BACKGROUND:Colorectal cancer (CRC) exhibits increased levels of arachidonic acid-derived pro-inflammatory derivatives indicating an uptake of dietary polyunsaturated fatty acids (PUFAs). OBJECTIVE:We aimed to investigate uptake of extrinsic fatty acids (FAs) in tumours and their relevance for CRC lipid metabolism and progression. DESIGN:Total FAs were quantified using gas chromatography-mass spectrometry in non-diseased mucosa and tumour tissue from patients with CRC of a discovery cohort (n=152), validated in an independent cohort (n=28) and associated with clinical, genomic and microbiome data. The genetic mouse tumour model Apc1638N was used to track the flux of stable isotope-labelled FAs in tumours from the intestinal lumen. The relationship between FA uptake and tumour progression was investigated in 2D and 3D cell models. RESULTS:Extrinsic long chain PUFAs, including arachidonic acid, accumulate in CRC, particularly in right-sided tumours, and in tumours of Apc1638N mice. The CRC-specific FA profiles were independent of sex, molecular subtypes, early-disease or late-disease onset. The absorption of FAs from the intestinal lumen in tumours was confirmed in specific pathogen-free Apc1638N mice. In the absence of the microbiome, in germ-free Apc1638N mice, fewer tumours were developed, and survival was increased. Inhibition of FA import or β-oxidation reduces cancer cell proliferation. CONCLUSION:Extrinsic FAs accumulate in CRC, verifying a central role of arachidonic acid-derived inflammatory mediators, but also suggesting a relevance of dietary FAs for cancer cell proliferation. It will be intriguing to explore to what extent targeting this flux pathway together with the interrelated microbiome opens new therapeutic avenues for CRC in humans.

The gut microbiota influences host metabolism, but the mechanisms of lipid uptake from food remain mysterious. Here we used stable isotope-labelled tracers in gnotobiotic mouse models, which revealed that host uptake of dietary lipids depends on microbial colonization. Systemic lipid metabolism modelling predicted that the gut microbiota restricts intestinal lipid absorption, and labelled lipid administration verified that the gut contents of microbiota-colonized mice contained up to 12-fold more lipids than those of germ-free animals. A combination of lipidomics and proteomics showed that gut microbes trigger Myd88 signalling, leading to a downregulation of hepatic Cyp7b1 activity and increased taurocholate production. Taurocholate stimulates phospholipase A1 activity in bile, causing the degradation of phosphatidylcholine that is essential for luminal micelle formation and lipid uptake. A diverse microbiome was associated with lower phosphatidylcholine content. This previously unrecognized host-gut microbiota interplay via enzymes in bile could provide future targets to modulate dietary lipid absorption.

Endoplasmic reticulum unfolded protein responses contribute to cancer development, with activating transcription factor 6 (ATF6) involved in microbiota-dependent tumorigenesis. Here we show the clinical relevance of ATF6 in individuals with early-onset and late colorectal cancer, and link ATF6 signalling to changes in lipid metabolism and intestinal microbiota. Transcriptional analysis in intestinal epithelial cells of ATF6 transgenic mice (nATF6IEC) identifies bacteria-specific changes in cellular metabolism enriched for fatty acid biosynthesis. Untargeted metabolomics and isotype labelling confirm ATF6-related enrichment of long-chain fatty acids in colonic tissue of humans, mice and organoids. FASN inhibition and microbiota transfer in germ-free nATF6IEC mice confirm the causal involvement of ATF6-induced lipid alterations in tumorigenesis. The selective expansion of tumour-relevant microbial taxa, including Desulfovibrio fairfieldensis, is mechanistically linked to long-chain fatty acid exposure using bioorthogonal non-canonical amino acid tagging, and growth analysis of Desulfovibrio isolates. We postulate chronic ATF6 signalling to select for tumour-promoting microbiota by altering lipid metabolism.

