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A high sucrose diet modifies brain oxylipins in a sex-dependent manner

Open AccessPublished:October 07, 2022DOI:https://doi.org/10.1016/j.plefa.2022.102506

      Highlights

      • Consumption of a high sucrose diet alters the brain free oxylipin profile.
      • The oxylipins changed by a high sucrose diet differ between sexes.
      • Regardless of diet, there are sex differences in the brain free oxylipin profile.

      Abstract

      Background

      Oxylipins have been implicated in many biological processes and diseases. Dysregulation of cerebral lipid homeostasis and altered lipid metabolites have been associated with the onset and progression of dementia. Although most dietary interventions have focused on modulation of dietary fats, the impact of a high sucrose diet on the brain oxylipin profile is unknown.

      Methods

      Male and female C57BL/6J mice were fed a high sucrose diet (HSD, 34%) in comparison to a control low sucrose diet (LSD, 12%) for 12 weeks beginning at 20 weeks of age. The profile of 53 free oxylipins was then measured in brain by ultra-high performance liquid chromatography tandem mass spectrometry. Serum glucose and insulin were measured enzymatically. We first assessed whether there were any effects of the diet on the brain oxylipin profile, then assessed for sex differences.

      Results

      There were no differences in fasting serum glucose between the sexes for mice fed a HSD or in fasting serum insulin levels for mice on either diet. The HSD altered the brain oxylipin profile in both sexes in distinctly different patterns: there was a reduction in three oxylipins (by 47–61%) and an increase in one oxylipin (16%) all downstream of lipoxygenase enzymes in males and a reduction in eight oxylipins (by 14–94%) mostly downstream of cyclooxygenase activity in females. 9-oxo-ODE and 6-trans-LTB4 were most influential in the separation of the oxylipin profiles by diet in male mice, whereas 5-HEPE and 12-HEPE were most influential in the separation by diet in female mice. Oxylipins 9‑hydroxy-eicosatetraenoic acid (HETE), 11-HETE, and 15-HETE were higher in the brains of females, regardless of diet.

      Conclusion

      A HSD substantially changes brain oxylipins in a distinctly sexually dimorphic manner. Results are discussed in terms of potential mechanisms and links to metabolic disease. Sex and diet effects on brain oxylipin composition may provide future targets for the management of neuroinflammatory diseases, such as dementia.

      Keywords

      1. Introduction

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      ]. While modulating dietary fats can change oxylipin levels within tissues, these changes do not necessarily occur in the same manner as their parent PUFAs within tissues [
      • Leng S.
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      Dietary LA and sex effects on oxylipin profiles in rat kidney, liver, and serum differ from their effects on PUFAs.
      ]. Brain oxylipins have been shown to be modified by changes in dietary fat [
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      The brain oxylipin profile is resistant to modulation by dietary n-6 and n-3 polyunsaturated fatty acids in male and female rats.
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      A walnut diet in combination with enriched environment improves cognitive function and affects lipid metabolites in brain and liver of aged NMRI mice.
      ]. Further, Chistyakov and colleagues reported that increasing the glucose concentration in cultures of astrocytes, an important glial cell in brain, modulated the oxylipins produced by the cells towards a pro-inflammatory state [
      • Chistyakov D.V.
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      High glucose shifts the oxylipin profiles in the astrocytes towards pro-inflammatory states.
      ]. However, to our knowledge, the effect of dietary sugars on oxylipins in the brain in vivo have not been studied and there is no published comprehensive profiling of brain oxylipins in response to a high sucrose dietary intervention. Yet, there is epidemiological data to suggest that a high sucrose diet (HSD) may be detrimental to cognitive function and lead to brain structural changes consistent with dementia [
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      A high-sucrose diet aggravates Alzheimer's disease pathology, attenuates hypothalamic leptin signaling, and impairs food-anticipatory activity in APPswe/PS1dE9 mice.
      ]. Further, we have demonstrated that a HSD alters the transcriptome of hippocampal microvessels in both male [
      • Nuthikattu S.
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      Inhibition of soluble epoxide hydrolase is protective against the multiomic effects of a high glycemic diet on brain microvascular inflammation and cognitive dysfunction.
      ] and female mice (unpublished work from our lab), implicating a role for sucrose in the multiomic regulation of brain memory centers.
      Multiple studies also indicate that there are effects of sex on the oxylipin content of plasma/serum, and of multiple tissues including kidney, liver, spleen, muscle, and brain [
      • Penner A.L.
      • Waytt V.
      • Winter T.
      • Leng S.
      • Duhamel T.A.
      • Aukema H.M.
      Oxylipin profiles and levels vary by skeletal muscle type, dietary fat and sex in young rats.
      ,
      • Gabbs M.
      • Zahradka P.
      • Taylor C.G.
      • Aukema H.M.
      Time course and sex effects of α-Linolenic acid-rich and DHA-rich supplements on human plasma oxylipins: a randomized double-blind crossover trial.
      ,
      • Leng S.
      • Winter T.
      • Aukema H.M.
      Dietary LA and sex effects on oxylipin profiles in rat kidney, liver, and serum differ from their effects on PUFAs.
      ,
      • Devassy J.G.
      • Yamaguchi T.
      • Monirujjaman M.
      • Gabbs M.
      • Ravandi A.
      • Zhou J.
      • Aukema H.M.
      Distinct effects of dietary flax compared to fish oil, soy protein compared to casein, and sex on the renal oxylipin profile in models of polycystic kidney disease.
      ,
      • Pauls S.D.
      • Ragheb M.
      • Winter T.
      • Leng S.
      • Taylor C.G.
      • Zahradka P.
      • Aukema H.M.
      Spleen oxylipin and polyunsaturated fatty acid profiles are altered by dietary source of polyunsaturated fatty acid and by sex.
      ,
      • Ferdouse A.
      • Leng S.
      • Winter T.
      • Aukema H.M.
      The brain oxylipin profile is resistant to modulation by dietary n-6 and n-3 polyunsaturated fatty acids in male and female rats.
      ,
      • Monirujjaman M.
      • Devassy J.G.
      • Yamaguchi T.
      • Sidhu N.
      • Kugita M.
      • Gabbs M.
      • Nagao S.
      • Zhou J.
      • Ravandi A.
      • Aukema H.M.
      Distinct oxylipin alterations in diverse models of cystic kidney diseases.
      ]. Our previous work has demonstrated that there is a sexually dimorphic response of the hippocampal microvascular transcriptome to the HSD [
      • Nuthikattu S.
      • Milenkovic D.
      • Norman J.E.
      • Rutledge J.
      • Villablanca A.
      The brain's microvascular response to high glycemia and to the inhibition of soluble epoxide hydrolase is sexually dimorphic.
      ]. This study is one in a series of studies investigating the effect of different chronic dietary stresses on the microvasculature of brain memory centers and the whole brain [
      • Nuthikattu S.
      • Milenkovic D.
      • Norman J.E.
      • Rutledge J.
      • Villablanca A.
      Inhibition of soluble epoxide hydrolase is protective against the multiomic effects of a high glycemic diet on brain microvascular inflammation and cognitive dysfunction.
      ,
      • Nuthikattu S.
      • Milenkovic D.
      • Rutledge J.
      • Villablanca A.
      The western diet regulates hippocampal microvascular gene expression: an integrated genomic analyses in female mice.
      ,
      • Nuthikattu S.
      • Milenkovic D.
      • Rutledge J.C.
      • Villablanca A.C.
      Lipotoxic injury differentially regulates brain microvascular gene expression in male mice.
      ,
      • Nuthikattu S.
      • Milenkovic D.
      • Rutledge J.C.
      • Villablanca A.C.
      Sex-dependent molecular mechanisms of lipotoxic injury in brain microvasculature: implications for dementia.
      ,
      • Aung H.H.
      • Altman R.
      • Nyunt T.
      • Kim J.
      • Nuthikattu S.
      • Budamagunta M.
      • Voss J.C.
      • Wilson D.
      • Rutledge J.C.
      • Villablanca A.C.
      Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways.
      ]. In the current study, we aim to assess the effects of a HSD on the brain oxylipin profile due to the paucity of data in this field, and the importance of gaining a better understanding of high glycemic stress in the brain. In addition, given the potency and diverse functions of the different oxylipins and those derived from different fatty acids, profiling of oxylipins in multiple tissues in a comprehensive manner is necessary to appreciate the overall biological effects. This essential data on brain oxylipin composition, sex, and diet effects, will be key to future studies on the function of oxylipins in the brain. We hypothesized that a HSD would modify brain oxylipins towards a neurodegenerative profile. We also anticipated that there may be sex differences in oxylipin profiles and responses to high and low sucrose diets. To test our hypotheses, in the present study we used a murine model, with males and females fed a HSD or a control, low sucrose diet (LSD), and examined the impact of the diets on the brain free oxylipin profiles as measured by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). We first looked at the comprehensive effect of the HSD on free oxylipin profiles in the brain, then addressed the effect of sex on these profiles.

      2. Methods

      2.1 Animals and diets

      Research was conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and ARRIVE 2.0 guidelines [

      The ARRIVE guidelines 2.0, ARRIVE Guidelines. (n.d.). https://arriveguidelines.org/arrive-guidelines (accessed August 29, 2022).

      ] and was approved by the Institutional Animal Care and Use Committee of the University of California, Davis (protocol number 20943, approval date April 18, 2019). Mice were housed in a temperature and humidity controlled environment with a 12 h light/dark cycle at the University of California, Davis Mouse Biology Program. Female mice were housed with up to three mice per cage; male mice were housed singly. Activity, water, and food intake were monitored by vivarium staff to ensure the wellbeing of the mice.
      Male and female C57Bl/6 J mice were purchased from Jackson Laboratories at 19-weeks of age (stock 000664). Upon receipt they were fed a standard chow diet (catalog number 0915 from Envigo Teklad Diets, Madison, WI) ad libitum and allowed to acclimate for one week prior to beginning study procedures. At 20 weeks of age, the diet was switched to one of two experimental diets provided ad libitum: a low sucrose diet (LSD 12%, catalog number TD.08485 from Envigo Teklad Diets, Madison, WI) or a high sucrose diet (HSD 34%, catalog number TD.05230, Envigo Teklad Diets, Madison, WI). The macronutrient content of the experimental diets is shown in Table 1. A more detailed composition of the diets can be found in Supplemental Table 1. The HSD and LSD differed primarily in their sucrose and corn starch content. To assess for chronic exposure to a HSD, the experimental diets were provided for 12 weeks prior to sacrifice, at which point mice were 32 weeks of age. We chose to provide the experimental diets for 12 weeks based on previous studies, which demonstrated that a Western-type diet given for up to 12 weeks can induce increased permeability in the blood-brain barrier, cognitive dysfunction, and significant changes in the expression of genes in brain microvasculature [
      • Nuthikattu S.
      • Milenkovic D.
      • Rutledge J.
      • Villablanca A.
      The western diet regulates hippocampal microvascular gene expression: an integrated genomic analyses in female mice.
      ,
      • Nuthikattu S.
      • Milenkovic D.
      • Rutledge J.C.
      • Villablanca A.C.
      Lipotoxic injury differentially regulates brain microvascular gene expression in male mice.
      ,
      • Nuthikattu S.
      • Milenkovic D.
      • Rutledge J.C.
      • Villablanca A.C.
      Sex-dependent molecular mechanisms of lipotoxic injury in brain microvasculature: implications for dementia.
      ,
      • Aung H.H.
      • Altman R.
      • Nyunt T.
      • Kim J.
      • Nuthikattu S.
      • Budamagunta M.
      • Voss J.C.
      • Wilson D.
      • Rutledge J.C.
      • Villablanca A.C.
      Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways.
      ,
      • Rutkowsky J.M.
      • Lee L.L.
      • Puchowicz M.
      • Golub M.S.
      • Befroy D.E.
      • Wilson D.W.
      • Anderson S.
      • Cline G.
      • Bini J.
      • Borkowski K.
      • Knotts T.A.
      • Rutledge J.C.
      Mouse metabolic phenotyping center imaging working group, reduced cognitive function, increased blood-brain-barrier transport and inflammatory responses, and altered brain metabolites in LDLr -/-and C57BL/6 mice fed a western diet.
      ].
      Table 1Experimental diet macronutrient content.
      DietFat (%kcal)Protein (%kcal)Carbohydrate (%kcal)Sucrose (g/kg)Cornstarch (g/kg)
      LSD1319.167.9120432.99
      HSD12.618.768.7341211.99
      There were four experimental groups of mice (n = 7 mice per experimental group): 1) male mice fed a LSD, 2) male mice fed a HSD, 3) female mice fed a LSD, and 4) female mice fed a HSD. At the completion of the dietary intervention period, mice were fasted for 8 h before being anesthetized with a combination of Ketamine and Xylazine. Blood was collected by ventricular puncture under anesthesia, then mice were euthanized. The brain was immediately harvested and preserved after euthanasia. Samples were subsequently immediately snap frozen in liquid nitrogen in an effort to preserve the lipid profiles and integrity. We estimate the total time for this process was less than 5 min. Samples remained in frozen storage (at −80 °C) for up to 23 months prior to extraction of the oxylipins in part due to institutional restrictions on research activities and laboratory access during COVID pandemic lockdown periods.

      2.2 Serum glucose and insulin analyses

      Serum was separated from whole blood by centrifugation. Serum samples were stored at −80 °C until analysis. Glucose was measured using enzymatic assays from Fisher Diagnostics (Middleton VA), and insulin was determined by electrochemiluminescence from Meso Scale Discovery (Rockville, MD) according to the manufacturer's instructions. The serum assays were performed by the UC Davis Mouse Metabolic Phenotyping Center (MMPC).

      2.3 Free oxylipin extraction from brain

      All measured oxylipins and internal standards with their abbreviations can be found in Supplemental Table 2.
      Brains (the right hemisphere of each) were homogenized in 200 µL of methanol containing 0.1% butylated hydroxytoluene and 0.1% acetic acid. The samples were spiked with 10 µL of deuterated surrogate standard solution containing 2 μM of d11–11(12)-EpETrE, d11–14,15-DiHETrE, d4–6-keto-PGF1α, d4–9-HODE, d4-LTB4, d4-PGE2, d4-TXB2, d6–20-HETE and d8–5-HETE in methanol. The samples were then precooled at −80 °C for 30 min and homogenized with a bead homogenizer (Nextadvance Bullet Blender Strom 24, Troy, NY, USA) using zirconia beads. The homogenized samples were centrifuged at 4 °C at a speed of 15,871 x g for 10 min. The supernatant was loaded onto 60 mg Waters Oasis HLB 3cc solid phase extraction (SPE) columns (Waters, Milford, MA, USA), pre-rinsed with one volume of ethyl acetate and two volumes of methanol, and pre-conditioned with two volumes of SPE buffer containing 5% methanol and 0.1% acetic acid in ultrapure Millipore water. The columns were rinsed twice with SPE buffer and subjected to 20 min of vacuum (≈15 psi). Oxylipins were eluted with 0.5 mL methanol and 1.5 mL ethyl acetate into 2 mL centrifuge tubes, dried under nitrogen and reconstituted in 100 µL methanol. Ten µL was analyzed by UPLC-MS/MS. Some samples were lost due to breakage of tubes during homogenization which resulted in analysis of brain oxylipins for n = 5 to 7 mice per group (male LSD n = 6, male HSD n = 5, female LSD n = 7, female HSD n = 5).

