A High-Phosphorus Diet Moderately Alters the Lipidome and Transcriptome in the Skeletal Muscle of Adult Mice.

A high phosphorus intake has been associated with various metabolic disorders, including chronic kidney disease, cardiovascular disease, and osteoporosis. Recent studies have demonstrated the effects of dietary phosphorus on lipid and glucose metabolism. This study investigated the impact of a high-phosphorus diet on mouse skeletal muscle lipid composition and gene transcription. Adult male mice (n = 12/group) received either a diet with an adequate (0.3%) or a high (1.2%) phosphorus concentration for 6 weeks. The lipidome analysis showed that among the 17 analyzed lipid classes, the concentrations of three classes were reduced in the high phosphorus group compared to the adequate phosphorus group. These classes were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and lysophosphatidylcholine (LPC) (p < 0.05). Out of the three hundred and twenty-three individual lipid species analyzed, forty-nine showed reduced concentrations, while three showed increased concentrations in the high phosphorus group compared to the adequate phosphorus group. The muscle transcriptome analysis identified 142 up- and 222 down-regulated transcripts in the high phosphorus group compared to the adequate phosphorus group. Gene set enrichment analysis identified that genes that were up-regulated in the high phosphorus group were linked to the gene ontology terms "mitochondria" and "Notch signaling pathway", whereas genes that were down-regulated were linked to the "PI3K-AKT pathway". Overall, the effects of the high-phosphorus diet on the muscle lipidome and transcriptome were relatively modest, but consistently indicated an impact on lipid metabolism.

Replacement of soybean oil by Hermetia illucens larvae fat in broiler diets alters the breast muscle lipidome and reduces lipid oxidation of the breast muscle during heat-processing.

Replacement of soybean oil by insect fat from Hermetia illucens (HI) has been reported to increase the proportions of saturated fatty acids (SFA) and decrease those of polyunsaturated fatty acids (PUFA) in total lipids of breast and thigh meat in broilers. Since the susceptibility of meat to oxidation is strongly dependent on its PUFA content, the present study hypothesised that replacement of soybean oil by HI larvae fat in broiler diets reduces the formation of lipid oxidation products, including oxidation products of cholesterol and phytosterols, in heat-processed breast muscle of broilers. To test this hypothesis, 100 male, 1-day-old Cobb 500 broilers were assigned to three groups and fed three different nutrient adequate diets, which varied only in the fat source (group HI-0: 0% HI larvae fat and 5% soybean oil; group HI-2.5: 2.5% HI larvae fat and 2.5% soybean oil; group HI-5.0: 5.0% HI larvae fat and 0% soybean oil), in a three-phase feeding system for 35 days. While the growth performance of the broilers was not different, the absolute and relative breast muscle weights were higher in group HI-5.0 than in group HI-0 (p < 0.05). The proportions of C12:0, C14:0, C14:1, C16:0, C16:1 and total SFA were higher and those of C18:1, C18:2 n-6, C18:3 n-3 and total PUFA were lower in breast muscle total lipids of group HI-5.0 than in groups HI-2.5 and HI-0 (p < 0.05). Lipidomic analysis of breast muscle revealed that the concentration of triacylglycerols was 46% and 53% lower in groups HI-2.5 and HI-5.0, respectively, than in group HI-0 (p < 0.05), whereas all other lipid classes detected did not differ among groups. Concentrations of thiobarbituric acid-reactive substances, 7α-hydroxycholesterol, 7ß-hydroxycholesterol and total cholesterol oxidation products in heat-processed breast muscle were lower in group HI-5.0 than in group HI-0 (p < 0.05). Concentrations of oxidation products of phytosterols in heat-processed breast muscle were generally much lower than those of cholesterol oxidation products and did not differ between the three groups of broilers. In conclusion, complete replacement of soybean oil with HI larvae fat in broiler diets strongly alters the fatty acid composition of breast muscle total lipids and reduce lipid oxidation of the breast muscle during heat-processing.

Quantification of bulk lipid species in human platelets and their thrombin-induced release.

