-FU) in vitro. Cell morphological changes, fatty acid composition, formation of PGE2, LTB4 and LXA4 and expression of COX-2, ALOX5, PGD synthase (PGDS), microsomal prostaglandin E synthase (mPGES) were assessed in LoVo and RKO cells when supplemented with PUFAs and 5-FU.

Results

PUFAs and 5-FU inhibited growth of LoVo and RKO cells to the same extent at the doses used and produced significant alterations in their shape. As expected, higher concentrations of supplemented PUFAs were noted in the cells compared to control. LA, GLA, AA, ALA and EPA supplementation to LoVo cells suppressed production of PGE₂, LTB₄,and ALOX5, mPGES expression, but enhanced that of LXA₄; whereas DHA enhanced PGE₂ and LXA₄ synthesis but decreased LTB₄ formation and COX-2, ALOX5, mPGES expression. In contrast, 5-FU enhanced formation of PGE₂, LTB₄ and mPGES expression, but suppressed LXA₄ synthesis and COX-2 expression. PGE₂, LTB₄ synthesis and ALOX5 expression was suppressed by LA, GLA, ALA and DHA; whereas AA, EPA and 5-FU enhanced PGE₂ but paradoxically AA decreased and EPA and 5-FU enhanced LTB4 synthesis in RKO cells. All the PUFAs tested enhanced, while 5-FU decreased LXA₄ formation in RKO cells; whereas GLA, AA, and 5-FU augmented while LA, ALA, EPA and DHA enhanced COX-2 expression in RKO cells.

Conclusions

Tumoricidal action of PUFAs on colorectal LoVo and RKO cancer cells in vitro was associated with increased formation of LXA₄, decreased synthesis of PGE₂ and LTB₄ and suppressed expression of COX-2, ALOX5, mPGES, whereas 5-FU produced contrasting actions on these indices.     

Comparative Analysis of Content of Omega-3 Polyunsaturated Fatty Acids in Food and Muscle Tissue of Fish from Aquaculture and Natural Habitats

Two fish species reared in aquaculture (pink salmon Oncorhynchus gorbuscha and whitefish Coregonus lavaretus) and ten fish species from natural habitats (whitefish C. lavaretus, tugun Coregonus tugun, broad whitefish Coregonus nasus, least cisco Coregonus sardinella, vendace Coregonus albula, boganid charr Salvelinus boganidae, charr Salvelinus alpinus complex, northern pike Esox lucius, sharp-snouted lenok Brachymystax lenok, and taimen Hucho taimen) have been studied. The content of two long-chain polyunsaturated omega-3 fatty acids (PUFAs), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), in the muscle tissue of the fish and in their food (intestine contents) are compared. In the aquacultures of whitefish and pink salmon, the total content of EPA and DHA is significantly higher in feed than in the muscle tissue of the fish, which indicates losses of PUFA in the two-link food chain of the aquaculture during their transfer to the upper trophic level. EPA and DHA losses in aquaculture, which are confirmed by numerous literature data, mean an inefficient usage of the available sources of PUFAs and the aggravation of the global deficit of these biochemicals in the human diet. A study of natural fish populations reveals the accumulation of EPA and DHA in their biomass compared to food in many cases, although opposite phenomena are also observed. An assumption on the presence of an optimal, physiologically adequate species-specific level of PUFA in the fish muscle tissue has been made based on our data and literature data. If the level of PUFAs in the muscles is lower than optimal, their accumulation (bioaccumulation) from food and/or de novo synthesis are observed. When the optimal level is exceeded, the content of EPA and DHA in biomass approaches maximum species-specific values; however, part of these PUFAs entering from food is not digested or is catabolized. According to the obtained data, the species of the order Salmoniformes have an optimal level of 2 to 6 mg/g of wet weight. It has been found that in aquaculture approaching to maximum values of EPA + DHA content was accompanied by their losses (scattering) in the food chains, while in natural ecosystems the maximum values of PUFA content in the fish biomass are achieved by their accumulation from the lower trophic level. Boganid charr S. boganidae had the highest content of EPA + DHA in the muscle tissue among all known fish species (32.78 mg/g of wet weight).     

