12-Hydroxyeicosatetraenoic acid is the only enzymatically produced HETE increased under brain ischemia.

Hydroxyeicosatetraenoic acids (HETE) are dramatically increased under brain ischemia and significantly affect post-ischemic recovery. However, the exact mechanism of HETE increase and their origin under ischemia are poorly understood. HETE might be produced de novo through lipoxygenase (LOX) -dependent synthesis with possible esterification into a lipid storage pool, or non-enzymatically through free radical oxidation of esterified arachidonic acid (20:4n6). Because HETE synthesized through LOX exhibit stereospecificity, chiral analysis allows separation of enzymatic from non-enzymatic pools. In the present study, we analyzed free HETE stereoisomers at 30 sec, 2 min, and 10 min of ischemia. Consistent with previous reports, we demonstrated a significant, gradual increase in all analyzed HETE over 10 min of brain ischemia, likely attributed to release of the esterified pool. The R/S ratio for 5-HETE, 8-HETE, and 15-HETE was not different from a racemic standard mix, indicating their non-enzymatic origin, which was in opposition to the inflamed tissue used as a positive control in our study. However, 12(S)-HETE was the predominant isoform under ischemia, indicating that ∼90 % of 12-HETE are produced enzymatically. These data demonstrate, for the first time, that 12-LOX is the major LOX isoform responsible for the enzymatic formation of the inducible HETE pool under ischemia. We also confirmed the requirement for enzyme inactivation with high-energy focused microwave irradiation (MW) for accurate HETE quantification and validated its application for chiral HETE analysis. Together, our data suggest that 12-LOX and HETE-releasing enzymes are promising targets for HETE level modulation upon brain ischemia.

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification.

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Exogenous oxygen is required for prostanoid induction under brain ischemia as evidence for a novel regulatory mechanism.

Previously, we and others reported a rapid and dramatic increase in brain prostanoids (PG), including prostaglandins, prostacyclins, and thromboxanes, under ischemia that is traditionally explained through the activation of esterified arachidonic acid (20:4n6) release by phospholipases as a substrate for cyclooxygenases (COX). However, the availability of another required COX substrate, oxygen, has not been considered in this mechanism. To address this mechanism for PG upregulation through oxygen availability, we analyzed mouse brain PG, free 20:4n6, and oxygen levels at different time points after ischemic onset using head-focused microwave irradiation (MW) to inactivate enzymes in situ before craniotomy. The oxygen half-life in the ischemic brain was 5.32 ± 0.45 s and dropped to undetectable levels within 12 s of ischemia onset, while there were no significant free 20:4n6 or PG changes at 30 s of ischemia. Furthermore, there was no significant PG increase at 2 and 10 min after ischemia onset compared to basal levels, while free 20:4n6 was increased ∼50 and ∼100 fold, respectively. However, PG increased ∼30-fold when ischemia was followed by craniotomy of nonMW tissue that provided oxygen for active enzymes. Moreover, craniotomy performed under anoxic conditions without MW did not result in PG induction, while exposure of these brains to atmospheric oxygen significantly induced PG. Our results indicate, for the first time, that oxygen availability is another important regulatory factor for PG production under ischemia. Further studies are required to investigate the physiological role of COX/PG regulation through tissue oxygen concentration.

A shotgun metagenomic analysis of the fecal microbiome in humans infected with Giardia duodenalis.

BACKGROUND: The mechanisms underlying the clinical outcome disparity during human infection with Giardia duodenalis are still unclear. In recent years, evidence has pointed to the roles of host factors as well as parasite's genetic heterogeneity as major contributing factors in the development of symptomatic human giardiasis. However, it remains contested as to how only a small fraction of individuals infected with G. duodenalis develop clinical gastrointestinal manifestations, whereas the majority of infected individuals remain asymptomatic. Here, we demonstrate that diversity in the fecal microbiome correlates with the clinical outcome of human giardiasis. METHODS: The genetic heterogeneity of G. duodenalis clinical isolates from human subjects with asymptomatic and symptomatic giardiasis was determined using a multilocus analysis approach. We also assessed the genetic proximity of G. duodenalis isolates by constructing phylogenetic trees using the maximum likelihood. Total genomic DNA (gDNA) from fecal specimens was utilized to construct DNA libraries, followed by performing paired-end sequencing using the HiSeq X platform. The Kraken2-generated, filtered FASTQ files were assigned to microbial metabolic pathways and functions using HUMAnN 3.04 and the UniRef90 diamond annotated full reference database (version 201901b). Results from HUMAnN for each sample were evaluated for differences among the biological groups using the Kruskal-Wallis non-parametric test with a post hoc Dunn test. RESULTS: We found that a total of 8/11 (72.73%) human subjects were infected with assemblage A (sub-assemblage AII) of G. duodenalis, whereas 3/11 (27.27%) human subjects in the current study were infected with assemblage B of the parasite. We also found that the parasite's genetic diversity was not associated with the clinical outcome of the infection. Further phylogenetic analysis based on the tpi and gdh loci indicated that those clinical isolates belonging to assemblage A of G. duodenalis subjects clustered compactly together in a monophyletic clade despite being isolated from human subjects with asymptomatic and symptomatic human giardiasis. Using a metagenomic shotgun sequencing approach, we observed that infected individuals with asymptomatic and symptomatic giardiasis represented distinctive microbial diversity profiles, and that both were distinguishable from the profiles of healthy volunteers. CONCLUSIONS: These findings identify a potential association between host microbiome disparity with the development of clinical disease during human giardiasis, and may provide insights into the mechanisms by which the parasite induces pathological changes in the gut. These observations may also lead to the development of novel selective therapeutic targets for preventing human enteric microbial infections.

Cyclooxygenase inhibition attenuates brain angiogenesis and independently decreases mouse survival under hypoxia.

Although cyclooxygenase (COX) role in cancer angiogenesis has been studied, little is known about its role in brain angioplasticity. In the present study, we chronically infused mice with ketorolac, a non-specific COX inhibitor that does not cross the blood-brain barrier (BBB), under normoxia or 50% isobaric hypoxia (10% O2 by volume). Ketorolac increased mortality rate under hypoxia in a dose-dependent manner. Using in vivo multiphoton microscopy, we demonstrated that chronic COX inhibition completely attenuated brain angiogenic response to hypoxia. Alterations in a number of angiogenic factors that were reported to be COX-dependent in other models were assayed at 24-hr and 10-day hypoxia. Intriguingly, hypoxia-inducible factor 1 was unaffected under COX inhibition, and vascular endothelial growth factor receptor type 2 (VEGFR2) and C-X-C chemokine receptor type 4 (CXCR4) were significantly but slightly decreased. However, a number of mitogen-activated protein kinases (MAPKs) were significantly reduced upon COX inhibition. We conclude that additional, angiogenic factor-independent mechanism might contribute to COX role in brain angioplasticity, probably including mitogenic COX effect on endothelium. Our data indicate that COX activity is critical for systemic adaptation to chronic hypoxia, and BBB COX is essential for hypoxia-induced brain angioplasticity. These data also indicate a potential risk for using COX inhibitors under hypoxia conditions in clinics. Further studies are required to elucidate a complete mechanism for brain long-term angiogenesis regulation through COX activity.

Blood-Brain Barrier Is the Major Site for a Rapid and Dramatic Prostanoid Increase upon Brain Global Ischemia.

We and others have demonstrated a rapid and dramatic increase in brain prostanoids upon decapitation-induced brain global ischemia and injury. However, the mechanism for this induction, including the cell types involved, are unknown. In the present study, we have validated and applied a pharmacological approach to inhibit prostanoid synthesis in the blood-brain barrier including endothelial cells. Our results indicate that a nonspecific cyclooxygenase (COX) inhibitor, ketorolac, does not pass the blood-brain barrier and does not enter red blood cells but penetrates endothelial cells. Ketorolac treatment did not affect basal prostanoid levels but completely prevented prostanoid induction upon global ischemia. These data indicate that basal prostanoids are synthesized in brain parenchyma cells, while inducible prostanoids are synthesized in the blood-brain barrier, most likely in endothelial cells. However, future studies with cell and COX isoform-specific gene ablation are needed to further validate this conclusion. These findings identify endothelial cells as a possible target for the development of pharmacological approaches to selectively attenuate inducible prostanoid pools without affecting basal levels under brain ischemia, trauma, surgery, and other related conditions.

Sterol Carrier Protein-2/Sterol Carrier Protein-x/Fatty Acid Binding Protein-1 Ablation Impacts Response of Brain Endocannabinoid to High-Fat Diet.

Brain endocannabinoids (EC) such as arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) primarily originate from serum arachidonic acid (ARA), whose level is regulated in part by a cytosolic ARA-binding protein, that is, liver fatty acid binding protein-1 (FABP1), not expressed in the brain. Ablation of the Fabp1 gene (LKO) increases brain AEA and 2-AG by decreasing hepatic uptake of ARA to increase serum ARA, thereby increasing ARA availability for uptake by the brain. The brain also expresses sterol carrier protein-2 (SCP-2), which is also a cytosolic ARA-binding protein. To further resolve the role of SCP-2 independent of FABP1, mice ablated in the Scp-2/Scp-x gene (DKO) were crossed with mice ablated in the Fabp1 gene (LKO) mice to generate triple knock out (TKO) mice. TKO impaired the ability of LKO to increase brain AEA and 2-AG. While a high-fat diet (HFD) alone increased brain AEA, TKO impaired this effect. Overall, these TKO-induced blocks were not attributable to altered expression of brain proteins in ARA uptake, AEA/2-AG synthesis, or AEA/2-AG degrading enzymes. Instead, TKO reduced serum levels of free ARA and/or total ARA and thereby decreased ARA availability for uptake to the brain and downstream synthesis of AEA and 2-AG therein. In summary, Scp-2/Scp-x gene ablation in Fabp1 null (LKO) mice antagonized the impact of LKO and HFD on brain ARA and, subsequently, EC levels. Thus, both FABP1 and SCP-2 participate in regulating the EC system in the brain.

Scp-2/Scp-x ablation in Fabp1 null mice differentially impacts hepatic endocannabinoid level depending on dietary fat.

Dysregulation of the hepatic endocannabinoid (EC) system and high fat diet (HFD) are associated with non-alcoholic fatty liver disease. Liver cytosol contains high levels of two novel endocannabinoid binding proteins-liver fatty acid binding protein (FABP1) and sterol carrier protein-2 (SCP-2). While Fabp1 gene ablation significantly increases hepatic levels of arachidonic acid (ARA)-containing EC and sex-dependent response to pair-fed high fat diet (HFD), the presence of SCP-2 complicates interpretation. These issues were addressed by ablating Scp-2/Scp-x in Fabp1 null mice (TKO). In control-fed mice, TKO increased hepatic levels of arachidonoylethanolamide (AEA) in both sexes. HFD impacted hepatic EC levels by decreasing AEA in TKO females and decreasing 2-arachidonoyl glycerol (2-AG) in WT of both sexes. Only TKO males on HFD had increased hepatic 2-AG levels. Hepatic ARA levels were decreased in control-fed TKO of both sexes. Changes in hepatic AEA/2-AG levels were not associated with altered amounts of hepatic proteins involved in AEA/2-AG synthesis or degradation. These findings suggested that ablation of the Scp-2/Scp-x gene in Fabp1 null mice exacerbated hepatic EC accumulation and antagonized the impact of HFD on hepatic EC levels-suggesting both proteins play important roles in regulating the hepatic EC system.

Comparative effects of high oleic acid vs high mixed saturated fatty acid obesogenic diets upon PUFA metabolism in mice.

Emerging evidence indicates that the fatty acid composition of obesogenic diets influences physiologic outcomes. There are scant data regarding how the content of non-essential fatty acids like monounsaturated fatty acids (MUFA) and saturated fatty acids (SFAs) impact the metabolism of polyunsaturated fatty acids (PUFAs). In this work, we tested the hypothesis that obesogenic diets enriched in oleic acid (OA; 18:1n-9) reduce polyunsaturated fatty acid (PUFA) levels vs an obesogenic diet enriched in SFAs. Adult male mice were fed for eight weeks either (1) a control 16% fat energy (en) diet with 5.7% en OA and 4.4% en SFA, (2) a 50% fat en diet with 33% en OA and 9.9% en SFA, or (3) a 50% en diet with a high SFA diet with 33% en SFA and 9.1% en OA. Dietary levels and intake of linoleic acid (LA; 18:2n-6) and α-linolenic acid (ALA; 18:3n-3) were constant between the experimental groups. Several peripheral organs (liver, heart, kidney, and adipose) were analyzed for lipid composition and oxylipin analysis was performed for liver and adipose. Our data demonstrate that a high OA diet reduced tissue content of LA and ALA (≥30%) in phospholipid and neutral lipid fractions, reduced the content of some LA-derived and ALA-derived oxylipins in liver and adipose, and conversely, elevated hepatic content of PGF2α. In all tissues examined, except for adipose, levels of arachidonic acid (ARA; 20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3) were either elevated or unaffected by the obesogenic diets. Our data indicate that the non-essential fatty content of obesogenic diets impacts PUFA content in peripheral tissues and influences the levels of bioactive oxylipins.

Fabp1 gene ablation inhibits high-fat diet-induced increase in brain endocannabinoids.

The endocannabinoid system shifts energy balance toward storage and fat accumulation, especially in the context of diet-induced obesity. Relatively little is known about factors outside the central nervous system that may mediate the effect of high-fat diet (HFD) on brain endocannabinoid levels. One candidate is the liver fatty acid binding protein (FABP1), a cytosolic protein highly prevalent in liver, but not detected in brain, which facilitates hepatic clearance of fatty acids. The impact of Fabp1 gene ablation (LKO) on the effect of high-fat diet (HFD) on brain and plasma endocannabinoid levels was examined and data expressed for each parameter as the ratio of high-fat diet/control diet. In male wild-type mice, HFD markedly increased brain N-acylethanolamides, but not 2-monoacylglycerols. LKO blocked these effects of HFD in male mice. In female wild-type mice, HFD slightly decreased or did not alter these endocannabinoids as compared with male wild type. LKO did not block the HFD effects in female mice. The HFD-induced increase in brain arachidonic acid-derived arachidonoylethanolamide in males correlated with increased brain-free and total arachidonic acid. The ability of LKO to block the HFD-induced increase in brain arachidonoylethanolamide correlated with reduced ability of HFD to increase brain-free and total arachidonic acid in males. In females, brain-free and total arachidonic acid levels were much less affected by either HFD or LKO in the context of HFD. These data showed that LKO markedly diminished the impact of HFD on brain endocannabinoid levels, especially in male mice.

FABP-1 gene ablation impacts brain endocannabinoid system in male mice.

Liver fatty acid-binding protein (FABP1, L-FABP) has high affinity for and enhances uptake of arachidonic acid (ARA, C20:4, n-6) which, when esterified to phospholipids, is the requisite precursor for synthesis of endocannabinoids (EC) such as arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). The brain derives most of its ARA from plasma, taking up ARA and transporting it intracellularly via cytosolic fatty acid-binding proteins (FABPs 3,5, and 7) localized within the brain. In contrast, the much more prevalent cytosolic FABP1 is not detectable in the brain but is instead highly expressed in the liver. Therefore, the possibility that FABP1 outside the central nervous system may regulate brain AEA and 2-AG was examined in wild-type (WT) and FABP1 null (LKO) male mice. LKO increased brain levels of AA-containing EC (AEA, 2-AG), correlating with increased free and total ARA in brain and serum. LKO also increased brain levels of non-ARA that contain potentiating endocannabinoids (EC*) such as oleoyl ethanolamide (OEA), PEA, 2-OG, and 2-PG. Concomitantly, LKO decreased serum total ARA-containing EC, but not non-ARA endocannabinoids. LKO did not elicit these changes in the brain EC and EC* as a result of compensatory up-regulation of brain protein levels of enzymes in EC synthesis (NAPEPLD, DAGLα) or cytosolic EC chaperone proteins (FABPs 3, 5, 7, SCP-2, HSP70), or cannabinoid receptors (CB1, TRVP1). These data show for the first time that the non-CNS fatty acid-binding protein FABP1 markedly affected brain levels of both ARA-containing endocannabinoids (AEA, 2-AG) as well as their non-ARA potentiating endocannabinoids. Fatty acid-binding protein-1 (FABP-1) is not detectable in brain but instead is highly expressed in liver. The possibility that FABP1 outside the central nervous system may regulate brain endocannabinoids arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) was examined in wild-type (WT) and FABP-1 null (LKO) male mice. LKO increased brain levels of arachidonic acid-containing endocannabinoids (AEA, 2-AG), correlating with increased free and total arachidonic acid in brain and serum. Read the Editorial Highlight for this article on page 371.

Mitochondrial Glycerol-3-Phosphate Acyltransferase-Dependent Phospholipid Synthesis Modulates Phospholipid Mass and IL-2 Production in Jurkat T Cells.

Changes in glycerophospholipid metabolism with age and disease can have a profound effect on immune cell activation and effector function. We previously demonstrated that glycerol-3-phosphate acyltransferase-1, the first and rate limiting step in de novo glycerophospholipid synthesis, plays a role in modulating murine T cell function. The resultant phenotype is characterized by decreased IL-2 production, increased propensity toward apoptosis, and altered membrane glycerophospholipid mass similar to that of an aged T cell. Since T cells in previous experiments were harvested from GPAT-1(-/-) mice, questions remained as to what extent the macro environment of the model influenced the observed cellular phenotype. Therefore, we generated and phenotypically characterized a mitochondrial glycerol-3-phosphate acyltransferase (GPAM) deficient Jurkat T cell. Furthermore, this line was used to probe possible mechanisms by which GPAT-1/GPAM regulates T cell function. We report here that many of the key dysfunctional characteristics of murine GPAT-1(-/-) T cells are recapitulated in the GPAMKD Jurkat T cell. We found striking decreased IL-2 production along with altered phospholipid mass and increased incidence of apoptosis. Since PtdOH is an indirect downstream product of GPAM, we attempted to rescue IL-2 production with PtdOH supplementation; however, this addition did not return IL-2 production to normal levels. Interestingly, we did find significantly decreased Zap-70 phosphorylation following stimulation, suggesting that GPAM deficiency may alter membrane based stimulatory signaling. These data show for the first time that GPAM deficiency results in an inherent defect in Jurkat T cell function and glycerophospholipid composition and that this defect cannot be rescued by addition of exogenous PtdOH.