Lipids play a central role in platelet physiology. Changes in the lipidome have already been described for basal and activated platelets. However, quantitative lipidomic data of platelet activation, including the released complex lipids, are unavailable. Here we describe an easy-to-use protocol based on flow-injection mass spectrometry for the quantitative analysis of bulk lipid species in basal and activated human platelets and their lipid release after thrombin activation. We provide lipid species concentrations of 12 healthy human donors, including cholesteryl ester (CE), ceramide (Cer), free cholesterol (FC), hexosylceramide (HexCer), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM) and triglycerides (TG). The assay exhibited good technical repeatability (CVs < 5% for major lipid species in platelets). Except for CE and TG, the inter-donor variability of the majority of lipid species concentrations in platelets was < 30% CV. Balancing of concentrations revealed the generation of LPC and loss of TG. Changes in lipid species concentrations indicate phospholipase-mediated release of arachidonic acid mainly from PC, PI, and PE but not from PS. Thrombin induced lipid release was mainly composed of FC, PS, PC, LPC, CE, and TG. The similarity of the released lipidome with that of plasma implicates that lipid release may originate from the open-canalicular system (OCS). The repository of lipid species concentrations determined with this standardized platelet release assay contribute to elucidating the physiological role of platelet lipids and provide a basis for investigating the platelet lipidome in patients with hemorrhagic or thrombotic disorders.

Dietary PUFA Preferably Modify Ethanolamine-Containing Glycerophospholipids of the Human Plasma Lipidome.

The content of polyunsaturated fatty acids (PUFA) in complex lipids essentially influences their physicochemical properties and has been linked to health and disease. To investigate the incorporation of dietary PUFA in the human plasma lipidome, we quantified glycerophospholipids (GPL), sphingolipids, and sterols using electrospray ionization coupled to tandem mass spectrometry of plasma samples obtained from a dietary intervention study. Healthy individuals received foods supplemented with different vegetable oils rich in PUFA. These included sunflower, linseed, echium, and microalgae oil as sources of linoleic acid (LA; FA 18:2 n-6), alpha-linolenic acid (ALA; FA 18:3 n-3), stearidonic acid (SDA; FA 18:4 n-3), and docosahexaenoic acid (DHA; FA 22:6 n-3). While LA and ALA did not influence the species profiles of GPL, sphingolipid, and cholesteryl ester drastically, SDA and DHA were integrated primarily in ethanolamine-containing GPL. This significantly altered phosphatidylethanolamine and plasmalogen species composition, especially those with 38-40 carbons and 6 double bonds. We speculate that diets enriched with highly unsaturated FA more efficiently alter plasma GPL acyl chain composition than those containing primarily di- and tri-unsaturated FA, most likely because of their more pronounced deviation of FA composition from typical western diets.

Sex, Food, and the Gut Microbiota: Disparate Response to Caloric Restriction Diet with Fiber Supplementation in Women and Men.

SCOPE: Dietary-based strategies are regularly explored in controlled clinical trials to provide cost-effective therapies to tackle obesity and its comorbidities. The article presents a complementary analysis based on a multivariate multi-omics approach of a caloric restriction intervention (CRD) with fiber supplementation to unveil synergic effects on body weight control, lipid metabolism, and gut microbiota. METHODS AND RESULTS: The study explores fecal bile acids (BAs) and short-chain fatty acids (SCFAs), plasma BAs, and fecal shotgun metagenomics on 80 overweight participants of a 12-week caloric restriction clinical trial (-500 kcal day-1 ) randomly allocated into fiber (10 g day-1 inulin + 10 g day-1 resistant maltodextrin) or placebo (maltodextrin) supplementation groups. The multi-omic data integration analysis uncovered the benefits of the fiber supplementation and/or the CRD (e.g., increase of Parabacteroides distasonis and fecal propionate), showing sex-specific effects on either adiposity and fasting insulin; effects thought to be linked to changes of specific gut microbiota species, functional genes, and bacterially produced metabolites like SCFAs and secondary BAs. CONCLUSIONS: This study identifies diet-microbe-host interactions helping to design personalised interventions. It also suggests that sex perspective should be considered routinely in future studies on dietary interventions efficacy. All in all, the study uncovers that the dietary intervention is more beneficial for women than men.

