Genetic screening and metabolomics identify glial adenosine metabolism as a therapeutic target in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and lacks disease-modifying therapies. We developed a Drosophila model for identifying novel glial-based therapeutic targets for PD. Human alpha-synuclein is expressed in neurons and individual genes are independently knocked down in glia. We performed a forward genetic screen, knocking down the entire Drosophila kinome in glia in alpha-synuclein expressing flies. Among the top hits were five genes (Ak1, Ak6, Adk1, Adk2, and awd) involved in adenosine metabolism. Knockdown of each gene improved locomotor dysfunction, rescued neurodegeneration, and increased brain adenosine levels. We determined that the mechanism of neuroprotection involves adenosine itself, as opposed to a downstream metabolite. We dove deeper into the mechanism for one gene, Ak1, finding rescue of dopaminergic neuron loss, alpha-synuclein aggregation, and bioenergetic dysfunction after glial Ak1 knockdown. We performed metabolomics in Drosophila and in human PD patients, allowing us to comprehensively characterize changes in purine metabolism and identify potential biomarkers of dysfunctional adenosine metabolism in people. These experiments support glial adenosine as a novel therapeutic target in PD.

Effect of 1-year lifestyle intervention with energy-reduced Mediterranean diet and physical activity promotion on the gut metabolome and microbiota: a randomized clinical trial.

BACKGROUND: The health benefits of the Mediterranean diet (MedDiet) have been linked to the presence of beneficial gut microbes and related metabolites. However, its impact on the fecal metabolome remains poorly understood. OBJECTIVES: Our goal was to investigate the weight-loss effects of a 1-y lifestyle intervention based on an energy-reduced MedDiet coupled with physical activity (intervention group), compared with an ad libitum MedDiet (control group), on fecal metabolites, fecal microbiota, and their potential association with cardiovascular disease risk factors. METHODS: A total of 400 participants (200 from each study group), aged 55-75 y, and at high cardiovascular disease risk, were included. Dietary and lifestyle information, anthropometric measurements, blood biochemical parameters, and stool samples were collected at baseline and after 1 y of follow-up. Liquid chromatography-tandem mass spectrometry was used to profile endogenous fecal metabolites, and 16S amplicon sequencing was employed to profile the fecal microbiota. RESULTS: Compared with the control group, the intervention group exhibited greater weight loss and improvement in various cardiovascular disease risk factors. We identified intervention effects on 4 stool metabolites and subnetworks primarily composed of bile acids, ceramides, and sphingosines, fatty acids, carnitines, nucleotides, and metabolites of purine and the Krebs cycle. Some of these were associated with changes in several cardiovascular disease risk factors. In addition, we observed a reduction in the abundance of the genera Eubacterium hallii group and Dorea, and an increase in alpha diversity in the intervention group after 1 y of follow-up. Changes in the intervention-related microbiota profiles were also associated with alterations in different fecal metabolite subnetworks and some cardiovascular disease risk factors. CONCLUSIONS: An intervention based on an energy-reduced MedDiet and physical activity promotion, compared with an ad libitum MedDiet, was associated with improvements in cardiometabolic risk factors, potentially through modulation of the fecal microbiota and metabolome. This trial was registered at https://www.isrctn.com/ as ISRCTN89898870 (https://doi.org/10.1186/ISRCTN89898870).

Olive oil consumption, plasma metabolites, and risk of type 2 diabetes and cardiovascular disease.

BACKGROUND: Olive oil consumption has been inversely associated with the risk of type 2 diabetes (T2D) and cardiovascular disease (CVD). However, the impact of olive oil consumption on plasma metabolites remains poorly understood. This study aims to identify plasma metabolites related to total and specific types of olive oil consumption, and to assess the prospective associations of the identified multi-metabolite profiles with the risk of T2D and CVD. METHODS: The discovery population included 1837 participants at high cardiovascular risk from the PREvención con DIeta MEDiterránea (PREDIMED) trial with available metabolomics data at baseline. Olive oil consumption was determined through food-frequency questionnaires (FFQ) and adjusted for total energy. A total of 1522 participants also had available metabolomics data at year 1 and were used as the internal validation sample. Plasma metabolomics analyses were performed using LC-MS. Cross-sectional associations between 385 known candidate metabolites and olive oil consumption were assessed using elastic net regression analysis. A 10-cross-validation (CV) procedure was used, and Pearson correlation coefficients were assessed between metabolite-weighted models and FFQ-derived olive oil consumption in each pair of training-validation data sets within the discovery sample. We further estimated the prospective associations of the identified plasma multi-metabolite profile with incident T2D and CVD using multivariable Cox regression models. RESULTS: We identified a metabolomic signature for the consumption of total olive oil (with 74 metabolites), VOO (with 78 metabolites), and COO (with 17 metabolites), including several lipids, acylcarnitines, and amino acids. 10-CV Pearson correlation coefficients between total olive oil consumption derived from FFQs and the multi-metabolite profile were 0.40 (95% CI 0.37, 0.44) and 0.27 (95% CI 0.22, 0.31) for the discovery and validation sample, respectively. We identified several overlapping and distinct metabolites according to the type of olive oil consumed. The baseline metabolite profiles of total and extra virgin olive oil were inversely associated with CVD incidence (HR per 1SD: 0.79; 95% CI 0.67, 0.92 for total olive oil and 0.70; 0.59, 0.83 for extra virgin olive oil) after adjustment for confounders. However, no significant associations were observed between these metabolite profiles and T2D incidence. CONCLUSIONS: This study reveals a panel of plasma metabolites linked to the consumption of total and specific types of olive oil. The metabolite profiles of total olive oil consumption and extra virgin olive oil were associated with a decreased risk of incident CVD in a high cardiovascular-risk Mediterranean population, though no associations were observed with T2D incidence. TRIAL REGISTRATION: The PREDIMED trial was registered at ISRCTN ( http://www.isrctn.com/ , ISRCTN35739639).

Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

Triglyceride-derived fatty acids reduce autophagy in a model of retinal angiomatous proliferation.

Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). VLDL receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids. Since fatty acid uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We found that excess circulating lipids restrained retinal autophagy, which contributed to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived fatty acid sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.

Changes in metabolomics profiles over ten years and subsequent risk of developing type 2 diabetes: Results from the Nurses' Health Study.

BACKGROUND: Metabolomics profiles were consistently associated with type 2 diabetes (T2D) risk, but evidence on long-term metabolite changes and T2D incidence is lacking. We examined the associations of 10-year plasma metabolite changes with subsequent T2D risk. METHODS: We conducted a nested T2D case-control study (n=244 cases, n=244 matched controls) within the Nurses' Health Study. Repeated metabolomics profiling (170 targeted metabolites) was conducted in participant blood specimens from 1989/1990 and 2000/2001, and T2D occurred between 2002 and 2008. We related 10-year metabolite changes (Δ-values) to subsequent T2D risk using conditional logistic models, adjusting for baseline metabolite levels and baseline levels and concurrent changes of BMI, diet quality, physical activity, and smoking status. FINDINGS: The 10-year changes of thirty-one metabolites were associated with subsequent T2D risk (false discovery rate-adjusted p-values [FDR]<0.05). The top three high T2D risk-associated 10-year changes were (odds ratio [OR] per standard deviation [SD], 95%CI): Δisoleucine (2.72, 1.97-3.79), Δleucine (2.53, 1.86-3.47), and Δvaline (1.93, 1.52-2.44); other high-risk-associated metabolite changes included alanine, tri-/diacylglycerol-fragments, short-chain acylcarnitines, phosphatidylethanolamines, some vitamins, and bile acids (ORs per SD between 1.31and 1.82). The top three low T2D risk-associated 10-year metabolite changes were (OR per SD, 95% CI): ΔN-acetylaspartic acid (0.54, 0.42-0.70), ΔC20:0 lysophosphatidylethanolamine (0.68, 0.56-0.82), and ΔC16:1 sphingomyelin (0.68, 0.56-0.83); 10-year changes of other sphingomyelins, plasmalogens, glutamine, and glycine were also associated with lower subsequent T2D risk (ORs per SD between 0.66 and 0.78). INTERPRETATION: Repeated metabolomics profiles reflecting the long-term deterioration of amino acid and lipid metabolism are associated with subsequent risk of T2D.

Postdiagnosis Loss of Skeletal Muscle, but Not Adipose Tissue, Is Associated with Shorter Survival of Patients with Advanced Pancreatic Cancer.

BACKGROUND: Pancreatic cancer is associated with development of cachexia, a wasting syndrome thought to limit survival. Few studies have longitudinally quantified peripheral tissues or identified biomarkers predictive of future tissue wasting. METHODS: Adipose and muscle tissue were measured by computed tomography (CT) at diagnosis and 50 to 120 days later in 164 patients with advanced pancreatic cancer. Tissue changes and survival were evaluated by Cox proportional hazards regression. Baseline levels of circulating markers were examined in relation to future tissue wasting. RESULTS: Compared with patients in the bottom quartile of muscle change per 30 days (average gain of 0.8 ± 2.0 cm2), those in the top quartile (average loss of 12.9 ± 4.9 cm2) had a hazard ratio (HR) for death of 2.01 [95% confidence interval (CI), 1.12-3.62]. Patients in the top quartile of muscle attenuation change (average decrease of 4.9 ± 2.4 Hounsfield units) had an HR of 2.19 (95% CI, 1.18-4.04) compared with those in the bottom quartile (average increase of 2.4 ± 1.6 Hounsfield units). Changes in adipose tissue were not associated with survival. Higher plasma branched chain amino acids (BCAA; P = 0.004) and lower monocyte chemoattractant protein-1 (MCP-1; P = 0.005) at diagnosis were associated with greater future muscle loss. CONCLUSIONS: In patients with advanced pancreatic cancer, muscle loss and decrease in muscle density in 2 to 4 months after diagnosis were associated with reduced survival. BCAAs and MCP-1 levels at diagnosis were associated with subsequent muscle loss. IMPACT: BCAAs and MCP-1 levels at diagnosis could identify a high-risk group for future tissue wasting.

