Incorporation Of Polyunsaturated Fatty Acids Research Articles

Systems biology of ferroptosis: A modeling approach

Ferroptosis is a recently discovered form of iron-dependent regulated cell death (RCD) that occurs via peroxidation of phospholipids containing polyunsaturated fatty acid (PUFA) moieties. Activating this form of cell death is an emerging strategy in cancer treatment. Because multiple pathways and molecular species contribute to the ferroptotic process, predicting which tumors will be sensitive to ferroptosis is a challenge. We thus develop a mathematical model of several critical pathways to ferroptosis in order to perform a systems-level analysis of the process. We show that sensitivity to ferroptosis depends on the activity of multiple upstream cascades, including PUFA incorporation into the phospholipid membrane, and the balance between levels of pro-oxidant factors (reactive oxygen species, lipoxogynases) and antioxidant factors (GPX4). We perform a systems-level analysis of ferroptosis sensitivity as an outcome of five input variables (ACSL4, SCD1, ferroportin, transferrin receptor, and p53) and organize the resulting simulations into ‘high’ and ‘low’ ferroptosis sensitivity groups. We make a novel prediction corresponding to the combinatorial requirements of ferroptosis sensitivity to SCD1 and ACSL4 activity. To validate our prediction, we model the ferroptotic response of an ovarian cancer stem cell line following single- and double-knockdown of SCD1 and ACSL4. We find that the experimental outcomes are consistent with our simulated predictions. This work suggests that a systems-level approach is beneficial for understanding the complex combined effects of ferroptotic input, and in predicting cancer susceptibility to ferroptosis.

Effects of the addition of docosahexaenoic acid and ?-tocopherol on quality of equine spermatozoa stored at 5°C

Plasma membrane composition has impact on phase transition from liquid crystal to gel state of cooled sperm cell. The incorporation of polyunsaturated fatty acids increases its fluidity and can contribute to sperm motility. The aim of this study was to compare the effect of adding docosahexaenoic acid (DHA) and ?-tocopherol (?-Toh) to the cooling extender, singly or combined, to the equine sperm parameters, submitted to cooling, up to 72 hours. Two ejaculates of ten stallions collected with artificial vagina were used, and evaluated for motility, plasma membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation, according to the following treatments: C; DHA; ?-Toh; DHA/?-Toh; EtOH 100: and EtOH 140 (corresponding to control; 10 ng mL-1 of DHA; 2 mM of ?-Toh; : 10 ng mL-1 of DHA + 2 mM of ?-Toh; 100 µL of ethanol and 140 µL of ethanol respectively). DHA treatment showed higher motility (68.2 ± 12.3; p < 0.05) when compared to control (62.1 ± 16.2), DHA/?-Toh (61.3 ± 12.7) and EtOH (58.1 ± 8.6) groups. In lipid peroxidation assay, the control group showed 2,506.2 ± 796.4 ng of MDA 108 spermatozoa-1, being significantly higher (p < 0.05) than the groups treated with DHA (2,036.0 ± 687.0), ?-Toh (1,890.8 ± 749.5) and DHA/?-Toh (1,821.1 ± 627.2). In conclusion, ?-Toh was effective in diminishing lipid peroxidation of equine sperm subjected to cooling, and DHA improved sperm motility and, in spite of being a polyunsaturated fatty acid with high susceptibility to peroxidation, reduced lipid peroxidation.

Comparing effects of polyunsaturated fatty acids derived from marine and plant sources on endothelial cell inflammation

AbstractHigh consumption of ω-3 long chain polyunsaturated fatty acids (PUFAs) has long-term health benefits. The principal dietary source of these ω-3 PUFAs (eicosapentaenoic acid, EPA and docosapentaenoic acid, DHA) is seafood, particularly oily fish. However current fish stocks are decreasing, indicating a need for alternative sources of bioactive PUFAs. Plant-derived ω-3 PUFAs (alpha-linolenic acid, ALA and stearidonic acid, SDA) may be able to provide land-based sustainable sources, but their functionality has been underexplored.Anti-inflammatory effects of ALA and SDA were compared to EPA and DHA in cultured EA.hy926 endothelial cells. Cells were treated with PUFAs (10, 25 and 50 μM) for 48 hours prior to stimulation with tumour necrosis factor for 24 hours. PUFAs incorporation was measured by gas chromatography and inflammatory responses were measured by ELISA, RT-PCR, western blot and flow cytometry. Adhesion of THP-1 monocytes to EA.hy926 cells was determined using a static adhesion assay.All PUFAs were incorporated into EA.hy926 cells in a dose-dependant manner (10 and 50 μM). Pre-treatment with ALA, SDA, EPA and DHA (50 μM) had differential effects on inflammatory responses in EA.hy926 cells depending on PUFA and response examined.EA.hy926 cells pre-treated with SDA had lower concentrations of soluble ICAM-1 (p < 0.05); however EPA and DHA resulted in greater reduction (p < 0.0001). EPA and DHA pre-treated EA.hy926 cells had significantly lower concentrations of IL-6 (p < 0.0001), IL-8 (p < 0.0001) and MCP-1 (p < 0.05, p < 0.01). ALA pre-treatment did not significantly affect any of the cytokines examined. Lower cell surface expression of ICAM-1 (p < 0.05), was seen for EA.hy926 cells pre-treated with SDA again to a lesser extent than EPA and DHA (p < 0.001, p < 0.0001), with no effect seen after ALA treatment.EA.hy926 cells pre-treated with ALA had significantly higher relative gene expression of NFKB (p < 0.05), as well as a tendency for more phosphorylated NFKBp65 protein (p < 0.06). In contrast, EA.hy926 cells pre-treated with DHA had significantly less phosphorylated NFKB (p < 0.0001). EA.hy926 cells with DHA treatment had significantly higher relative gene expression of PPARa (p < 0.05). SDA and EPA had no effect on expression of either of the genes or proteins examined.Finally pre-treatment with ALA, SDA and DHA all resulted in reduced adhesion of THP-1 monocytes to EA.hy926, however this effect not observed with EPA.Marine derived PUFA, particularly DHA, resulted in potent anti-inflammatory effects within this endothelial cell model. Of the two plant derived PUFAs, SDA treatment lead to some anti-inflammatory effects, which were not seen after treatment with ALA.

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.

Performance, fatty acids profile and oxidative stability of meat of rabbits fed different lipid sources

Abstract To evaluate the use of different lipid sources in diets of growing rabbits, 60 New Zealand White rabbits were evaluated on performance, carcass traits, meat composition, fatty acid profile and oxidative stability of refrigerated and frozen meat. The experiment was distributed in a randomized block design, in five treatments, with six replicates and two animals per experimental unit (one male and one female). The treatments consisted of five isonutritive diets, with 2.5% soybean oil (SO), sunflower oil (SFO), cottonseed oil (CO), beef tallow (BT) and poultry fat (PF). There was no effect (P > 0.05) of dietary lipid sources on animal performance, carcass traits and meat composition (P > 0.05). Dietary inclusion of beef tallow resulted in a lower proportion of polyunsaturated fatty acids in meat compared to vegetable oils. The values of thiobarbituric acid-reactive substances (TBARS) indicated higher oxidation in the frozen meat of the animals fed with sunflower oil, cottonseed oil and bovine tallow in their diets. In conclusion, vegetable oils and animal fats can be included in growing rabbit diets without impair performance and meat quality parameters. Dietary inclusion of soybean oil is more advantageous because it promotes the incorporation of polyunsaturated fatty acids and greater lipid stability of refrigerated and frozen rabbit meat.

Very low inadequate dietary intakes of essential n-3 polyunsaturated fatty acids (PUFA) in pregnant and lactating French women: The INCA2 survey

The French National survey INCA2 pointed out that the majority of the French population (children, adolescents, adults and elderly) ingest low quantities of n-3 polyunsaturated fatty acid (PUFA) in the form of both precursor (alpha-linolenic acid, ALA) and long-chain (mainly docosahexaenoic acid, DHA). However, we don't know whether such inadequate n-3 PUFA consumption is also found again in pregnant and lactating women. Dietary lipid and PUFA intakes were determined from 28 pregnant and 21 lactating French women by using the most recent set of national robust data on food (National Survey INCA2 performed in 2006 and 2007), and compared with that of 742 women of childbearing age. Main results showed that mean daily intakes of n-3 PUFA were very low in this French woman population because no pregnant and lactating women met recommended dietary intakes (RDIs). Moreover, some of them ingested quantities 4 times (ALA) to 10 times (DHA) lower than RDIs. Very similar dietary intakes were observed in women of childbearing age. French pregnant and lactating women did not change their dietary habits to favor ALA and n-3 long-chain PUFA consumption via rich-ALA vegetable oils and fish and oily fish consumption, and have low n-3 PUFA dietary consumption typical of French women of childbearing age. Such PUFA intakes could have adverse impact on long-chain n-3 PUFA incorporation in brain membranes of fetus and infants, but also on cognitive and visual development of infants during the first years of life.