No Effect of Dietary Fish Oil Supplementation on the Recruitment of Brown and Brite Adipocytes in Mice or Humans under Thermoneutral Conditions.

SCOPE: Brown and brite adipocytes within the mammalian adipose organ provide non-shivering thermogenesis and thus, have an exceptional capacity to dissipate chemical energy as heat. Polyunsaturated fatty acids (PUFA) of the n3-series, abundant in fish oil, have been repeatedly demonstrated to enhance the recruitment of thermogenic capacity in these cells, consequently affecting body adiposity and glucose tolerance. These effects are scrutinized in mice housed in a thermoneutral environment and in a human dietary intervention trial. METHODS AND RESULTS: Mice are housed in a thermoneutral environment eliminating the superimposing effect of mild cold-exposure on thermogenic adipocyte recruitment. Dietary fish oil supplementation in two different inbred mouse strains neither affects body mass trajectory nor enhances the recruitment of brown and brite adipocytes, both in the presence and absence of a ß3-adrenoreceptor agonist imitating the effect of cold-exposure on adipocytes. In line with these findings, dietary fish oil supplementation of persons with overweight or obesity fails to recruit thermogenic adipocytes in subcutaneous adipose tissue. CONCLUSION: Thus, the authors' data question the hypothesized potential of n3-PUFA as modulators of adipocyte-based thermogenesis and energy balance regulation.

Specific Wheat Fractions Influence Hepatic Fat Metabolism in Diet-Induced Obese Mice.

Low whole grain consumption is a risk factor for the development of non-communicable diseases such as type 2 diabetes. Dietary fiber and phytochemicals are bioactive grain compounds, which could be involved in mediating these beneficial effects. These compounds are not equally distributed in the wheat grain, but are enriched in the bran and aleurone fractions. As little is known on physiological effects of different wheat fractions, the aim of this study was to investigate this aspect in an obesity model. For twelve weeks, C57BL/6J mice were fed high-fat diets (HFD), supplemented with one of four wheat fractions: whole grain flour, refined white flour, bran, or aleurone. The different diets did not affect body weight, however bran and aleurone decreased liver triglyceride content, and increased hepatic n-3 polyunsaturated fatty acid (PUFA) concentrations. Furthermore, lipidomics analysis revealed increased PUFA concentration in the lipid classes of phosphatidylcholine (PC), PC-ether, and phosphatidylinositol in the plasma of mice fed whole grain, bran, and aleurone supplemented diets, compared to refined white flour. Furthermore, bran, aleurone, and whole grain supplemented diets increased microbial α-diversity, but only bran and aleurone increased the cecal concentrations of short-chain fatty acids. The effects on hepatic lipid metabolism might thus at least partially be mediated by microbiota-dependent mechanisms.

A Multi-omics Approach to Unraveling the Microbiome-Mediated Effects of Arabinoxylan Oligosaccharides in Overweight Humans.

