The estrogen signaling pathway reprograms prostate cancer cell metabolism and supports proliferation and disease progression.

Just as the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet, the incomplete understanding of ERα functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for the treatment of this disease. Using two human PCa tissue microarray cohorts, we first demonstrated that nuclear ERα expression was heterogeneous among patients, being only detected in half of tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single-cell RNA-Seq analyses revealed that estrogens partially mimic the androgen transcriptional response and induce specific biological pathways linked to proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed the regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprograms PCa metabolism, is associated with disease progression, and could be targeted for therapeutic purposes.

Isocitrate dehydrogenase 1 sustains a hybrid cytoplasmic-mitochondrial tricarboxylic acid cycle that can be targeted for therapeutic purposes in prostate cancer.

The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models, in vitro human PCa cell lines, and human patient-derived organoids (PDOs) were used to study the expression and activity of IDH enzymes in the normal prostate and PCa. Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography-mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry-based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.

Multiple metabolic pathways fuel the truncated tricarboxylic acid cycle of the prostate to sustain constant citrate production and secretion.

OBJECTIVE: The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa), this phenotype is reprogrammed, making it an interesting therapeutic target. However, how the truncated prostate TCA cycle works is still not completely understood. METHODS: We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. RESULTS: First, mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate, thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis, several key nutrients were shown to allow the completion of the prostate TCA cycle, revealing a much more complex metabolic profile than originally anticipated. Indeed, along with the known pathway of aspartate replenishing oxaloacetate, glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids, aspartate entered the TCA cycle at the malate entry point, upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate-oxaloacetate-citrate pathway, but also by at least three additional pathways: glutaminolysis, reductive carboxylation, and aspartate-malate conversion. CONCLUSIONS: Our results add a significant new dimension to the prostate citrate-secretory phenotype, with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa.

Docosahexaenoic acid-rich algae oil supplementation on breast milk fatty acid profile of mothers who delivered prematurely: a randomized clinical trial.

Preterm infants are deficient in long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), a fatty acid (FA) associated with an increase in bronchopulmonary dysplasia (BPD). In two previous randomized control trials, DHA supplementation did not reduce the risk of BPD. We examined the breast milk FA profile, collected 14 days after birth, of mothers who delivered before 29 weeks of gestation and who were supplemented with DHA-rich algae oil or a placebo within 72 h after birth as part of the MOBYDIck trial. Milk FA were analyzed by gas chromatography. The total amount of FA (mg/mL) was similar in both groups but the supplementation increased DHA (expressed as % of total FA, mean ± SD, treatment vs placebo, 0.95 ± 0.44% vs 0.34 ± 0.20%; P < 0.0001), n-6 docosapentaenoic acid (DPA) (0.275 ± 0.14% vs 0.04 ± 0.04%; P < 0.0001) and eicosapentaenoic acid (0.08 ± 0.08% vs 0.07 ± 0.07%; P < 0.0001) while decreasing n-3 DPA (0.16 ± 0.05% vs 0.17 ± 0.06%; P < 0.05). Supplementation changed the ratio of DHA to arachidonic acid (1.76 ± 1.55% vs 0.60 ± 0.31%; P < 0.0001) and n-6 to n-3 FA (0.21 ± 0.06% vs 0.17 ± 0.04%; P < 0.0001). DHA-rich algae supplementation successfully increased the DHA content of breast milk but also included secondary changes that are closely involved with inflammation and may contribute to changing clinical outcomes.

A FACS-Free Purification Method to Study Estrogen Signaling, Organoid Formation, and Metabolic Reprogramming in Mammary Epithelial Cells.

Few in vitro models are used to study mammary epithelial cells (MECs), and most of these do not express the estrogen receptor α (ERα). Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs. These MECs remain differentiated for up to six days with >85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland's morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ERα signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favoring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed mass spectrometry (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ERα reprograms cell metabolism in mammary organoids. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis.

Drinkable lecithin nanovesicles to study the biological effects of individual hydrophobic macronutrients and food preferences.

