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Our laboratory previously reported that docosahexaenoic acid (DHA) differentially activates p38 mitogen-activated protein kinase (MAPK) in growing and quiescent human endothelial cells, which represent the dysfunctional and healthy states in vivo, respectively. Since endothelial nitric oxide synthase (eNOS) activity differs between healthy and dysfunctional endothelial cells, and p38 MAPK reportedly regulates both the activity and expression of eNOS, we hypothesized that the beneficial actions of DHA on endothelial cells are due to eNOS activation by p38 MAPK. The contribution of mitogen- and stress-activated protein kinase (MSK), a p38 MAPK substrate, was also investigated. Growing and quiescent EA.hy926 cells, prepared on Matrigel®-coated plates, were incubated with inhibitors of p38MAPK or MSK before adding DHA. eNOS phosphorylation and levels were quantified by Western blotting. Treatment with 20 µM DHA activated eNOS in both growth states whereas 125 µM DHA suppressed eNOS activation in growing cells. Quiescent cells had higher basal levels of eNOS than growing cells, while 125 µM DHA decreased eNOS levels in both growth states. p38 MAPK inhibition enhanced eNOS activation in quiescent cells but suppressed it in growing cells. Interestingly, 125 µM DHA counteracted these effects of p38 MAPK inhibition in both growth states. MSK was required for eNOS activation in both growth states, but it only mediated eNOS activation by DHA in quiescent cells. MSK thus affects eNOS via a pathway independent of p38MAPK. Quiescent cells were also more resistant to the apoptosis-inducing effect of 125 µM DHA compared to growing cells. The growth state-dependent regulation of p38MAPK and eNOS by DHA provides novel insight into the molecular mechanisms by which DHA influences endothelial cell function.

Peripheral artery disease (PAD) has high morbidity and mortality rates. A metabolomics approach was employed to determine whether consumption of bean-rich diets for 8 weeks would impact the metabolomic profile of individuals with PAD. Serum and urine, collected from 54 participants with clinical PAD at baseline and after 8 weeks on 0.3 cups beans/day (n = 19), 0.6 cups beans/day (n = 20), or control (n = 23) diet, and the beans were extracted and analyzed using LC-QTOF-MS. As a result, PGE2 p-acetamidophenyl ester, PGF2α diethyl amide and 5-l-glutamyl-l-alanine were significantly changed in the serum or urine of bean groups compared with control. Significant changes (P < 0.05) in the profile and/or levels of 22 flavonoids present in bean extracts showed the potential importance of the mixture of beans used in this study. In a subset of participants taking metoprolol, after 8 weeks the bean-rich diets significantly elevated metoprolol in the serum while reducing it in urine compared with baseline. In addition, the diets significantly enhanced the urinary excretion of metformin. In conclusion, several biochemical pathways including prostaglandins and glutathione were affected by bean consumption. Significant changes in the metabolism of metoprolol and metformin with bean consumption suggested the presence of diet-drug interactions that may require adjustment of the prescribed dose. ClinicalTrials.gov Identifier: NCT01382056. Novelty: Bean consumption by people with PAD alters the levels of certain metabolites in serum and urine. Different bean types (black, red kidney, pinto, navy) have unique flavonoid profiles. Metabolomics revealed potential diet–drug interactions as serum and/or urinary levels of metoprolol and metformin are modified by bean consumption.

Angiotensin-converting enzyme 2 (ACE2) is a target of interest for both COVID-19 and cardiovascular disease management. Even though lower ACE2 levels may be beneficial in SARS-CoV-2 infectivity, maintaining the ACE1/ACE2 balance is also crucial for cardiovascular health. So far, reports describing conditions capable of altering ACE2 protein levels, especially via dietary components, are limited. In this study, the effects of omega-3 polyunsaturated fatty acids (n3-PUFA) on the protein levels of ACE1 and ACE2 in rodent tissues, human endothelial and kidney cell lines, and human plasma were examined. The ability of n3-PUFA to affect the entry of the SARS-CoV-2 pseudovirus into cells was also tested. Docosahexaenoic acid (DHA), and in some cases eicosapentaenoic acid (EPA), but not α-linoleic acid (ALA), reduced both ACE1 and ACE2 (non-glycosylated p100 and glycosylated p130 forms) in the heart, aorta, and kidneys of obese rats, as well as in human EA.hy926 endothelial and HEK293 kidney cells. Dietary supplementation with either DHA or ALA had no effect on plasma soluble ACE2 levels in humans. However, treatment of HEK293 cells with 80 and 125 µM DHA for 16 h inhibited the entry of the SARS-CoV-2 pseudovirus. These results strongly suggest that DHA treatment may reduce the ability of SARS-CoV-2 to infect cells via a mechanism involving a decrease in the absolute level of ACE2 protein as well as its glycosylation. Our findings warrant further evaluation of long-chain n3-PUFA supplements as a novel option for restricting SARS-CoV-2 infectivity in the general population.

Monocytes play a large role in chronic inflammatory conditions such as obesity, atherosclerosis and infection. Marine-derived omega-3 fatty acids such as docosahexaenoic acid (DHA) beneficially alter immune function and attenuate chronic inflammation in part by modifying gene expression. Comparisons with plant-derived omega-3 α-linolenic acid (ALA) on immune cell gene expression and function are limited.Transcriptome analysis was performed on THP-1 human monocytes treated with ALA, DHA or vehicle for 48 hr using fold change analysis, principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), variable importance analysis (VIP), and ingenuity pathway analysis (IPA). Candidate genes were validated by qPCR. Functional assays evaluated the transcriptomic predictions. Expression of candidate transcripts identified in THP-1 cells were examined in PBMC from clinical trial (OXBIO; NCT03583281) participants consuming ALA- or DHA-rich oil supplements.ALA and DHA-treated monocytes presented distinct transcriptomic profiles as per VIP and PLS-DA. Both fatty acids were predicted to reduce cellular cholesterol content, while ALA would uniquely increase response to infection and chemotactic signals. Functional assays revealed ALA and DHA decreased cholesterol content. DHA significantly decreased the response to infection and chemotaxis, but ALA had no effect. Candidate transcripts responded similarly in PBMC from n-3 PUFA supplemented women with obesity.ALA and DHA differentially alter the transcription profiles and functions associated with the response to infection, chemotaxis, and cholesterol metabolism in mononuclear immune cells. Thus, they may uniquely affect related disease processes contributing to obesity, atherosclerosis, and the response to infection.

Background Differences in health effects of dietary α-linolenic acid (ALA) and DHA are mediated at least in part by differences in their effects on oxylipins. Objectives Time course and sex differences of plasma oxylipins in response to ALA- compared with DHA-rich supplements were examined. Methods Healthy men and women, aged 19-34 y and BMI 18-28 kg/m2, were provided with capsules containing ∼4 g/d of ALA or DHA in a randomized double-blind crossover study with >6-wk wash-in and wash-out phases. Plasma PUFA and oxylipin (primary outcome) concentrations at days 0, 1, 3, 7, 14, and 28 of supplementation were analyzed by GC and HPLC-MS/MS, respectively. Sex differences, supplementation and time effects, and days to plateau were analyzed. Results ALA supplementation doubled ALA concentrations, but had no effects on ALA oxylipins after 28 d, whereas DHA supplementation tripled both DHA and its oxylipins. Increases in DHA oxylipins were detected as early as day 1, and a plateau was reached by days 5-7 for 11 of 12 individual DHA oxylipins and for total DHA oxylipins. Nine individual DHA oxylipins reached a plateau in females with DHA supplementation, compared with only 4 in males. A similar time course and sex difference pattern occurred with EPA and its oxylipins with DHA supplementation. DHA compared with ALA supplementation also resulted in higher concentrations of 4 individual arachidonic acids, 1 linoleic acid, and 1 dihomo-γ-linolenic acid oxylipin, despite not increasing the concentrations of these fatty acids, further demonstrating that oxylipins do not always reflect their precursor PUFA. Conclusions DHA compared with a similar dose of ALA has greater effects on both n-3 and n-6 oxylipins in young, healthy adults, with differences in response to DHA supplementation occurring earlier and being greater in females. These findings can help explain differences in dietary effects of ALA and DHA.This study was registered at clinicaltrials.gov as NCT02317588.