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega‐3 (also called n‐3) long‐chain polyunsaturated fatty acids (≥C20; LC‐PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega‐3 LC‐PUFAs, i.e. eicosapentaenoic acid (20:5 n‐3, EPA) and docosahexaenoic acid (22:6 n‐3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega‐3 LC‐PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non‐native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.     

2-Dodecylcyclobutanone, a radiolytic product of palmitic acid, is genotoxic in primary human colon cells and in cells from preneoplastic lesions

The irradiation of fat results in the formation of 2-alkylcyclobutanones, a new class of food contaminants. Results of previous in vitro studies with primary human colon cells and in vivo experiments with rats fed with 2-alkylcyclobutanones indicated that these radiolytic derivatives may be genotoxic and enhance the progression of colon tumors. The underlying mechanisms of these effects, however, are not clearly understood. Therefore we performed additional investigations to elucidate the genotoxic potential of 2-dodecylcyclobutanone (2dDCB) that is generated from palmitic acid. In particular, we explored the relative sensitivities of human colon cells, representing different stages of tumor development and healthy colon tissues, respectively. HT29clone19A cells, LT97 adenoma cells and primary human epithelial cells were exposed to 2dDCB (150-2097 microM). We determined cytotoxic effects using trypan blue exclusion. Genotoxicity, reflected as strand breaks, was assessed using the alkaline version of the comet assay and chromosomal abnormalities were investigated by 24-color fluorescence-in-situ-hybridization. 2dDCB was cytotoxic in a time- and dose-dependent manner in LT97 adenoma cells and in freshly isolated primary cells but not in the human colon tumor cell line. Associated with this was a marked induction of DNA damage by 2dDCB in LT97 adenoma cells and in freshly isolated colonocytes, whereas in the HT29clone19A cells no strand breaks were detectable. A long-term incubation of LT97 adenoma cells with lower concentrations of 2dDCB revealed cytogenetic effects. In summary, 2dDCB was clearly genotoxic in healthy human colon epithelial cells and in cells representing preneoplastic colon adenoma. These findings provide additional evidence that this compound may be regarded as a possible risk factor for processes in colon carcinogenesis related to initiation and progression.     

Relationship between diet and plasma long-chain n-3 PUFAs in older people: impact of apolipoprotein E genotype

The main risk factors for Alzheimer''s disease, age and the ϵ4 allele of the APOE gene (APOE4), might modify the metabolism of n-3 PUFAs and in turn, their impact on cognition. The aim of this study was to investigate the association between dietary fat and plasma concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in elderly persons, taking the APOE4 genotype into account. The sample was composed of 1,135 participants from the Three-City study aged 65 years and over, of whom 19% were APOE4 carriers. Mean plasma proportions of EPA [1.01%, standard deviation (SD) 0.60] and DHA (2.41%, SD 0.81) did not differ according to APOE4. In multivariate models, plasma EPA increased with frequency of fish consumption (P < 0.0001), alcohol intake (P = 0.0006), and female gender (P = 0.02), and decreased with intensive consumption of n-6 oils (P = 0.02). The positive association between fish consumption and plasma DHA was highly significant whatever the APOE genotype (P < 0.0001) but stronger in APOE4 noncarriers than in carriers (P = 0.06 for interaction). Plasma DHA increased significantly with age (P = 0.009) in APOE4 noncarriers only. These findings suggest that dietary habits, gender, and APOE4 genotype should be considered when designing interventions to increase n-3 PUFA blood levels in older people.     