Ferroptosis has recently emerged as a promising strategy to combat therapy-resistant cancers. As lipid peroxidation is a key trigger of ferroptotic cell death, enhancing cancer cell susceptibility through the supply of highly peroxidisable fatty acids represents a novel therapeutic approach. Conjugated linolenic acids (CLnAs) fulfill this requirement, exhibiting a peroxidation propagation rate eight times higher than their non-conjugated counterpart, α-linolenic acid. This study evaluates jacaric acid (JA), a plant-derived CLnA, as a ferroptotic inducer, both as a monotherapy and in combination with RAS-selective lethal 3 (RSL3), a canonical ferroptosis inducer, in 2D and 3D breast cancer cell models. JA treatment significantly reduced cell viability across all models, primarily through lipid peroxidation driven by JA incorporation into cellular lipids rather than alterations in anti-ferroptotic gene expression. Moreover, JA synergistically enhanced RSL3 cytotoxicity under 2D and several 3D conditions. Similar effects were observed with punicic acid, another plant-derived CLnA isomer. Our study exploits a common feature of cancer metabolism, increased fatty acid uptake, to turn it into a vulnerability. The incorporation of JA into breast cancer cells creates a highly peroxidisable environment that increases cancer cell sensitivity to RSL3, potentially reducing required doses and minimising side effects.

Increasing energy expenditure in brown adipose (BAT) tissue by cold-induced lipolysis is discussed as a potential strategy to counteract imbalanced lipid homeostasis caused through unhealthy lifestyle and cardiometabolic disease. Yet, it is largely unclear how liberated fatty acids (FA) are metabolized. We investigated the liver and BAT lipidome of mice housed for 1 week at thermoneutrality, 23 degrees C and 4 degrees C using quantitative mass spectrometry-based lipidomics. Housing at temperatures below thermoneutrality triggered the generation of phosphatidylethanolamine (PE) in both tissues. Particularly, the concentrations of PE containing polyunsaturated fatty acids (PUFA) in their acyl chains like PE 18:0_20:4 were increased at cold. Investigation of the plasma's FA profile using gas chromatography coupled to mass spectrometry revealed a negative correlation of PUFA with unsaturated PE in liver and BAT indicating a flux of FA from the circulation into these tissues. Betaadrenergic stimulation elevated intracellular levels of PE 38:4 and PE 40:6 in beige wildtype adipocytes, but not in adipose triglyceride lipase (ATGL)-deficient cells. These results imply an induction of PE synthesis in liver, BAT and thermogenic adipocytes after activation of the beta-adrenergic signaling cascade.

Microbes, immune cells, and enterocytes interact to regulate the intake of lipids in the mouse gut.

Ferroptosis is a cell death pathway that can be promoted by peroxidizable polyunsaturated fatty acids in cancer cells. Here, we investigated the mechanisms underlying the toxicity of punicic acid (PunA), an isomer of conjugated linolenic acids (CLnAs) bearing three conjugated double bonds highly prone to peroxidation, on prostate cancer (PCa) cells. PunA induced ferroptosis in PCa cells and triggered massive lipidome remodeling, more strongly in PC3 androgen-negative cells than in androgen-positive cells. The greater sensitivity of androgen-negative cells to PunA was associated with lower expression of glutathione peroxidase 4 (GPX4). We then identified the phospholipase PLA2G7 as a PunA-induced ferroptosis suppressor in PCa cells. Overexpressing PLA2G7 decreased lipid peroxidation levels, suggesting that PLA2G7 hydrolyzes hydroperoxide-containing phospholipids, thus preventing ferroptosis. Importantly, overexpressing both PLA2G7 and GPX4 strongly prevented PunA-induced ferroptosis in androgen-negative PCa cells. This study shows that PLA2G7 acts complementary to GPX4 to protect PCa cells from CLnA-induced ferroptosis.