Fundamental nutritional studies on bioactive molecules require minimizing exposure to confounding foreign elements, like solvents. Herein, aqueous formulations of lecithin nanovesicles are proposed to study three individual trans fatty acids relevant to human nutrition: elaidic acid, trans-vaccenic acid and trans-palmitoleic acid. This proof-of-concept study describes the encapsulation of fatty acids, in vivo bioavailability, and the use of nanovesicles in behavioral experiments. The oral bioavailability of the encapsulated molecules and the selective exposure of animals to each trans-fatty acid of interest were confirmed in healthy rats. Behavioral studies also evidenced that nanovesicles can be used to evaluate the palatability of the lipids and investigate food preferences in mice. Altogether this study shows that lecithin nanovesicles offer an elegant tool to efficiently deliver hydrophobic molecules to animal models. This approach paves the way for future studies deconvoluting the nutritional effects of trans-fatty acids.

F2-isoprostanes and fatty acids profile in early pregnancy complicated by pre-existing diabetes.

BACKGROUND: Diabetes and pregnancy are both associated with oxidative stress, characterized by an increase of F2-isoprostanes from the non-enzymatic oxidation of arachidonic acid, a n - 6 polyunsaturated fatty acid (PUFA). We hypothesized that pregnant women with pre-existing diabetes will be characterized by elevated levels of specific F2-isoPs isomers and altered PUFA composition in plasma early pregnancy when compared to normoglycemic controls. METHODS: Plasma samples from 23 women with uncomplicated pregnancies and 11 women with pre-existing diabetes in pregnancy were collected between 12 and 18 weeks of pregnancy (MIROS cohort). Six F2-isoprostanes isomers were measured by high-performance liquid chromatography coupled to tandem mass spectrometry. Fatty acids concentrations in plasmatic phospholipids were measured by gas chromatography coupled to a flame ionization detector. RESULTS: F2-isoprostanes, specifically the 8-iso-15(R)-PGF2α levels, were 67% higher in diabetic than normoglycemic pregnancies (p = 0.026). The total n - 6 PUFA and arachidonic acid level did not differ between study groups. In contrast, total n - 3 level was 32% lower in diabetic pregnancies than in controls (p = 0.002); EPA(20:5) and DHA(22:6) being specifically reduced (p = 0.035 and p = 0.003 respectively). Delta-6-desaturase (D6D) activity index, calculated using fatty acid ratios, was 9% lower in pre-existing diabetes than in controls (p = 0.042). CONCLUSIONS: Pre-existing diabetes in early pregnancy displays a distinctive F2-isoprostanes profile when compared to other pathologies of pregnancy, such as preeclampsia, as previously assessed in the same cohort.

Fatty acid composition of birds and game hunted by the Eastern James Bay Cree people of Québec.

BACKGROUND: Indigenous peoples have traditionally relied on foods hunted and gathered from their immediate environment. The Eastern James Bay Cree people consume wild game and birds, and these are believed to provide health as well as cultural benefits. OBJECTIVE: To determine the fatty acid (FA) composition of traditional game and bird meats hunted in the Eastern James Bay area. DESIGN: Harvested traditional game and birds were analysed for FA composition. A total of 52 samples from six wildlife species were collected in the areas of Chisasibi, Waswanipi and Mistissini, of which 35 were from birds (white partridge and Canada goose) and 17 were from land animals (beaver, moose, caribou and black bear). RESULTS: Alpha-linolenic acid (ALA) was the most common n-3 polyunsaturated fatty acid (PUFA) in all samples except for the black bear flesh, in which it was docosapentaenoic acid (DPAn-3). In white partridge, beaver and caribou flesh, PUFAs (mainly n-6) were the most common category of fats while in goose, moose and black bear flesh, monounsaturated fatty acids (MUFAs) predominated. In all species, saturated fatty acids (SFAs) were the second most important FAs. It would appear that in the land animals and birds that were analysed, the SFA content was lower and the PUFA content was higher than store-bought meats giving them a more heart-healthy profile. CONCLUSIONS: These results showed that the FA composition of game species consumed by the James Bay Cree population is consistent with a beneficial diet and that traditional foods should continue to be promoted among the Cree people to provide better physical health as well as social and spiritual benefits.

High-fat diet aggravates amyloid-beta and tau pathologies in the 3xTg-AD mouse model.