Liver X receptors in lipid signalling and membrane homeostasis.

Liver X receptors α and β (LXRα and LXRβ) are nuclear receptors with pivotal roles in the transcriptional control of lipid metabolism. Transcriptional activity of LXRs is induced in response to elevated cellular levels of cholesterol. LXRs bind to and regulate the expression of genes that encode proteins involved in cholesterol absorption, transport, efflux, excretion and conversion to bile acids. The coordinated, tissue-specific actions of the LXR pathway maintain systemic cholesterol homeostasis and regulate immune and inflammatory responses. LXRs also regulate fatty acid metabolism by controlling the lipogenic transcription factor sterol regulatory element-binding protein 1c and regulate genes that encode proteins involved in fatty acid elongation and desaturation. LXRs exert important effects on the metabolism of phospholipids, which, along with cholesterol, are major constituents of cellular membranes. LXR activation preferentially drives the incorporation of polyunsaturated fatty acids into phospholipids by inducing transcription of the remodelling enzyme lysophosphatidylcholine acyltransferase 3. The ability of the LXR pathway to couple cellular sterol levels with the saturation of fatty acids in membrane phospholipids has implications for several physiological processes, including lipoprotein production, dietary lipid absorption and intestinal stem cell proliferation. Understanding how LXRs regulate membrane composition and function might provide new therapeutic insight into diseases associated with dysregulated lipid metabolism, including atherosclerosis, diabetes mellitus and cancer.

Fish oil supplementation reverses behavioral and neurochemical alterations induced by swimming exercise in rats

Diet and exercise are known to affect learning and memory. However, the effects of these interventions in the brain under development remains to be better investigated as the effects of high-intensity exercise. Moreover, it is still unclear how long the influence of diet and exercise lasts after the interventions are ceased. To investigate this, juvenile Wistar rats (30 days old) were supplemented with fish oil rich in polyunsaturated fatty acids (PUFAs) and performed swimming training for 50 days, 45 min per day, 5 times/week. The animals were assessed for locomotor activity with the open field test and for spatial memory with the object location task. To investigate neurochemical parameters such as fatty acids incorporation within the plasma membrane and brain-derived neurotrophic factor (BDNF) levels, the animals were euthanized, and the hippocampus dissected. These investigations were made at the end of the supplementation and exercise protocols and 21 days after the protocol has ended. Results indicate that high-intensity exercise impaired the spatial memory and decreased the levels of BDNF. Although supplementation led to PUFAs incorporation in plasma membrane, it did not prevent the harmful effect of exercise on memory. After 21 days of interruption, we observed that the supplementation reversed not only the deleterious effect of exercise on memory but also increased the BDNF levels. These results point to a complex influence of diet and exercise on spatial memory of juvenile rats, persisting after 21 days of interruption.

A randomised trial of the effect of omega-3 polyunsaturated fatty acid supplements on the human intestinal microbiota

ObjectiveOmega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter the mouse intestinal microbiome compatible with antineoplastic...

ER phospholipid composition modulates lipogenesis during feeding and in obesity.

Sterol regulatory element-binding protein 1c (SREBP-1c) is a central regulator of lipogenesis whose activity is controlled by proteolytic cleavage. The metabolic factors that affect its processing are incompletely understood. Here, we show that dynamic changes in the acyl chain composition of ER phospholipids affect SREBP-1c maturation in physiology and disease. The abundance of polyunsaturated phosphatidylcholine in liver ER is selectively increased in response to feeding and in the setting of obesity-linked insulin resistance. Exogenous delivery of polyunsaturated phosphatidylcholine to ER accelerated SREBP-1c processing through a mechanism that required an intact SREBP cleavage-activating protein (SCAP) pathway. Furthermore, induction of the phospholipid-remodeling enzyme LPCAT3 in response to liver X receptor (LXR) activation promoted SREBP-1c processing by driving the incorporation of polyunsaturated fatty acids into ER. Conversely, LPCAT3 deficiency increased membrane saturation, reduced nuclear SREBP-1c abundance, and blunted the lipogenic response to feeding, LXR agonist treatment, or obesity-linked insulin resistance. Desaturation of the ER membrane may serve as an auxiliary signal of the fed state that promotes lipid synthesis in response to nutrient availability.

