Fasting enriches liver triacylglycerol with n-3 polyunsaturated fatty acids: implications for understanding the adipose–liver axis in serum docosahexaenoic acid regulation

We investigated the effect of short-term fasting on coordinate changes in the fatty acid composition of adipose triacylglycerol (TAG), serum non-esterified fatty acids (NEFA), liver TAG, and serum TAG and phospholipids in mice fed ad libitum or fasted for 16 h overnight. In contrast to previous reports under conditions of maximal lipolysis, adipose tissue TAG was not preferentially depleted of n-3 PUFA or any specific fatty acids, nor were there any striking changes in the serum NEFA composition. Short-term fasting did, however, increase the hepatic proportion of n-3 PUFA, and almost all individual species of n-3 PUFA showed relative and absolute increases. The relative proportion of n-6 PUFA in liver TAG also increased but to a lesser extent, resulting in a significant decrease in the n-6:n-3 PUFA ratio (from 14.3 ± 2.54 to 9.6 ± 1.20), while the proportion of MUFA decreased significantly and SFA proportion did not change. Examination of genes involved in PUFA synthesis suggested that hepatic changes in the elongation and desaturation of precursor lipids could not explain this effect. Rather, an increase in the expression of fatty acid transporters specific for 22:6n-3 and other long-chain n-3 and n-6 PUFA likely mediated the observed hepatic enrichment. Analysis of serum phospholipids indicated a specific increase in the concentration of 22:6n-3 and 16:0, suggesting increased specific synthesis of DHA-enriched phospholipid by the liver for recirculation. Given the importance of blood phospholipid in distributing DHA to neural tissue, these findings have implications for understanding the adipose–liver–brain axis in n-3 PUFA metabolism.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0490-2) contains supplementary material, which is available to authorized users.     

Neuropathological Responses to Chronic NMDA in Rats Are Worsened by Dietary n-3 PUFA Deprivation but Are Not Ameliorated by Fish Oil Supplementation

Background

Dietary long-chain n-3 polyunsaturated fatty acid (PUFA) supplementation may be beneficial for chronic brain illnesses, but the issue is not agreed on. We examined effects of dietary n-3 PUFA deprivation or supplementation, compared with an n-3 PUFA adequate diet (containing alpha-linolenic acid [18:3 n-3] but not docosahexaenoic acid [DHA, 22:6n-3]), on brain markers of lipid metabolism and excitotoxicity, in rats treated chronically with NMDA or saline.

Methods

Male rats after weaning were maintained on one of three diets for 15 weeks. After 12 weeks, each diet group was injected i.p. daily with saline (1 ml/kg) or a subconvulsive dose of NMDA (25 mg/kg) for 3 additional weeks. Then, brain fatty acid concentrations and various markers of excitotoxicity and fatty acid metabolism were measured.

Results

Compared to the diet-adequate group, brain DHA concentration was reduced, while n-6 docosapentaenoic acid (DPA, 22:5n-6) concentration was increased in the n-3 deficient group; arachidonic acid (AA, 20:4n-6) concentration was unchanged. These concentrations were unaffected by fish oil supplementation. Chronic NMDA increased brain cPLA₂ activity in each of the three groups, but n-3 PUFA deprivation or fish oil did not change cPLA₂ activity or protein compared with the adequate group. sPLA₂ expression was unchanged in the three conditions, whereas iPLA₂ expression was reduced by deprivation but not changed by supplementation. BDNF protein was reduced by NMDA in N-3 PUFA deficient rats, but protein levels of IL-1β, NGF, and GFAP did not differ between groups.

Conclusions

N-3 PUFA deprivation significantly worsened several pathological NMDA-induced changes produced in diet adequate rats, whereas n-3 PUFA supplementation did not affect NMDA induced changes. Supplementation may not be critical for this measured neuropathology once the diet has an adequate n-3 PUFA content.     

Is Western Diet-Induced Nonalcoholic Steatohepatitis in Ldlr-/- Mice Reversible?

Background

Nonalcoholic fatty liver disease (NAFLD) is a major public health burden in western societies. The progressive form of NAFLD, nonalcoholic steatohepatitis (NASH), is characterized by hepatosteatosis, inflammation, oxidative stress, and hepatic damage that can progress to fibrosis and cirrhosis; risk factors for hepatocellular carcinoma. Given the scope of NASH, validating treatment protocols (i.e., low fat diets and weight loss) is imperative.