Interest in bile acids (BAs) is growing due to their emerging role as signaling molecules and their association with various diseases such as colon cancer and metabolic syndrome. Analyzing BAs requires chromatographic separation of isomers, often with long run times, which hinders BA analysis in large studies. Here, we present a high-throughput method based on liquid chromatography-tandem mass spectrometry to quantify BAs in mouse samples. After acidic protein precipitation in the presence of a comprehensive mixture of stable isotope-labeled internal standards (SIL-ISs), BAs are separated on a biphenyl column by gradient elution at basic pH. Quantification is performed using a six-point calibration curve. Except for the separation of β- and ω-muricholic acid (MCA) species, a rapid separation of 27 BA species was achieved in a run time of 6.5 min. Plasma quality controls (QCs) were used to evaluate intra- and inter-day precision. The CV was less than 10

Lipid composition is conserved within sub-cellular compartments to maintain cell function. Lipidomic analyses of liver, muscle, white and brown adipose tissue (BAT) mitochondria revealed substantial differences in their glycerophospholipid (GPL) and free cholesterol (FC) contents. The GPL to FC ratio was 50-fold higher in brown than white adipose tissue mitochondria. Their purity was verified by comparison of proteomes with ER and mitochondria-associated membranes. A lipid signature containing PC and FC, calculated from the lipidomic profiles, allowed differentiation of mitochondria from BAT of mice housed at different temperatures. Elevating FC in BAT mitochondria prevented uncoupling protein (UCP) 1 function, whereas increasing GPL boosted it. Similarly,STARD3overexpression facilitating mitochondrial FC import inhibited UCP1 function in primary brown adipocytes, whereas a knockdown promoted it. We conclude that the mitochondrial GPL/FC ratio is key for BAT function and propose that targeting it might be a promising strategy to promote UCP1 activity.

Cold-induced lipolysis is widely studied as a potential therapeutic strategy to combat metabolic disease, but its effect on lipid homeostasis in humans remains largely unclear. Blood plasma comprises an enormous repertoire in lipids allowing insights into whole body lipid homeostasis. So far, reported results originate from studies carried out with small numbers of male participants. Here, the blood plasma's lipidome of 78 male and 93 female volunteers, who were exposed to cold below the shivering threshold for 2 h, was quantified by comprehensive lipidomics using high-resolution mass spectrometry. Short-term cold exposure increased the concentrations in 147 of 177 quantified circulating lipids and the response of the plasma's lipidome was sex-specific. In particular, the amounts of generated glycerophospholipid and sphingolipid species differed between the sexes. In women, the BMI could be related with the lipidome's response. A logistic regression model predicted with high sensitivity and specificity whether plasma samples were from male or female subjects based on the cold-induced response of phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) species. In summary, cold exposure promotes lipid synthesis by supplying fatty acids generated after lipolysis for all lipid classes. The plasma lipidome, i.e. PC, LPC and SM, shows a sex-specific response, indicating a different regulation of its metabolism in men and women. This supports the need for sex-specific research and avoidance of sex bias in clinical trials.

There is general consent that with decreasing bone mineral density the amount of marrow adipose tissue increases. While image-based techniques, claim an increase in saturated fatty acids responsible for this effect, this study shows an increase in both saturated and unsaturated fatty acids in the bone marrow. Using fatty acid methyl ester gas chromatography-mass spectrometry, characteristic fatty acid patterns for patients with normal BMD (N = 9), osteopenia (N = 12), and osteoporosis (N = 9) have been identified, which differ between plasma, red bone marrow and yellow bone marrow. Selected fatty acids, e.g. FA10:0, FA14:1, or FA16:1 n-7 in the bone marrow or FA18:0, FA18:1 n-9, FA18:1 n-7, FA20:0, FA20:1 n-9, or FA20:3 n-6 in the plasma, correlated with osteoclast activity, suggesting a possible mechanism how these fatty acids may interfere with BMD. Although several fatty acids correlated well with the osteoclast activity and BMD, there was not a single fatty acid contained in our fatty acid profile that can be claimed for controlling BMD, a fact that may be attributed to the genetic heterogeneity of the patients.