Plasma Metabolites Associated with Frequent Red Wine Consumption: A Metabolomics Approach within the PREDIMED Study.

SCOPE: The relationship between red wine (RW) consumption and metabolism is poorly understood. It is aimed to assess the systemic metabolomic profiles in relation to frequent RW consumption as well as the ability of a set of metabolites to discriminate RW consumers. METHODS AND RESULTS: A cross-sectional analysis of 1157 participants is carried out. Subjects are divided as non-RW consumers versus RW consumers (>1 glass per day RW [100 mL per day]). Plasma metabolomics analysis is performed using LC-MS. Associations between 386 identified metabolites and RW consumption are assessed using elastic net regression analysis taking into consideration baseline significant covariates. Ten-cross-validation (CV) is performed and receiver operating characteristic curves are constructed in each of the validation datasets based on weighted models. A subset of 13 metabolites is consistently selected and RW consumers versus nonconsumers are discriminated. Based on the multi-metabolite model weighted with the regression coefficients of metabolites, the area under the curve is 0.83 (95% CI: 0.80-0.86). These metabolites mainly consisted of lipid species, some organic acids, and alkaloids. CONCLUSIONS: A multi-metabolite model identified in a Mediterranean population appears useful to discriminate between frequent RW consumers and nonconsumers. Further studies are needed to assess the contribution of these metabolites in health and disease.

Plasma Metabolites Associated with Coffee Consumption: A Metabolomic Approach within the PREDIMED Study.

Few studies have examined the association of a wide range of metabolites with total and subtypes of coffee consumption. The aim of this study was to investigate associations of plasma metabolites with total, caffeinated, and decaffeinated coffee consumption. We also assessed the ability of metabolites to discriminate between coffee consumption categories. This is a cross-sectional analysis of 1664 participants from the PREDIMED study. Metabolites were semiquantitatively profiled using a multiplatform approach. Consumption of total coffee, caffeinated coffee and decaffeinated coffee was assessed by using a validated food frequency questionnaire. We assessed associations between 387 metabolite levels with total, caffeinated, or decaffeinated coffee consumption (≥50 mL coffee/day) using elastic net regression analysis. Ten-fold cross-validation analyses were used to estimate the discriminative accuracy of metabolites for total and subtypes of coffee. We identified different sets of metabolites associated with total coffee, caffeinated and decaffeinated coffee consumption. These metabolites consisted of lipid species (e.g., sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine) or were derived from glycolysis (alpha-glycerophosphate) and polyphenol metabolism (hippurate). Other metabolites included caffeine, 5-acetylamino-6-amino-3-methyluracil, cotinine, kynurenic acid, glycocholate, lactate, and allantoin. The area under the curve (AUC) was 0.60 (95% CI 0.56-0.64), 0.78 (95% CI 0.75-0.81) and 0.52 (95% CI 0.49-0.55), in the multimetabolite model, for total, caffeinated, and decaffeinated coffee consumption, respectively. Our comprehensive metabolic analysis did not result in a new, reliable potential set of metabolites for coffee consumption.

Altered exocrine function can drive adipose wasting in early pancreatic cancer.

Malignancy is accompanied by changes in the metabolism of both cells and the organism1,2. Pancreatic ductal adenocarcinoma (PDAC) is associated with wasting of peripheral tissues, a metabolic syndrome that lowers quality of life and has been proposed to decrease survival of patients with cancer3,4. Tissue wasting is a multifactorial disease and targeting specific circulating factors to reverse this syndrome has been mostly ineffective in the clinic5,6. Here we show that loss of both adipose and muscle tissue occurs early in the development of pancreatic cancer. Using mouse models of PDAC, we show that tumour growth in the pancreas but not in other sites leads to adipose tissue wasting, suggesting that tumour growth within the pancreatic environment contributes to this wasting phenotype. We find that decreased exocrine pancreatic function is a driver of adipose tissue loss and that replacement of pancreatic enzymes attenuates PDAC-associated wasting of peripheral tissues. Paradoxically, reversal of adipose tissue loss impairs survival in mice with PDAC. When analysing patients with PDAC, we find that depletion of adipose and skeletal muscle tissues at the time of diagnosis is common, but is not associated with worse survival. Taken together, these results provide an explanation for wasting of adipose tissue in early PDAC and suggest that early loss of peripheral tissue associated with pancreatic cancer may not impair survival.