BACKGROUND: Omega-3 fatty acids, including DHA and α-linolenic acid (ALA), are proposed to improve metabolic health by reducing obesity-associated inflammation. Their effects are mediated in part by conversion to oxylipins. ALA is relatively understudied, and direct comparisons to other omega-3 fatty acids are limited. OBJECTIVES: We compared the effects of equal doses of ALA and DHA on plasma oxylipins and markers of metabolic health in women with obesity. METHODS: We carried out a randomized, double-blind, crossover clinical trial where women aged 20–51 with a BMI of 30–51 kg/m(2) were supplemented with 4 g/day of ALA or DHA for 4 weeks in the form of ALA-rich flaxseed oil or DHA-rich fish oil. The primary outcome, the plasma oxylipin profile, was assessed at Days 0 and 28 of each phase by HPLC-MS/MS. Plasma fatty acids, inflammatory markers, and the monocyte glucose metabolism were key secondary outcomes. Data were analyzed using a mixed model. RESULTS: Compared to the baseline visit, there were higher plasma levels of nearly all oxylipins derived from DHA (3.8-fold overall; P

Healthy and functional endothelial cells play important roles in maintaining vascular homeostasis, whereas endothelial dysfunction initiates and exacerbates vascular disease progression. Interventional studies with dietary fatty acids have shown that these molecules have varying effects on vascular function. It is hypothesized that the actions of dietary fatty acids on vascular function may be mediated in part through endothelial cells. This review summarizes the results of studies that have examined the acute and chronic effects of dietary fatty acids on endothelial function and vascular properties in humans, as well as the potential mechanisms by which n-3 polyunsaturated fatty acids regulate endothelial function. Altogether, this article provides an extensive review of how fatty acids contribute to vascular function through their ability to modulate endothelial cells and discusses relationships between dietary fatty acids and endothelial cells in the context of vascular dysfunction.

BACKGROUND: Novel oils high in monounsaturated fatty acids (MUFAs) and low in saturated fatty acids (SFAs) are an alternative to partially hydrogenated oils high in trans-unsaturated fatty acids. There is widespread use of high-MUFA oils across the food industry; however, limited knowledge of their cardiovascular impact exists. OBJECTIVES: We investigated the effects of diets containing canola oil, high-oleic acid canola oil (HOCO), and a control oil blend (diet formulated to emulate a Western fat profile) on lipids, lipoproteins, and apolipoproteins (apos), as secondary outcomes of the trial. METHODS: In a multi-center, double-blind, randomized, 3-period crossover, controlled feeding trial, men (n = 44) and women (n = 75) with a mean age of 44 y, mean body mass index (BMI; in kg/m(2)) of 31.7, and an increased waist circumference plus ≥1 metabolic syndrome criteria consumed prepared, weight-maintenance diets containing canola oil [17.5% MUFAs, 9.2% polyunsaturated fatty acids (PUFAs), 6.6% SFAs], HOCO (19.1% MUFAs, 7.0% PUFAs, 6.4% SFAs), or control oil (10.5% MUFAs, 10.0% PUFAs, 12.3% SFAs) for 6 wk with ≥4-wk washouts. Fasting serum lipids were assessed at baseline and 6 wk. Diet effects were examined using a repeated measures mixed model. RESULTS: Compared with the control, canola and HOCO diets resulted in lower endpoint total cholesterol (TC; −4.2% and −3.4%; P

Dietary PUFA and their effects on adipose tissue have been well studied, but oxylipins, the oxygenated metabolites of PUFA, have been sparsely studied in adipose tissue. To determine the oxylipin profile and to examine their potential importance in various adipose sites, female and male rats were provided control, high linoleic acid (LA), or high LA and high α-linolenic acid (LA + ALA) diets for six weeks. Analysis of gonadal (GAT), mesenteric (MAT), perirenal (PAT), and subcutaneous adipose tissues (SAT) revealed higher numbers of oxylipins in MAT and SAT, primarily due to 20–22 carbon cytochrome P450 oxylipins, as well as metabolites of cyclooxygenase derived oxylipins. LA oxylipins made up 75–96% of the total oxylipin mass and largely determined the total relative amounts between depots (GAT > MAT > PAT > SAT). However, when the two most abundant LA oxylipins (TriHOMEs) were excluded, MAT had the highest mass of oxylipins and exhibited the most sex differences. These differences existed despite comparable PUFA composition between depots. Dietary LA increased oxylipins derived from n-6 PUFA, and the addition of ALA generally returned n-6 PUFA oxylipins to levels similar to control and elevated some n-3 oxylipins. These data on oxylipin profiles in adipose depots from different anatomical sites and the effects of diet and sex provide fundamental knowledge that will aid future studies investigating the physiological effects of adipose tissue.

Partial replacement of saturated fatty acids (SFA) with unsaturated fatty acids is recommended to reduce cardiovascular disease (CVD) risk. Monounsaturated fatty acids (MUFA), including oleic acid, are associated with lower CVD risk. Measurement of flow-mediated dilation of the brachial artery (FMD) is the gold standard for measuring endothelial function and predicts CVD risk. This study examined the effect of partially replacing SFA with MUFA from conventional canola oil and high-oleic acid canola oil on FMD. Participants (n = 31) with an elevated waist circumference plus ≥1 additional metabolic syndrome criterion completed FMD measures as part of the Canola Oil Multi-Centre Intervention Trial 2 (COMIT-2), a multi-center, double-blind, three-period crossover, controlled feeding randomized trial. Diet periods were 6 weeks, separated by ≥4-week washouts. Experimental diets were provided during all feeding periods. Diets only differed by the fatty acid profile of the oils: canola oil (CO; 17.5% energy from MUFA, 9.2% polyunsaturated fatty acids (PUFA), 6.6% SFA), high-oleic acid canola oil (HOCO; 19.1% MUFA, 7.0% PUFA, 6.4% SFA), and a control oil blend (CON; 11% MUFA, 10% PUFA, 12% SFA). Multilevel models were used to examine the effect of the diets on FMD. No significant between-diet differences were observed for average brachial artery diameter (CO: 6.70 ± 0.15 mm, HOCO: 6.57 ± 0.15 mm, CON: 6.73 ± 0.14 mm; p = 0.72), peak brachial artery diameter (CO: 7.11 ± 0.15 mm, HOCO: 7.02 ± 0.15 mm, CON: 6.41 ± 0.48 mm; p = 0.80), or FMD (CO: 6.32 ± 0.51%, HOCO: 6.96 ± 0.49%, CON: 6.41 ± 0.48%; p = 0.81). Partial replacement of SFA with MUFA from CO and HOCO had no effect on FMD in participants with or at risk of metabolic syndrome.

Oxylipins are oxygenated derivatives of polyunsaturated fatty acids, generated by COX, LOX and CYP enzymes, that regulate various aspects of endothelial cell physiology. Although 15-LOX and its products are positively associated with atherosclerosis, the relevant mechanisms have not been explored. The current study examined the effects of PD146176 (PD), a putative 15-LOX inhibitor, on EA.hy926 endothelial cell functions in the growing and confluent states. The effects of PD on endothelial cell oxylipin production (profiled by LC/MS/MS), cell viability, proliferation, eNOS activity, ICAM-1 and VE-cadherin levels were assessed. The contribution of signaling pathways relevant to endothelial function (p38 MAPK, Akt, PPARα) were also investigated. PD treatment for 30 min did not block formation of individual 15-LOX oxylipins, but 20 μM PD stimulated the accumulation of total LOX and COX products, while reducing several individual CYP products generated by epoxygenase. At 20 μM, the accumulated total oxylipins were primarily LOX-derived (86%) followed by COX (12%) and CYP (2%). PD altered cell functions by upregulating p38 MAPK and PPARα and downregulating Akt in a dose-dependent fashion. These observations suggest a link between PD-induced changes in oxylipins and altered endothelial cell functions via specific signaling pathways. In conclusion, the results of this study imply that PD does not function as a 15-LOX inhibitor in EA.hy926 endothelial cells, and instead inhibits CYP epoxygenase. These findings suggest that the cellular function changes induced by PD may be contingent upon its ability to modulate total oxylipin production, particularly by the LOX and CYP families.