Inhibition by eicosapentaenoic acid of IL-1β-induced PGHS-2 expression in human microvascular endothelial cells: involvement of lipoxygenase-derived metabolites and p38 MAPK pathway

Prostaglandin H synthase 2 (PGHS-2), a highly inducible isoenzyme, is responsible for overproduction of the prostaglandins (PGs) in inflammatory sites.We established that among fish oil polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), greatly decreased interleukin-1β (IL-1β)-induced PGHS-2 expression in human pulmonary microvascular endothelial cells (HPMECs). Lipoxygenase products 12 (S)-hydroperoxyeicosapentaenoic acid ((S)-HpEPE), 15 (S)-HpEPE and leukotriene (LT) D5 reproduced similar inhibitory effect, suggesting that they may be the intermediate metabolites responsible for PGHS-2 down-regulation by EPA. Accordingly, the EPA effect is prevented by nordihydroguaiaretic acid (NDGA) and by REV 5901, nonspecific and specific 5-lipoxygenase inhibitors, respectively. Besides, inhibition of cyclooxygenase activity by ibuprofen, indomethacin or aspirin was not able to prevent this effect. Moreover, cyclooxygenase metabolites of EPA (PGs D3, E3 and I3) markedly potentiate IL-1β-induced PGHS-2 expression, probably by increasing intracellular cAMP levels. Peroxisome proliferator-activated receptors (PPARs) are known to be activated by fatty acids (FAs) such as EPA. We found here that HPMECs express only weak amounts of PPARα and PPARγ whose activation by synthetic agonists, Wy-14,643 and ciglitazone, does not cause any inhibition of IL-1β-induced PGHS-2 expression. This finding ruled out the involvement of PPARs in the EPA inhibitory effect. In addition, we established that EPA, which failed to inhibit nuclear factor-κB (NF-κB) activation, suppressed p38 mitogen-activated protein kinase (MAPK) phosphorylation in stimulated HPMECs.Our data demonstrate that EPA, unlike DHA, down-regulates PGHS-2 expression in HPMECs probably through its 5-lipoxygenase-dependent metabolites and advocates a beneficial role for this FA in limiting inflammatory response.     

Contrasting effects of membrane enrichment with polyunsaturated fatty acids on phospholipid composition and cholesterol efflux from cholesterol-loaded J774 mouse or primary human macrophages

A high consumption of polyunsaturated fatty acids (PUFAs), particularly n-3 PUFAs, is atheroprotective. PUFAs incorporation into membrane phospholipids alters the functionality of membrane proteins. We studied the consequences of the in vitro supplementation of several PUFAs on the FA profiles and on ABCA1-dependent cholesterol efflux capacities from cholesterol-loaded macrophages. Arachidonic acid (AA, C20:4 n-6) and, to a lesser extent, eicosapentaenoic acid (EPA, C20:5 n-3), dose-dependently impaired cholesterol efflux from cholesterol-loaded J774 mouse macrophages without alterations in ABCA1 expression, whereas docosahexaenoic acid (DHA, C22:6 n-3) had no impact. AA cells exhibited higher proportions of arachidonic acid and adrenic acid (C22:4 n-6), its elongation product. EPA cells exhibited slightly higher proportions of EPA associated with much higher proportions of docosapentaenoic acid (C22:5 n-3), its elongation product and with lower proportions of AA. Conversely, both EPA and DHA and, to a lesser extent, AA decreased cholesterol efflux from cholesterol-loaded primary human macrophages (HMDM). The differences observed in FA profiles after PUFA supplementations were different from those observed for the J774 cells. In conclusion, we are the first to report that AA and EPA, but not DHA, have deleterious effects on the cardioprotective ABCA1 cholesterol efflux pathway from J774 foam cells. Moreover, the membrane incorporation of PUFAs does not have the same impact on cholesterol efflux from murine (J774) or human (HMDM) cholesterol-loaded macrophages. This finding emphasizes the key role of the cellular model in cholesterol efflux studies and may partly explain the heterogeneous literature data on the impact of PUFAs on cholesterol efflux.     