Methods

We evaluated the efficacy of two diets, a non-purified chow (NP) and purified (low-fat low-cholesterol, LFLC) diet to reverse western diet (WD)-induced NASH and fibrosis in Ldlr^-/- mice.

Results

Mice fed WD for 22–24 weeks developed robust hepatosteatosis with mild fibrosis, while mice maintained on the WD an additional 7–8 weeks developed NASH with moderate fibrosis. Returning WD-fed mice to the NP or LFLC diets significantly reduced body weight and plasma markers of metabolic syndrome (dyslipidemia, hyperglycemia) and hepatic gene expression markers of inflammation (Mcp1), oxidative stress (Nox2), fibrosis (Col1A, LoxL2, Timp1) and collagen crosslinking (hydroxyproline). Time course analyses established that plasma triglycerides and hepatic Col1A1 mRNA were rapidly reduced following the switch from the WD to the LFLC diet. However, hepatic triglyceride content and fibrosis did not return to normal levels 8 weeks after the change to the LFLC diet. Time course studies further revealed a strong association (r² ≥ 0.52) between plasma markers of inflammation (TLR2 activators) and hepatic fibrosis markers (Col1A, Timp1, LoxL2). Inflammation and fibrosis markers were inversely associated (r² ≥ 0.32) with diet-induced changes in hepatic ω3 and ω6 polyunsaturated fatty acids (PUFA) content.

Conclusion

These studies establish a temporal link between plasma markers of inflammation and hepatic PUFA and fibrosis. Low-fat low-cholesterol diets promote reversal of many, but not all, features associated with WD-induced NASH and fibrosis in Ldlr^-/- mice.     

从人和动物血浆及红细胞膜脂肪酸谱探讨脂质代谢实验模型动物选取

目的比较大鼠、家兔和人血浆及红细胞膜脂肪酸谱的异同,为脂质代谢模型动物的选取提供可靠依据。方法采用氯仿-甲醇快速提取大鼠、家兔和人血浆及细胞膜中的脂肪,并用碱法甲基化,经薄层层析纯化的甲基酯用100 mm×0.25 mm气相色谱毛细管柱测定脂肪酸组成。结果血浆中总MUFA、9c18：1、9c12c18：2 n-6和α-18：3 n-3等脂肪酸含量较高;而红细胞膜中总PUFA、AA、22：4n-6、DPA和DHA等脂肪酸含量较高;人和大鼠16：0、18：0、18：1、α-18：3n-3、AA、EPA、DHA、MUFA、n-3PUFA和n-6/n-3值均较为接近,但和家兔存在显著差异。结论与家兔相比,大鼠和人血浆及红细胞膜脂肪酸谱较接近,因此,在选取脂质代谢实验动物时,应优先考虑大鼠为模型动物,以得到与人体实际更接近的结果。     

N-3 PUFA improved post-menopausal depression induced by maternal separation and chronic mild stress through serotonergic pathway in rats—effect associated with lipid mediators

Early life maternal separation (MS) increases the vulnerability to depression in rats with chronic mild stress (CMS). N-3 polyunsaturated fatty acids (PUFA) improved depressive behaviors in rats with acute stress; however, their effects on rats with MS+CMS were not apparent. The purpose of the present study was to investigate the hypothesis that lifetime n-3 PUFA supplementation improves post-menopausal depression through the serotonergic and glutamatergic pathways while modulating n-3 PUFA-derived metabolites. Female rats were fed diets of either 0% n-3 PUFA during lifetime or 1% energy n-3 PUFA during pre-weaning, post-weaning, or lifetime periods. Rats were allocated to non-MS or MS groups and underwent CMS after ovariectomy. N-3 PUFA increased brain n-3 PUFA-derived endocannabinoid/oxylipin levels, and reversed depressive behaviors. N-3 PUFA decreased blood levels of adrenocorticotropic hormone and corticosterone, and brain expressions of corticotropin-releasing factor and miRNA-218, which increased the expression of the glucocorticoid receptor. N-3 PUFA decreased the expression of tumor necrosis factor-α, interleukin (IL)-6, IL-1β, and prostaglandin E₂, while increased the expression of miRNA-155. N-3 PUFA also increased brainstem serotonin levels and hippocampal expression of the serotonin-1A receptor, cAMP response element-binding protein (CREB), phospho-CREB, and brain-derived neurotrophic factor. However, n-3 PUFA did not affect brain expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subtype 1, N-methyl-D-aspartate receptor subtype 2B, or miRNA-132. Moreover, n-3 PUFA exposure during lifetime caused greater effects than pre- and post-weaning periods. The present study suggested that n-3 PUFA improved depressive behaviors through serotonergic pathway while modulating the metabolites of n-3 PUFA in post-menopausal depressed rats with chronic stress.     