Lipidomic study of the impact of eicosapentaenoic acid (EPA) on abca1-mediated cholesterol efflux from human macrophages

Aim: A diet rich in n-3 polyunsaturated fatty acids (PUFAs) is cardioprotective. PUFAs incorporation into membrane phospholipids (PLs) modifies the composition, which may influence membrane protein functions. We reported in human macrophages that the incorporation of eicosapentaenoic acid (EPA) (C20:5, 70 μM) reduced the anti-atherogenic ABCA1 pathway. We conducted a more complete analysis of the PLs modifications.

Eicosapentaenoic acid membrane incorporation impairs cholesterol efflux from cholesterol-loaded human macrophages by reducing the cholesteryl ester mobilization from lipid droplets

A diet containing a high n−3/n−6 polyunsaturated fatty acids (PUFA) ratio has cardioprotective properties. PUFAs incorporation into membranes influences the function of membrane proteins. We investigated the impact of the membrane incorporation of PUFAs, especially eicosapentaenoic acid (EPA) (C20:5 n−3), on the anti-atherogenic cholesterol efflux pathways. We used cholesteryl esters (CE)-loaded human monocyte-derived macrophages (HMDM) to mimic foam cells exposed to the FAs for a long period of time to ensure their incorporation into cellular membranes. Phospholipid fraction of EPA cells exhibited high levels of EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n−3), which was associated with a decreased level of arachidonic acid (AA) (C20:4 n−6). EPA 70μM reduced ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 30% without any alteration in ABCA1 expression. The other tested PUFAs, DPA, docosahexaenoic acid (DHA) (C22:6 n−3), and AA, were also able to reduce ABCA1 functionality while the monounsaturated oleic FA slightly decreased efflux and the saturated palmitic FA had no impact. Moreover, EPA also reduced cholesterol efflux to HDL mediated by the Cla-1 and ABCG1 pathways. EPA incorporation did not hinder efflux in free cholesterol-loaded HMDM and did not promote esterification of cholesterol. Conversely, EPA reduced the neutral hydrolysis of cytoplasmic CE by 24%. The reduced CE hydrolysis was likely attributed to the increase in cellular TG contents and/or the decrease in apo E secretion after EPA treatment. In conclusion, EPA membrane incorporation reduces cholesterol efflux in human foam cells by reducing the cholesteryl ester mobilization from lipid droplets.

Abstract 5374: Polyunsaturated fatty acids (PUFAs) alter the epigenetic landscape in MDA-MB-231 triple negative breast carcinoma (TNBC)

Abstract Breast cancer is the second leading cause of cancer-related deaths in US women; 10-20% of which are triple-negative breast cancer (TNBC) subtypes and have the poorest short-term prognosis. This is probably due to an epithelial to mesenchymal transition (EMT) that tumors undergo, de-differentiating into cancer stem cells. Epigenetic reprogramming is proposed to be required for malignant transformation and invasion, particularly in cells undergoing EMT transitions such as TNBC. Therefore, altering the epigenetic landscape in metastatic TNBC lines would provide researchers with a unique opportunity to sensitize tumors to treatment and induce apoptosis. Epigenetic research chiefly focuses on inhibitors of co-repressors histone deacetylase (HDACi) and DNA methyltransferases (DNMTi). Short chain fatty acids, have proven to be effective HDACi’s, suggesting a possible role for other fats in epigenetic regulation. Research has shown that several omega-3 polyunsaturated fatty acids (PUFAs), specifically docosahexaenoic acid (DHA; C22:6, n-3) and eicosapentaenoic acid (EPA; C20:5, n-3), inhibit tumorigenesis whereas the omega-6 PUFA linoleic acid (LA; 1C8:2, n-6) promotes neoplastic growth; supporting the association of elevated cancer incidence with n-6 PUFA dietary intake. We initiated studies to evaluate the impact of omega-3 fatty acids on tumor epigenetic pathways. Therefore we monitored epigenetic alterations in MDA-MB-231 TNBC cells in response to both omega-3 and omega-6 PUFAs in a time and dose-dependent manner. DHA, EPA, and arachidonic acid (AA; 20:5, n-6) all elicit apoptosis in a similar manner, indicating that initiating apoptosis is not omega-6 or omega-3 specific. We now show that PUFA incorporation and the resulting attenuated metabolism are not sufficient to induce apoptosis in MDA-MB-231 TNBC at 3 and 12 hour timepoints. Additionally, plasma membrane integrity was not appreciably altered at 3 or 12 hour time points. These data, combined with a lack apoptosis at 12 hours, indicate that the level of intact PUFA in cell membranes does not initiate apoptosis. Moreover, mitochondrial membrane potential was universally diminished early and throughout the time course and, although not limiting, decreased ATP levels were observed at 12 hours (relative to vehicle control). Furthermore, activity of HDAC classes (I, IIa and IIb) were altered early at 3 and 48 hours, but typically punctuated with a recovery to basal levels noted at the 12 hour time point. Chromatin condensation was observable primarily in 12 and 48 hour time points, although it was noted in DHA and EPA treatments at 3 hours. Collectively, these data demonstrate that PUFA-mediated epigenetic changes occur on a time scale proceeding apoptotic induction, indicating that PUFAs may be useful as an epigenetically-targeted cancer therapeutic agent or adjuvant therapy. Citation Format: Amy M. Chattin, Mykelti O'Brien, Ronald S. Pardini. Polyunsaturated fatty acids (PUFAs) alter the epigenetic landscape in MDA-MB-231 triple negative breast carcinoma (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5374. doi:10.1158/1538-7445.AM2017-5374