Influences of stearidonic acid-enriched soybean oil on the blood and organ biochemical parameters in rats

In this study, we administered various diets of stearidonic acid (SDA, 18:4n?3) soybean oil to rats and examined the subsequent blood and organ biochemical parameters. Male Wistar rats (seven rats/group, six groups total) were fed diets supplemented with a test oil for 4 weeks. Diets containing test oils were: FFC diet (fish-oil-free control diet), C diet (control group, assuming a Japanese diet), SDA25 diet (25% 18:4n?3 soybean oil in the C diet), SDA50 (50% 18:4n?3 soybean oil in the C diet), ALA diet (34% flaxseed oil in the C diet), and EPA+DHA diet (34% fish oil in the C diet). The intake of 18:4n?3 showed increased relative efficiency of 20:5n?3 accretions in serum and liver triacylglycerol and significantly decreased the serum triacylglycerol level in rats. The results suggested that the consumption of 18:4n?3 soybean oil may modify the lipid and fatty acid profiles of body fats, even when EPA and DHA derived from fish is consumed.     

Amphibia as a Vector of Transfer of Long-Chain Polyunsaturated Omega-3 Fatty Acids from Aquatic to Terrestrial Ecosystems

The flow of long-chain polyunsaturated fatty acids (PUFAs) of the omega-3 family, namely, eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), exported by amphibian metamorphs from water to terrestrial ecosystems in the Medveditsa River floodplain, was quantified for the first time. The total biomass export by three amphibian species (Pelobates fuscus, Bombina bombina, and Pelophylax ridibundus) per unit area of the lake surface was 0.594 g/m² per year (as a mean for 2 years). The biomass flow per unit area of land was 0.726 g/ha per year (0.302 g/ha per year for organic carbon) in 2015–2016. The average annual total removal of EPA + DHA by amphibians from the floodplain lake was 1.47 mg/m² of water surface area. Due to the high content of EPA and DHA in biomass, amphibians are potentially a valuable food for terrestrial predators having no access to other sources of essential PUFAs.     

Influence of miRNA-106b and miRNA-135a on butyrate-regulated expression of p21 and Cyclin D2 in human colon adenoma cells

Epigenetic and posttranslational modifications of the expression of cell cycle-relevant genes or proteins like p21, e.g., by miRNAs are crucial mechanisms in the development or prevention of colon cancer. The present study investigated the influence of butyrate and trichostatin A (TSA) as histone deacetylase inhibitors on the expression of colon cancer-relevant miRNA (miR-135a, miR-135b, miR-24, miR-106b, miR-let-7a) in LT97 colon adenoma cells as a model of an early stage of colon carcinogenesis. The impact of distinct miRNAs (miR-106b, miR-135a) on butyrate-mediated regulation of p21 and Cyclin D2 gene and protein expression as well as the effect on LT97 cell proliferation (non-transfected, miR-106b and miR-135a mimic transfected) was analyzed. Butyrate and partial TSA reduced the expression of miR-135a, miR-135b, miR-24 and miR-let-7a (~0.5-fold, 24 h) and miR-24, miR-106b and miR-let-7a (~0.5–0.7-fold, 48 h) in LT97 cells. Levels of p21 mRNA and protein were significantly increased by butyrate and TSA (~threefold and 4.5-fold, respectively, 24 h) in non-transfected but not in miR-106b transfected LT97 cells. Levels of Cyclin D2 mRNA were significantly reduced by butyrate and TSA (~0.3-fold, 24 h) in non-transfected and miR-135a-transfected LT97 cells, whereas protein levels were predominantly not influenced. MiR-106b and miR-135a significantly reduced butyrate-/TSA-mediated inhibition of LT97 cell proliferation (72 h). These results indicate that butyrate is able to modify colon cancer-relevant miRNAs like miR-106b and miR-135a which are involved in the regulation of cell cycle-relevant genes like p21 and might influence inhibition of adenoma cell proliferation.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0500-4) contains supplementary material, which is available to authorized users.     