Long-term consumption of dietary fiber is generally considered beneficial for weight management and metabolic health, but the results of interventions vary greatly depending on the type of dietary fibers involved. This study provides a comprehensive evaluation of the effects of a specific dietary fiber consisting of a wheat-bran extract enriched in arabinoxylan-oligosaccharides (AXOS) in a human intervention trial. An integrated multi-omics analysis has been carried out to evaluate the effects of an intervention trial with an AXOS-enriched diet in overweight individuals with indices of metabolic syndrome. Microbiome analyses were performed by shotgun DNA sequencing in feces; in-depth metabolomics using nuclear magnetic resonance in fecal, urine, and plasma samples; and massive lipid profiling using mass spectrometry in fecal and serum/plasma samples. In addition to their bifidogenic effect, we observed that AXOS boost the proportion of Prevotella species. Metagenome analysis showed increases in the presence of bacterial genes involved in vitamin/cofactor production, glycan metabolism, and neurotransmitter biosynthesis as a result of AXOS intake. Furthermore, lipidomics analysis revealed reductions in plasma ceramide levels. Finally, we observed associations between Prevotella abundance and short-chain fatty acids (SCFAs) and succinate concentration in feces and identified a potential protective role of Eubacterium rectale against metabolic disease given that its abundance was positively associated with plasma phosphatidylcholine levels, thus hypothetically reducing bioavailability of choline for methylamine biosynthesis. The metagenomics, lipidomics, and metabolomics data integration indicates that sustained consumption of AXOS orchestrates a wide variety of changes in the gut microbiome and the host metabolism that collectively would impact on glucose homeostasis. (This study has been registered at ClinicalTrials.gov under identifier NCT02215343)IMPORTANCE The use of dietary fiber food supplementation as a strategy to reduce the burden of diet-related diseases is a matter of study given its cost-effectiveness and the positive results demonstrated in clinical trials. This multi-omics assessment, on different biological samples of overweight subjects with signs of metabolic syndrome, sheds light on the early and less evident effects of short-term AXOS intake on intestinal microbiota and host metabolism. We observed a deep influence of AXOS on gut microbiota beyond their recognized bifidogenic effect by boosting concomitantly a wide diversity of butyrate producers and Prevotella copri, a microbial species abundant in non-Westernized populations with traditional lifestyle and diets enriched in fresh unprocessed foods. A comprehensive evaluation of hundreds of metabolites unveiled new benefits of the AXOS intake, such as reducing the plasma ceramide levels. Globally, we observed that multiple effects of AXOS consumption seem to converge in reversing the glucose homeostasis impairment.

The gut microbiota drives the impact of bile acids and fat source in diet on mouse metabolism.

BACKGROUND: As the gut microbiota contributes to metabolic health, it is important to determine specific diet-microbiota interactions that influence host metabolism. Bile acids and dietary fat source can alter phenotypes of diet-induced obesity, but the interplay with intestinal microorganisms is unclear. Here, we investigated metabolic consequences of diets enriched in primary bile acids with or without addition of lard or palm oil, and studied gut microbiota structure and functions in mice. RESULTS: In combination with bile acids, dietary lard fed to male C57BL/6N mice for a period of 8 weeks enhanced fat mass accumulation in colonized, but not in germ-free mice when compared to palm oil. This was associated with impaired glucose tolerance, lower fasting insulin levels, lower counts of enteroendocrine cells, fatty liver, and elevated amounts of hepatic triglycerides, cholesteryl esters, and monounsaturated fatty acids. Lard- and bile acid-fed mice were characterized by shifts in dominant gut bacterial communities, including decreased relative abundances of Lachnospiraceae and increased occurrence of Desulfovibrionaceae and the species Clostridium lactatifermentans and Flintibacter butyricus. Metatranscriptomic analysis revealed shifts in microbial functions, including lipid and amino acid metabolism. CONCLUSIONS: Caution is required when interpreting data from diet-induced obesity models due to varying effects of dietary fat source. Detrimental metabolic consequences of a diet enriched with lard and primary bile acids were dependent on microbial colonization of the host and were linked to hepatic lipid rearrangements and to alterations of dominant bacterial communities in the cecum.

Analysis of hepatic transcript profile and plasma lipid profile in early lactating dairy cows fed grape seed and grape marc meal extract.

