Ability Of Polyunsaturated Fatty Acids Research Articles

Functions of aldolase in lipid synthesis of Schizochytrium sp. by gene disruption to switch carbon metabolism

BackgroundAs a key rate-limiting enzyme in the glycolytic pathway of cells, aldolase affects the distribution of intracellular carbon flux and determines the overall ability of subsequent cell metabolism, which are mainly reported in the medical related researches, but rarely involved microorganisms. In this study, the aldolase gene of Schizochytrium limacinum SR21 (ALDOA) was knocked out to explore the effect of regulating carbon flux on cell growth and lipid synthesis.ResultsThe knockout of ALDOA showed an adverse effect on cell growth and total lipids production, which was decreased by 9.6% and 23.2%, respectively, but helped to improve the synthetic ability of polyunsaturated fatty acids (PUFAs), in which the proportion of docosahexaenoic acid (DHA) increased by 22.9%. Analysis of phospholipomics, real-time quantitative PCR and metabolomics revealed that the knockout of ALDOA weakened the glycolysis pathway and tricarboxylic acid cycle to inhibit cell growth, and lowered the Kennedy pathway to reduce the production of total lipids and the synthesis of phospholipids to affect cell metabolism. Correspondingly, the knockout of ALDOA enhanced the metabolic flux of the pentose phosphate pathway to provide more reducing power for PUFAs accumulation and improved the glycerophosphorylcholine acylation pathway to promote the accumulation of DHA.ConclusionsALDOA knockout redistributes the carbon metabolic flux in cells, by weakening the glycolysis, tricarboxylic acid cycle and glyceride synthesis pathway to inhibit cell growth and total lipid production, and strengthening the pentose phosphate pathway and glycerophosphorylcholine acylation pathway to increase the synthesis of PUFAs and DHA accumulation. This study provides a new idea for identifying the aldolase function in microorganisms and a metabolic strategy to improve DHA accumulation in Schizochytrium.

Modulation of Fatty Acid-Related Genes in the Response of H9c2 Cardiac Cells to Palmitate and n-3 Polyunsaturated Fatty Acids.

While high levels of saturated fatty acids are associated with impairment of cardiovascular functions, n-3 polyunsaturated fatty acids (PUFAs) have been shown to exert protective effects. However the molecular mechanisms underlying this evidence are not completely understood. In the present study we have used rat H9c2 ventricular cardiomyoblasts as a cellular model of lipotoxicity to highlight the effects of palmitate, a saturated fatty acid, on genetic and epigenetic modulation of fatty acid metabolism and fate, and the ability of PUFAs, eicosapentaenoic acid, and docosahexaenoic acid, to contrast the actions that may contribute to cardiac dysfunction and remodeling. Treatment with a high dose of palmitate provoked mitochondrial depolarization, apoptosis, and hypertrophy of cardiomyoblasts. Palmitate also enhanced the mRNA levels of sterol regulatory element-binding proteins (SREBPs), a family of master transcription factors for lipogenesis, and it favored the expression of genes encoding key enzymes that metabolically activate palmitate and commit it to biosynthetic pathways. Moreover, miR-33a, a highly conserved microRNA embedded in an intronic sequence of the SREBP2 gene, was co-expressed with the SREBP2 messenger, while its target carnitine palmitoyltransferase-1b was down-regulated. Manipulation of the levels of miR-33a and SREBPs allowed us to understand their involvement in cell death and hypertrophy. The simultaneous addition of PUFAs prevented the effects of palmitate and protected H9c2 cells. These results may have implications for the control of cardiac metabolism and dysfunction, particularly in relation to dietary habits and the quality of fatty acid intake.

Abstract 4536: Polyunsaturated fatty acids alter the epigenetic landscape in triple-negative breast cancer (TNBC)