Eicosapentaenoic acid induces neovasculogenesis in human endothelial progenitor cells by modulating c-kit protein and PI3-K/Akt/eNOS signaling pathways

Human endothelial progenitor cells (hEPCs) derived from bone marrow play a crucial in the prevention of ischemic injuries in the course of postnatal neovasculogenesis. Frequent fish oil (FO) consumption is reportedly associated with a significantly lower incidence of cardiovascular disease. However, the molecular mechanisms of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) are not well elucidated, and the beneficial effect of FO consumption on neovasculogenesis has not been demonstrated yet. In the current study, we investigated the effects of EPA/DHA and FO consumption on neovasculogenesis by using vascular tube formation assay, Western blotting, real-time polymerase chain reaction, immunohistochemical staining and Doppler imaging in both in vitro and in vivo models. The results demonstrate that EPA and DHA dose-dependently enhance the neovasculogenesis and cell migration of hEPCs in vitro. The mechanisms of action included up-regulation of the c-kit protein as well as the phosphorylation of the ERK1/2, Akt and endothelial nitric oxide synthase signaling molecules in hEPCs. Furthermore, EPA significantly suppressed the expression of microRNA 221 in vitro. In experimental animal models, FO consumption significantly induced the formation of new blood vessels (neovasculogenesis) and prevented ischemia. Taken together, it is suggested that FO consumption enhances neovasculogenesis mainly through the effects of EPA in hEPCs, thereby exerting a preventive effect against ischemic injury.     

Long-Chain Omega-3 Polyunsaturated Fatty Acids Have Developmental Effects on the Crop Pest,the Cabbage White Butterfly Pieris rapae

Nutritional enhancement of crops using genetic engineering can potentially affect herbivorous pests. Recently, oilseed crops have been genetically engineered to produce the long-chain omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at levels similar to that found in fish oil; to provide a more sustainable source of these compounds than is currently available from wild fish capture. We examined some of the growth and development impacts of adding EPA and DHA to an artificial diet of Pieris rapae, a common pest of Brassicaceae plants. We replaced 1% canola oil with EPA: DHA (11:7 ratio) in larval diets, and examined morphological traits and growth of larvae and ensuing adults across 5 dietary treatments. Diets containing increasing amounts of EPA and DHA did not affect developmental phenology, larval or pupal weight, food consumption, nor larval mortality. However, the addition of EPA and DHA in larval diets resulted in progressively heavier adults (F _{4, 108} = 6.78; p = 0.011), with smaller wings (p < 0.05) and a higher frequency of wing deformities (R = 0.988; p = 0.001). We conclude that the presence of EPA and DHA in diets of larval P. rapae may alter adult mass and wing morphology; therefore, further research on the environmental impacts of EPA and DHA production on terrestrial biota is advisable.     

N-3 polyunsaturated fatty acids alleviate high glucose-mediated dysfunction of endothelial progenitor cells and prevent ischemic injuries both in vitro and in vivo

Hyperglycemia is associated with a reduced number of endothelial progenitor cells (EPCs) that impairs vascular function. Circulating EPCs play important roles in postnatal neovasculogenesis and the prevention of ischemic injury. Frequent consumption of fish oil (FO) that is abundant with eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) is reportedly associated with an alleviation of diabetic complications and a lowered incidence of cardiovascular disease. The aim of this study was to examine whether N-3 polyunsaturated fatty acids such as EPA and DHA would reverse the high glucose-mediated dysfunction of EPCs in vitro and thereby prevent the ischemic injury that occurs under the hyperglycemic conditions in Type 2 diabetes (T2D) db^−/− mice. The results demonstrate that EPA and DHA alleviate high glucose-mediated impairment of tubular formation in EPCs through a rescue of neovasculogenic capability. The molecular mechanisms underlying the effects of EPA and DHA include the activation of the extracellular signal-regulated kinase 1/2, Akt/endothelial nitric oxide synthase (eNOS) and AMP-activated kinase (AMPK) signaling cascades as well as the phosphorylation of the downstream FOXO3a protein in EPCs. Moreover, EPA and DHA up-regulate the expression of c-kit, erythroid 2-related factor and heme oxygenase-1 proteins. Daily consumption of FO at dosages of 4% and 6% (wt/wt) significantly increased the level of bone marrow-derived and circulating EPCs, induced a recovery of blood flow and prevented ischemic injuries in a T2D db^−/− mouse model. The effects of FO consumption were exerted the activation of Akt/eNOS and AMPK signaling cascades without any effect on the plasma VEGF level in vivo.     

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.