BACKGROUND: It was recently reported that dairy cows fed a polyphenol-rich grape seed and grape marc meal extract (GSGME) during the transition period had an increased milk yield, but the underlying reasons remained unclear. As polyphenols exert a broad spectrum of metabolic effects, we hypothesized that feeding of GSGME influences metabolic pathways in the liver which could account for the positive effects of GSGME in dairy cows. In order to identify these pathways, we performed genome-wide transcript profiling in the liver and lipid profiling in plasma of dairy cows fed GSGME during the transition period at 1 week postpartum. RESULTS: Transcriptomic analysis of the liver revealed 207 differentially expressed transcripts, from which 156 were up- and 51 were down-regulated, between cows fed GSGME and control cows. Gene set enrichment analysis of the 155 up-regulated mRNAs showed that the most enriched gene ontology (GO) biological process terms were dealing with cell cycle regulation and the most enriched Kyoto Encyclopedia of Genes and Genomes pathways were p53 signaling and cell cycle. Functional analysis of the 43 down-regulated mRNAs revealed that a great part of these genes are involved in endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) and inflammatory processes. Accordingly, protein folding, response to unfolded protein, unfolded protein binding, chemokine activity and heat shock protein binding were identified as one of the most enriched GO biological process and molecular function terms assigned to the down-regulated genes. In line with the transcriptomics data the plasma concentrations of the acute phase proteins serum amyloid A (SAA) and haptoglobin were reduced in cows fed GSGME compared to control cows. Lipidomic analysis of plasma revealed no differences in the concentrations of individual species of major and minor lipid classes between cows fed GSGME and control cows. CONCLUSIONS: Analysis of hepatic transcript profile in cows fed GSGME during the transition period at 1 week postpartum indicates that polyphenol-rich feed components are able to inhibit ER stress-induced UPR and inflammatory processes, both of which are considered to contribute to liver-associated diseases and to impair milk performance in dairy cows, in the liver of dairy cows during early lactation.

Bile Acid Metabolome after an Oral Lipid Tolerance Test by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

CONTEXT: Besides their role in intestinal resorption of lipids, bile acids are regarded as endocrine and metabolic signaling molecules. The detailed profile of bile acid species in peripheral blood after an oral lipid tolerance test (OLTT) is unknown. OBJECTIVE: We quantified the regulation of 18 bile acids after OLTT in healthy individuals. MATERIAL AND METHODS: 100 volunteers were characterized by anthropometric and laboratory parameters and underwent OLTT. Venous blood was drawn in the fasted state (0 h) and at 2h, 4h, and 6 h after OLTT. Serum concentrations of 18 bile acids were measured by LC-MS/MS. RESULTS: All of the 6 taurine-conjugated bile acids (TUDCA, THDCA, TCA, TCDCA, TDCA, TLCA) and all of the 6 glycine-conjugated bile acids (GUDCA, GHDCA, GCA, GCDCA, GDCA, GLCA) rose significantly at 2h and remained elevated during OLTT. Of the primary bile acids, CA remained unchanged, whereas CDCA significantly decreased at 4h. Of the secondary bile acids, DCA, UDCA and HDCA were not altered, whereas LCA decreased. There was a significant positive correlation between the intestinal feed-back regulator of bile acid synthesis FGF-19 and bile acids. This correlation seems to depend on all of the six taurine-conjugated bile acids and on GCA, GDCA, and GCDCA. Females and users of hormonal contraception displayed higher levels of taurine-conjugated bile acids. CONCLUSIONS: The novelty of the study is based on the identification of single bile acids during OLTT. LC-MS/MS-based quantification of bile acids in serum provides a reliable tool for future investigation of endocrine and metabolic effects of bile acids.

Effect of high versus low doses of fat and vitamin A dietary supplementation on fatty acid composition of phospholipids in mice.