Table S1: Primersequences used for human and murine cell lines. Table S2: Upstream regulators of genes with increased expression in MDA-MB-231 cells co-cultured with murine adipose tissue (HFD). Table S3: Upstream regulators of genes with decreased expression in MDA-MB-231 cells co-cultured with murine adipose tissue (HFD). Table S4: Characterization of the free fatty acid profile of ACMs<30 and ACMs>40. Table S5: Enriched GO terms (Biological Process) in genes with {greater than or equal to}1.5-fold downregulation in MDAMB-231 cells co-cultured with adipose tissue of HFD mice vs control. Table S6: Enriched GO terms (Biological Process) in genes with {greater than or equal to}1.5-fold upregulation in MDA-MB231 cells co-cultured with adipose tissue of HFD mice vs control. Fig. S1: Pathway Analysis and gene expression data of co-cultured MDA-MB-231 cells. Fig. S2: ACM increases PPAR target gene expression in murine E0771 cells. Fig. S3: ANGPTL4 mRNA is increased in human triple negative breast cancer and associated with reduced survival. Fig. S4: PPAR target gene expression is dependent on PPARα. Fig. S5: Free fatty acids activate PPAR signaling in TNBC cells. Fig. S6: Incubation of TNBC cells with ACM or BSA-OA decreases de novo FA synthesis. Fig. S7: Cultivation of TNBC cells with ACM or BSA-OA induces the expression of β-oxidation genes. Fig. S8: Treatment with oleic acid increases intracellular lipid droplet formation in TNBC cells. Fig S9: ACM promotes proliferation and migration of E0771 cells. Fig. S10: ANGPTL4 knockdown in MDA-MB-231 cells does not affect cell proliferation. Fig. S11: MMP2 is upregulated in MDA-MB-231 cells upon ACM cultivation.

The induction of endoplasmic reticulum unfolded protein responses (UPR ER ) contributes to cancer development and progression. We recently linked microbiota-related triggers to the tumor-promoting role of signal transducer activating transcription factor 6 (ATF6) in the colon. Here we substantiate the clinical relevance of ATF6 and related bacterial genera in colorectal cancer patient cohorts. Spatial and longitudinal bacterial profiling in ATF6 transgenic mice (nATF6 IEC ) identified tumor-initiating and tumor-progressing shifts in the mucosa-associated microbiota. Transcriptional analysis in intestinal epithelial cells (IEC) of germ-free and specific pathogen-free nATF6 IEC mice defined bacteria-specific changes in cellular metabolism enriched for fatty acid biosynthesis. Untargeted metabolomics, isotope-labeling in intestinal organoids and FASN inhibition confirmed ATF6-mediated involvement of long-chain fatty acids in tumorigenesis. Multi-omics data integration identified a bacteria-lipid network characterized by fatty acid efflux, catabolism and detoxification. We postulate chronic ATF6 signaling to drive a clinically relevant pathologic response altering lipid metabolism to select for a tumor-promoting microbiota. Graphical Abstract Chronic ATF6 signaling in the colonic epithelium alters lipid metabolism to select a tumor-promoting microbiota nATF6 expression is induced in intestinal epithelial cells in transgenic mice (nATF6 IEC ) Biallelic nATF6 IEC germ-free mice (grey circle) and floxed or monoallelic specific pathogen-free mice (black circle) remain in a state of homeostasis (yellow circle), while biallelic specific pathogen-free mice (black circle) develop spontaneous colon tumorigenesis (red circle) Mechanistically, biallelic nATF6 IEC mice in the presence of an intestinal microbiota alter colonic lipid metabolism, including the upregulation of Fasn The microbial lipid-response to the altered lipid milieu results in dysbiosis and the subsequent formation of colon adenomas Inhibition of FASN in biallelic nATF6 IEC mice prevents colon tumor formation (yellow arrow) Created with BioRender.com nATF6: activated activating transcription factor 6; LCFA: long-chain fatty acids; SAFA: saturated fatty acids; Fasn: fatty acid synthase; C75 i.p.: intraperitoneal injection of the Fasn inhibitor C75.

Einleitung Die Darm-Leber-Achse wird mit Lebererkrankungen und deren Pathophysiologie in Verbindung gebracht, obwohl die zugrundeliegenden Mechanismen noch unklar sind. Wir haben den funktionellen Beitrag des Darmmikrobioms und seiner Metaboliten zur Leberregeneration in einem präklinischen Mausmodell und in Patientenproben untersucht.