Two weeks of docosahexaenoic acid (DHA) supplementation increases synthesis-secretion kinetics of n-3 polyunsaturated fatty acids compared to 8 weeks of DHA supplementation

Docosahexaenoic acid (DHA, 22:6n-3) must be consumed in the diet or synthesized from n-3 polyunsaturated fatty acid (PUFA) precursors. However, the effect of dietary DHA on the metabolic pathway is not fully understood. Presently, 21-day-old Long Evans rats were weaned onto one of four dietary protocols: 1) 8 weeks of 2% ALA (ALA), 2) 6 weeks ALA followed by 2 weeks of 2% ALA + 2% DHA (DHA), 3) 4 weeks ALA followed by 4 weeks DHA and 4) 8 weeks of DHA. After the feeding period, ²H₅-ALA and ¹³C₂₀-eicosapentaenoic acid (EPA, 20:5n-3) were co-infused and blood was collected over 3 h for determination of whole-body synthesis-secretion kinetics. The synthesis-secretion coefficient (ml/min, means ± SEM) for EPA (0.238±0.104 vs. 0.021±0.001) and DPAn-3 (0.194±0.060 vs. 0.020±0.008) synthesis from plasma unesterified ALA, and DPAn-3 from plasma unesterified EPA (2.04±0.89 vs. 0.163±0.025) were higher (P<.05) after 2 weeks compared to 8 weeks of DHA feeding. The daily synthesis-secretion rate (nmol/d) of DHA from EPA was highest after 4 weeks of DHA feeding (843±409) compared to no DHA (70±22). Liver gene expression of ELOVL2 and FADS2 were lower (P<.05) after 4 vs. 8 weeks of DHA. Higher synthesis-secretion kinetics after 2 and 4 weeks of DHA feeding suggests an increased throughput of the PUFA metabolic pathway. Furthermore, these findings may lead to novel dietary strategies to maximize DHA levels while minimizing dietary requirements.     

Chronic dietary n-6 PUFA deprivation leads to conservation of arachidonic acid and more rapid loss of DHA in rat brain phospholipids

To determine how the level of dietary n-6 PUFA affects the rate of loss of arachidonic acid (ARA) and DHA in brain phospholipids, male rats were fed either a deprived or adequate n-6 PUFA diet for 15 weeks postweaning, and then subjected to an intracerebroventricular infusion of ³H-ARA or ³H-DHA. Brains were collected at fixed times over 128 days to determine half-lives and the rates of loss from brain phospholipids (J_out). Compared with the adequate n-6 PUFA rats, the deprived n-6-PUFA rats had a 15% lower concentration of ARA and an 18% higher concentration of DHA in their brain total phospholipids. Loss half-lives of ARA in brain total phospholipids and fractions (except phosphatidylserine) were longer in the deprived n-6 PUFA rats, whereas the J_out was decreased. In the deprived versus adequate n-6 PUFA rats, the J_out of DHA was higher. In conclusion, chronic n-6 PUFA deprivation decreases the rate of loss of ARA and increases the rate of loss of DHA in brain phospholipids. Thus, a low n-6 PUFA diet can be used to target brain ARA and DHA metabolism.     