Abstract Breast cancer is the second leading cause of cancer-related deaths in US women. The lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor II (HER2) in basal-like breast cancer subtypes, commonly referred to as triple-negative breast cancer (TNBC), account for 10-20% of cases and are associated with the poorest short-term prognosis due to a lack of targeted therapies. Epigenetic therapies are currently of interest to target TNBC, as many theorize epigenetic reprogramming is required for malignant transformation and invasion. Research has shown omega-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA; C22:6, n-3) and eicosapentaenoic acid (EPA; C20:5, n-3) inhibit tumorigenesis, whereas linoleic acid (LA; C18:2, n-6) has been shown to promote neoplastic growth; supporting the association of increased cancer incidence with omega-6 PUFA dietary intake. Arachidonic acid (AA; C20:4, n-6) is associated with pro-inflammatory signaling processes, and therefore tumorigenesis; however, conflicting results exist in the literature across tumor types. PUFAs are bioactive nutrients known to have epigenetic effects within normal cells and recently, the ability of PUFA to alter DNA methylation and histone acetylation was reported in HCT-116 colon carcinoma (Cho, Y. et al, 2014). Current research focuses on the potential of dietary intervention in TNBC as an epigenetic therapy. This study monitors epigenetic alterations in MDA-MB-231 TNBC in response to DHA, LA, EPA and AA. DHA, EPA and AA all elicit similar levels of apoptosis in MDA-MB-231, while LA promotes tumorigenesis. Initial results demonstrate promoter methylation changes (p<0.05) with a decrease in PTGS2 (prostaglandin-endoperoxidase synthase 2), and SOCS1(suppressor of cytokine signaling 1) and an increase in CDX2 (caudal type homeobox 2), HIC1 (hypermethylated in cancer 1), SPARC (Secreted Protein, Acidic, Cysteine-Rich (Osteonectin)), TSC1 (tuberous sclerosis 1), and WIF1 (WNT inhibitory factor 1) post DHA treatment. Studies are on-going to assess if the promoter methylation changes result in transcriptome and proteome level fluctuations. We are also investigating global gDNA methylation alterations, including 5-methylcytosine, in response to treatment. DHA, EPA, LA and AA exhibit dose-dependent effects on histone acetylation and HDAC class I/II activity. There is an increase in HDAC class I/II activity with the EC75, but not with the EC50 treatment for all PUFAs. Histone 3 global acetylation is decreased for all PUFA treatments at the EC75 versus EC50 dose. However, only LA and DHA have decreased H3 acetylation with an EC75 dose whereas EPA and AA have increased acetylation levels compared to the control. Research is ongoing to define a possible mechanism, but current data suggests PUFA may be useful as epigenetically-targeted cancer therapeutic agent or adjuvant. Citation Format: Amy M. Chattin, Mykelti O’Brien, Ronald S. Pardini. Polyunsaturated fatty acids alter the epigenetic landscape in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4536.

LA and ALA prevent glucose intolerance in obese male rats without reducing reactive lipid content, but cause tissue-specific changes in fatty acid composition.

While the cause of Type 2 diabetes remains poorly defined, the accumulation of reactive lipids within white adipose tissue, skeletal muscle, and liver have been repeatedly implicated as underlying mechanisms. The ability of polyunsaturated fatty acids (PUFAs) to prevent the development of insulin resistance has gained considerable interest in recent years; however, the mechanisms-of-action remain poorly described. Therefore, we determined the efficacy of diets supplemented with either linoleic acid (LA) or α-linolenic acid (ALA) in preventing insulin resistance and reactive lipid accumulation in key metabolic tissues of the obese Zucker rat. Obese Zucker rats displayed impaired glucose homeostasis and reduced n-3 and n-6 PUFA content in the liver and epididymal white adipose tissue (EWAT). After the 12-wk feeding intervention, both LA- and ALA-supplemented diets prevented whole body glucose and insulin intolerance; however, ALA had a more pronounced effect. These changes occurred in association with n-3 and n-6 accumulation in all tissues studied, albeit to different extents (EWAT > liver > muscle). Triacylglycerol (TAG), diacylglycerol (DAG), ceramide, and sphingolipid accumulation were not attenuated in obese animals supplemented with either LA or ALA, suggesting that preservation of glucose homeostasis occurred independent of changes in reactive lipid content. However, PUFA-supplemented diets differentially altered the fatty acid composition of TAGs, DAGs, and PLs in a tissue-specific manner, suggesting essential fatty acid metabolism differs between tissues. Together, our results indicate that remodeling of the fatty acid composition of various lipid fractions may contribute to the improved glucose tolerance observed in obese rats fed PUFA-supplemented diets.