OBJECTIVES: Angiotensin-converting enzyme 2 (ACE2) is a transmembrane protein located on the surface of endothelial cells that promotes vasodilation by promoting the hydrolysis of angiotensin II. However, ACE2 also serves as the cellular receptor for SARS-CoV-2. Infection by SARS-CoV-2 can promote endothelial dysfunction which in turn is associated with greater infection severity. Long chain omega-3 fatty acids (n3 PUFA) like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are reported to prevent endothelial dysfunction. Thus, we hypothesize that treatment with n3 PUFA may suppress the actions of SARS-CoV-2 on endothelial cells, potentially through endothelial ACE2. The objective of the study was therefore to investigate whether changes in ACE2 levels occur as part of the endothelial response to n3 PUFA treatment. METHODS: Male fa/fa Zucker rats were randomised into 4 PUFA-based diet groups: α-linoleic acid (ALA), EPA, DHA, and linoleic acid (LA), for 8 weeks. EA.hy926 cells were cultured to sub-confluent and quiescent states and treated with various doses of DHA, EPA, and ALA for 8 or 24 h. ACE2 levels in tissues and cells were quantified by Western blotting. RESULTS: In contrast to ALA and EPA, DHA treatment significantly reduced ACE2 levels in rat heart, aorta, and kidney but not lungs after 8 weeks of diet intervention. EPA only showed this reduction compared to ALA in kidney. Interestingly, when applied to human endothelial cells in culture, DHA decreased ACE2 levels of growing EA.hy926 cells even at relatively low doses, but no significant effect was observed in quiescent cells. EPA also had an effect, but only at an extremely high dose. CONCLUSIONS: DHA reduced ACE2 levels in key organs and human endothelial cells, which should produce beneficial effects by lowering the susceptibility of cells to SARS-CoV-2. FUNDING SOURCES: St Boniface Hospital Foundation - Research Without Borders and University of Manitoba - GETS

OBJECTIVES: One of the earliest events in atherosclerotic plaque formation is the migration of monocytes to damaged blood vessels, and the accumulation of cholesterol in monocyte-derived macrophages. The omega-3 fatty acid docosahexaenoic acid (DHA) is known to inhibit this process. While there is limited evidence suggesting α-linolenic acid (ALA) has a similar effect, ALA has not been directly compared to DHA. The primary objective of this study was to compare the gene expression profiles of monocytes that have been exposed to either ALA or DHA and examine the effect of these fatty acids on monocyte cholesterol content and migration in a cell culture model. METHODS: Transcriptome analysis was performed on total mRNA isolated from human THP-1 monocytes treated with ALA, DHA or vehicle for 48 h. Candidate genes identified via fold change and Ingenuity Pathway Analysis were validated by qPCR. Functional assays to measure total cholesterol content and migration were then performed on monocytes treated with ALA or DHA. RESULTS: Transcriptome analysis identified a series of genes associated with cholesterol metabolism and cell migration altered by ALA and DHA treatment. Changes in mRNA levels for candidate genes were validated by qPCR, with similar expression patterns as in the transcriptome analysis. Based on these data, both fatty acids were predicted to reduce cholesterol synthesis, ALA would increase migration and DHA would have no effect. Functional assays were then performed and revealed that ALA and DHA decreased cholesterol content to a similar extent. Additionally, contrary to our predictions, DHA significantly decreased migration, while ALA had no effect. CONCLUSIONS: The results suggest ALA and DHA may influence monocyte migration through distinct gene pathways, while cholesterol metabolism may be regulated by a common mechanism. Furthermore, only DHA treatment reduced monocyte migration in functional assays, while both fatty acids reduced cholesterol content. Due to the critical role of monocyte migration and cholesterol content in the pathophysiology of atherosclerosis, it may be concluded from this study that both DHA and ALA may exert protective effects involving different mechanisms as they relate to individuals at risk for cardiovascular disease. FUNDING SOURCES: CIHR, NSERC

Diets varying in SFA and MUFA content can impact glycaemic control; however, whether underlying differences in genetic make-up can influence blood glucose responses to these dietary fatty acids is unknown. We examined the impact of dietary oils varying in SFA/MUFA content on changes in blood glucose levels (primary outcome) and whether these changes were modified by variants in the stearoyl-CoA desaturase (SCD) gene (secondary outcome). Obese men and women participating in the randomised, crossover, isoenergetic, controlled-feeding Canola Oil Multicenter Intervention Trial II consumed three dietary oils for 6 weeks, with washout periods of ˜6 weeks between each treatment. Diets studied included a high SFA/low MUFA Control oil (36·6 % SFA/28·2 % MUFA), a conventional canola oil (6·2 % SFA/63·1 % MUFA) and a high-oleic acid canola oil (5·8 % SFA/74·7 % MUFA). No differences in fasting blood glucose were observed following the consumption of the dietary oils. However, when stratified by SCD genotypes, significant SNP-by-treatment interactions on blood glucose response were found with additive models for rs1502593 (P = 0·01), rs3071 (P = 0·02) and rs522951 (P = 0·03). The interaction for rs3071 remained significant (P = 0·005) when analysed with a recessive model, where individuals carrying the CC genotype showed an increase (0·14 (sem 0·09) mmol/l) in blood glucose levels with the Control oil diet, but reductions in blood glucose with both MUFA oil diets. Individuals carrying the AA and AC genotypes experienced reductions in blood glucose in response to all three oils. These findings identify a potential new target for personalised nutrition approaches aimed at improving glycaemic control.

The arachidonate 12-lipoxygenase (ALOX12) enzyme catalyzes polyunsaturated fatty acids and facilitates generation of bioactive lipid mediators associated with various biological processes and disease pathologies. The human genome assembly revealed that the ALOX12 gene overlaps an antisense non-coding gene designated as ALOX12-antisense 1 (ALOX12-AS1). This arrangement indicates that the uncharacterized ALOX12-AS1 long non-coding RNA (lncRNA) may bind to the sense coding ALOX12 mRNA to form an antisense-sense duplex providing the basis of a novel ALOX12 regulatory mechanism. Therefore, this study was designed to determine whether the interaction of ALOX12-AS1 with ALOX12 mRNA functions as an anti-sense/sense duplex-mediated regulatory mechanism controlling the cellular content of ALOX12. Our findings indicate that two major isoforms of ALOX12-AS1 lncRNA are ubiquitously expressed in a variety of primary adult human tissues and different transformed cell types. RNA-FISH revealed cell-type-specific cytosolic as well as nuclear and nucleolar localization of the lncRNA. Interestingly, phorbol ester-induced nucleo-cytoplasmic translocation of the lncRNA in monocytic THP-1 cells resulted in a reduction of ALOX12 protein without a concomitant change in its mRNA level. This indicated ALOX12-AS1 operates via an antisense-sense duplex-mediated translational downregulation mechanism. This deduction was validated by demonstrating sense/antisense duplex formation and an association of the duplex with ribosomal proteins in HEK293 cells. Overall, this study revealed a hitherto unknown mechanism of antisense lncRNA-mediated translational downregulation of ALOX12 that adds to the existing regulatory mechanisms for the modulation of potent bioactive lipid mediators that contribute to both health and disease.