Markers of Endogenous Desaturase Activity and Risk of Coronary Heart Disease in the CAREMA Cohort Study

Background

Intakes of n-3 polyunsaturated fatty acids (PUFAs), especially EPA (C20∶5n-3) and DHA (C22∶6n-3), are known to prevent fatal coronary heart disease (CHD). The effects of n-6 PUFAs including arachidonic acid (C20∶4n-6), however, remain unclear. δ-5 and δ-6 desaturases are rate-limiting enzymes for synthesizing long-chain n-3 and n-6 PUFAs. C20∶4n-6 to C20∶3n-6 and C18∶3n-6 to C18∶2n-6 ratios are markers of endogenous δ-5 and δ-6 desaturase activities, but have never been studied in relation to incident CHD. Therefore, the aim of this study was to investigate the relation between these ratios as well as genotypes of FADS1 rs174547 and CHD incidence.

Methods

We applied a case-cohort design within the CAREMA cohort, a large prospective study among the general Dutch population followed up for a median of 12.1 years. Fatty acid profile in plasma cholesteryl esters and FADS1 genotype at baseline were measured in a random subcohort (n = 1323) and incident CHD cases (n = 537). Main outcome measures were hazard ratios (HRs) of incident CHD adjusted for major CHD risk factors.

Results

The AA genotype of rs174547 was associated with increased plasma levels of C204n-6, C20∶5n-3 and C22∶6n-3 and increased δ-5 and δ-6 desaturase activities, but not with CHD risk. In multivariable adjusted models, high baseline δ-5 desaturase activity was associated with reduced CHD risk (P for trend = 0.02), especially among those carrying the high desaturase activity genotype (AA): HR (95% CI) = 0.35 (0.15–0.81) for comparing the extreme quintiles. High plasma DHA levels were also associated with reduced CHD risk.

Conclusion

In this prospective cohort study, we observed a reduced CHD risk with an increased C20∶4n-6 to C20∶3n-6 ratio, suggesting that δ-5 desaturase activity plays a role in CHD etiology. This should be investigated further in other independent studies.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.     

Estrogen and n-3 polyunsaturated fatty acid supplementation have a synergistic hypotriglyceridemic effect in ovariectomized rats

The n-3 polyunsaturated fatty acids (PUFAs), EPA and DHA, as well as estrogen have been shown to decrease circulating levels of triglyceride (TG), but their underlying mode of action is unclear. The purpose of this study was to determine the effects of n-3 PUFA consumption and estrogen injection on TG metabolism. Rats (n = 48) were fed a modified AIN-93G diet with 0, 1, or 2 % EPA + DHA relative to the total energy intake during 12 weeks. At 8 weeks, rats were ovariectomized (OVX), and after a 1-week recovery, rats were injected with either 17β-estradiol-3-benzoate (E₂) or corn oil for the last 3 weeks. The n-3 PUFA consumption and E₂ injection independently decreased the hepatic expressions of sterol regulatory element-binding protein 1, acetyl-CoA carboxylase 1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2) (P < 0.05). There were interactions between n-3 PUFA consumption and E₂ injection on hepatic expression of FAS and DGAT2. In addition, n-3 PUFA consumption and E₂ injection up-regulated the expression of AMP-activated protein kinase (AMPK), phosphorylated AMPK, peroxisomal proliferator-activated receptor α, and carnitine palmitoyltransferase 1 in liver and skeletal muscle. E₂ injection increased the expression of estrogen receptor α and β in skeletal muscle and liver, but n-3 PUFA consumption increased the expression of both receptors only in skeletal muscle. The present study suggests that the hypotriglyceridemic effects of n-3 PUFA consumption and E₂ injection could be due to the down-regulation of hepatic TG synthesis and up-regulation of TG oxidation in liver and skeletal muscle in OVX rats.     