      2.4 Mass spectrometry analysis of oxylipins

      Oxylipins were analyzed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) using an Agilent 1290 Infinity UPLC system coupled to an Agilent 6460 Triple Quadropole mass-spectrometer (Agilent Technologies, Santa Clara, CA, USA) and equipped with an Agilent ZORBAX Eclipse Plus C18 column (2.1 × 150 mm, 1.8 μm particle size; Agilent Technologies, Santa Clara, CA, USA; Cat #959,759–902). The column temperature was maintained at 45 °C. The temperature of the auto-sampler was set at 4 °C and the sample injection volume was 10 µL. The mobile phase consisted of solvent A (water containing 0.1% acetic acid) and solvent B (Acetonitrile: methanol 80:15 containing 0.1% acetic acid). The mobile phase gradient program was a 20 min run as follows: 1) 0–2 min, 35% B, 0.25 mL/min; 2) 2–12 min, 35 to 85% B, 0.25 mL/min; 3) 12–15 min, 85% B, 0.25 mL/min; 4) 15.1–17 min, 85% to 100% B, 0.4 mL/min; 5) 17.1–19 min, 100 to 35% B, 0.4 mL/min; and 6) 19–20 min, 35% B, 0.3 mL/min. Electrospray ionization (negative mode) was used as the ion source with the experimental parameters as follows: Gas temperature, 300 °C, Gas flow, 10 L/min; Sheath gas temperature, 350 °C; Sheath gas flow, 11 mL/min; Nebulizers 35 psi; Capillary gas, 4000 V/−4000 V. Optimization parameters and parent and product ion monitoring pairs are also described in Supplemental Table 3. We have provided the raw mass spectra for the standard, blank, and a representative sample for 15-HETE in Supplemental Figure 1. Any oxylipins for which there were no discernible peaks or where there was a result of not detected for 50% or more of all groups were removed from analyses. The oxylipins that were removed from analyses for these reasons were as follows: Resolvin E1, 17-HDoHE, 17(18)-EpETE, 14(15)-EpETE, 11(12)-EpETE, 8(9)-EpETE, 14,15-DiHETE, 11,12-DiHETE, 8,9-DiHETE, 15-HEPE, 8-HEPE, LTB3, PGE3, 8,9-DiHETrE, 20-HETE, LXA4, LTC4, LTE4, 20-OH-LTB4, 20-COOH-LTB4, PGB2, and 13-oxo-ODE. For the remaining oxylipins, any non-detects were replaced by 1/5 of the minimum positive value of that variable to estimate the limit of detection. The peaks for PGD1 and PGE1 were indistinguishable, therefore they were combined and are referred to as PGD/E1. For many samples, PGD2 signals were above the standard curve; the values were included in the analyses.

      2.5 Statistical analysis

      Statistical analyses of bodyweight and serum parameters were done using Prism (GraphPad Software, San Diego, CA). Any outliers were determined by ROUT with Q = 1%. Pairwise analyses were done using t-test (with Welch's correction if variances were not equal) or Mann-Whitney test (if not normally distributed). Significance was determined at p < 0.05.
      Statistical analyses of oxylipins as well as the generation of heatmaps were done using MetaboAnalyst 5.0 [,
      • Pang Z.
      • Chong J.
      • Zhou G.
      • de Lima Morais D.A.
      • Chang L.
      • Barrette M.
      • Gauthier C.
      • Jacques P.-É.
      • Li S.
      • Xia J.
      MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights.
      ]. Partial least squares discriminate analysis (PLS-DA) and hierarchical clustering heatmaps were completed using log transformed data. All pairwise comparisons of oxylipins were done on data without transformation using the non-parametric Wilcoxon rank-sum test, since data were not normally distributed for all oxylipins. Boxplots were generated using Prism (GraphPad Software, San Diego, CA). Significance was determined at p < 0.05 based on the raw p-value, when the FDR p-value was below 0.05, this was indicated. Reported percent differences were calculated based on the medians of the groups.

      3. Results

      At the completion of the study, there were no differences in body weight, fasting serum glucose, or fasting serum insulin levels between male mice fed a LSD or HSD, or between female mice fed a LSD or HSD. However, males weighed more than females regardless of diet, and had significantly higher serum glucose levels than females on the LSD. There were no differences in fasting serum glucose between the sexes for mice fed a HSD. There were also no significant sex differences in fasting serum insulin levels for mice on either diet. These data are summarized in Supplemental Table 4.

      3.1 The effects of a HSD and LSD on brain oxylipins in male mice

      To address the effect of diet on the brain free oxylipin profile we performed two types of analyses. Specifically, we first used PLS-DA, which is a method that reduces the variables used to predict to a smaller set of predictors, and thus informs about a difference. PLS-DA uses partial least squares regression against a dummy matrix that indicates class membership of samples [
      • Cho H.-W.
      • Kim S.B.
      • Jeong M.K.
      • Park Y.
      • Miller N.G.
      • Ziegler T.R.
      • Jones D.P.
      Discovery of metabolite features for the modelling and analysis of high-resolution NMR spectra.
      ,
      • Barker M.
      • Rayens W.
      Partial least squares for discrimination.
      ]. From this analysis, the variable importance in projection (VIP) scores were calculated; these inform about the relative contribution a variable makes to the model and to the PLS-DA difference [
      • Cho H.-W.
      • Kim S.B.
      • Jeong M.K.
      • Park Y.
      • Miller N.G.
      • Ziegler T.R.
      • Jones D.P.
      Discovery of metabolite features for the modelling and analysis of high-resolution NMR spectra.
      ]. Next, we performed pairwise analyses of the individual oxylipins using the Wilcoxon rank-sum test to compare the HSD and LSD fed mice.
      For male mice, PLS-DA analysis of animals fed the HSD compared to the LSD demonstrated that the diets generated distinct brain free oxylipin profiles (Fig. 1A). The VIP scores of the top 10 oxylipins for components 1 and 2 can be found in Fig. 1B-C. The VIP scores indicated that 9-oxo-ODE and 6-trans-LTB4 were most influential in the separation of the oxylipin profiles of male mice on the two diets. Furthermore, pairwise comparisons indicated that a HSD reduced the levels of three oxylipins and increased the level of one oxylipin in males when compared to a LSD (Fig. 2). These oxylipins were all downstream of LOX metabolism of omega 6 fatty acids. Specifically, there was a change in the linoleic acid-derived oxylipin 9-oxo-ODE (61% decrease) and arachidonic acid-derived oxylipins 15-HETE (16% increase), 15-oxo-ETE (53% decrease), and 8,15-DIHETE (47% decrease).
      Fig 1
      Fig. 1PLS-DA of the brain free oxylipin profile of LSD and HSD fed male mice. (A) PLS-DA two dimensional scores plot of the brain free oxylipin profile of male mice fed a LSD (light blue) or a HSD (dark blue). (B) VIP scores and relative levels for the oxylipins with the top 10 highest VIP scores for component 1. (C) VIP scores and relative levels for the oxylipins with the top 10 highest VIP scores for component 2. Groups are indicated as follows: M HSD - male mice fed a HSD, M LSD - male mice fed a LSD. N = 5–6 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      Fig 2
      Fig. 2Diagram of oxylipins changed by a HSD in male mice. Boxplots of oxylipins which were significantly different between male mice fed a HSD (dark blue) and LSD (light blue) are shown in a diagram demonstrating the pathways producing the oxylipin from their parent fatty acid. The box extends from the 25th to the 75th percentile, with the middle line indicating the median. The whiskers extend to the minimum and maximum values, with individual values indicated by black dots. LOX: lipoxygenase enzyme; COX: cyclooxygenase enzyme; * p < 0.05. N = 5–6 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

      3.2 The effects of a HSD and LSD on brain oxylipins in female mice

      Using the same approach as we did for our analysis in male mice, PLS-DA analysis of female mice fed a HSD compared to a LSD demonstrated that the brain free oxylipin profile of female mice on the two diets was distinctly different from each other. (Fig. 3A). The VIP scores of the top 10 oxylipins for components 1 and 2 can be seen in Fig. 3B-C and indicate that 5-HEPE and 12-HEPE were most influential in the separation of the oxylipin profiles of female mice fed the two diets. Pairwise comparisons of oxylipin content demonstrated that eight oxylipins were lower in the HSD-fed group when compared to the LSD-fed group (Fig. 4). In females, the majority of the oxylipins altered by a HSD were downstream of COX metabolism of the omega 6 fatty acids. Compared to the LSD, the following oxylipins were significantly reduced by the HSD in females: 12-HEPE (93% decrease) (derived from eicosapentanoic acid), PGD/E1 (35% decrease) (derived from dihomo-gamma linolenic acid), PGD2 (31% decrease), PGE2 (38% decrease), PGF2a (36% decrease), PGJ2 (70% decrease), TXB2 (33% decrease), and LTB4 (14% decrease) (all derived from arachidonic acid).
      Fig 3
      Fig. 3PLS-DA of the brain free oxylipin profile of LSD and HSD fed female mice. (A) PLS-DA two dimensional scores plot of the brain free oxylipin profile of female mice fed a LSD (light pink) or a HSD (dark pink). (B) VIP scores and relative levels for the oxylipins with the top 10 highest VIP scores for component 1. (C) VIP scores and relative levels for the oxylipins with the top 10 highest VIP scores for component 2. Groups are indicated as follows: F HSD - female mice fed a HSD, F LSD - female mice fed a LSD. N = 5–7 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      Fig 4
      Fig. 4Diagram of oxylipins changed by a HSD in female mice. Boxplots of oxylipins which were significantly different between female mice fed a HSD (dark pink) and a LSD (light pink) are shown in a diagram demonstrating the pathways producing the oxylipin from their parent fatty acid. The box extends from the 25th to the 75th percentile, with the middle line indicating the median. The whiskers extend to the minimum and maximum values, with individual values indicated by black dots. LOX: lipoxygenase enzyme; COX: cyclooxygenase enzyme; * p < 0.05; **p < 0.01. N = 5–7 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

      3.3 Hierarchical clustering and analysis of sex effects on the brain oxylipin profiles and response to diet

      To assess the effect of sex more directly on brain oxylipins, we performed hierarchical clustering of all four study groups (male and females on the HSD and LSD). Hierarchical clustering analysis confirmed that both diet (the HSD) and sex contributed to differences in the oxylipin profile (Fig. 5). By PLS-DA, we further demonstrated that the brain free oxylipin profiles clustered separately by sex for the LSD (Fig. 6A) and the HSD (Fig. 7A). The VIP scores for the top 10 oxylipins for components 1 and 2 of the PLS-DAs of male and female mice on the LSD can be found in Fig. 6B-C. For the LSD, VIP scores indicated that 9-oxo-ODE, 9-HOTrE, and LTD4 were the most influential oxylipins in separating males and females by PLS-DA. Alternatively, for mice on the HSD, VIP scores indicated that the three most influential oxylipins in separating males and females were 16(17)-EpDPE, 12-HEPE, and 7(8)-EpDPE. The VIP scores for components 1 and 2 of the PLS-DAs of male and female mice on the HSD can be found in Fig. 7B-C.
      Fig 5
      Fig. 5Hierarchical clustering heatmap of brain free oxylipins for LSD and HSD fed male and female mice. All analyzed oxylipins are shown. Groups are indicated as follows: M HSD - male mice fed a HSD, M LSD - male mice fed a LSD, F HSD - female mice fed a HSD, F LSD - female mice fed a LSD. N = 5–7 per group.
      Fig 6
      Fig. 6PLS-DA of the brain free oxylipin profile of male and female mice fed a LSD. (A) PLS-DA two dimensional scores plot of the brain free oxylipin profile of male (light blue) and female (light pink) mice fed a LSD. (B) VIP scores and relative levels for the oxylipins with the top 10 highest VIP scores for component 1. (C) VIP scores and relative levels for the oxylipins with the top 10 highest VIP scores for component 2. Groups are indicated as follows: F LSD - female mice fed a LSD, M LSD - male mice fed a LSD. N = 6–7 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      Fig 7
      Fig. 7PLS-DA of the brain free oxylipin profile of male and female mice fed a HSD. (A) PLS-DA two dimensional scores plot of the brain free oxylipin profile of male (dark blue) and female (dark pink) mice fed a HSD. (B) VIP scores and relative levels for the oxylipins with the top 10 highest VIP scores for component 1. (C) VIP scores and relative levels for the oxylipins with the top 10 highest VIP scores for component 2. Groups are indicated as follows: F HSD - female mice fed a HSD, M HSD - male mice fed a HSD. N = 5 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      As detailed above, our data indicated that the brain free oxylipin profile in mice differed depending on whether they are fed the HSD or the LSD, and this occurred in both males and females. In addition, the oxylipins that are altered by the HSD further differed between the sexes. However, the levels of three oxylipins, 9-HETE, 11-HETE, 15-HETE, were higher in females than males, regardless of diet. The difference between males and females for these three oxylipins was more pronounced in the mice consuming the LSD, with females having median values more than twice that of males. Diagrams of the oxylipins derived from arachidonic acid which differed between males and females can be found in Fig. 8A, and those that differed between males and females derived from other PUFAs can be found in Fig. 8B.
      Fig 8
      Fig. 8Male and female differences in oxylipins. Boxplots of oxylipins which were significantly different between male and female mice fed a LSD (light blue or light pink, respectively) or HSD (dark blue or dark pink, respectively) are shown in a diagram demonstrating the pathways producing the oxylipin from (A) arachidonic acid and (B) other parent fatty acids. The box extends from the 25th to the 75th percentile, with the middle line indicating the median. The whiskers extend to the minimum and maximum values, with individual values indicated by black dots. LOX: lipoxygenase enzyme; NE: non-enzymatic; COX: cyclooxygenase enzyme; CYP : cytochrome p450 enzyme ; * p < 0.05; **p < 0.01; # FDR adjusted p < 0.05. N = 5–7 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      Fig 8
      Fig. 8Male and female differences in oxylipins. Boxplots of oxylipins which were significantly different between male and female mice fed a LSD (light blue or light pink, respectively) or HSD (dark blue or dark pink, respectively) are shown in a diagram demonstrating the pathways producing the oxylipin from (A) arachidonic acid and (B) other parent fatty acids. The box extends from the 25th to the 75th percentile, with the middle line indicating the median. The whiskers extend to the minimum and maximum values, with individual values indicated by black dots. LOX: lipoxygenase enzyme; NE: non-enzymatic; COX: cyclooxygenase enzyme; CYP : cytochrome p450 enzyme ; * p < 0.05; **p < 0.01; # FDR adjusted p < 0.05. N = 5–7 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      A comprehensive summary of the data for all detected oxylipins, and how they were modified by diet and sex, can be found in Table 2.
      Table 2Summary of Diet and Sex Effects for all analyzed brain free oxylipins.
      SexMaleFemale
      DietLSDHSDLSDHSD
      Linoleic acid derived oxylipins
      Lipoxygenase products
      9-HODE4.775 (0.275)2.603 (1.584)3.103 (1.721)1.515 (1.285)
      13-HODE7.368 (1.987)2.820 (3.717)5.172 (1.900)
      p < 0.05 compared to males on the same diet (sex effect).
      2.593 (1.720)
      9-oxo-ODE0.978 (0.208)0.385 (0.599)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      0.003 (0.162)
      p < 0.05 compared to males on the same diet (sex effect).
      0.535 (0.931)
      9,10,13-TriHOME0.940 (0.152)0.630 (0.308)0.460 (0.237)
      p < 0.05 compared to males on the same diet (sex effect).
      0.350 (0.300)
      9,12,13-TriHOME1.405 (0.464)0.929 (0.634)0.695 (0.635)0.414 (0.233)
      Cytochrome P450 products
      9(10)-EpOME0.100 (0.182)0.622 (1.325)0.165 (1.171)0.012 (0.046)
      12(13)-EpOME0.481 (2.806)0.944 (1.243)2.643 (2.747)0.388 (1.130)
      9,10-DiHOME0.377 (0.136)0.417 (0.455)0.006 (0.037)
      p < 0.05 compared to males on the same diet (sex effect).
      0.006 (0.000)
      12,13-DiHOME0.759 (0.558)0.348 (0.429)0.251 (0.329)0.269 (0.425)
      Dihomo-γ-linolenic acid derived oxylipins
      Lipoxygenase products
      15(S)-HETrE2.807 (0.801)2.462 (0.851)1.288 (0.794)
      p < 0.05 compared to males on the same diet (sex effect).
      1.678 (1.442)
      Cyclooxygenase products
      PGD/E11.898 (0.495)2.180 (0.119)1.625 (0.411)1.064 (0.285)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      ,
      p < 0.05 compared to males on the same diet (sex effect).
      Arachidonic acid derived oxylipins
      Non-enzymatic
      9-HETE91.157 (10.252)89.999 (15.108)198.919 (75.763)
      FDR adjusted p < 0.05 compared to males on the same diet (sex effect).
      160.388 (49.810)
      p < 0.05 compared to males on the same diet (sex effect).
      Lipoxygenase products
      8-HETE10.353 (3.727)11.116 (2.973)11.331 (2.051)15.661 (4.729)
      p < 0.05 compared to males on the same diet (sex effect).
      5-HETE14.459 (1.505)11.326 (4.283)12.517 (3.055)10.645 (5.160)
      11-HETE91.283 (10.369)90.098 (15.162)198.871 (76.009)
      FDR adjusted p < 0.05 compared to males on the same diet (sex effect).
      160.436 (49.949)
      p < 0.05 compared to males on the same diet (sex effect).
      12-HETE100.504 (60.291)106.693 (26.330)52.045 (26.647)52.103 (33.257)
      15-HETE104.504 (8.101)120.780 (20.092)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      260.897 (35.824)
      FDR adjusted p < 0.05 compared to males on the same diet (sex effect).
      226.901 (89.966)
      p < 0.05 compared to males on the same diet (sex effect).
      5-oxo-ETE4.113 (2.871)2.643 (3.734)4.120 (2.053)2.832 (3.849)
      12-oxo-ETE1.174 (0.373)1.845 (0.957)0.988 (0.520)1.258 (1.153)
      15-oxo-ETE5.985 (3.147)2.820 (0.513)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      7.117 (3.964)15.887 (9.614)
      p < 0.05 compared to males on the same diet (sex effect).
      5,15-DiHETE2.307 (0.494)2.757 (1.285)2.388 (1.144)1.956 (0.423)
      8,15-DiHETE58.707 (11.712)30.881 (11.588)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      33.712 (32.395)20.591 (1.756)
      LTB40.587 (0.253)0.246 (0.079)0.263 (0.045)0.227 (0.080)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      LTD40.085 (0.176)0.066 (0.117)0.001 (0.000)
      p < 0.05 compared to males on the same diet (sex effect).
      0.001 (0.000)
      6-trans-LTB40.393 (0.462)0.032 (0.051)0.004 (0.278)0.004 (0.018)
      Cyclooxygenase products
      PGD2
      PGD2 signal was above the standard curve for many samples.
      253.530 (85.033)315.903 (5.885)266.072 (65.440)182.586 (44.286)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      ,
      p < 0.05 compared to males on the same diet (sex effect).
      PGE247.609 (20.925)51.192 (6.883)46.971 (28.662)29.114 (4.261)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      ,
      p < 0.05 compared to males on the same diet (sex effect).
      PGF2a74.048 (17.837)79.351 (6.582)86.942 (15.597)55.641 (6.288)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      ,
      p < 0.05 compared to males on the same diet (sex effect).
      PGJ21.565 (0.727)2.159 (0.244)2.243 (0.894)0.676 (0.352)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      ,
      p < 0.05 compared to males on the same diet (sex effect).
      15-deoxy-PGJ20.566 (0.231)0.673 (0.268)0.744 (0.514)0.412 (0.198)
      6-keto-PGF1a70.623 (23.335)58.411 (15.504)51.995 (18.767)42.614 (11.666)
      p < 0.05 compared to males on the same diet (sex effect).
      TXB261.604 (12.347)64.275 (9.773)72.375 (20.592)48.588 (3.849)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      ,
      p < 0.05 compared to males on the same diet (sex effect).
      Cytochrome P450 products
      5(6)-EpETrE21.134 (11.172)21.893 (1.349)24.105 (10.127)30.866 (20.630)
      8(9)-EpETrE21.368 (8.369)21.365 (3.488)23.656 (7.610)21.446 (10.357)
      11(12)-EpETrE19.129 (8.212)19.314 (3.742)21.527 (7.591)19.062 (9.988)
      14(15)-EpETrE23.054 (6.692)25.247 (10.502)25.643 (7.719)16.308 (2.540)
      5,6-DiHETrE0.079 (0.086)0.003 (0.014)0.003 (0.050)0.003 (0.030)
      11,12-DiHETrE0.435 (0.171)0.277 (0.152)0.335 (0.263)0.298 (0.187)
      14,15-DiHETrE1.129 (0.161)0.943 (0.153)1.281 (0.674)0.951 (0.179)
      α-linolenic acid derived oxylipins
      Lipoxygenase products
      9-HOTrE1.696 (0.401)1.080 (1.183)0.013 (0.259)
      p < 0.05 compared to males on the same diet (sex effect).
      0.013 (0.000)
      13-HOTrE1.445 (0.266)0.880 (0.512)0.731 (0.810)0.656 (0.387)
      Eicosapentaenoic acid derived oxylipins
      Lipoxygenase products
      5-HEPE0.173 (0.240)0.065 (0.160)0.059 (0.561)0.008 (0.000)
      12-HEPE0.607 (0.453)0.469 (0.252)0.404 (0.171)0.027 (0.000)
      p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      ,
      p < 0.05 compared to males on the same diet (sex effect).
      Cyclooxygenase products
      PGD30.555 (0.301)0.447 (0.123)0.472 (0.127)0.298 (0.270)
      Cytochrome P450 products
      5,6-DiHETE8.906 (4.868)6.145 (2.040)6.647 (4.994)5.155 (0.875)
      17,18-DiHETE4.557 (2.764)1.455 (2.751)3.902 (3.718)0.455 (3.287)
      Docosahexaenoic acid derived oxylipins
      Cytochrome P450 products
      7(8)-EpDPE2.676 (4.341)1.949 (2.608)2.697 (1.755)9.486 (9.969)
      10(11)-EpDPE1.227 (1.489)1.412 (0.149)2.914 (1.761)7.826 (9.095)
      p < 0.05 compared to males on the same diet (sex effect).
      13(14)-EpDPE2.480 (0.868)3.853 (1.099)3.365 (1.183)5.285 (11.738)
      16(17)-EpDPE2.599 (1.546)0.686 (0.730)1.973 (2.208)4.486 (10.677)
      p < 0.05 compared to males on the same diet (sex effect).
      19(20)-EpDPE4.864 (1.827)4.622 (0.611)6.221 (4.129)10.164 (11.956)
      16,17-DiHDPA0.189 (0.104)0.182 (0.103)0.203 (0.113)0.154 (0.157)
      19,20-DiHDPA0.913 (0.311)0.937 (0.193)1.280 (0.422)0.976 (0.112)
      Median (interquartile range) all units: pmol/g tissue.
      d p < 0.05 compared to LSD fed mice of the same sex (diet effect).
      s p < 0.05 compared to males on the same diet (sex effect).
      s+ FDR adjusted p < 0.05 compared to males on the same diet (sex effect).
      1 PGD2 signal was above the standard curve for many samples.

      4. Discussion

      The current study addressed, for the first time and in a comprehensive manner, the effects of a HSD on the brain oxylipin profile. Oxylipins are a series of bioactive metabolites produced from the oxygenation of PUFAs, which are involved in many biological processes including inflammation, cardiovascular diseases (CVD), and stroke [
      • Gabbs M.
      • Leng S.
      • Devassy J.G.
      • Monirujjaman M.
      • Aukema H.M.
      Advances in our understanding of oxylipins derived from dietary PUFAs.
      ,
      • Tourdot B.E.
      • Ahmed I.
      • Holinstat M.
      The emerging role of oxylipins in thrombosis and diabetes.
      ,
      • Gleim S.
      • Stitham J.
      • Tang W.H.
      • Martin K.A.
      • Hwa J.
      An eicosanoid-centric view of atherothrombotic risk factors.
      ,
      • Nayeem M.A.
      Role of oxylipins in cardiovascular diseases.
      ,
      • Caligiuri S.P.B.
      • Parikh M.
      • Stamenkovic A.
      • Pierce G.N.
      • Aukema H.M.
      Dietary modulation of oxylipins in cardiovascular disease and aging.
      ,
      • Caligiuri S.P.B.
      • Aukema H.M.
      • Ravandi A.
      • Lavallée R.
      • Guzman R.
      • Pierce G.N.
      Specific plasma oxylipins increase the odds of cardiovascular and cerebrovascular events in patients with peripheral artery disease.
      ,
      • Pauls S.D.
      • Du Y.
      • Clair L.
      • Winter T.
      • Aukema H.M.
      • Taylor C.G.
      • Zahradka P.
      Impact of age, menopause, and obesity on oxylipins linked to vascular health.
      ]. For this reason, they have important implications to health and disease. The most well-known group of oxylipins are the eicosanoids, which are derived from arachidonic acid. They have been shown to play an important role within the central nervous system in a variety of functions, including in neurodegenerative diseases [
      • Tassoni D.
      • Kaur G.
      • Weisinger R.S.
      • Sinclair A.J.
      The role of eicosanoids in the brain.
      ]. Oxylipins are amenable to dietary manipulation and have been suggested to be a target in cardiovascular diseases and age-related degenerative disorders [
      • Caligiuri S.P.B.
      • Parikh M.
      • Stamenkovic A.
      • Pierce G.N.
      • Aukema H.M.
      Dietary modulation of oxylipins in cardiovascular disease and aging.
      ]. However, most studies have only examined the manipulation of dietary fats.
      By comprehensively profiling brain oxylipins after a high sucrose dietary intervention, we report for the first time a number of novel findings: 1) dietary changes other than modulation of dietary PUFA content can impact brain oxylipin profiles, 2) compared to a low sucrose diet, a high sucrose diet can modify the brain's oxylipin profile in a fundamental way, 3) sex is an important factor affecting the brain oxylipin response to a HSD, and 4) the manner in which a HSD alters the brain oxylipin profile is distinct, unique, and sexually dimorphic with no oxylipins modified in common between males and females. We discuss our findings in the context of diet, sex, potential mechanisms, and implications for cardiovascular disease and dementia.

      4.1 Effect of a high sucrose diet on the brain's oxylipin profile in males and females

      Some of the changes induced by the HSD in males could be seen as beneficial in the context of brain health. For example, there was a significant reduction in 8,15-DiHETE levels in male mice fed the HSD. 8,15-DiHETE is produced, in part, through the action of 5-LOX, an enzyme that has been implicated in neurodegenerative disorders and suggested as a treatment target for AD [
      • Czapski G.A.
      • Czubowicz K.
      • Strosznajder J.B.
      • Strosznajder R.P.
      The lipoxygenases: their regulation and implication in Alzheimer's disease.
      ]. Reductions in the levels of these oxylipins could suggest a lower activity of 5-LOX. However, 5-LOX has been found to be higher in peripheral blood mononuclear cells of AD patients compared to controls [
      • Di Francesco A.
      • Arosio B.
      • Gussago C.
      • Dainese E.
      • Mari D.
      • D'Addario C.
      • Maccarrone M.
      Involvement of 5-lipoxygenase in Alzheimer's disease: a role for DNA methylation.
      ]. Further, the amount of 15-HETE was increased by a HGD in males. 15-HETE is believed to be neuroprotective, has been shown to be important for angiogenesis and recovery after stroke, and has been found to be lower in the brains of the APP/tau mouse model of AD when compared to wild types [
      • Chen L.
      • Zhu Y.-M.
      • Li Y.-N.
      • Li P.-Y.
      • Wang D.
      • Liu Y.
      • Qu Y.-Y.
      • Zhu D.-L.
      • Zhu Y.-L.
      The 15-LO-1/15-HETE system promotes angiogenesis by upregulating VEGF in ischemic brains.
      ,
      • Wang D.
      • Liu Y.
      • Chen L.
      • Li P.
      • Qu Y.
      • Zhu Y.
      • Zhu Y.
      Key role of 15-LO/15-HETE in angiogenesis and functional recovery in later stages of post-stroke mice.
      ,
      • Xu R.
      • Wang S.
      • Li W.
      • Liu Z.
      • Tang J.
      • Tang X.
      Activation of peroxisome proliferator-activated receptor-γ by a 12/15-lipoxygenase product of arachidonic acid: a possible neuroprotective effect in the brain after experimental intracerebral hemorrhage.
      ,
      • Tajima Y.
      • Ishikawa M.
      • Maekawa K.
      • Murayama M.
      • Senoo Y.
      • Nishimaki-Mogami T.
      • Nakanishi H.
      • Ikeda K.
      • Arita M.
      • Taguchi R.
      • Okuno A.
      • Mikawa R.
      • Niida S.
      • Takikawa O.
      • Saito Y.
      Lipidomic analysis of brain tissues and plasma in a mouse model expressing mutated human amyloid precursor protein/tau for Alzheimer's disease.
      ]. In these studies, the changes in the levels of these oxylipins could be neuroprotective. However, more research is needed to better discern the functional consequences of the oxylipin changes we observed in the brain of male mice following the HSD, as inferences from prior work would suggest an overall deleterious effect of the HSD.
      In female mice, the HSD-induced changes in the brain oxylipin profile in a manner suggesting a generally detrimental consequence. Reduced levels of 5-HEPE and 12-HEPE were important in separating the effects of the HSD, and pairwise analyses found a significantly lower level of 12-HEPE, PGD2, PGE2, PGF2a, and PGJ2 with the HSD. Higher levels of plasma 12-HEPE have been associated with better performance on the Trails-B cognitive test [
      • Shinto L.
      • Lahna D.
      • Murchison C.F.
      • Dodge H.
      • Hagen K.
      • David J.
      • Kaye J.
      • Quinn J.F.
      • Wall R.
      • Silbert L.C.
      Oxidized products of Omega-6 and Omega-3 long chain fatty acids are associated with increased white matter hyperintensity and poorer executive function performance in a cohort of cognitively normal hypertensive older adults.
      ], and both 5- and 12-HEPE were found to be lower in the plasma of patients with AD [
      • Borkowski K.
      • Pedersen T.L.
      • Seyfried N.T.
      • Lah J.J.
      • Levey A.I.
      • Hales C.M.
      • Dammer E.B.
      • Blach C.
      • Louie G.
      • Kaddurah-Daouk R.
      • Newman J.W.
      Alzheimer's disease metabolomics consortium, association of plasma and CSF cytochrome P450, soluble epoxide hydrolase, and ethanolamide metabolism with Alzheimer's disease.
      ]. Multiple studies have found lower levels of some of the 2 series prostaglandins in association with AD processes. PGD2 levels were lower in the brains of the APP/tau AD mouse model when compared to wild type [
      • Tajima Y.
      • Ishikawa M.
      • Maekawa K.
      • Murayama M.
      • Senoo Y.
      • Nishimaki-Mogami T.
      • Nakanishi H.
      • Ikeda K.
      • Arita M.
      • Taguchi R.
      • Okuno A.
      • Mikawa R.
      • Niida S.
      • Takikawa O.
      • Saito Y.
      Lipidomic analysis of brain tissues and plasma in a mouse model expressing mutated human amyloid precursor protein/tau for Alzheimer's disease.
      ]. PGF2a levels were seen to be lower in the plasma of patients with AD [
      • Borkowski K.
      • Pedersen T.L.
      • Seyfried N.T.
      • Lah J.J.
      • Levey A.I.
      • Hales C.M.
      • Dammer E.B.
      • Blach C.
      • Louie G.
      • Kaddurah-Daouk R.
      • Newman J.W.
      Alzheimer's disease metabolomics consortium, association of plasma and CSF cytochrome P450, soluble epoxide hydrolase, and ethanolamide metabolism with Alzheimer's disease.
      ]. Others found that PGF2a and PGE2 were decreased in the cerebrospinal fluid of AD patients and that levels of these oxylipins were positively correlated with mini-mental state examination scores [
      • Do K.V.
      • Hjorth E.
      • Wang Y.
      • Jun B.
      • Kautzmann M.-A.I.
      • Ohshima M.
      • Eriksdotter M.
      • Schultzberg M.
      • Bazan N.G.
      Cerebrospinal fluid profile of lipid mediators in Alzheimer's disease.
      ]. Further, postmortem brain tissue from AD patients were found to have reduced capacity for PGE2 and PGF2a synthesis compared to controls [
      • Wong P.T.
      • McGeer P.L.
      • McGeer E.G.
      Decreased prostaglandin synthesis in postmortem cerebral cortex from patients with Alzheimer's disease.
      ]. A potential connection between these reduced levels of prostaglandins and AD was provided in a study by Toyomoto and colleagues, which found PGE2 and PGJ2 were able to induce secretion of nerve growth factor (NGF), from astrocytes in vitro [
      • Toyomoto M.
      • Ohta M.
      • Okumura K.
      • Yano H.
      • Matsumoto K.
      • Inoue S.
      • Hayashi K.
      • Ikeda K.
      Prostaglandins are powerful inducers of NGF and BDNF production in mouse astrocyte cultures.
      ]. Therefore, based on prior studies, the reductions in oxylipins in females consuming the HSD could be consistent with a profile indicative of a greater propensity for neurodegeneration.
      It is important to note that there were additional oxylipins altered by the HSD in both males and females for which there are few studies in the literature to elucidate their role in brain health. Although not studied in the context of brain health, some of the oxylipins altered by the HSD in our study have been implicated in CVD and metabolic disease. For example, 15-oxo-ETE which was decreased by a HSD in males, has previously been demonstrated to increase monocyte adhesion to the endothelium, an early step in the pathogenesis of atherosclerosis [
      • Ma G.
      • Pan B.
      • Ren S.
      • Guo C.
      • Guo Y.
      • Wei L.
      • Zheng L.
      • Chen B.
      15-oxoeicosatetraenoic acid mediates monocyte adhesion to endothelial cell.
      ]. 9-oxo-ODE, which was also decreased by a HSD in males, was previously shown to be higher in concentration in the plasma of individuals with metabolic syndrome than in controls, and associated with an adverse lipoprotein profile [
      • Tricò D.
      • Di Sessa A.
      • Caprio S.
      • Chalasani N.
      • Liu W.
      • Liang T.
      • Graf J.
      • Herzog R.I.
      • Johnson C.D.
      • Umano G.R.
      • Feldstein A.E.
      • Santoro N.
      Oxidized derivatives of linoleic acid in pediatric metabolic syndrome: is their pathogenic role modulated by the genetic background and the gut microbiota?.
      ]. The connections of these oxylipins to processes in cardiovascular disease and metabolic disease, would suggest that reductions in the amount of these oxylipins could be beneficial to brain health, although more research in this area is needed.