To investigate potential dietary risk factors of Alzheimer's disease (AD), triple transgenic (3xTg-AD) mice were exposed from 4 to 13 months of age to diets with a low n-3:n-6 polyunsaturated fatty acid (PUFA) ratio incorporated in either low-fat (5% w/w) or high-fat (35% w/w) formulas and compared with a control diet. The n-3:n-6 PUFA ratio was decreased independently of the dietary treatments in the frontal cortex of 3xTg-AD mice compared to non-transgenic littermates. Consumption of a high-fat diet with a low n-3:n-6 PUFA ratio increased amyloid-beta (Abeta) 40 and 42 concentrations in detergent-insoluble extracts of parieto-temporal cortex homogenates from 3xTg-AD mice. Low n-3:n-6 PUFA intake ratio increased insoluble tau regardless of total fat consumption, whereas high-fat diet incorporating a low n-3:n-6 PUFA ratio also increased soluble tau compared to controls. Moreover, the high-fat diet decreased cortical levels of the postsynaptic marker drebrin, while leaving presynaptic proteins synaptophysin, SNAP-25 and syntaxin 3 unchanged. Overall, these results suggest that high-fat consumption combined with low n-3 PUFA intake promote AD-like neuropathology.

Environmental contaminants and redox status of coenzyme Q10 and vitamin E in Inuit from Nunavik.

The Inuit are heavily exposed to potentially prooxidant contaminants such as methylmercury (MeHg) and polychlorinated biphenyls (PCB) through their traditional diet. This diet is also an abundant source of n-3 polyunsaturated fatty acids (n-3 PUFA), selenium, and antioxidants, which might reduce cardiovascular risk. Although Inuit from Nunavik have low concentrations of plasma oxidized low-density lipoprotein (OxLDL) and elevated glutathione-related antioxidant defenses, the variance in OxLDL was predicted by PCB and blood glutathione, leaving the issue of contaminant-associated oxidative stress unresolved. The objective of the study was to assess oxidative stress in these Inuit by measuring the plasma concentrations and redox states of alpha-tocopherol and coenzyme Q10 (CoQ10), 2 sensitive biomarkers of oxidative stress, in relation to exposure. Plasma lipophilic antioxidants were determined by high-performance liquid chromatography-coupled electrochemical detection; and their relations to PCB, MeHg, n-3 PUFA, selenium, and OxLDL were assessed by multivariate analyses. Ubiquinol-10, ubiquinone-10, and ubiquinone-10 to CoQ10(total) ratio were elevated as compared with white populations but showed no associations with PCB, MeHg, or n-3 PUFA. Ubiquinol-10 (beta = .23, P = .007) and CoQ10(total) (beta = .27, P = .009) were predicted by blood selenium; and alpha-tocopherol, by PCB (beta = 4.12, P = .0002), n-3 PUFA (beta = 9.16, P = .02), and OxLDL (beta = 3.04, P = .05). Unexpectedly, the alpha-tocopheryl quinone to alpha-tocopherol ratio, in the reference range, was negatively predicted by PCB (beta = -0.41, P = .02). Using sensitive biomarkers of redox alterations, we found no evidence for MeHg- or PCB-associated oxidative stress in these Inuit. However, despite robust blood antioxidant defenses, the unusually elevated ubiquinone-10 to CoQ10(total) ratio (0.21 +/- 0.11) suggests some form of oxidative stress of unknown origin.

Seasonal mercury exposure and oxidant-antioxidant status of James Bay sport fishermen.

The effects of a moderate seasonal exposure to methylmercury on plasma low-density lipoprotein (LDL) oxidation and cardiovascular risk indices are not known. The objective of the study was to assess the effects of a seasonal exposure to mercury at similar dose reported to increase cardiovascular risk through fish consumption. Effects on lipoprotein cholesterol and fatty acid profiles, LDL oxidation, and blood oxidant-antioxidant balance were to be assessed in sport fishermen presenting normal blood selenium and omega-3 fatty acid contents. Thirty-one healthy James Bay sport fishermen were assessed for within-subject longitudinal seasonal variations in hair and blood mercury, plasma oxidized LDL, lipophilic antioxidants, homocysteine, blood selenium, and glutathione peroxidase and reductase activities determined before and after the fishing season and compared by matched-pair tests. Hair mercury doubled during the fishing season (2.8+/-0.4 microg/g, P<.0001). Baseline blood selenium, homocysteine, and erythrocyte fatty acid profiles did not change. Plasma high-density lipoprotein cholesterol increased (+5%, P=.05), whereas very low-density lipoprotein cholesterol and oxidized LDL decreased (-8%, P=.05; -18%, P=.008). Blood glutathione peroxidase (+9.7%, P=.001), glutathione reductase (+7.2%, P<.0001), and total glutathione (+45% P<.0001) increased during the fishing season. Plasma total coenzyme Q10 (+13%, P=.02), ubiquinone-10 (+67%, P=.03), and beta-carotene (+46%, P=.01) also increased, whereas vitamin E status was unaffected. Pairwise correlations revealed no association between mercury exposure and any of the biomarkers investigated. In contrast, strong predictors of cardiovascular risk such as high-density lipoprotein cholesterol, oxidized LDL, and glutathione peroxidase improved during the fishing season despite elevated methylmercury exposure. The beneficial effects of seasonal fishing activity and fish consumption on cardiovascular health may suppress detrimental effects of concomitant moderate methylmercury exposure.