Dietary docosahexaenoic and eicosapentaenoic acid: Emerging mediators of inflammation

The inflammatory response is designed to help fight and clear infection, remove harmful chemicals, and repair damaged tissue and organ systems. Although this process, in general, is protective, the failure to resolve the inflammation and return the target tissue to homeostasis can result in disease, including the promotion of cancer. A plethora of published literature supports the contention that dietary n-3 polyunsaturated fatty acids (PUFA), and eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in particular, are important modulators of a host's inflammatory/immune responses. The following review describes a mechanistic model that may explain, in part, the pleiotropic anti-inflammatory and immunosuppressive properties of EPA and DHA. In this review, we focus on salient studies that address three overarching mechanisms of n-3 PUFA action: (i) modulation of nuclear receptor activation, i.e., nuclear factor-κB (NF-κB) suppression; (ii) suppression of arachidonic acid–cyclooxygenase-derived eicosanoids, primarily prostaglandin E₂-dependent signaling; and (iii) alteration of the plasma membrane micro-organization (lipid rafts), particularly as it relates to the function of Toll-like receptors (TLRs), and T-lymphocyte signaling molecule recruitment to the immunological synapse (IS). We propose that lipid rafts may be targets for the development of n-3 PUFA-containing dietary bioactive agents to down-modulate inflammatory and immune responses and for the treatment of autoimmune and chronic inflammatory diseases.     

Polyunsaturated fatty acids in fish tissues more closely resemble algal than terrestrial diet sources

The River Continuum Concept implies that consumers in headwater streams have greater dietary access to terrestrial basal resources, but recent studies have highlighted the dietary importance of high-quality algae. Algae provide consumers with physiologically important omega-3 (n-3) polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). However, terrestrial plants and most benthic stream algae lack the long-chain (LC) n-3 PUFA docosahexaenoic acid (DHA, 22:6n-3), which is essential for neural development in fish and other vertebrates. We sampled subalpine streams to investigate how the PUFA composition of neural (brain and eyes), muscle, and liver tissues of freshwater fish is related to their potential diets (macroinvertebrates, epilithon, fresh and conditioned terrestrial leaves). The PUFA composition of consumers was more similar to epilithon than to terrestrial leaves. Storage lipids of eyes most closely resembled dietary PUFA (aquatic invertebrates and algae). However, DHA and arachidonic acid (ARA, 20:4n-6) were not directly available in the diet but abundant in organs. This implies that algal PUFA were selectively retained or were produced internally via enzymatic PUFA conversion by aquatic consumers. This field study demonstrates the nutritional importance of algal PUFA for neural organs in aquatic consumers of headwater regions.

     

Diets enriched in menhaden fish oil, seal oil, or shark liver oil have distinct effects on the lipid and fatty-acid composition of guinea pig heart

The purpose of this investigation was to determine whether diets supplemented with oils from three different marine sources, all of which contain high proportions of long-chain n-3 polyunsaturated fatty acids (PUFA), result in qualitatively distinct lipid and fatty acid profiles in guinea pig heart. Albino guinea pigs (14 days old) were fed standard, nonpurified guinea pig diets (NP) or NP supplemented with menhaden fish oil (MO), harp seal oil (SLO) or porbeagle shark liver oil (PLO) (10%, w/w) for 4-5 weeks. An n-6 PUFA control group was fed NP supplemented with corn oil (CO). All animals appeared healthy, with weight gains marginally lower in animals fed the marine oils. Comparison of relative organ weights indicated that only the livers responded to the diets, and that they were heavier only in the marine-oil fed guinea pigs. Heart total cholesterol levels were unaffected by supplementing NP with any of the oils, whereas all increased the triacylglycerol (TAG) content. The fatty-acid profiles of totalphospholipid (TPL), TAG and free fatty acid (FFA) fractions of heart lipids showed that feeding n-3 PUFA significantly altered the proportions of specific fatty-acid classes. For example, all marine-oil-rich diets were associated with increases in total monounsaturated fatty acids in TPL (p < 0.05), and with decreases in total saturates in TAG (p < 0.05). Predictably, the n-3 PUFA enriched regimens significantly increased the cardiac content of n-3 PUFA and decreased that of n-6 PUFA, although the extent varied among the diets. As a result, n-6/n-3 ratios were significantly lower in all myocardial lipid classes of marine-oil-fed guinea pigs. Analyses of the profiles of individual PUFA indicated that quantitatively, the fatty acids of the three marine oils were metabolized and/or incorporated into TPL, TAG and FFA in a diet-specific manner. In animals fed MO-enriched diets in which eicosapentaenoic acid (EPA) > docosahexacnoic acid (DHA), ratios of DHA /EPA in the hearts were 1.2, 2.2 and 1.5 in TPL, TAG and FFA, respectively. In SLO-fed guinea pigs in which dietary EPA DHA, ratios of DHA/EPA were 0.9, 3.4 and 2.1 in TPL, TAG and FFA, respectively. Feeding NP + PLO (DHA/EPA = 4.8), resulted in values for DHA/EPA in cardiac tissue of 2.1, 10.6 and 2.9 in TPL, TAG and FFA, respectively. In the TAG and FFA, proportions of n-3 docosapentaenoic acid (n-3 DPA) were equal to or higher than EPA in the SLO- and PLO-fed animals. The latter group exhibited the greatest difference between the DHA/n-3 DPA ratio in the diet and in cardiac TAG and FFA fractions (7, 3.4 and 3.1, respectively). Quantitative analysis indicated that 85% of the n-3 PUFA were in TPL, 7-11% were in TAG, and 2-6% were FFA. Specific patterns of distribution of EPA, DPA and DHA depended on the dietary oil. Both the qualitative and quantitative results of this study demonstrated that in guinea pigs, n-3 PUFA in different marine oils are metabolized and/or incorporated into cardiac lipids in distinct manners. In support of the concept that the diet-induced alterations reflect changes specifically in cardiomyocytes, we observed that direct supplementation of cultured guinea pig myocytes for 2-3 weeks with EPA or DHA produced changes in the PUFA profiles of their TPL that were qualitatively similar to those observed in tissue from the dietary study. The factors that regulate specific deposition of n-3 PUFA from either dietary oils or individual PUFA are not yet known, however the differences that we observed could in some manner be related to cardiac function and thus their relative potentials as health-promoting dietary fats.     