Abstract 38: Saturated free fatty acids activate a c-Src-JNK1 axis within lipid rafts: Implications for the development of insulin resistance, obesity, and obesity-associated cancer

Abstract Obesity is a risk factor for the development of insulin resistance/type 2 diabetes and several types of cancer, including cancers of the colon, breast, liver, endometrium and esophagus. Obese individuals have increased levels of free fatty acids (FFAs) in their serum, and so we are investigating signaling pathways that are activated by FFAs in vitro to gain insight into pathways that may trigger insulin resistance and the activation of oncogenes in obese individuals in vivo. Obesity is associated with chronic activation of c-Jun NH2-terminal kinase (JNK)1 in liver, fat and muscle tissue, and studies using JNK1-deficient mice demonstrate that JNK activation is important for adiposity, peripheral insulin resistance and the production of the proinflammatory cytokines, but the mechanism of JNK1 activation in obesity is unclear. Our data demonstrate that JNK1 is activated by FFAs in vitro in a c-Src-dependent manner, and that activation of c-Src occurs within lipid rafts. Src-deficient murine embryonic fibroblasts (MEFs) and SYF MEFs, which are deficient in c-Src family kinases c-Src, c-Yes and c-Fyn, do not activate JNK1 after exposure to FFAs in vitro, while NIH 3T3 MEFs and SYF MEFs reconstituted with c-Src activate JNK1 normally. In contrast, SYF MEFs reconstituted with c-Yes and c-Fyn do not activate JNK1. Knockdown of c-Src with shRNA in HEK 293T cells also inhibits FFA-induced activation of JNK1, and inhibition of c-Src with pharmacologic inhibitors has the same effect in J774 mouse macrophages. Pharmacologic inhibition of c-Src also prevents the FFA-induced upregulation of tumor necrosis factor (TNF)-α mRNA in macrophages. Importantly, c-Src is activated within lipid rafts after FFA treatment, as seen by both detergent-dependent and detergent-independent methods of raft isolation. Interestingly, omega-3 polyunsaturated fatty acids (PUFAs), which can protect against the onset of obesity, will also block JNK activation following treatment with saturated FFAs, and we suspect that this effect may be due to the ability of PUFAs to exclude c-Src from lipid rafts. Finally, preliminary data suggests that c-Src may be aberrantly activated in liver tissue from obese mice that were fed a high-fat diet. Experiments are currently underway to verify the role of c-src in the activation of JNK1 in vivo by acutely infusing mice with a free fatty acid emulsion in the presence of a pharmacologic inhibitor of c-src. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 38.

Differential modulation of the glutamate transporters GLT1, GLAST and EAAC1 by docosahexaenoic acid

At present, the ability of polyunsaturated fatty acids (PUFAs) to regulate individual glutamate transporter subtypes is poorly understood and very little information exists on the mechanism(s) by which PUFAs achieve their effects on the transport process. Here we investigate the effect of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the activity of the mammalian glutamate transporter subtypes, GLT1, GLAST and EAAC1 individually expressed in human embryonic kidney (HEK) cells. Exposure of cells to 100 μM DHA increased the rate of d-[ 3H]aspartate uptake by over 72% of control in HEK GLT1 cells, and by 45% of control in HEK EAAC1 cells. In contrast, exposure of HEK GLAST cells to 200 μM DHA resulted in almost 40% inhibition of d-[ 3H]aspartate transport. Removal of extracellular calcium increased the inhibitory potential of DHA in HEK GLAST cells. In contrast, in the absence of extracellular calcium, the stimulatory effect of DHA on d-[ 3H]aspartate uptake in HEK GLT1 and HEK EAAC1 cells was abolished, and significant inhibition of the transport process by DHA was observed. Inhibition of CaM kinase II or PKC had no effect on the ability of DHA to inhibit transport into HEK GLAST cells but abolished the stimulatory effect of DHA on d-[ 3H]aspartate transport into HEK GLT1 and HEK EAAC1 cells. Inhibition of PKA had no effect on the modulation of d-[ 3H]aspartate transport by DHA in any of the cell lines. We conclude that DHA differentially modulates the GLT1, GLAST and EAAC1 glutamate transporter subtypes via different mechanisms. In the case of GLT1 and EAAC1, DHA appears to stimulate d-[ 3H]aspartate uptake via a mechanism requiring extracellular calcium and involving CaM kinase II and PKC, but not PKA. In contrast, the inhibitory effect of DHA on GLAST does not require extracellular calcium and does not involve CaM kinase II, PKC or PKA.