From the Editor’s desk...: July 2017

From the Editor’s desk...: July 2017

Oral feeding with polyunsaturated fatty acids fosters hematopoiesis and thrombopoiesis in healthy and bone marrow-transplanted mice

Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1+ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure.

Impaired hepatic lipid synthesis from polyunsaturated fatty acids in TM6SF2 E167K variant carriers with NAFLD

Carriers of the transmembrane 6 superfamily member 2 E167K gene variant (TM6SF2EK/KK) have decreased expression of the TM6SF2 gene and increased risk of NAFLD and NASH. Unlike common 'obese/metabolic' NAFLD, these subjects lack hypertriglyceridemia and have lower risk of cardiovascular disease. In animals, phosphatidylcholine (PC) deficiency results in a similar phenotype. PCs surround the core of VLDL consisting of triglycerides (TGs) and cholesteryl-esters (CEs). We determined the effect of the TM6SF2 E167K on these lipids in the human liver and serum and on hepatic gene expression and studied the effect of TM6SF2 knockdown on hepatocyte handling of these lipids. Liver biopsies were taken from subjects characterized with respect to the TM6SF2 genotype, serum and liver lipidome, gene expression and histology. In vitro, after TM6SF2 knockdown in HuH-7 cells, we compared incorporation of different fatty acids into TGs, CEs, and PCs. The TM6SF2EK/KK and TM6SF2EE groups had similar age, gender, BMI and HOMA-IR. Liver TGs and CEs were higher and liver PCs lower in the TM6SF2EK/KK than the TM6SF2EE group (p<0.05). Polyunsaturated fatty acids (PUFA) were deficient in liver and serum TGs and liver PCs but hepatic free fatty acids were relatively enriched in PUFA (p<0.05). Incorporation of PUFA into TGs and PCs in TM6SF2 knockdown hepatocytes was decreased (p<0.05). Hepatic expression of TM6SF2 was decreased in variant carriers, and was co-expressed with genes regulated by PUFAs. Hepatic lipid synthesis from PUFAs is impaired and could contribute to deficiency in PCs and increased intrahepatic TG in TM6SF2 E167K variant carriers.

Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ

Peroxisome proliferator-activated receptor δ (PPARδ) regulates many genes involved in lipid metabolism. Hepatic lysophosphatidylcholine acyltransferase 3 (LPCAT3) has critical functions in triglycerides transport and endoplasmic reticulum stress response due to its unique ability to catalyze the incorporation of polyunsaturated fatty acids into phospholipids. Previous studies identified liver X receptor as the transcription factor controlling LPCAT3 expression in mouse liver tissue. Here we show that the hepatic LPCAT3 gene is transcriptionally regulated by PPARδ. Adenovirus-mediated knockdown of PPARδ in cultured hepatic cells and liver tissue reduced LPCAT3 mRNA levels, and exogenous overexpression of PPARδ increased LPCAT3 mRNA expression. Activation of PPARδ in HepG2, Huh7, and Hepa 1-6 cells with its specific agonists increased LPCAT3 mRNA levels in all three hepatic cell lines. Through conducting sequence analysis, LPCAT3 promoter assays, and direct DNA binding assays, we have mapped the functional PPAR-responsive element to a proximal region from -135 to -123 of the LPCAT3 promoter that plays an essential role in mediating PPARδ-induced transactivation of the LPCAT3 gene. Finally, we have provided in vivo evidence showing that activation of PPARδ by agonist L165041 in mice increased hepatic LPCAT3 mRNA abundance and LPCAT enzymatic activity, which is associated with increased incorporations of arachidonate into liver phosphatidylcholine and phosphatidylethanolamine. Furthermore, transient liver-specific knockdown of LPCAT3 in mice affected PPARδ-mediated activation of several hepatic genes involving in FA metabolism. Altogether, our new findings identify LPCAT3 as a direct PPARδ target gene and suggest a novel function of PPARδ in regulation of phospholipid metabolism through LPCAT3.