Composition of galactolipids,betaine lipids and triglyceride-associated fatty acids of the symbiotic dinoflagellate Zooxanthella (Brandtodinium) nutricula: A glimpse into polyunsaturated fatty acids available to its polycystine radiolarian host

Zooxanthella nutricula is a photosynthetic dinoflagellate symbiont of polycystine radiolarians. As such, it is hypothesized to provide fixed organic carbon, including in the form of acylglycerolipids and sterols, to its non-photosynthetic host. We have previously characterized the sterols of Z. nutricula that may be transferred to its host and, in the present study, have turned our attention to three classes of fatty acid-containing lipids, chloroplast-associated galactolipids, betaine lipids, which are non-phosphorylated phospholipid analogs present in many eukaryotes, and triglycerides. Zooxanthella nutricula was observed using positive-ion electrospray/mass spectrometry (ESI/MS) and ESI/MS/MS to produce the galactolipids mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) enriched in octadecapentaenoic (18:5(n-3)) and octadecatetraenoic (18:4(n-3)) acid to place it within a group of peridinin-containing dinoflagellates in a C₁₈/C₁₈ (sn-1/sn-2 fatty acid regiochemistry) cluster, as opposed to another cluster with C₂₀/C₁₈ MGDG and DGDG, where the C₂₀ fatty acid is eicosapentaenoic acid (20:5(n-3)) and the C₁₈ fatty acid is either 18:5(n-3) or 18:4(n-3). Zooxanthella nutricula was also observed to produce 38:10 (total number of fatty acid carbons:total number of double bonds), 38:6, and 44:7 diacylglycerylcarboxyhydroxymethylcholine (DGCC) as the sole type of betaine lipid. Although it is more difficult to determine which fatty acids are present in the sn-1 and sn-2 positions on the glycerol backbone of DGCC using ESI/MS/MS, gas chromatography/mass spectrometry (GC/MS)-based examination indicated the putatively DGCC-associated polyunsaturated fatty acid (PUFA) docosahexaenoic acid (22:6(n-3)). Coupled with the C₁₈ PUFAs of MGDG and DGDG, and fatty acids associated with triglycerides (also examined via GC/MS), Z. nutricula could serve as a rich source of PUFAs for its radiolarian host. These data demonstrate that Z. nutricula produces a similar set of PUFA-containing lipids as Symbiodinium microadriaticum, a photosynthetic dinoflagellate symbiont of cnidarians, indicating a metabolic commonality in these phylogenetically discrete dinoflagellate symbionts with unrelated host organisms.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Conversion of alpha-linolenic acid to longer-chain polyunsaturated fatty acids in human adults

The principal biological role of alpha-linolenic acid (alphaLNA; 18:3n-3) appears to be as a precursor for the synthesis of longer chain n-3 polyunsaturated fatty acids (PUFA). Increasing alphaLNA intake for a period of weeks to months results in an increase in the proportion of eicosapentaenoic acid (EPA; 20:5n-3) in plasma lipids, in erythrocytes, leukocytes, platelets and in breast milk but there is no increase in docosahexaenoic acid (DHA; 22:6n-3), which may even decline in some pools at high alphaLNA intakes. Stable isotope tracer studies indicate that conversion of alphaLNA to EPA occurs but is limited in men and that further transformation to DHA is very low. The fractional conversion of alphaLNA to the longer chain n-3 PUFA is greater in women which may be due to a regulatory effect of oestrogen. A lower proportion of alphaLNA is used for beta-oxidation in women compared with men. Overall, alphaLNA appears to be a limited source of longer chain n-3 PUFA in humans. Thus, adequate intakes of preformed long chain n-3 PUFA, in particular DHA, may be important for maintaining optimal tissue function. Capacity to up-regulate alphaLNA conversion in women may be important for meeting the demands of the fetus and neonate for DHA.     

Metabolic Engineering Plant Seeds with Fish Oil-Like Levels of DHA

Background

Omega-3 long-chain (≥C₂₀) polyunsaturated fatty acids (ω3 LC-PUFA) have critical roles in human health and development with studies indicating that deficiencies in these fatty acids can increase the risk or severity of cardiovascular and inflammatory diseases in particular. These fatty acids are predominantly sourced from fish and algal oils, but it is widely recognised that there is an urgent need for an alternative and sustainable source of EPA and DHA. Since the earliest demonstrations of ω3 LC-PUFA engineering there has been good progress in engineering the C₂₀ EPA with seed fatty acid levels similar to that observed in bulk fish oil (∼18%), although undesirable ω6 PUFA levels have also remained high.

Methodology/Principal Findings

The transgenic seed production of the particularly important C₂₂ DHA has been problematic with many attempts resulting in the accumulation of EPA/DPA, but only a few percent of DHA. This study describes the production of up to 15% of the C₂₂ fatty acid DHA in Arabidopsis thaliana seed oil with a high ω3/ω6 ratio. This was achieved using a transgenic pathway to increase the C₁₈ ALA which was then converted to DHA by a microalgal Δ6-desaturase pathway.

Conclusions/Significance

The amount of DHA described in this study exceeds the 12% level at which DHA is generally found in bulk fish oil. This is a breakthrough in the development of sustainable alternative sources of DHA as this technology should be applicable in oilseed crops. One hectare of a Brassica napus crop containing 12% DHA in seed oil would produce as much DHA as approximately 10,000 fish.     

Change in fatty acid composition of serum lipids in obese females after short-term weight-reducing regimen with the addition of n-3 long chain polyunsaturated fatty acids in comparison to controls

Short-term weight-reducing regimens were shown to influence fatty acid composition of serum lipids unfavorably. Adding long chain n-3 polyunsaturated fatty acids (n-3 LC PUFA) to a low-calorie diet (LCD) could avoid these changes. The aim of this study was to examine the effect of a short-term in-patient weight-reducing regimen including LCD with yogurt enriched by low doses of n-3 PUFA (n-3 LCD). The enriched yogurt contained 790 mg of fish oil, predominantly eicosapentaenoic (20:5n-3; EPA) and docosahexaenoic (22:6n-3; DHA). Forty obese women were randomly assigned to the group consuming LCD and joghurt either with or without n-3 enrichment. Following the 3-week diet in the n-3 LCD group a significantly higher increase in the proportion of n-3 LC PUFA (sum of n-3 FA, EPA and DHA) in serum lipids was confirmed. In phospholipids (PL) a significant difference in the sum of n-6 fatty acids was found, a decrease in the n-3 LCD group and an increase in LCD group. Significantly higher increase in the PL palmitate (16:0) was shown in the LCD group. The results suggest that low doses of n-3 fatty acid enrichment can help to avoid unfavorable changes in fatty acid composition in serum lipids after a short-term weight-reducing regimen.