Dietary contaminants and oxidative stress in Inuit of Nunavik.

The aim of the present study was to investigate the potential deleterious effects of dietary contaminants such as polychlorinated biphenyls (PCBs) and methylmercury (MeHg) on different molecules sensitive to oxidative stress, namely, plasma oxidized low-density lipoproteins (OxLDLs), plasma homocysteine (Hcy), blood glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione (GSH). We also planned to assess the potential beneficial effects of long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs) and selenium (Se) that are also present in the traditional Inuit diet. A total of 99 participants were studied. Plasma levels of PCBs, blood levels of Se and MeHg, plasma lipids (triacylglycerols, total, LDL-, and high-density lipoprotein cholesterol [LDL-C and HDL-C, respectively], apolipoprotein B-LDL), erythrocyte n-3 PUFAs, OxLDL, Hcy, blood GPx, GSH, and GR have been determined. Mean concentrations of MeHg, Se, and PCBs were respectively 10- to 14-fold, 8- to 15-fold, and 16- to 18-fold higher than reported in white population consuming little or no fish. Multivariate analyses show that variance in plasma OxLDL concentrations was predicted by LDL-C (P = .007), HDL-C (P = .005), and PCBs (P = .006). The level of LDL oxidation, represented as the ratio OxLDL/apolipoprotein B-LDL, was predicted by LDL-C (P = .0002), HDL-C (P = .002), and GSH (P = .005). Concentration of plasma Hcy was positively predicted by age (P = .02) but negatively by body mass index (P = .04) and Se (P = .005). Glutathione was predicted by the smoking status (P = .004) and the level of LDL oxidation (P = .005), whereas GR was only predicted by the smoking status (P = .0009). The variance of GPx was not predicted by any contaminant or other physiological parameter. Dietary MeHg showed no association with the examined oxidative biomarkers, whereas PCB level was a predictor of the plasma concentration of OxLDL, although this concentration remained very low. The level of GPx activity in Inuit was higher than levels previously reported to be protective in whites. Homocysteine was negatively predicted by Se, suggesting a possible beneficial effect of Se. Moreover, n-3 PUFAs were highly correlated with dietary contaminants, but had no relationships with oxidative biomarkers. This study suggests that, in adult Inuit, contaminated traditional diet seems to have no direct oxidative effects on molecules involved in oxidative stress.

Postmortem brain fatty acid profile of levodopa-treated Parkinson disease patients and parkinsonian monkeys.

Fatty acids play a critical role in brain function but their specific role in the pathophysiology of Parkinson disease (PD) and levodopa-induced motor complications is still unknown. From a therapeutic standpoint, it is important to determine the relation between brain fatty acids and PD because the brain fatty acid content depends on nutritional intake, a readily manipulable environmental factor. Here, we report a postmortem analysis of fatty acid profile by gas chromatography in the brain cortex of human patients (12 PD patients and nine Controls) as well as in the brain cortex of monkeys (four controls, five drug-naive MPTP monkeys and seven levodopa-treated MPTP monkeys). Brain fatty acid profile of cerebral cortex tissue was similar between PD patients and Controls and was not correlated with age of death, delay to autopsy or brain pH. Levodopa administration in MPTP monkeys increased arachidonic acid content (+7%; P < 0 .05) but decreased docosahexaenoic acid concentration (-15%; P < 0.05) and total n-3:n-6 polyunsaturated fatty acids ratio (-27%; P < 0.01) compared to drug-naive MPTP animals. Interestingly, PD patients who experienced motor complications to levodopa had higher arachidonic acid concentrations in the cortex compared to Controls (+13.6%; P < 0.05) and to levodopa-treated PD patients devoid of motor complications (+14.4%; P < 0.05). Furthermore, PD patients who took an above-median cumulative dose of levodopa had a higher relative amount of saturated fatty acids but lower monounsaturated fatty acids in their brain cortex (P < 0.01). These results suggest that changes in brain fatty acid relative concentrations are associated with levodopa treatment in PD patients and in a non-human primate model of parkinsonism.