Objective: Cardiovascular disease, a major cause of mortality and morbidity, exhibits sexual dimorphism since the onset of cardiovascular disease occurs later in women than in men. The loss of cardioprotection in older women may be due to an increase in arterial stiffness after menopause. Free fatty acid metabolites of polyunsaturated fatty acids, called oxylipins, are known to impact vessel function and may be responsible for the vascular benefits of polyunsaturated fatty acids. The objectives of this study were to compare the plasma oxylipin profiles of young females (20–55 years), older females (55 + ), and older males (55 + ) and to identify associations between oxylipins and cardiovascular disease risk factors, such as obesity and arterial stiffness. Approach and Results: We quantified plasma oxylipins by high-performance liquid chromatography–tandem mass spectrometry in archived samples taken from completed clinical trials. We identified 3 major 12-lipoxygenase products, 12-hydroxy-eicosatetraenoic acid, 12-hydroxy-eicosapentaenoic acid, and 14-hydroxy-docosahexaenoic acid, that are present at high levels in young females compared with older females and males. These oxylipins also decreased with obesity and displayed robust negative associations with arterial stiffness as assessed by brachial-ankle pulse wave velocity. According to multiple linear regression modeling, these associations were maintained even after correcting for body mass index category combined with either age, menopausal status, or estradiol levels. Using linear discriminant analysis, the combination of these 3 oxylipins effectively distinguished participants according to both brachial-ankle pulse wave velocity risk group and age. Conclusions: Higher 12-lipoxygenase oxylipin plasma concentrations associated with lower arterial stiffness in premenopausal females may be an important contributing factor to sex differences in cardiovascular disease. Registration: URL: https://www.clinicaltrials.gov ; Unique identifiers: NCT01661543, NCT01562171, NCT01890330, NCT02571114 and NCT02317588.

Endothelial cells, which help to maintain vascular homeostasis, can be functionally modulated by polyunsaturated fatty acids. Previously, we reported that docosahexaenoic acid (DHA) reduced the viability of confluent EA.hy926 endothelial cells with caspase-3 activation. This study therefore examined the molecular mechanism by which DHA affects the viability of confluent cells, with a focus on the interaction between caspase-9, caspase-8, caspase-3, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) by Western blotting. Our results revealed that DHA induces apoptosis of confluent cells through both intrinsic and extrinsic pathways, which requires activation of p38 MAPK, and involves activation of JNK, caspase-9, caspase-8 and caspase-3 with the exception that cleavage of caspase-8 was incomplete and truncated BID was not detected at the maximum time (8 h) examined. Apoptosis induced by high levels of DHA in healthy endothelial cells is achieved through positive feedback loops linking these MAPKs to multiple caspases, as well as negative feedback from p38 MAPK to JNK. However, only p38 MAPK is crucial in apoptosis induction in comparison with JNK or any other caspase examined. This study has expanded the knowledge on the molecular mechanism of DHA-induced apoptosis in human endothelial cells and has also implied the differential roles of MAP kinases and caspases in apoptosis.

Background: Progesterone receptor membrane component 1 (PGRMC1) is a non-canonical progesterone (P4) binding protein. PGRMC1 is elevated in a variety of cancers and its phosphorylation state associated with hormone responsiveness in breast cancer. Metabolic reprogramming is a key factor for tumor growth during malignancies. Recently, we reported that the P4-inducedWarburg effectinHEK293cells is associated with altered post-translational modifications (PTMs) of PGRMC1, including phosphorylation, SUMOylation, and ubiquitination, which were linked to rapid proteasomal degradation of the protein. The previous study also identified hexokinase (HK) as a potential novel interacting partner of PGRMC1. HKs catalyze the first essential step of glucose metabolism and directly couple glycolysis to mitochondrial respiration. Therefore, in the present study, P4’s effects on glycolysis and PTMs of PGRMC1 as well as its interaction with HKs were compared between HEK293 and HepG2 cells to unravel the signaling pathways that mediate cell-type-specific metabolic reprogramming.Methods: P4-induced glucose metabolism in wild-type and PGRMC1-deficient cells wasassessed using the Seahorse flux analyzer, while PTMs of PGRMC1/HKs and protein-protein interaction were studied using immunoprecipitation, isoelectric focussing, phosphomimetics, and mass spectrometry.Translocation of HKs to different subcellular organelles were studied using subcellular fractionation, and the cell-type-specific effect of PGRMC1-deficiency on endoplasmic reticulum (ER) and mitochondria; ultrastructure were examined by electron microscopy. Results:P4 treatment caused a rapid increase in glycolysis in HEK293 cells, whereas it decreased glycolysis in HepG2 cells. In addition, PGRMC1 was not degraded in HepG2 cells which is in contrast to HEK293 cells where rapid proteasomal degradation of PGRMC1 occurredfollowing P4 treatment. Besides, PGRMC1 half-life and PTMs under basal condition were found cell-type-specific and the P4-induced PTMsdiffered between the two cell types. Furthermore, we observed cell-type-specific interaction of HKs with PGRMC1, and differential translocation of HK1/2 to the ER, mitochondria and nuclear compartments following P4 treatment. PGRMC1 deficiency altered ER structure in HepG2 cells. Thus, multiple factors underlying the cell-type-specific P4-PGRMC1-mediated metabolic reprogrammingwere identified. Conclusions: These findings provide a hitherto unknown novel P4-induced cell-type-specific PGRMC1-HK signaling mechanism that contributes to the molecular basis of P4-induced metabolic reprogramming, with important applications for hormone responsiveness in cancer.

Consuming pulses (dry beans, dry peas, chickpeas, lentils) over several weeks can improve vascular function and decrease cardiovascular disease risk; however, it is unknown whether pulses can modulate postprandial vascular responses. The objective of this study was to compare different bean varieties (black, navy, pinto, red kidney) and white rice for their acute postprandial effects on vascular and metabolic responses in healthy individuals.The study was designed as a single-blinded, randomized crossover trial with a minimum 6 days between consumption of the food articles. Vascular tone (primary endpoint), haemodynamics and serum biochemistry (secondary endpoints) were measured in 8 healthy adults before and at 1, 2, and 6 h after eating ¾ cup of beans or rice. Blood pressure and pulse wave velocity (PWV) were lower at 2 h following red kidney bean and pinto bean consumption compared to rice and navy bean, respectively (p 0.05). There was greater vasorelaxation 6 h following consumption of darker-coloured beans, as shown by decreased vascular tone: PWV was lower after consuming black bean compared to pinto bean, augmentation pressure was lower after consuming black bean compared to rice and pinto bean, and wave reflection magnitude was lower after consuming red kidney bean and black bean compared to rice, navy bean, and pinto bean (p 0.05). LDL-cholesterol concentrations were lower 6 h after black bean consumption compared to rice (p 0.05).Overall, red kidney and black beans, the darker-coloured beans, elicited a positive effect on the tensile properties of blood vessels, and this acute response may provide insight for how pulses modify vascular function.

OBJECTIVES: Docosahexaenoic acid (DHA) can be obtained directly from the diet or produced by elongation and desaturation of α-linolenic acid (ALA). Both are proposed to reduce inflammation associated with obesity, however, fewer studies have investigated ALA. The objective of this study was to evaluate the gene expression changes in monocytes induced by each fatty acid and to compare the predicted functional outcomes. METHODS: RNA was extracted from THP-1 monocytes treated with ALA, DHA or vehicle for 48 h and then transcriptomics profiles were assessed by microarray. Multiple tools were used for data interpretation, including fold change analysis, Principal Component Analysis (PCA), Variable Importance Projection (VIP), Ingenuity Pathway Analysis (IPA) and Network Analyst. RESULTS: We found that the ALA and DHA treatments produced distinct profiles with many individual genes making small contributions to the separation between groups. Relative to vehicle treatment, many downregulated targets were similarly affected by both ALA and DHA. Several of these downregulated genes are involved in cholesterol synthesis and are regulated by miR-335–5p, a microRNA upregulated by both treatments. Consistently, IPA predicted similar pathways and functions are decreased by ALA and DHA, most notably cholesterol biosynthesis. In contrast, ALA and DHA upregulated unique gene sets and in agreement IPA predicted each treatment would activate distinct pathways and functions. ALA was strongly and uniquely predicted to increase infection responses while only DHA was predicted to increase oxidative phosphorylation. Finally, analysis of the protein-protein interaction network involving the genes modified by each fatty acid treatment allowed us to predict the most functionally important gene targets, which will be tested in future studies. CONCLUSIONS: These analyses have revealed both unique and overlapping effects of ALA and DHA on the monocyte gene expression profile, providing further evidence that they have distinct bioactivities. Many novel predictions were made and these will form the basis for future studies investigating the effects of ALA and DHA on human physiology. FUNDING SOURCES: Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research.

Oxylipins, which are circulating bioactive lipids generated from polyunsaturated fatty acids (PUFAs) by cyclooxygenase, lipooxygenase and cytochrome P450 enzymes, have diverse effects on endothelial cells. Although studies of the effects of oxylipins on endothelial cell function are accumulating, a review that provides a comprehensive compilation of current knowledge and recent advances in the context of vascular homeostasis is lacking. This is the first compilation of the various in vitro, ex vivo and in vivo reports to examine the effects and potential mechanisms of action of oxylipins on endothelial cells. The aggregate data indicate docosahexaenoic acid-derived oxylipins consistently show beneficial effects related to key endothelial cell functions, whereas oxylipins derived from other PUFAs exhibit both positive and negative effects. Furthermore, information is lacking for certain oxylipin classes, such as those derived from α-linolenic acid, which suggests additional studies are required to achieve a full understanding of how oxylipins affect endothelial cells.

Background Various foods are known to have beneficial effects on health when consumed whole; however, there is a trend towards preparing foods from processed ingredients, and it remains unclear whether the benefits of the whole food are retained. The purpose of this study was therefore to examine whether different processing techniques affect the lowering of cholesterol and the vascular effects of black beans (Phaseolus vulgaris L.). Results Beans were prepared by overnight soaking and boiling - the standard method - and by micronization, extrusion, or dehulling and boiling, and they were then fine milled. Beans prepared by the standard method were also coarse milled. These five materials were incorporated into semi-purified diets (30% wt/wt) and fed to spontaneously hypertensive rats for 4 weeks. Body weight, blood pressure, and aorta morphology were unaltered by the diets. Fasting total cholesterol was significantly reduced in rats fed micronized beans compared with extruded beans (both fine-milled) or the bean-free diet, while boiling combined with coarse milling lowered low-density lipoprotein (LDL) cholesterol. The lack of cholesterol lowering in rats fed extruded bean compared to micronized was not explained by the amount or composition of dietary fiber or resistant starch. Differences in the polyphenolic profile as determined by high-performance liquid chromatography (HPLC) were also unable to explain the variations in cholesterol-lowering capacity. Conclusion The present study demonstrates that processing of black beans alters the health effects observed with the whole pulse, and suggests that products prepared with processed ingredients will need to be tested empirically to establish whether the biological effects are maintained in vivo. © 2020 Society of Chemical Industry.

As the largest secondary lymphoid organ, the spleen plays an important role in immune responses. The role of arachidonic acid (ARA) and its 20-carbon eicosanoids in modulating immune function has long been of interest. However, recent advances have enabled the identification of numerous other n-6 and n-3 polyunsaturated fatty acid (PUFA)-derived oxylipins. Here, we investigate the effects of diet and sex on the spleen nonesterified oxylipin profiles and phospholipid and neutral lipid PUFA composition in Sprague-Dawley rats supplemented with oils rich in α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or linoleic acid. Dietary ALA, EPA, and DHA resulted in lower levels of ARA and ARA oxylipins. Oxylipins derived from other n-6 PUFA were also reduced despite no or opposite effect on their PUFA levels. Each diet also resulted in higher levels of oxylipins almost exclusively derived from the supplemented PUFA, despite PUFA in the same biosynthetic pathway also often being increased. Further, while oxylipin differences often reflected changes to phospholipid PUFA, there were instances where they corresponded more closely to changes in neutral lipid PUFA. With respect to sex effects, >50% of lipoxygenase ARA-derived oxylipins were higher in males in at least one diet group, while multiple DHA oxylipins were lower in males only in rats provided the DHA diet. This fundamental description of oxylipin composition in the spleen, including the influence of diet and sex and the relationship to PUFA composition, will help inform future studies examining the functions of these oxylipins under physiological and pathological conditions.

Chronic inflammation, mediated in large part by proinflammatory macrophage populations, contributes directly to the induction and perpetuation of metabolic diseases, including obesity, insulin resistance and type 2 diabetes. Polyunsaturated fatty acids (PUFAs) can have profound effects on inflammation through the formation of bioactive oxygenated metabolites called oxylipins. The objective of this study was to determine if exposure to the dietary omega-3 PUFA α-linolenic acid (ALA) can dampen the inflammatory properties of classically activated (M1-like) macrophages derived from the human THP-1 cell line and to examine the accompanying alterations in oxylipin secretion. We find that ALA treatment leads to a reduction in lipopolysaccharide (LPS)-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α production. Although ALA is known to be converted to longer-chain PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), DHA oxylipins were reduced overall by ALA treatment, as was LPS-induced secretion of EPA oxylipins. In contrast, we observed profound increases in oxylipins directly derived from ALA. Lipoxygenase products of linoleic acid were also dramatically increased, and LPS-induced production of AA oxylipins, particularly prostaglandin D2, was reduced. These results suggest that ALA may act to dampen the inflammatory phenotype of M1-like macrophages by a unique set of mechanisms distinct from those used by the long-chain omega-3 fatty acids EPA and DHA. Thus, there is strong rationale for investigating the functions of ALA oxylipins and lesser-known LA oxylipins since they hold promise as anti-inflammatory agents.

Dietary EPA and DHA given together alter oxylipins in adipose tissue. To compare the separate effects of individual dietary n-3 PUFA on oxylipins in different adipose depots (gonadal, mesenteric, perirenal, subcutaneous) in males and females, rats were provided diets containing higher levels of α-linolenic acid (ALA), EPA or DHA. Each n-3 PUFA enhanced its respective oxylipins the most, while effects on other n-3 oxylipins varied. For example: in perirenal and subcutaneous depots, more DHA oxylipins were higher with dietary ALA than with EPA; dietary EPA uniquely decreased 14-hydroxy-docosahexaenoic acid, in contrast to increasing many other DHA oxylipins. The n-3 PUFAs also reduced oxylipins from n-6 PUFAs in order of effectiveness: DHA > EPA > ALA. Diet by sex interactions in all depots except the perirenal depot resulted in higher oxylipins in males given DHA, and higher oxylipins in females given the other diets. Diet and sex effects on oxylipins did not necessarily reflect effects on either their tissue phospholipid or neutral lipid PUFA precursors. These varying diet and sex effects on oxylipins in the different adipose sites indicate that they may have distinct effects on adipose function.

Hormonal dysregulation plays a significant role in the metabolic switching during malignant transformation. Progesterone Receptor Membrane Component 1 (PGRMC1) is a single-pass transmembrane receptor activated by the binding of progesterone (P4), a sex hormone. In a previous study, P4 treatment caused rapid (within 30 min) induction of aerobic glycolysis in transformed HEK293 cells, a hallmark malignant phenotype known as the Warburg effect. This metabolic reprogramming was associated with the proteasomal degradation of a 70 kilodalton (kDa) PGRMC1. PGRMC1 interacts with a variety of proteins, including G protein-coupled receptors (GPCRs) and P4-PGRMC1 signaling modulates cyclic adenosine monophosphate (cAMP) production. Therefore, we hypothesized that the P4-induced Warburg effect and proteasomal degradation of PGRMC1 involve G proteins and β-Arrestins (ARRBs). In the present study, we investigated P4-induced aerobic glycolysis, proteasomal degradation of p70 PGRMC1, as well as abundance and subcellular translocation of PGRMC1 along with two key glycolytic enzymes Hexokinase 1 (HK1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in six Gα subunit (Gsix) proteins or ARRB1/2-deficient HEK293 cells. Loss of ARRB1/2 or Gsix proteins inhibited P4-induced p70 PGRMC1 degradation but failed to prevent the P4-induced Warburg effect. Also, deficiency of ARRB1/2 or Gsix proteins differentially affected the basal as well as P4-induced abundance and subcellular translocation of PGRMC1, HK1, and GAPDH proteins. Overall, the findings indicate that P4-PGRMC1-mediated metabolic reprogramming in HEK293 cells depends on β-Arrestins and Gα proteins suggesting the involvement of an underlying GPCR signal transduction pathway.

Introduction Consumption of omega-3 polyunsaturated fatty acids (n-3 PUFAs) has been reported to provide health benefits, but it remains unknown whether the fatty acids themselves or their oxygenated metabolites, oxylipins, are responsible for the beneficial effects. Purpose This paper describes the design and rationale of a randomized, double-blinded, cross-over study comparing the effects of α-linolenic acid (ALA)-rich flax oil and docosahexaenoic acid (DHA)-rich fish oil supplementation on circulating oxylipin profiles in females with obesity, in relation to obesity-induced inflammation. Methods and analysis Pre-menopausal females (n = 24) aged 20-55 with a BMI ≥30, will consume capsules containing flaxseed oil (4 g ALA/day) or fish oil (4 g DHA + 0.8 g EPA/day) during 4-week supplementation phases, with a minimum 4-week washout. The primary outcome is alterations in plasma oxylipin profiles. Secondary outcomes include effects of supplementation on circulating markers of inflammation, adipokines, plasma fatty acid composition, blood lipid profile, anthropometrics, oxylipin and cytokine profiles of stimulated immune cells, monocyte glucose metabolism, blood pressure and pulse wave velocity. Ethics and significance This trial has been approved by the University of Manitoba Biomedical Research Ethics Board and the St. Boniface Hospital Research Review Committee. The study will provide information regarding the effects of ALA and DHA supplementation on oxylipin profiles in obese but otherwise healthy females. Additionally, it will improve our understanding of the response of circulating inflammatory mediators originating from immune cells, adipose tissue and the liver to n-3 PUFA supplementation in relation to the metabolic features of obesity.

The present study investigated the feasibility, tolerability, and adherence of daily consumption of whole pulses (dried beans, peas, lentils, chickpeas) by individuals with peripheral artery disease participating in an 8-week study. Study questionnaires and semi-structured interviews for 26 participants were used to determine prestudy pulse consumption and participants' experiences with respect to adherence, positive and negative effects, bowel routine, satiety, and enjoyment of the foods. Although the majority of participants rarely consumed pulses prior to the study, there was a high rate of adherence to daily consumption of the study foods for 8 weeks despite comments regarding study fatigue during the latter part of the study. Participants had no gastrointestinal side effects (42%) or experienced flatulence that resolved by week 4 (23%), whereas 62% reported improvements in their bowel pattern. By week 8 greater satiety was noted by some participants (19%), with the categories "less afternoon snacking" and "not snacking" receiving more responses. The key finding of this study was that consumption of pulses is a viable approach for this population; however, the frequency of consumption that is tolerable in the long term should be integrated with the dose and timeframe required to achieve and maintain health benefits.

The unique characteristics and healthful reputation of caffeine-free rooibos tea (RT) make it an ideal carrier for vitamin D3 supplementation, and a potential base for the addition of Saskatoon berry syrup (SBS), a natural flavor additive. The objective of this study was to determine the effect of vitamin D3 fortification and SBS addition on the flavor profile, consumer acceptability, and antioxidant properties of RT. Six formulations (RT, RT with SBS, RT with SBS and vitamin D3 , RT with vitamin D3 , green tea [GT], and GT with SBS) were evaluated by 12 trained panelists and 114 consumers. The formulations were also assessed for antioxidant capacity, physical characteristics, and untargeted phytochemical content. Sensory results revealed that the mean intensity values for berry and sweet attributes were significantly higher (P < 0.05) while bitter and astringent attributes were significantly lower when SBS was added to RT samples compared to those without syrup. Acceptability of flavor, aftertaste, and overall acceptability were also significantly higher for the RT with SBS. The addition of SBS to RT significantly increased the antioxidant capacities which may increase the related health benefits of RT. SBS contributed several polyphenols, particularly flavonoids, to the tea. Vitamin D3 added to RT formulations did not significantly affect the sensory attributes, acceptability, or antioxidant content. For the development of a functional vitamin D3 fortified iced-tea beverage that can be consumed as part of the daily diet, SBS could be a favorable flavoring additive that may provide additional health benefits.

Questions remain regarding the potential negative effects of dietary high protein (HP) on kidney health, particularly in the context of obesity in which the risk for renal disease is already increased. To examine whether some of the variability in HP effects on kidney health may be due to source of protein, obese fa/fa Zucker rats were given HP (35% of energy from protein) diets containing either casein, soy protein, or a mixed source of animal and plant proteins for 12 weeks. Control lean and obese rats were given diets containing casein at normal protein (15% of energy from protein) levels. Body weight and blood pressure were measured, and markers of renal structural changes, damage, and function were assessed. Obesity alone resulted in mild renal changes, as evidenced by higher kidney weights, proteinuria, and glomerular volumes. In obese rats, increasing the protein level using the single, but not mixed, protein sources resulted in higher renal fibrosis compared with the lean rats. The mixed-protein HP group also had lower levels of serum monocyte chemoattractant protein-1, even though this diet further increased kidney and glomerular size. Soy and mixed-protein HP diets also resulted in a small number of damaged glomeruli, while soy compared with mixed-protein HP diet delayed the increase in blood pressure over time. Since obesity itself confers added risk of renal disease, an HP diet from mixed-protein sources that enables weight loss but has fewer risks to renal health may be advantageous.

We recently reported that loss of calcium/calmodulin-dependent protein kinase kinase-2 (CAMKK2), a serine/threonine kinase activated by intracellular calcium, in mice leads to tissue-specific aberrant turnover of transferrin (TF), a receptor-mediated iron-transporter that supplies iron to tissues. Iron dyshomeostasis is associated with the pathogenesis of several diseases, making TF transport relevant to health. In this study, we used hemizygous CAMKK2 hypomorphic human endothelial cell (EA.hy926) clones to demonstrate that cells with reduced CAMKK2 exhibit increased TF uptake and transcytosis, and decreased intracellular trafficking to subcellular organelles compared to wild-type. The abnormal TF trafficking in CAMKK2 hypomorphic cells correlated with a reduction in intracellular iron content and defective glucose metabolism including glycolysis and mitochondrial respiration. CAMKK2 deficiency also caused reduction in GAPDH and VDAC1 protein level which correlated to defective bioenergetics function. These findings have identified a novel mechanistic link between abnormal calcium signaling, iron dyshomeostasis and metabolic dysfunction involving CAMKK2.

Background Development of instruments capable of detecting early stage vascular disease has increased interest in employing arterial stiffness (e.g. pulse wave velocity (PWV), augmentation index (AIx)) and endothelial dysfunction (e.g. reactive hyperemia index (RHI)) to diagnose atherosclerotic disease before occurrence of a cardiovascular event. However, amongst the equipment designed for this purpose, there is insufficient information regarding each of these parameters to establish appropriate cutoffs to distinguish between healthy and unhealthy blood vessels. To address these limitations, the study was designed to establish the upper arterial stiffness and endothelial function thresholds in a healthy population, by comparing the outputs from different instruments capable of measuring PWV, AIx and RHI. Methods A systematic comparison of PWV, AIx and RHI was conducted to determine the inter-relationships between these parameters of vascular functionality. Outputs were obtained non-invasively using three instruments, the VP-1000 (VP), SphygmoCor (SC), and EndoPAT (EP), in 40 apparently healthy males and females. Results Correlations were found between the brachial-ankle PWV and radial-ankle PWV (by VP and SC), and PWV (VP) with AIx (SC). The interchangeability of these outputs was demonstrated by the Bland Altman test, making it feasible to extrapolate cut-offs for radial-ankle PWV and AIx equivalent to brachial-ankle PWV that signify healthy vessels. In contrast, RHI showed no association with AIx, suggesting these endothelial and arterial parameters are functionally distinct. Conclusions It was concluded that it is possible to compare the vascular function outputs of different instruments and identify healthy from unhealthy vessels, even though the approaches for quantifying the underlying physiological processes may differ. In this way, non-invasive determination of arterial function could be a new paradigm for detecting existing early stage asymptomatic atherosclerotic disease in individuals using techniques that are amenable to the clinical setting. Electronic supplementary material The online version of this article (10.1186/s12872-019-1167-3) contains supplementary material, which is available to authorized users.

Hepatic steatosis, an early stage of non-alcoholic fatty liver disease, is commonly present in obesity and type 2 diabetes, and is associated with reduced hepatic omega-3 polyunsaturated fatty acid (n3-PUFA) status that impacts on the anti-inflammatory and insulin sensitizing functions of n3-PUFA. Our objective was to directly compare plant- and marine-based n3-PUFA (&alpha, linoleic acid (ALA)), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)) for their effects on hepatic steatosis, markers of hepatic inflammation and fibrosis, and insulinemia in obese rats. Fa/fa Zucker rats were provided diets containing ALA, EPA, DHA, or linoleic acid (LA, n6-PUFA) for eight weeks and compared to baseline fa/fa rats and lean Zucker rats fed LA-rich diet for eight weeks. Both DHA and EPA groups had liver lipid similar to baseline, however, DHA was more effective than EPA for reducing hepatic fatty acid synthase (FAS), increasing the proportion of smaller lipid droplets, reversing early fibrotic damage, and reducing fasting hyperinsulinemia. EPA was more effective for reducing FoxO1. Dietary ALA did not attenuate hepatic steatosis, most inflammatory markers or FAS. In summary, amongst the n3-PUFA, DHA was the most effective for elevating hepatic DHA levels, and preventing progression of hepatic steatosis via reductions in FAS and a marker of fibrosis.

OBJECTIVES: Our laboratory previously reported that docosahexaenoic acid (DHA) activates p38 mitogen-activated protein kinase (MAPK) differently in growing and quiescent human endothelial cells, which represent the atherogenic and healthy states in vivo, respectively. Endothelial nitric oxide synthase (eNOS) activity differs between these two states. Since eNOS can be regulated by p38MAPK and mitogen-stimulated kinase (MSK) is a p38MAPK substrate involved in cell proliferation and inflammation, we hypothesized that DHA's atheroprotective actions require eNOS activation via the p38MAPK/MSK pathway. Thus, our objective was to investigate the role of p38MAPK/MSK in the eNOS response to DHA and determine whether the proposed pathway is growth state-sensitive. METHODS: EA.hy926 cells were cultured on Matrigel-coated plates to sub-confluent and quiescent states and treated with DHA ± SB202190 or SB747651A, inhibitors of p38MAPK and MSK, respectively. eNOS activation was quantified by Western blot detection of Ser1177 phosphorylation. RESULTS: eNOS activation by DHA in EA.hy926 cells was concentration-, time-, and growth state-dependent. p38MAPK inhibition suppressed eNOS activity in sub-confluent cells and increased eNOS activity in quiescent cells, while MSK inhibition suppressed eNOS activity in both growth states. eNOS activity remained suppressed with DHA treatment under MSK inhibition and showed no dose- or growth state-dependent effects. In contrast, when p38MAPK was inhibited, high dose DHA activated eNOS in sub-confluent cells, but dose-dependently decreased the elevated eNOS activity of quiescent cells. CONCLUSIONS: eNOS activity in endothelial cells is modulated by DHA via p38MAPK and MSK. The growth state-dependent regulation of p38MAPK and eNOS by DHA provides novel insight into the molecular mechanisms of DHA's atheroprotective actions in relation to health status. FUNDING SOURCES: Research Manitoba, St Boniface Hospital Foundation, University of Manitoba - GETS

In this study, we hypothesize that the biologically active isomers of conjugated linoleic acid (CLA), cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) CLA, have different effects on early and late stages 3T3-L1 preadipocyte differentiation. Both c9-t11 and t10-c12CLA stimulated early stage pre-adipocyte differentiation (day 2), while t10-c12CLA inhibited late differentiation (day 8) as determined by lipid droplet numbers and both perilipin-1 levels and phosphorylation state. At day 8, the adipokines adiponectin, chemerin and adipsin were all reduced in t10-c12CLA treated cells versus control cells. Immunofluorescence microscopy showed perilipin-1 was present solely on lipid droplets on day 8 in t10-c12 treated 3T3-L1 cells, whereas preilipin-1 was also located in the perinuclear region in control and c9-t11 treated cells. The t10-c12CLA isomer also decreased levels of hormone-sensitive lipase and inhibited lipolysis. These findings indicate that the decrease in lipid droplets caused by t10-c12CLA is the result of an inhibition of lipid droplet production during adipogenesis rather than a stimulation of lipolysis. Additionally, treatment with Gö6976 blocked the effect of t10-c12CLA on perilipin-1 phosphorylation, implicating PKCα in perilipin-1 phosphorylation, and thus a regulator of triglyceride catabolism. These data are supported by evidence that t10-c12CLA activated PKCα. These are the first data to show that CLA isomers can affect lipid droplet dynamics in adipocytes through PKCα.

Background Trans-10, cis-12 (t10-c12) CLA treatment reduces lipid accumulation in differentiating mouse and human adipocytes, and decreases fat mass in mice, yet the mechanism of action remains unknown. Objective This study investigated the effect of the cis-9, trans-11 (c9-t11) and t10-c12 CLA isomers on the Wnt/β-catenin pathway, which has been reported to inhibit adipogenesis by down-regulating PPARγ. Results We observed that t10-c12 CLA treatment of 3T3-L1 adipocytes increases the levels of β-catenin and Ser-675 phosphorylated β-catenin due to inhibition of its degradation. These changes in β-catenin were not linked to either the Wnt/β-catenin agonist Wnt10b or other upstream effectors such as SFRP-5. Paradoxically, the presence of higher amounts of β-catenin did not elevate cyclin D1 levels, which is recognized as a critical target gene. Neither of the CLA isomers affected the localization of β-catenin in the cytosol and nucleus as determined by immunofluorescence microscopy. However, subcellular fractionation suggested the level of cytosolic β-catenin was reduced in t10-c12 CLA treated cells. Immunoprecipitation revealed that t10-c12 CLA increased the interaction of β-catenin and PPARγ. Conclusions t10-c12-CLA inhibits adipocyte differentiation by increasing β-catenin stability in 3T3-L1 adipocytes, thus enhancing sequestration of PPARγ in an inactive complex, which prevents progression of adipogenesis.

High protein diets are commonly promoted for the management of metabolic syndrome parameters. However, the current literature is complex with positive, detrimental and no effects being demonstrated. This conflicting evidence has prevented the development of concrete recommendations regarding the effectiveness and safety of high protein diets for managing metabolic syndrome. Furthermore, evidence that different dietary sources of protein have distinct metabolic effects has been obtained, but additional research is needed to extend and clarify the limited information currently available. In particular, future studies are required to determine whether specific protein sources can elicit effects at normal to moderately increased protein intake as a means of avoiding any potential risks associated with high protein diets.

OBJECTIVES: One of the earliest events in atherosclerotic plaque formation is the adhesion and migration of monocytes to damaged blood vessels. The fish-oil derived omega-3 fatty acid docosahexaenoic acid (DHA) has been shown to inhibit this process. There is limited evidence suggesting α-linolenic acid (ALA) has a similar effect, however, ALA has not been directly compared to DHA. Therefore, the primary objective of this study was to compare the gene expression profiles of monocytes that have been exposed to either ALA or DHA and subsequently examine the effect of these fatty acids on monocyte adhesion in a cell culture model. METHODS: Whole transcriptome analysis was performed on total mRNA isolated from a human THP-1 monocyte cell line treated with ALA, DHA or vehicle for 48 h. Candidate genes identified via fold change and Ingenuity Pathway Analysis were validated by qPCR. An adhesion assay was subsequently performed on monocytes treated with ALA or DHA to corroborate the predicted outcomes of our analysis. RESULTS: Transcriptome analysis identified a series of genes associated with cell adhesion and migration. The change in mRNA levels for each of the candidate genes was validated by qPCR, and in all cases a similar expression pattern was obtained as observed with the gene expression analysis. Based on these data, it was predicted that these processes would be upregulated in response to ALA but not DHA. The functional assay revealed that ALA increased or had no effect on monocyte adhesion, while DHA was found to significantly decrease adhesion. CONCLUSIONS: The results suggest ALA and DHA influence adhesion and migration through distinct gene pathways. Furthermore, the functional assays indicated that DHA treatment but not ALA reduces adhesion. Due to the critical role of monocyte adhesion and migration in the pathophysiology of atherosclerosis, it may be concluded from this study both DHA and ALA may exert protective effects in different ways as they relate to individuals at risk for cardiovascular disease. FUNDING SOURCES: Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research.

OBJECTIVES: Docosahexaenoic acid (DHA) is known for its protective effects against cardiovascular disease, which has been the leading cause of death worldwide for 2 decades. The molecular mechanisms responsible for DHA's atheroprotective effects, however, remain largely unknown. DHA has been found to activate p38 mitogen-activated protein kinase (MAPK) differently in growing and quiescent human endothelial cells, which represent dysfunctional and healthy states in vivo, respectively. This study was designed to characterize the activation pattern of p38MAPK in endothelial cells in response to DHA treatment and to identify possible downstream targets by which DHA exerts its protective effects. METHODS: EA.hy926 cells were cultured on Matrigel-coated plates to sub-confluent, confluent, and quiescent states. The cells were treated with DHA to establish concentration (10 μM to 150 μM) and time course (10 min to 24 h) curves, with or without SB202190, a p38MAPK-specific inhibitor. The activation of p38MAPK and its downstream targets was quantified by Western blotting. Histone H3 modifications upon DHA treatment were tested with an ELISA-based kit. RESULTS: The activation of p38MAPK by DHA in EA.hy926 cells was concentration-, time-, and growth state-dependent. Upon p38MAPK inhibition, activation of mitogen and stress activated kinase 1 (MSK1), a downstream target of p38MAPK, declined. This reduction was attenuated by low concentrations of DHA in quiescent endothelial cells but not confluent or sub-confluent cells. Since MSK1 can act on histone H3 and other chromatin binding proteins like cAMP response element-binding protein (CREB), H3 modifications were monitored. In the confluent state, DHA caused a 6-fold increase in total H3, with a concomitant decrease in most methylation, acetylation, and phosphorylation marks, including H3K9me1/3, H3K27me2, H3K36me1/3, H3K9ac, H3K18ac, H3S10ph, and H3S28ph. CONCLUSIONS: This study showed that DHA may exert its effects in endothelial cells via the p38MAPK signalling pathway, and MSK1 may be a downstream effector possibly leading to epigenetic changes. The results provide novel insights regarding DHA's atheroprotective actions and identify new therapeutic targets with potential for treating atherosclerosis. FUNDING SOURCES: Research Manitoba, St Boniface Hospital Foundation-Research Without Borders.

OBJECTIVES: This study was designed to investigate whether unconventional prefoldin RPB5 interactor (URI)-1 mediates hepatic accumulation of triglyceride (TG) in response to a diet with trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA) in lean or genetically obese mice. URI-1 belongs to the prefoldin family of proteins that have been shown to coordinate nutrient availablility by transcriptional regulation of genes involved in glucose and lipid metabolism. Thus, it was hypothesized that URI-1 in liver is involved in increased fatty acid uptake and accumulation leading to fatty liver. METHODS: C57BL/6 and db/db mice were randomly assigned to two diet groups, control (CTL) and t10,c12 CLA (0.4% w/w). After 4 weeks, the mice were weighed and euthanized. Livers were dissected, weighed and stored at –80°C. Liver lysates were prepared from the tissue for Western blotting to measure hepatic protein levels of URI-1 and FABP1. The amount of lipid in the livers was determined using the LabAssay™ Triglyceride kit, a colorimetric TG assay. RESULTS: The liver to body weight ratio of db/db and C57BL/6 mice fed t10,c12 CLA increased by 90% and 52%, respectively, compared to their counterparts fed the CTL diet. Likewise, the hepatic TG concentration (mg TG/mg protein) was increased 38% and 5-fold, respectively, in CLA-fed db/db and C57BL/6 mice compared to CTL db/db and C57BL/6 mice. Western blotting showed that FABP1 levels were approximately 2-fold greater in the db/db t10,c12 CLA group relative to the db/db CTL group, and may contribute to increased fatty acid uptake. Furthermore, URI-1 protein levels were elevated 4-fold in db/db and C57BL6 mice fed t10,c12 CLA compared to their respective CTL groups. Lastly, correlation analysis revealed that URI-1 levels were significantly correlated with hepatic TG concentrations (r = 0.61) and liver/body weight ratio (r = 0.64). CONCLUSIONS: This study revealed a relationship between hepatic TG accumulation and URI-1, a protein associated with hepatocellular carcinoma (HCC) and cirrhosis. This study provides a basis for in vitro experiments exploring the causative role of URI-1 in propagating hepatic TG accumulation, and ultimately the progression of fatty liver disease to HCC and cirrhosis. FUNDING SOURCES: University Collaborative Research Project, NSERC Discovery, and University of Manitoba Graduate Enhancement of Tri-Council Stipends.

Edible legume seeds, such as lentils, have been shown to modulate the structural and functional properties of hypertensive blood vessels, however, the effects of dried beans have not been similarly evaluated. To determine whether beans could attenuate hypertension-induced vascular changes (remodeling and stiffness) in relation to their phytochemical content, spontaneously hypertensive rats (SHR) were fed diets containing black beans (BB, high phytochemical content as indicated by their dark seed coat colour) or navy (white) beans (NB, low phytochemical content) for eight weeks. An additional follow-up phase was included to determine how long the alterations in vascular properties are maintained after bean consumption is halted. Assessments included blood pressure (BP), pulse wave velocity (PWV), vessel compliance (small-artery) and morphology (large-artery), and body composition. Neither BBs nor NBs altered BP or PWV in SHR. SHR-BB demonstrated greater medial strain (which is indicative of greater elasticity) at higher intraluminal pressures (80 and 140 mmHg) compared to SHR-NB. BB consumption for 8 weeks enhanced vascular compliance compared to SHR-NB, as demonstrated by a rightward shift in the stress&ndash, strain curve, but this improvement was lost within 2 weeks after halting bean consumption. BB and NB increased lean mass after 8 weeks, but halting BB consumption increased fat mass. In conclusion, regular consumption of BBs may be appropriate as a dietary anti-hypertensive strategy via their positive actions on vascular remodeling and compliance.