Dietary fat and vitamin A provide important precursors for potent bioactive ligands of nuclear hormone receptors, which regulate various enzymes involved in lipid homeostasis, metabolism and inflammation. We determined the effects of dietary fat and dietary vitamin A on hepatic expression of two fatty acid metabolizing enzymes, elongase 6 (ELOVL6) and stearoyl-coenzyme A desaturase 1 (SCD1) and the concentration of saturated fatty acids (SAFA) and monounsaturated fatty acid (MUFA) of phospholipids in serum and liver. Mice (n = 6) were fed 4 weeks with diets containing 2, 5 and 25 % of fat or vitamin A (0, 2,500 and 326,500 RE/kg as retinyl palmitate). MUFAs and SAFAs were measured using GC and ESI-MS/MS. Hepatic expression of metabolizing enzymes was determined using QRT-PCR. ELOVL6 was significantly down-regulated in response to a high-fat diet (p < 0.001) and significantly up-regulated in response to low-fat diet (p < 0.05). SCD1 expression was significantly lower in high- versus low-fat diet (p < 0.05). The vitamin A content in the diet did not influence the hepatic expression of both enzymes. In plasma, the amounts of MUFAs bound to phospholipids significantly decreased in response to a high-fat diet and increased after a low-fat diet. This tendency was also observed in the liver for various phospholipids sub-classes. In summary, this study shows that fat content in the diet has a stronger impact than the content of vitamin A on hepatic gene expression of SCD1 and ELOVL6 and thereby on MUFA and SAFA concentrations in liver and plasma.

Sphingomyelin and phosphatidylcholine contrarily affect the induction of apoptosis in intestinal epithelial cells.

SCOPE: The major alimentary sources for the plasma membrane lipid sphingomyelin (SM) are dairy products, eggs, and meat. We recently reported that the SM metabolite ceramide induces cathepsin D mediated apoptosis in murine intestinal epithelial cells (IECs) and increases inflammation in acute colitis. We investigated the impact of SM and phosphatidylcholine on apoptosis in human IECs and point out BH3-interacting death agonist (BID) as link between cathepsin D and apoptosis. METHODS AND RESULTS: HT-29 and isolated human IECs were stimulated with SM or phosphatidylcholine. SM treatment resulted in increased apoptosis. Phosphatidylcholine showed contrary effects. Western revealed higher amounts of cathepsin D and BID activation upon lipid stimulation. Western blotting revealed BID activation through SM in both an induced and a spontaneous mouse model of colitis. CONCLUSION: Dietary phospholipids may induce or abolish apoptosis in IECs and seem to play a role in the pathogenesis of inflammatory bowel diseases. This nutritional factor might be considered when evaluating the pathogenesis of inflammatory bowel diseases. Effects of SMase- and SM treatment on inflammation in dextran sulfate sodium induced animal models of colitis and in vitro experiments are discussed as controversial. Variable sources of SM, feeding techniques, and mouse strains might play a role.

Effects of lycopene on the initial state of atherosclerosis in New Zealand White (NZW) rabbits.

BACKGROUND: Lycopene is the main carotenoid in tomatoes, where it is found in high concentrations. Strong epidemiological evidence suggests that lycopene may provide protection against cardiovascular diseases. We therefore studied the effects of lycopene on diet-induced increase in serum lipid levels and the initiation of atherosclerosis in New Zealand White (NZW) rabbits. METHODOLOGY/PRINCIPAL FINDINGS: The animals, divided into four groups of 9 animals each, were fed either a standard diet, a high-cholesterol diet containing 0.5% cholesterol, a high-cholesterol diet containing placebo beadlets, or a high-cholesterol diet plus 5 mg/kg body weight/day of lycopene (in the form of lycopene beadlets), for a period of 4 weeks. We found significantly elevated lycopene plasma levels in the animal group treated with lycopene beadlets. Compared to the high-cholesterol and the placebo group, this was associated with a significant reduction of 50% in total cholesterol and LDL cholesterol serum levels in the lycopene group. The amount of cholesteryl ester in the aorta was significantly decreased by lycopene. However, we did not observe a significant decrease in the extent of aortic surface lipid accumulation in the lycopene group. In addition, no differences in the intima-media thickness among groups were observed. Endothelial-dependent and endothelial-independent vasodilation in isolated rabbit aortic and carotid rings did not differ among any of the animal groups. CONCLUSIONS: Lycopene supplementation for 4 weeks increased lycopene plasma levels in the animals. Although we found strongly reduced total and LDL cholesterol serum levels as well as significantly lower amounts of cholesteryl ester in the aortae in the lycopene-treated group, no significant differences in initial lesions in the aortae were detected.

The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p.

The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Ta-king advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings.