Walnuts change lipoprotein composition suppressing TNFa-stimulated cytokine production by diabetic adipocyte
Walnut consumption can provide both vascular and metabolic health benefits, and walnut-induced changes in lipoprotein particle chemical payloads may be responsible for these health benefits. To explore this possibility with a focus on metabolic health, this study investigated the impact of walnut consumption on lipoprotein lipid composition and changes in LDL anti-inflammatory properties, as reported by inflamed adipocyte. Hypercholesterolemic, postmenopausal females were treated with 40 g/day (i.e. 1.6 servings/day; n =15) of walnuts for 4 weeks. Fatty acids and their oxygenated metabolites, i.e. oxylipins, were quantified in isolated lipoproteins. Human primary adipocytes were exposed to LDL and TNFa-stimulated adipokine production was measured. Walnut treatment elevated \xce\xb1-linolenic acid and its epoxides in all lipoproteins and depleted mid-chain alcohols in VLDL and LDL, but not HDL. Walnuts also reduced TNFa-induced diabetic adipocyte production of IL-6 (\xe2\x88\x9248% p =0.0006) and IL-8 (\xe2\x88\x9230%p =0.01), changes inversely correlated with levels of \xce\xb1-linolenic acid-derived epoxides but not \xce\xb1-linolenic acid itself.In conclusion, modest walnut consumption can alter lipoprotein lipid profiles and enhance their ability to inhibit TNFa-dependent pro-inflammatory responses in human diabetic primary adipocytes. Moreover, this study suggests the oxylipins, rather than the parent fatty acids, mediate LDL action of adipocytes.
Introduction:<!>Study design and analysis strategy:<!>Lipoprotein isolation:<!>Lipoprotein fatty acids and oxylipins analysis:<!>Adipocyte culture:<!>Measurement of adipokines levels:<!>Generation of DIL-LDL and LDL uptake assay:<!>Intracellular lipid accumulation:<!>Statistical analysis:<!>Walnuts impact lipoprotein composition:<!>Walnuts consumption changes functionality of low density lipoproteins in the context of adipocyte response to TNFa:<!>LDL oxylipins, but not their parent fatty acids, are strongly correlated with the anti-inflammatory effects:<!>Discussion:<!>Limitations:<!>Conclusion
<p>The consumption of walnuts and other tree nuts has been associated with a lower risk for cardiovascular disease [1]. Specifically, walnut intake has been associated with lower type-II diabetes risk [2], while tree nuts can lower LDL cholesterol and triglycerides [1, 3] and attenuate endothelial dysfunctions induced by hypercholesterolemia [4] and high-fat meals [5]. In subjects with hypercholesterolemia, walnut interventions can also decrease the production of the pro-inflammatory cytokines interleukin-6 (IL-6), IL-1b and TNFa by peripheral blood mononuclear cells [6]. Adipose inflammation is a significant consequence of obesity that negatively impacts tissue glucose handling and insulin sensitivity [7]. Importantly, elevated oxidized LDL (ox-LDL) are associated with type II diabetes [8], adipocytes actively take up ox-LDL [9, 10], the plasma levels of ox-LDL has been negatively correlated with plasma levels of the adipose generated cytokine adiponectin in humans [11] and walnut consumption can reduce LDL oxidation [4, 12, 13]. Walnuts are a rich source of linoleic acid (LA), α-linolenic acid (aLA) [14] and numerous bioactive phenolics [1, 15–19]. While each of these components have bioactive potential, it has been postulated that aLA, an omega-3 fatty acid, is a major factor responsible for the anti-inflammatory effects in a manner analogous to the anti-inflammatory effects of fish oil being attributed to the long chain omega-3 fatty acids [1].</p><p>The bioactivity of PUFAs can be associated with their direct action, as well as through the action of their metabolic products. Major products of PUFA metabolism are the oxylipins, a superclass of oxygenated metabolites. These compounds are formed by: cyclooxygenases (COX) – producing prostaglandins and thromboxanes; lipoxygenases (LOX) – producing hydroperoxides and their associated downstream products (e.g. mid-chain alcohols); cytochrome P450 (CYP) – producing epoxides and omega-hydroxides; autooxidation, producing peroxide associated products including isoprostanes and certain mid-chain alcohols [20, 21]. The structures of representative alkaline-stable arachidonic acid-derived oxylipins are presented in the Figure 1. Oxylipins are the primary mediators of PUFA action, regulating processes including inflammation, glucose metabolism and cardiovascular homeostasis (broadly reviewed in [22–29]). Different classes of oxylipins manifest distinct properties, and these effects are influenced by the parent fatty acid from which they are derived [22]. For instance, epoxides are generally anti-inflammatory [23, 24] and can suppress the production of pro-inflammatory cytokines [25, 26]. Moreover, elevated plasma concentrations of epoxides have been linked with better cardiovascular outcomes in diverse animal models [26]. On the other hand, many mid-chain alcohols are pro-inflammatory [27], with links to low-grade systemic inflammation and pro-inflammatory cytokine production in adipose tissue [30]. While much is known regarding the action of these compounds in general, their sources and mechanisms of production and delivery in vivo are as of yet poorly understood.</p><p>While oxylipins are generally considered to be autocrine and paracrine in nature, they are transported through plasma in esterified forms within lipoprotein particles at high abundances [31, 32]. The trafficking of these particle associated metabolites, and their impact on lipoprotein-metabolizing tissues are not well understood [28, 29]. Clinical trials have demonstrated that PUFA intake influences the oxylipin composition of lipoproteins. Supplementation with marine omega-3 PUFAs results in increased docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)-derived oxylipins at the expense of arachidonic acid (AA) metabolites [31–33]. Moreover, marine-PUFAs induce changes in oxylipin composition differently among different lipoproteins classes [31]. While the effect of fish oil supplementation on the oxylipin profile has been the subject of several studies [31–33], to the best of our knowledge, the impact of tree nuts on the lipoprotein esterified oxylipin profiles has not been reported.</p><p>Previously, in a randomized controlled trial walnut treatment improved endothelial dysfunction in hyper-triglyceridemic women, an effect associated with increases in plasma non-esterified fatty acid epoxides [34]. In the current study, using samples from the study above, we investigated walnut-induced changes in the lipoprotein oxylipin and fatty acids composition. Given the known pathological association of ox-LDL, we also tested the impact of walnut-induced changes to LDL on human primary adipocytes, a model lipoprotein-metabolizing tissue which actively takes up LDL and ox-LDL [9, 10]. The production of pro- and anti-inflammatory adipokines were assessed as the primary outcome. Since walnuts have been reported to have beneficial effect in pathological conditions associated with the metabolic syndrome [4, 5, 34] and elevated ox-LDL [4, 12, 13], adipocytes with diabetic and non-diabetic phenotypes were used. Moreover, since low-grade systemic inflammation accompanies the development of obesity and diabetes and is associated with elevated pro-inflammatory cytokine production, cell culture experiments were performed with or without TNFa, as a surrogate for an inflammatory environment.</p><p>Previous studies have clearly demonstrated that dietary PUFAs can improve select health outcomes [32, 35, 36], and alter lipoprotein particles composition [32]. Moreover, lipoprotein composition has been associated with diverse physiological outcomes [37]. In the present study, we used a dietary intervention to link and examine the food-lipoprotein- inflammatory physiology axis.</p><!><p>This study is an ancillary study of a randomized controlled trial on the impact of walnut consumption on vascular function in hypercholesterolemic postmenopausal women [34] after 4 weeks of 40 g/day (i.e. 1.6 servings/day; n =18) or 5 g/day (i.e. 0.2 servings/day;n =12). In a subset of individuals (40 g/day, n =15 and 5 g/day n =5) samples were probed for potential changes in plasma lipids and lipoproteins (the baseline subject characteristics are presented in the Table S1). The study was registered with ClinincalTrials.gov (NCT01235390) with the study protocol approved by the Institutional Review Board of the University of California, Davis. Subject inclusion and exclusion criteria and the feeding protocol were previously described [34]. The original study observed improvements in microvascular function with the intervention of 4 weeks of 40 g/day of walnuts compared to 5 g/day. The 5 g/day level was chosen in order to provide a "placebo" comparative group that was predicted to not improve the primary outcome of microvascular function. However, in the current subset of individuals, 5 g/day of walnut showed a lipoprotein lipidomic response when analyzed as a changed from baseline (Figure S1 and Table S2). Moreover, the low and variable number of subject in the treatment groups significantly limits the power of the analysis and the placebo-controlled approach does not allow for control of the subject effect. For those reasons the current study has been analyzed using a change from baseline approach.</p><!><p>Lipoproteins were isolated by size exclusion chromatography as described by [38] using a 10 × 300 cm Superose 6 (GE Healthcare; Uppsala Sweden). The isocratic mobile phase, 20 mM TBS + 0.1% EDTA + 0.1% BHT, was degassed with helium and run at 0.5 mL/min. A 200 μL aliquot of plasma were injected and 500 μL fractions were collected as shown in Figure S2 for: VLDL, fractions 16–18 (3 total; 8.0 to 9.0 mL); LDL, fractions 23–26 (4 total; 11.5 to 13.0 mL); HDL, fractions 31–34 (4 total; 15.5 to 17 mL).</p><!><p>Total fatty acids and alkaline stable oxylipins were isolated using a liquid-liquid extraction and quantified using GC-MS for fatty acids and ultra-performance LC-MS/MS for oxylipins. Lipids were isolated using a liquid-liquid extraction with cyclohexane/isopropanol/ammonium acetate after enrichment with a suite of deuterated and rare compounds used as analytical surrogates, [39, 40]. Fatty acids were transformed into methyl esters using methanolic transesterification. The fatty acid methyl esters were separated on 30 m × 0.25 mm, 0.25 μm DB-225 ms (Agilent Technologies; Santa Clara, CA), detected with a 5973 A mass selective detector (Agilent) using electron impact ionization and selected ion monitoring, and quantified against authentic standards. Results were corrected for the recoveries of analytical surrogates as previously described [40]. Esterified oxylipins were transformed into oxylipin free acids by base hydrolysis, and isolated by subsequent hydrophilic/lipophilic interaction solid phase extraction. Alkaline stable oxylipins were separated on a 2.1 × 150, 1.7_μm Acquity BEH column (Waters Corp, Milford MA) and detected by negative mode electrospray ionization and multi-reaction monitoring on a Sciex 4000 QTRAP [40] . Concentrations were calibrated using analytical standards as previously reported [40, 41].</p><!><p>Primary human diabetic and non-diabetic subcutaneous pre-adipocytes as well as culture media and hormones used for differentiation were purchased from Lonza (Basel, Switzerland). Both diabetic and non-diabetic cells were purchased as a single batch. Pre-adipocytes were grown in a 96 well plate format in PGM-2 Basal Medium (Lonza; Basel, Switzerland) supplemented with 10% FBS,50 μg/mL of gentamicin and 37 ng/mL of amphotericin. Confluent cells were differentiated for 14 days in a PGM-2 Basal Medium (Lonza; Basel, Switzerland) supplemented with differentiation hormone mixture (PGM™−2 SingleQuots™; Lonza; Basel, Switzerland) containing insulin, indomethacin, 3-isobutyl-1-methylxanthine and dexamethasone. All assays were performed on differentiated cells, after 6 h of treatment with 30 μg/mL of LDL (based on the protein content) from the walnut study (n =5 for each treatment group) [34] or phosphate buffered saline vehicle, in the presence or absence of 3 ng/mL TNFa [42]. Each replication in the cell culture experiment represents LDL isolated from a different subject.</p><!><p>The concentrations of IL-6, IL-8, MCP-1, PAI-1, IL-1b, leptin, adiponectin, and resistin were assessed in culture media using a Luminex multiplexing platform (ThermoFisher; Waltham, MA) with kits purchased from EMD Millipore.</p><!><p>Isolated LDL were combined with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DIL; Molecular Probes, Eugene, OR, USA) according to [43]. Generated Dil-LDL (30 μg/ml) were incorporated into culture media without FBS and applied on cells for 1 h. Next, the culture media was discarded and the amount of DIL dye in the culture dish was measured at 580 nm using Synergy MX plate reader (Biotek; Winooski, VT, USA).</p><!><p>Intracellular lipid accumulation was measured using Oil Red O staining (Lonza; Basel, Switzerland). Briefly, cells were fixed with 10% formalin for 30 min. Next, the formalin was removed and the cells were stained with 2 mg/mL Oil Red O in 60% isopropanol for 1 hour. Further excess Oil Red O was discarded and cells washed three times with distilled water. Bound Oil Red O was measured at 500 nm using Synergy MX plate reader (Biotek; Winooski, VT, USA).</p><!><p>All statistical tests were performed using JMP Pro 13.1 (JMP, SAS institute, Carry, NC). Prior to analysis outliers were removed using the robust Huber M test and missing data were imputed using multivariate normal imputation. Data imputation was performed to facilitate multivariate data analysis and did not significantly change univariate analysis results like t-tests. Both treatment groups (5 g/day and 40 g/day) were analyzed independently as change from the baseline. Treatment effects on lipoprotein composition were evaluated by t-test, multivariate ANOVA (MANOVA) and partial least square discriminant analysis (PLS-DA). The t-test was performed separately for each metabolite with the visit as a fixed effect, adjusted for subject effect. For MANOVAs, variables were auto scaled [44] and grouped by hierarchical clustering using the Ward minimal variance method. MANOVA model was built with the metabolite in each cluster as a fixed effect and subject as a random effect. MANOVA and t-test p-values cutoffs were corrected for multiple comparisons using the false discovery rate (FDR) approach of Benjamini and Hochberg [45] with q =0.1. PLA-DA was used to facilitate visualization of the treatment effects across all lipoprotein particles. PLS-DA model was built using the nonlinear iterative partial least squares algorithm with K-Fold variation method (k =7) and included 204 variables (i.e. 68 detected fatty acids and oxylipins across the HDL, LDL and VLDL). Prior to the PLS-DA the paired data for each subject at baseline and after 4 weeks of treatment were scaled such that their means were centered on zero (i.e. subject centered) to reduce the subject effect and emphasizes the treatment effect using the following equations:
baselineSubjected Centered= baseline −( baseline + final 2)
finalSubjected Centered= final −( baseline + final 2)</p><p>Low density lipoprotein-dependent effects on adipocyte adipokine production were evaluated using MANOVA. Prior to analysis, variables were clustered as above and a Tukey post-hoc test allowed multiple comparisons adjustment. Correlations between culture media adipokine levels and LDL metabolite concentrations were analyzed separately for TNFa exposed and non-exposed cells using either Pearson's or Spearman's analysis as indicated on associated figures. Data from both treatments as well as final and baseline time points were used for the analysis.</p><!><p>While 40 g/day of walnuts did not alter the lipoproteins triglycerides, cholesterol, phospholipids (Figure S2) or protein levels, it significantly and differentially altered the lipoprotein fatty acid and oxylipin compositions. These changes are shown in aggregate in Figure 2, and individually in tabular form for HDL (Table 1 and Table S3a), LDL (Table 2 and Table S3b) and VLDL (Table 3 and Table S3c). Of the fatty acids, aLA (C18:3n3), and LA (C18:2n6) were increased in all lipoproteins, while C22:5n3 was increased in both LDL and VLDL and C18:3n6 was increased in VLDL but decreased in LDL. The saturated fatty acids stearic acid (C18:0) and palmitic acid (C16:0) were elevated only in VLDL, while the monounsaturated palmitoleic acid (C16:1n7) and oleic acid (C18:1n9) were decreased only in LDL. Of the oxylipins, mid-chain alcohols from all parent fatty acids, apart from aLA, were decreased in VLDL (VLDL clusters 1, 4 and 7 in the Table 3). On the other hand, in LDL only AA and DGLA-derived mid-chain alcohols decreased (LDL cluster 3 in the Table 2). Epoxides of α-linolenic acid were increased across all lipoproteins (HDL cluster 1 in the Table 1, LDL cluster 1 in the Table 2 and VLDL cluster 3 in the Table 3), whereas walnuts increased AA and DHA derived epoxides specifically in the HDL compartment (Figure 3). It is important to mention that while walnut treatment did not affect fasting complex lipid levels, it significantly decreased postprandial cholesterol (−14%, p =0.0007) and phospholipids (−16%, p =0.009) in LDL (Data not shown).</p><!><p>Human primary adipocytes with and without TNFa stimulation, provided a model system to probe the impact of lipoprotein particle structure on inflammatory signaling. LDL isolated after 4 weeks of 40 g/day walnut consumption prevented the TNFa stimulated IL-6 and IL-8 production in human primary diabetic adipocytes (HPDA) (Figure 4A). Notably, the non-diabetic primary adipocytes did not show a TNFa-stimulated change in IL-6 and IL-8 (data not shown). HPDA cells treated with TNFa, in the presence of LDL from the baseline (week 0), increased the levels of IL-6 and IL-8 in the media (48% and 30% respectively). LDL isolated after walnut treatment counteracted this effect, lowering IL-6 and IL-8 levels to that of uninflamed cells. Walnut treatment did not affect other measured adipokines (i.e. adiponectin, resistin, leptin, IL-1b, MCP-1 and PAI-1), and neither walnut treatment nor TNFa effected LDL uptake or the adipocyte lipid accumulation (data not shown).</p><!><p>To investigate associations between anti-inflammatory effects and altered fatty acids and their metabolites in LDL, correlations between pro-inflammatory adipokines, parent fatty acids and the sum of the metabolite classes derived from those lipids were performed. This analysis allowed the visualization of changes in the correlation structure between these variables in cells with or without TNFa stimulation (Figure 4B).</p><p>In non-inflamed adipocytes, there was no correlation between the levels of IL-6, IL-8 and aLA, LA, AA and the sum of their corresponding epoxides, diols and alcohols. On the other hand, in the inflamed adipocytes (n =14), aLA-derived epoxides (EpODEs) showed negative correlations with IL-6 (r =−0.66; p =0.0097) and IL-8 (r =−0.62; p =0.018), which were stronger than with aLA at r =0.5; p =0.07 and r =−0.46; p =0.1, respectively. Moreover, no correlations with the aLA derived alcohols (HOTEs) were observed. Arachidonic acid-derived alcohols (HETEs) were positively correlated with the levels of IL-6 (r =0.54; p =0.045) and IL-8 (r =0.54; p =0.045), while AA-derived epoxides (EpETrEs) and AA itself showed no correlation with IL-6 or IL8. Similarly to AA metabolites, LA alcohols positively correlated with IL-6 (r =0.52; p =0.055) and IL-8 (r =0.54; p =0.053) but other LA derivatives showed no relationships.</p><p>To investigate the associations of LDL lipids and oxylipins with adipokine production in more detail, we generated a Sperman ρ correlation network between all measured adipokines from the diabetic adipocytes and all measured lipids and oxylipins, in the presence or absence of TNFa (Figure S3).</p><!><p>The daily consumption of walnuts has been reported to have impacts on select metabolic systems, and has been associated with diverse beneficial health outcomes including improvements in inflammatory markers and select cardiovascular outcomes (reviewed in [46]). The causal links between walnut consumption and health benefits are still poorly understood, but they have generally been associated with their high levels of the omega 3 fatty acid aLA [46]. Speculation regarding the impact of phenolic bioactives have also been made[47, 48], however the implications of in vivo metabolism on these compounds are poorly understood.</p><p>Low grade systemic inflammation is related to cardio-metabolic disorders and is characterized by increased levels of circulating pro-inflammatory cytokines like IL-6 and IL-8 (reviewed in [49, 50]). In obesity, adipocytes significantly contribute to increased pools of pro-inflammatory cytokines [50]. Moreover, lipoprotein clearance is reduced in obese individuals, increasing LDL and ox-LDL levels, which have been positively associated with the levels of adipose-derived pro inflammatory cytokines, called adipokines [51, 52]. LDL is associated with adipocyte inflammation [53–55], and the level of ox-LDL has been shown to negatively correlate with plasma concentrations of the anti-inflammatory adipokine adiponectin in humans. Moreover, the direct treatment of 3T3-L1 adipocytes with ox-LDL induces production of adipokines including resistin and visfatin [52]. Negative effect of LDL has long been associated with the LDL cholesterol load and its ability to promote foam cell formation [56], however in the current study, a modest intake of walnuts induced positive changes in LDL function without altering cholesterol loads. Here we show that LDL isolated from subjects after 4 weeks of40 g/day (i.e. 1.6 servings/day) walnut consumption reduced the TNFa-stimulated production of the pro-inflammatory cytokines IL-6 and IL-8 by human primary diabetic adipocytes in culture. Notably, the levels of IL-6 and IL-8 in the inflamed adipocytes showed strong negative associations with the LDL composition of aLA-derived epoxy fatty acids and much less strongly with their parent fatty acid, suggesting the oxylipin load to be one of the mediators of the LDL effect. The levels of IL-6 are reported to increase with hypertriglyceridemia [57] and long-term (90 days) consumption of 15 mL/day of walnut oil has been reported to reduce triglycerides and cholesterol levels in patients with Type 2 Diabetes Mellitus [58]. Our findings suggest that walnut consumption may correct the inflammatory status without correction of hyperlipidemia. Of note, in a mouse model, IL-6 levels were reported to fluctuate between fasting and postprandial state and muscle tissue was shown to be responsible for this fluctuation [59], future studies should also focus on VLDL-mediated oxylipin delivery to the skeletal muscle and their impact on cytokine production.</p><p>We have previously reported that PUFA-rich diets as well as pathological states like metabolic syndrome or statin-resistant hypertriglyceridemia change lipoprotein oxylipins and fatty acids composition [31, 32]. Walnuts are rich in LA as well as aLA. In addition, walnuts provide a substantial amount of phenolics, vitamins, minerals, and amino acids that on their own may be of benefit, however, their specific role, let alone potential interactive effects with walnut-derived fatty acids has yet to be defined [60]. Fish oil supplementation, abundant in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), causes increases in all LOX and CYP EPA- and DHA-derived oxylipins, at the expense of oxylipins derived from other PUFAs [31–33]. On the contrary, in the current study, short term walnut treatment increased the CYP-derived epoxy-fatty acids of aLA in lipoproteins, but not aLA derived mid-chain alcohols generated by LOX metabolism. Moreover, walnut treatment reduced all PUFAs mid-chain alcohols in lipoprotein which is not accompanied by decrease in corresponding epoxy-fatty acids. Importantly, walnut treatment decreased 9-HETE, a mid-chain alcohol autooxidation marker [20], suggesting that the changes in lipoprotein oxylipins caused by walnut are more than just an effect of parent PUFA enrichment. The presence of antioxidant or lipoxygenase inhibitors within the food matrix along with the lipids are therefore likely important contributors to the observed effects [61]. Consistent with this idea, phenolic compounds that are present in walnuts, like caffeic acid, have been shown to inhibit 5-lipoxygenase [62].</p><p>Walnut-induced changes in oxylipins seem to be lipoprotein specific. AA derived epoxides are uniquely enriched in HDL. HDL is involved in reverse cholesterol transport but also recirculate phospholipids and cholesterol esters [63, 64], lipid classes that are rich in oxylipins. This lipoprotein specific action of walnuts should not be surprising, considering the distinct physiological function of lipoproteins, as well as the distinct origin of their lipid content. While VLDL is secreted by the liver, HDL obtain their lipids from tissues in the periphery [63, 64]. The enrichment of AA, aLA and LA epoxides within HDL is noteworthy, as we have previously reported that the non-esterified forms were positively associated with microvascular function [34]. Moreover, in isolation, AA derived epoxides can act as vasodilators [65]. HDL has been reported to induce nitric oxide production, and promote apoptosis and anti-inflammatory effects, while limiting LDL oxidation within the vasculature to lower cardiovascular risk, with dysfunctional HDL increasing disease risk [66]. Due in part to technological limitations, there has been limited research describing the origin of oxylipins in lipoproteins, despite their apparent physiological function. The observed specific increase in the HDL AA epoxide pool may reflect changes in peripheral tissue oxylipin metabolism. While the functional arm of this study used LDL to assess walnut-mediated impacts on peripheral tissue inflammation, an exploration of the anti-inflammatory effect of HDL on periphery is also warranted.</p><!><p>This study was originally design as a "placebo"-controlled, however, due to the apparent placebo effect, low n, and high variability of the placebo group this study was analyzed as change from the baseline. The properties of the placebo group also prevented us from analyzing the walnut treatment as a dose response, with the baseline as 0 g/day, placebo as 5 g/day and the treatment as 40 g/day. This study was performed in a group of hypercholesterolemic woman and therefore extrapolation of the findings to other subject should be done with caution. Also, cell culture experiments were performed on a single batch of primary cell line and are an approximation of the in vivo condition and therefore these results should be treated as a proof of principal, demonstrating a shift in potential interactions, and not as a demonstrated physiological response.</p><p>As epoxy PUFAs were not directly added to cells, the LDL aLA-derived epoxide levels and the interference with TNFa-stimulated cytokine production is only an association and not a demonstrated physiological action. The current findings justify future studies to evaluate the direct action of aLA-derived metabolites on adipocyte inflammatory responses. Additionally, lipoproteins consist of the complex matrix of lipids, lipid mediators and additional factors like mRNA. Therefore, focusing on one component of LDL rather than the whole composition could be misleading.</p><!><p>This study demonstrates that the consumption of a modest amount of walnuts for 4 weeks differentially changed lipoprotein fatty acid and oxylipin composition. This treatment affected LDL functionality in the context of adipocyte inflammation. Specifically, LDL oxylipin concentrations, but not those of their parent fatty acids, were strongly correlated with the inhibition of a pro-inflammatory cytokines secretion by adipocytes. These findings provide a functional demonstration of a food-lipoprotein-inflammatory physiology axis positively influenced by the daily consumption of walnuts.</p>