Overexpression and hyperactivity of breast cancer-associated fatty acid synthase (oncogenic antigen-519) is insensitive to normal arachidonic fatty acid-induced suppression in lipogenic tissues but it is selectively inhibited by tumoricidal alpha-linolenic and gamma-linolenic fatty acids: a novel mechanism by which dietary fat can alter mammary tumorigenesis.

Activity and expression of fatty acid synthase (FAS), a critical enzyme in the de novo biosynthesis of fatty acids in mammals, is exquisitely sensitive to nutritional regulation of lipogenesis in liver or adipose tissue. Surprisingly, a number of studies have demonstrated hyperactivity and overexpression of FAS (oncogenic antigen-519) in a biologically aggressive subset of human breast carcinomas, suggesting that FAS-dependent neoplastic lipogenesis is unresponsive to nutritional regulation. We have assessed the role of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) on the enzymatic activity and protein expression of tumor-associated FAS in SK-Br3 human breast cancer cells, an experimental paradigm of FAS-overexpressing tumor cells in which FAS enzyme constitutes up to 28%, by weight, of the cytosolic proteins. Of the omega-3 PUFAs tested, alpha-linolenic acid (ALA) dramatically reduced FAS activity in a dose-dependent manner (up to 61%). omega-3 PUFA docosahexaenoic acid (DHA) demonstrated less marked but still significant inhibitory effects on FAS activity (up to 37%), whereas eicosapentaenoic acid (EPA) was not effective. Of the omega-6 fatty acids tested, gamma-linolenic acid (GLA) was the most effective dose-dependent inhibitor of FAS activity, with a greater than 75% FAS activity reduction. Remarkably, omega-6 PUFAs linoleic acid (LA) and arachidonic acid (ARA), suppressors of both hepatic and adipocytic FAS-dependent lipogenesis, had no significant inhibitory effects on the activity of tumor-associated FAS in SK-Br3 breast cancer cells. Western blotting studies showed that down-regulation of FAS protein expression tightly correlated with previously observed inhibition of FAS activity, suggesting that ALA-, DHA-, and GLA-induced changes in FAS activity resulted from effects at the protein level. We investigated whether the FAS inhibitory effect of GLA and omega-3 PUFAs correlated with a cytotoxic effect related to a peroxidative mechanism. Measurement of cell viability by MTT assay indicated a significant cellular toxicity after ALA and GLA exposures. Furthermore, we observed a significant correlation between the ability of PUFAs to repress FAS and cause cell toxicity. In the presence of anti-oxidants (vitamin E), ALA and GLA dramatically lost their ability to inhibit FAS activity. Interestingly, a combination of ALA and GLA was FAS inhibitory in an additive manner, and this FAS repression was only partially reversible by vitamin E. In examining the molecular mechanisms underlying resistance of breast cancer-associated FAS to normal dietary fatty acid-induced suppression, a dramatic decrease of FAS accumulation was found after exposure of SK-Br3 cells to mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (MAPK ERK1/2) inhibitor U0126, phosphatidylinositol-3'-kinase (PI-3'K) blocker LY294002, and/or anti-HER-2/neu antibody trastuzumab. Interestingly, a long-term exposure to pharmacological inhibitors of FAS activity cerulenin [(2S,3R) 2,3-epoxy-4-oxo-7E,10E-dodecadienamide] or C75 also resulted in a significant reduction of FAS accumulation. These data indicate that: a) GLA- and omega-3 PUFA-induced repression of tumor-associated FAS may result, at least in part, from a non-specific cytotoxic effect due to peroxidative mechanisms; b) alternatively, GLA and omega-3 PUFAs have a suppressive effect on FAS expression and activity that can result in the accumulation of toxic fluxes of the FAS substrate malonyl-CoA; c) GLA- and/or omega-3 PUFA-induced repression of tumor-associated FAS may represent a novel mechanism of PUFA-induced cytotoxicity clinically useful against breast carcinomas carrying overexpression of FAS enzyme; d) fundamental differences in the ability of FAS gene to respond to normal fatty acid's regulatory actions in lipogenic tissues may account for the observed extremely high levels of FAS in breast carcinoma; and e) FAS overexpression in SK-Br3 breast cancer cells is driven by increases in HER-2/neu signaling, acting in major part through a constitutive downstream art through a constitutive downstream activation of the MAPK ERK1/2 and PI-3'K/AKT transduction cascades.