Plasma Metabolites From Choline Pathway and Risk of Cardiovascular Disease in the PREDIMED (Prevention With Mediterranean Diet) Study.

BACKGROUND: The relationship between plasma concentrations of betaine and choline metabolism and major cardiovascular disease (CVD) end points remains unclear. We have evaluated the association between metabolites from the choline pathway and risk of incident CVD and the potential modifying effect of Mediterranean diet interventions. METHODS AND RESULTS: We designed a case-cohort study nested within the PREDIMED (Prevention With Mediterranean Diet) trial, including 229 incident CVD cases and 751 randomly selected participants at baseline, followed up for 4.8 years. We used liquid chromatography-tandem mass spectrometry to measure, at baseline and at 1 year of follow-up, plasma concentrations of 5 metabolites in the choline pathway: trimethylamine N-oxide, betaine, choline, phosphocholine, and α-glycerophosphocholine. We have calculated a choline metabolite score using a weighted sum of these 5 metabolites. We used weighted Cox regression models to estimate CVD risk. The multivariable hazard ratios (95% confidence intervals) per 1-SD increase in choline and α-glycerophosphocholine metabolites were 1.24 (1.05-1.46) and 1.24 (1.03-1.50), respectively. The baseline betaine/choline ratio was inversely associated with CVD. The baseline choline metabolite score was associated with a 2.21-fold higher risk of CVD across extreme quartiles (95% confidence interval, 1.36-3.59; P<0.001 for trend) and a 2.27-fold higher risk of stroke (95% confidence interval, 1.24-4.16; P<0.001 for trend). Participants in the higher quartiles of the score who were randomly assigned to the control group had a higher risk of CVD compared with participants in the lower quartile and assigned to the Mediterranean diet groups (P=0.05 for interaction). No significant associations were observed for 1-year changes in individual plasma metabolites and CVD. CONCLUSIONS: A metabolite score combining plasma metabolites from the choline pathway was associated with an increased risk of CVD in a Mediterranean population at high cardiovascular risk. CLINICAL TRIAL REGISTRATION: URL: http://www.controlled-trials.com. Unique identifier: ISRCTN35739639.

Metabolites of Glutamate Metabolism Are Associated With Incident Cardiovascular Events in the PREDIMED PREvención con DIeta MEDiterránea (PREDIMED) Trial.

BACKGROUND: Glutamate metabolism may play a role in the pathophysiology of cardiometabolic disorders. However, there is limited evidence of an association between glutamate-related metabolites and, moreover, changes in these metabolites, and risk of cardiovascular disease (CVD). METHODS AND RESULTS: Plasma levels of glutamate and glutamine were measured at baseline and 1-year follow-up in a case-cohort study including 980 participants (mean age 68 years; 46% male) from the PREvención con DIeta MEDiterránea (PREDIMED) randomized trial, which assessed a Mediterranean diet intervention in the primary prevention of CVD. During median 4.8 years of follow-up, there were 229 incident CVD events (nonfatal stroke, nonfatal myocardial infarction, or CVD death). In fully adjusted models, per 1-SD, baseline glutamate was associated with 43% (95% CI: 16% to 76%) and 81% (39% to 137%) increased risk of composite CVD and stroke alone, respectively, and baseline glutamine-to-glutamate ratio with 25% (6% to 40%) and 44% (25% to 58%) decreased risk of composite CVD and stroke alone, respectively. Associations appeared linear for stroke (both Plinear trend≤0.005). Among participants with high baseline glutamate, the interventions lowered CVD risk by 37% compared to the control diet; the intervention effects were not significant when baseline glutamate was low (Pinteraction=0.02). No significant effect of the intervention on year-1 changes in metabolites was observed, and no effect of changes themselves on CVD risk was apparent. CONCLUSIONS: Baseline glutamate was associated with increased CVD risk, particularly stroke, and glutamine-to-glutamate ratio was associated with decreased risk. Participants with high glutamate levels may obtain greater benefits from the Mediterranean diet than those with low levels. CLINICAL TRIAL REGISTRATION: URL: www.controlled-trials.com. Unique identifier: ISRCTN 35739639.

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells.

The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A. We observed increased intracellular concentrations of methylthioadenosine (MTA, the metabolite cleaved by MTAP) in cells harboring MTAP deletions. Furthermore, MTA specifically inhibited PRMT5 enzymatic activity. Administration of either MTA or a small-molecule PRMT5 inhibitor showed a modest preferential impairment of cell viability for MTAP-null cancer cell lines compared with isogenic MTAP-expressing counterparts. Together, our findings reveal PRMT5 as a potential vulnerability across multiple cancer lineages augmented by a common "passenger" genomic alteration.