      4.2 Potential mechanisms of HSD induced changes in the brain oxylipin profile

      Our study describes changes induced by the high sucrose diet, but the question of how these changes occur remains to be elucidated, and the mechanisms could differ between males and females. The high sucrose diet, through its fructose content, could increase de novo lipogenesis, the products of which are saturated and monounsaturated fatty acids [
      • Softic S.
      • Cohen D.E.
      • Kahn C.R.
      Role of dietary fructose and hepatic de novo lipogenesis in fatty liver disease.
      ]. This could in turn lead to a change in membrane lipid composition with a reduction in the percentage of PUFAS, as has been shown to happen in the liver of ducks overfed with carbohydrates [
      • Molee W.
      • Bouillier-Oudot M.
      • Auvergne A.
      • Babilé R.
      Changes in lipid composition of hepatocyte plasma membrane induced by overfeeding in duck.
      ]. We were unable to find any publications addressing whether brain membrane lipids are altered by high sucrose intake, but in theory, a reduction in the PUFA content for release by phospholipases and subsequent production of oxylipins could also help explain why some oxylipins were reduced by the high sucrose diet. However, the sex specific responses of the oxylipin profile to the HSD would suggest that other mechanisms are also involved.
      Another potential mechanism for the changes in the brain oxylipin profile induced by the HSD is that the high sucrose intake could lead to changes in enzyme levels or activity through signaling mechanisms. To our knowledge, there have been no reports directly implicating high sucrose consumption with changes in oxylipin producing enzyme expression or activity. However, there is evidence to suggest that a high sucrose diet could cause changes in the enzymes that produce oxylipins indirectly. Mammalian target of rapamycin (mTOR) has been implicated in mediating the detrimental effects of a high sucrose diet on Alzheimer's disease pathology. In a study by Orr et al., the authors demonstrated that 3xTg-AD mice exhibited increased mTOR activity along with increased amyloid β and tau pathology after a high sucrose intake; administration of the mTOR inhibitor, rapamycin, reduced the amyloid β and tau pathology [
      • Orr M.E.
      • Salinas A.
      • Buffenstein R.
      • Oddo S.
      Mammalian target of rapamycin hyperactivity mediates the detrimental effects of a high sucrose diet on Alzheimer's disease pathology.
      ]. Others have demonstrated that mTOR activation suppresses the COX2 prostaglandin synthesis in adipose [
      • Zhang X.
      • Luo Y.
      • Wang C.
      • Ding X.
      • Yang X.
      • Wu D.
      • Silva F.
      • Yang Z.
      • Zhou Q.
      • Wang L.
      • Wang X.
      • Zhou J.
      • Boyd N.
      • Spafford M.
      • Burge M.
      • Yang X.O.
      • Liu M.
      Adipose mTORC1 suppresses prostaglandin signaling and beige adipogenesis via the CRTC2-COX-2 pathway.
      ]. This same mechanism could occur in brain and would provide a potential explanation for the reduced prostaglandin levels we see in females after consumption of the high sucrose diet.

      4.3 Sex as an important modifier of the brain oxylipin response to a HSD

      In our study, not only did the HSD diet itself modify brain oxylipins when compared to a LSD, but males and females had distinct responses to the HSD. Pairwise comparisons found that males on a HSD demonstrated changes in oxylipins downstream of LOX enzymes which are involved in the metabolism of PUFAs to hydroxy fatty acids and their metabolites (such as leukotrienes, lipoxins, resolvins, and protectins) [
      • Gabbs M.
      • Leng S.
      • Devassy J.G.
      • Monirujjaman M.
      • Aukema H.M.
      Advances in our understanding of oxylipins derived from dietary PUFAs.
      ]. On the other hand, pairwise comparisons indicated that females on a HSD had significantly reduced levels of oxylipins which were mostly downstream of COX activity. COX catalyzes the generation of prostanoids from PUFAs, including prostaglandins and thromboxanes [
      • Gabbs M.
      • Leng S.
      • Devassy J.G.
      • Monirujjaman M.
      • Aukema H.M.
      Advances in our understanding of oxylipins derived from dietary PUFAs.
      ]. The mediators produced downstream of COX and LOX consist of both pro- and anti-inflammatory components, and often depend on which PUFA is being metabolized [
      • Gabbs M.
      • Leng S.
      • Devassy J.G.
      • Monirujjaman M.
      • Aukema H.M.
      Advances in our understanding of oxylipins derived from dietary PUFAs.
      ]. Therefore, it is difficult to draw any specific conclusion about the implications of reduction of activity of one pathway or another. However, this finding does indicate that the metabolic mechanism by which a HSD impacts males and females is distinctly different. Interestingly, inhibition of both COXs and LOXs have been suggested as potential targets for AD therapeutics [
      • Czapski G.A.
      • Czubowicz K.
      • Strosznajder J.B.
      • Strosznajder R.P.
      The lipoxygenases: their regulation and implication in Alzheimer's disease.
      ,
      • Cudaback E.
      • Jorstad N.L.
      • Yang Y.
      • Montine T.J.
      • Keene C.D.
      Therapeutic implications of the prostaglandin pathway in Alzheimer's disease.
      ,
      • Chen F.
      • Ghosh A.
      • Lin J.
      • Zhang C.
      • Pan Y.
      • Thakur A.
      • Singh K.
      • Hong H.
      • Tang S.
      5-lipoxygenase pathway and its downstream cysteinyl leukotrienes as potential therapeutic targets for Alzheimer's disease.
      ,
      • Bitto A.
      • Giuliani D.
      • Pallio G.
      • Irrera N.
      • Vandini E.
      • Canalini F.
      • Zaffe D.
      • Ottani A.
      • Minutoli L.
      • Rinaldi M.
      • Guarini S.
      • Squadrito F.
      • Altavilla D.
      Effects of COX1-2/5-LOX blockade in Alzheimer transgenic 3xTg-AD mice.
      ]. Although the consequences of these differences remain to be fully elucidated, the stark sex difference in the sexually dimorphic response of brain oxylipins to a HSD highlights the importance of performing studies in both sexes. This essential data on brain oxylipin composition, and sex and diet effects, will be key to future studies on the function of oxylipins in the brain.

      4.4 Sex effects on the oxylipin profile and potential mechanisms

      We found three oxylipins for which brain unesterified levels were elevated in females when compared to males regardless of diet, namely 9-HETE, 11-HETE, and 15-HETE. 9-HETE is produced through the non-enzymatic oxidation of arachidonic acid. 11-HETE and 15-HETE have been reported to be generated from arachidonic acid by both COX and LOX enzymes [
      • Gabbs M.
      • Leng S.
      • Devassy J.G.
      • Monirujjaman M.
      • Aukema H.M.
      Advances in our understanding of oxylipins derived from dietary PUFAs.
      ,
      • Setty B.N.
      • Stuart M.J.
      • Walenga R.W.
      Formation of 11-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid in human umbilical arteries is catalyzed by cyclooxygenase.
      ,
      • Thuresson E.D.
      • Lakkides K.M.
      • Smith W.L.
      Different catalytically competent arrangements of arachidonic acid within the cyclooxygenase active site of prostaglandin endoperoxide H synthase-1 lead to the formation of different oxygenated products.
      ]. To our knowledge, there is no data on the effects of 9-HETE and 11-HETE in the context of the brain literature. 15-HETE is believed to be neuroprotective [
      • Chen L.
      • Zhu Y.-M.
      • Li Y.-N.
      • Li P.-Y.
      • Wang D.
      • Liu Y.
      • Qu Y.-Y.
      • Zhu D.-L.
      • Zhu Y.-L.
      The 15-LO-1/15-HETE system promotes angiogenesis by upregulating VEGF in ischemic brains.
      ,
      • Wang D.
      • Liu Y.
      • Chen L.
      • Li P.
      • Qu Y.
      • Zhu Y.
      • Zhu Y.
      Key role of 15-LO/15-HETE in angiogenesis and functional recovery in later stages of post-stroke mice.
      ,
      • Xu R.
      • Wang S.
      • Li W.
      • Liu Z.
      • Tang J.
      • Tang X.
      Activation of peroxisome proliferator-activated receptor-γ by a 12/15-lipoxygenase product of arachidonic acid: a possible neuroprotective effect in the brain after experimental intracerebral hemorrhage.
      ,
      • Tajima Y.
      • Ishikawa M.
      • Maekawa K.
      • Murayama M.
      • Senoo Y.
      • Nishimaki-Mogami T.
      • Nakanishi H.
      • Ikeda K.
      • Arita M.
      • Taguchi R.
      • Okuno A.
      • Mikawa R.
      • Niida S.
      • Takikawa O.
      • Saito Y.
      Lipidomic analysis of brain tissues and plasma in a mouse model expressing mutated human amyloid precursor protein/tau for Alzheimer's disease.
      ]. Although more data are needed, it seems that these findings suggest that females may have a more protective brain oxylipin profile, than males.
      Membrane fatty acid composition could contribute to observed differences in the oxylipin profile. However, the sex differences we observed indicate that this cannot be the only mechanism of observed differences. For example, we observed lower levels of the arachidonic acid derived 2 series prostaglandins in females than in males on the HSD. However, there are also multiple arachidonic acid derived oxylipins for which the oxylipin levels are higher in females than in males on the HSD, specifically 8-HETE, 9-HETE, 11-HETE, 15-HETE, and 15-oxo-ETE. This would suggest that the observed sex differences are not simply based on substrate availability. While differences in brain fatty acid composition could be a contributing factor to the sex differences in the oxylipin profiles, other factors are likely also in play.
      Another possible mechanism behind the sex differences we observed in the brain oxylipin profile is differences in the expression and/or activity of the enzymes producing oxylipins from PUFAs. Others have demonstrated sex differences of oxylipin generating enzymes in various tissues such as higher levels of COX2 in males, different cellular localization of 5-LOX leading to lower activity in males, higher expression of soluble epoxide hydrolase (sEH) in males, and differences in levels of isoforms of CYP enzymes [
      • Gerges S.H.
      • El-Kadi A.O.S.
      Sex differences in eicosanoid formation and metabolism: a possible mediator of sex discrepancies in cardiovascular diseases.
      ]. Differences have also been found in brain tissue. For instance, COX2 has been found to be higher in males after traumatic brain injury [
      • Günther M.
      • Plantman S.
      • Davidsson J.
      • Angéria M.
      • Mathiesen T.
      • Risling M.
      COX-2 regulation and TUNEL-positive cell death differ between genders in the secondary inflammatory response following experimental penetrating focal brain injury in rats.
      ]; and soluble epoxide hydrolase (sEH) has been found to be higher in male cerebral microvessels [
      • Zhang W.
      • Iliff J.J.
      • Campbell C.J.
      • Wang R.K.
      • Hurn P.D.
      • Alkayed N.J.
      Role of soluble epoxide hydrolase in the sex-specific vascular response to cerebral ischemia.
      ]. The sex differences in sEH expression have been implicated as a mechanism behind the sexual dimorphism in endothelial dysfunction after stroke, reviewed in [
      • Davis C.M.
      • Fairbanks S.L.
      • Alkayed N.J.
      Mechanism of the sex difference in endothelial dysfunction after stroke.
      ]. Others have demonstrated that testosterone and estradiol mediate differences in the expression and activity of oxylipin producing enzymes [
      • Gerges S.H.
      • El-Kadi A.O.S.
      Sex differences in eicosanoid formation and metabolism: a possible mediator of sex discrepancies in cardiovascular diseases.
      ,
      • Pace S.
      • Sautebin L.
      • Werz O.
      Sex-biased eicosanoid biology: impact for sex differences in inflammation and consequences for pharmacotherapy.
      ]. Therefore, differences in enzyme expression due to the hormonal milieu is another plausible explanation for the sex differences in oxylipin profiles.

      4.5 Study limitations and research gaps

      Our study is, to our knowledge, the first comprehensive evaluation and profiling of brain oxylipins in response to a HSD. It revealed some previously undescribed differences between the LSD and the HSD, shedding light onto the metabolic consequences of a HSD in the brain. The existing data on the consequences of oxylipin changes in brain are sparse, underscoring both the contribution of our study and the need for further research in this area. In addition, although all precautions were taken for expediency and immediate tissue preservation in our work, we cannot discount the potential for an effect of the harvesting procedure on lipid profiles and integrity.
      Further, many of the studies connecting oxylipins to dementia and brain health have limitations, including our own studies which were not coupled to functional memory assays in mice. Another challenge in interpreting results stems from the analyses of different tissues, and the relatively few studies that have examined brain. While some prior studies were conducted using serum/plasma oxylipin levels and not brain levels, the current study did not measure the serum/plasma oxylipin profile. Serum/plasma oxylipin levels may not change in the same manner as levels in brain, as it has been reported that changes in oxylipin levels in brain and plasma do not always respond the same to dietary modification [
      • Taha A.Y.
      • Hennebelle M.
      • Yang J.
      • Zamora D.
      • Rapoport S.I.
      • Hammock B.D.
      • Ramsden C.E.
      Regulation of rat plasma and cerebral cortex oxylipin concentrations with increasing levels of dietary linoleic acid.
      ]. Furthermore, studies based on in vitro models do not consider the complexity of the whole organism, and while they may inform cellular processes, they do not necessarily predict what will happen in vivo. For example, high glucose concentrations in astrocyte cultures increased many of the 2 series prostaglandins and TXB2 secretion [
      • Chistyakov D.V.
      • Goriainov S.V.
      • Astakhova A.A.
      • Sergeeva M.G.
      High glucose shifts the oxylipin profiles in the astrocytes towards pro-inflammatory states.
      ]; however, in the current in vivo study, the HSD nutritional intervention did not alter the levels of these oxylipins in the brains of males, and reduced them in females. Future studies using oxylipin profiling of brain tissue for whole organism models of dementia and cognitive decline, and disaggregating by males and females, could help elucidate this complex area of research.
      In conclusion, this study demonstrates a high sucrose diet can impact the brain free oxylipin profile quite significantly when compared to a low sucrose diet, and revealed for the first time, dramatic sex effects in the response of the brain to a high sucrose dietary stress. Our data indicates that the HSD impacts the LOX pathway in the brain of males, while primarily affecting the COX pathway in the brain of females. Based on the previous literature, it appears that the HSD may be more detrimental in females. However, certain oxylipins which were elevated in females regardless of diet may be protective, namely 9-HETE, 11-HETE, and 15-HETE. As oxylipins are an important group of fatty acid metabolites that can be modulated by diet and have been implicated in cardiovascular disease and age-related degeneration, they are gaining recognition as potential targets for dietary interventions for prevention. Findings from this study informing about specific brain oxylipins profiles, diet effects, and sex differences may help guide future studies. In addition, understanding how diet changes the oxylipin composition of the brain may provide future targets for the management of cardiovascular disease and neuroinflammation - a precursor to neurodegenerative brain diseases such as dementia.

      Funding

      This work was supported by an award from the Nora Eccles Treadwell Foundation , San Francisco, CA [ A20–0111 ], the Richard A. and Nora Eccles Harrison Endowed Chair in Diabetes Research (J.C.R.), and the Frances Lazda Endowed Chair in Women's Cardiovascular Medicine (A.C.V.).

      CRediT authorship contribution statement

      Jennifer E. Norman: Data curation, Formal analysis, Validation, Visualization, Writing – original draft, Writing – review & editing. Saivageethi Nuthikattu: Project administration, Methodology, Investigation, Data curation, Validation, Writing – review & editing. Dragan Milenkovic: Supervision, Writing – review & editing. John C. Rutledge: Conceptualization, Funding acquisition, Resources, Supervision. Amparo C. Villablanca: Conceptualization, Funding acquisition, Resources, Methodology, Supervision, Writing – original draft, Writing – review & editing.

      Declaration of Competing Interest

      The authors declare no conflicts of interest.

      Acknowledgments

      We thank Dr. Jennifer Rutkowsky, Nikita Patel, Anthony Pam, Taarini Hariharan, Nejma Wais, Ryan Vinh, and Corey Buckley for technical assistance in this project. We are grateful for the technical support and services provided to our research by the UC Davis Mouse Metabolic Phenotyping Center (funded by U24DK092993). Additionally, we thank the laboratory of Dr. Ameer Taha at UC Davis for performance of the brain oxylipin and fatty acid extractions and measurements on a recharge basis.

      Appendix. Supplementary materials

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