Abstract 11869: Identification of Human Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor Delta in Hepatic Cells and in Liver Tissue

Peroxisome proliferator-activated receptor delta (PPARD) is a transcription factor regulating many important genes involved in lipid and glucose metabolism. Hepatic lysophosphatidylcholine acyltransferase 3 (LPCAT3) plays critical functions in TG secretion and ER stress response due to its unique ability to catalyze the incorporation of polyunsaturated fatty acids into phospholipids. Previous studies identified liver X receptor as the transcription factor controlling LPCAT3 expression in liver tissue. Here we showed for the first time that the hepatic LPCAT3 gene is transcriptionally regulated by PPARD in vitro and in vivo . Treatments of HepG2, Huh-7, and Hepa 1-6 cells with PPARD specific agonists (L-165041 and GW0742) increased LPCAT3 mRNA levels 2-3 fold of control. Adenovirus-mediated knockdown of PPARD in HepG2 cells reduced the basal level of LPCAT3 mRNA by approximately 40% compared to control and further attenuated the induction of PPARD agonist on LPCAT3 gene expression. To demonstrate the direct regulation of PPARD on LPCAT3 gene transcription, we analyzed human LPCAT3 promoter sequence and identified two putative PPAR responsive elements (PPRE-1, -171; PPRE-2, -727) within 1 kb of the proximal LPCAT3 promoter. LPCAT3 promoter assays demonstrated strong inductions of the reporter activity by PPARD agonists. Mutational analysis of the human LPCAT3 promoter revealed that the PPRE-1 is involved in conferring responsiveness to PPARD agonist mediated transcriptional activity. Direct binding of PPARD to PPRE-1 sequence was demonstrated by chromatin immunoprecipitation and gel retardation assays. Finally, we provided in vivo evidence showing that activation of PPARD by L-165041 in mice increased LPCAT3 mRNA abundance in the liver. Altogether, these new findings identify LPCAT3 as a direct PPARD target gene and suggest a novel function of PPARD in regulation of phospholipid metabolism through LPCAT3.

Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase.

The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4 °C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid + docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36 h, at 40 °C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids.

TTC39B deficiency stabilizes LXR reducing both atherosclerosis and steatohepatitis.

SummaryCellular mechanisms that mediate steato-hepatitis, an increasingly prevalent condition in the Western world for which no therapies are available1, are poorly understood. Despite the fact its synthetic agonists induce fatty liver, the Liver X receptor (LXR) transcription factor remains a target of interest because of its anti-atherogenic, cholesterol removal and anti-inflammatory activities. We discovered that tetratricopeptide repeat (TPR) domain protein 39B (Ttc39b, C9orf52) (T39), a high density lipoprotein (HDL) gene discovered in human genome wide association studies (GWAS)2, promotes the ubiquitination and degradation of LXR. Chow-fed T39-/- mice displayed increased HDL cholesterol levels associated with increased enterocyte ATP binding cassette transporter A1 (Abca1) expression and increased LXR protein without change in LXR mRNA. When challenged with a high fat/high cholesterol/bile salt (HF/HC/BS) diet, T39-/- mice or mice with hepatocyte-specific T39 deficiency showed increased hepatic LXR protein and target gene expression, and unexpectedly protection from steato-hepatitis and death. Western Type Diet (WTD)-fed Low density lipoprotein receptor (Ldlr)-/-T39-/- mice showed decreased fatty liver, increased HDL, decreased LDL and reduced atherosclerosis. In addition to increasing hepatic Abcg5/8 expression and limiting dietary cholesterol absorption, T39 deficiency inhibited hepatic sterol regulatory element binding protein 1 (SREBP-1, ADD1) processing. This was explained by an increase in microsomal phospholipids containing polyunsaturated fatty acids (PUFA), linked to an LXRα-dependent increase in expression of enzymes mediating PC biosynthesis and incorporation of PUFA into phospholipids. The preservation of endogenous LXR protein activates a beneficial profile of gene expression that promotes cholesterol removal and inhibits lipogenesis. T39 inhibition could be an effective strategy for reducing both steato-hepatitis and atherosclerosis.