Shotgun metagenomics and systemic targeted metabolomics highlight indole-3-propionic acid as a protective gut microbial metabolite against influenza infection.

The gut-to-lung axis is critical during respiratory infections, including influenza A virus (IAV) infection. In the present study, we used high-resolution shotgun metagenomics and targeted metabolomic analysis to characterize influenza-associated changes in the composition and metabolism of the mouse gut microbiota. We observed several taxonomic-level changes on day (D)7 post-infection, including a marked reduction in the abundance of members of the Lactobacillaceae and Bifidobacteriaceae families, and an increase in the abundance of Akkermansia muciniphila. On D14, perturbation persisted in some species. Functional scale analysis of metagenomic data revealed transient changes in several metabolic pathways, particularly those leading to the production of short-chain fatty acids (SCFAs), polyamines, and tryptophan metabolites. Quantitative targeted metabolomics analysis of the serum revealed changes in specific classes of gut microbiota metabolites, including SCFAs, trimethylamine, polyamines, and indole-containing tryptophan metabolites. A marked decrease in indole-3-propionic acid (IPA) blood level was observed on D7. Changes in microbiota-associated metabolites correlated with changes in taxon abundance and disease marker levels. In particular, IPA was positively correlated with some Lactobacillaceae and Bifidobacteriaceae species (Limosilactobacillus reuteri, Lactobacillus animalis) and negatively correlated with Bacteroidales bacterium M7, viral load, and inflammation markers. IPA supplementation in diseased animals reduced viral load and lowered local (lung) and systemic inflammation. Treatment of mice with antibiotics targeting IPA-producing bacteria before infection enhanced viral load and lung inflammation, an effect inhibited by IPA supplementation. The results of this integrated metagenomic-metabolomic analysis highlighted IPA as an important contributor to influenza outcomes and a potential biomarker of disease severity.

Discovery, Structure-Activity Relationships, and In Vivo Activity of Dihydropyridone Agonists of the Bile Acid Receptor TGR5.

A novel series of potent agonists of the bile acid receptor TGR5 bearing a dihydropyridone scaffold was developed from a high-throughput screen. Starting from a micromolar hit compound, we implemented an extensive structure-activity-relationship (SAR) study with the synthesis and biological evaluation of 83 analogues. The project culminated with the identification of the potent nanomolar TGR5 agonist 77A. We report the GLP-1 secretagogue effect of our lead compound ex vivo in mouse colonoids and in vivo. In addition, to identify specific features favorable for TGR5 activation, we generated and optimized a three-dimensional quantitative SAR model that contributed to our understanding of our activity profile and could guide further development of this dihydropyridone series.

Roux-en-Y gastric bypass induces hepatic transcriptomic signatures and plasma metabolite changes indicative of improved cholesterol homeostasis.

BACKGROUND & AIMS: Roux-en-Y gastric bypass (RYGB), the most effective surgical procedure for weight loss, decreases obesity and ameliorates comorbidities, such as non-alcoholic fatty liver (NAFLD) and cardiovascular (CVD) diseases. Cholesterol is a major CVD risk factor and modulator of NAFLD development, and the liver tightly controls its metabolism. How RYGB surgery modulates systemic and hepatic cholesterol metabolism is still unclear. METHODS: We studied the hepatic transcriptome of 26 patients with obesity but not diabetes before and 1 year after undergoing RYGB. In parallel, we measured quantitative changes in plasma cholesterol metabolites and bile acids (BAs). RESULTS: RYGB surgery improved systemic cholesterol metabolism and increased plasma total and primary BA levels. Transcriptomic analysis revealed specific alterations in the liver after RYGB, with the downregulation of a module of genes implicated in inflammation and the upregulation of three modules, one associated with BA metabolism. A dedicated analysis of hepatic genes related to cholesterol homeostasis pointed towards increased biliary cholesterol elimination after RYGB, associated with enhancement of the alternate, but not the classical, BA synthesis pathway. In parallel, alterations in the expression of genes involved in cholesterol uptake and intracellular trafficking indicate improved hepatic free cholesterol handling. Finally, RYGB decreased plasma markers of cholesterol synthesis, which correlated with an improvement in liver disease status after surgery. CONCLUSIONS: Our results identify specific regulatory effects of RYGB on inflammation and cholesterol metabolism. RYGB alters the hepatic transcriptome signature, likely improving liver cholesterol homeostasis. These gene regulatory effects are reflected by systemic post-surgery changes of cholesterol-related metabolites, corroborating the beneficial effects of RYGB on both hepatic and systemic cholesterol homeostasis. IMPACT AND IMPLICATIONS: Roux-en-Y gastric bypass (RYGB) is a widely used bariatric surgery procedure with proven efficacy in body weight management, combatting cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD). RYGB exerts many beneficial metabolic effects, by lowering plasma cholesterol and improving atherogenic dyslipidemia. Using a cohort of patients undergoing RYGB, studied before and 1 year after surgery, we analyzed how RYGB modulates hepatic and systemic cholesterol and bile acid metabolism. The results of our study provide important insights on the regulation of cholesterol homeostasis after RYGB and open avenues that could guide future monitoring and treatment strategies targeting CVD and NAFLD in obesity.

Multiple Selection Criteria for Probiotic Strains with High Potential for Obesity Management.

Since alterations of the gut microbiota have been shown to play a major role in obesity, probiotics have attracted attention. Our aim was to identify probiotic candidates for the management of obesity using a combination of in vitro and in vivo approaches. We evaluated in vitro the ability of 23 strains to limit lipid accumulation in adipocytes and to enhance the secretion of satiety-promoting gut peptide in enteroendocrine cells. Following the in vitro screening, selected strains were further investigated in vivo, single, or as mixtures, using a murine model of diet-induced obesity. Strain Bifidobacterium longum PI10 administrated alone and the mixture of B. animalis subsp. lactis LA804 and Lactobacillus gasseri LA806 limited body weight gain and reduced obesity-associated metabolic dysfunction and inflammation. These protective effects were associated with changes in the hypothalamic gene expression of leptin and leptin receptor as well as with changes in the composition of gut microbiota and the profile of bile acids. This study provides crucial clues to identify new potential probiotics as effective therapeutic approaches in the management of obesity, while also providing some insights into their mechanisms of action.

NASH-related increases in plasma bile acid levels depend on insulin resistance.

BACKGROUND & AIMS: Plasma bile acids (BAs) have been extensively studied as pathophysiological actors in non-alcoholic steatohepatitis (NASH). However, results from clinical studies are often complicated by the association of NASH with type 2 diabetes (T2D), obesity, and insulin resistance (IR). Here, we sought to dissect the relationship between NASH, T2D, and plasma BA levels in a large patient cohort. METHODS: Four groups of patients from the Biological Atlas of Severe Obesity (ABOS) cohort (Clinical Trials number NCT01129297) were included based on the presence or absence of histologically evaluated NASH with or without coincident T2D. Patients were matched for BMI, homeostatic model assessment 2 (HOMA2)-assessed IR, glycated haemoglobin, age, and gender. To study the effect of IR and BMI on the association of plasma BA and NASH, patients from the HEPADIP study were included. In both cohorts, fasting plasma BA concentrations were measured. RESULTS: Plasma BA concentrations were higher in NASH compared with No-NASH patients both in T2D and NoT2D patients from the ABOS cohort. As we previously reported that plasma BA levels were unaltered in NASH patients of the HEPADIP cohort, we assessed the impact of BMI and IR on the association of NASH and BA on the combined BA datasets. Our results revealed that NASH-associated increases in plasma total cholic acid (CA) concentrations depend on the degree of HOMA2-assessed systemic IR, but not on ß-cell function nor on BMI. CONCLUSIONS: Plasma BA concentrations are elevated only in those NASH patients exhibiting pronounced IR. LAY SUMMARY: Non-alcoholic steatohepatitis (NASH) is a progressive liver disease that frequently occurs in patients with obesity and type 2 diabetes. Reliable markers for the diagnosis of NASH are needed. Plasma bile acids have been proposed as NASH biomarkers. Herein, we found that plasma bile acids are only elevated in patients with NASH when significant insulin resistance is present, limiting their utility as NASH markers.

Characterization of one anastomosis gastric bypass and impact of biliary and common limbs on bile acid and postprandial glucose metabolism in a minipig model.

The alimentary limb has been proposed to be a key driver of the weight-loss-independent metabolic improvements that occur upon bariatric surgery. However, the one anastomosis gastric bypass (OAGB) procedure, consisting of one long biliary limb and a short common limb, induces similar beneficial metabolic effects compared to Roux-en-Y Gastric Bypass (RYGB) in humans, despite the lack of an alimentary limb. The aim of this study was to assess the role of the length of biliary and common limbs in the weight loss and metabolic effects that occur upon OAGB. OAGB and sham surgery, with or without modifications of the length of either the biliary limb or the common limb, were performed in Gottingen minipigs. Weight loss, metabolic changes, and the effects on plasma and intestinal bile acids (BAs) were assessed 15 days after surgery. OAGB significantly decreased body weight, improved glucose homeostasis, increased postprandial GLP-1 and fasting plasma BAs, and qualitatively changed the intestinal BA species composition. Resection of the biliary limb prevented the body weight loss effects of OAGB and attenuated the postprandial GLP-1 increase. Improvements in glucose homeostasis along with changes in plasma and intestinal BAs occurred after OAGB regardless of the biliary limb length. Resection of only the common limb reproduced the glucose homeostasis effects and the changes in intestinal BAs. Our results suggest that the changes in glucose metabolism and BAs after OAGB are mainly mediated by the length of the common limb, whereas the length of the biliary limb contributes to body weight loss.NEW & NOTEWORTHY Common limb mediates postprandial glucose metabolism change after gastric bypass whereas biliary limb contributes to weight loss.

Circulating biomarkers of nitric oxide bioactivity and impaired muscle vasoreactivity to exercise in adults with uncomplicated type 1 diabetes.

AIMS/HYPOTHESIS: Early compromised endothelial function challenges the ability of individuals with type 1 diabetes to perform normal physical exercise. The exact mechanisms underlying this vascular limitation remain unknown, but may involve either formation or metabolism of nitric oxide (NO), a major vasodilator, whose activity is known to be compromised by oxidative stress. METHODS: Muscle microvascular reactivity (near-infrared spectroscopy) to an incremental exhaustive bout of exercise was assessed in 22 adults with uncomplicated type 1 diabetes (HbA1c 64.5 ± 15.7 mmol/mol; 8.0 ± 1.4%) and in 21 healthy individuals (18-40 years of age). NO-related substrates/metabolites were also measured in the blood along with other vasoactive compounds and oxidative stress markers; measurements were taken at rest, at peak exercise and after 15 min of recovery. Demographic characteristics, body composition, smoking status and diet were comparable in both groups. RESULTS: Maximal oxygen uptake was impaired in individuals with type 1 diabetes compared with in healthy participants (35.6 ± 7.7 vs 39.6 ± 6.8 ml min-1 kg-1, p < 0.01) despite comparable levels of habitual physical activity (moderate to vigorous physical activity by accelerometery, 234.9 ± 160.0 vs 280.1 ± 114.9 min/week). Compared with non-diabetic participants, individuals with type 1 diabetes also displayed a blunted exercise-induced vasoreactivity (muscle blood volume at peak exercise as reflected by ∆ total haemoglobin, 2.03 ± 5.82 vs 5.33 ± 5.54 µmol/l; interaction 'exercise' × 'group', p < 0.05); this was accompanied by lower K+ concentration (p < 0.05), reduced plasma L-arginine (p < 0.05)-in particular when HbA1c was high (mean estimation: -4.0, p < 0.05)-and lower plasma urate levels (p < 0.01). Nonetheless, exhaustive exercise did not worsen lipid peroxidation or other oxidative stress biomarkers, and erythrocytic enzymatic antioxidant resources were mobilised to a comparable extent in both groups. Nitrite and total nitrosation products, which are potential alternative NO sources, were similarly unaltered. Graphical abstract CONCLUSIONS/INTERPRETATION: Participants with uncomplicated type 1 diabetes displayed reduced availability of L-arginine, the essential substrate for enzymatic nitric oxide synthesis, as well as lower levels of the major plasma antioxidant, urate. Lower urate levels may reflect a defect in the activity of xanthine oxidase, an enzyme capable of producing NO from nitrite under hypoxic conditions. Thus, both canonical and non-canonical NO production may be reduced. However, neither of these changes exacerbated exercise-induced oxidative stress. TRIAL REGISTRATION: clinicaltrials.gov NCT02051504.

Simultaneous determination of di(2-ethylhexyl) phthalate and diisononylcyclohexane-1,2-dicarboxylate and their monoester metabolites in four labile blood products by liquid chromatography tandem mass spectrometry.

Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer that is largely used for PVC blood bags. The migration of DEHP from medical devices into labile blood products (LBP) is a well-known situation. While DEHP has beneficial effects on the storage of red blood cells, it can have toxicological impact due to its potential reprotoxic effects (classified group 1B). Since July 1st, 2015, the French law prohibits the use of tubing made in DEHP-plasticized PVC in paediatric, neonatal and maternity wards. This provision, which could extend in several years more widely to medical devices used for drugs infusion, dialysis, feeding and blood bags, has led manufacturers to replace DEHP to alternative plasticizers such as diisononylcyclohexane-1,2-dicarboxylate (DINCH). In this paper, a liquid chromatography-tandem mass spectrometry (LCMS/MS) method has been developed and validated for the determination of DEHP, DINCH and their corresponding monoester metabolites (MEHP and MINCH) in four labile blood products (LBP): whole blood (WB), red cells concentrate (RCC), plasma and platelet concentrate (PC). Due to strong contamination of blank LBP by DEHP because of its ubiquitous presence in working environment and despite the attention paid to avoid contamination of solvents and glassware, a trap chromatographic column was implemented between the solvent mixing chamber and the injector of the LC system. This set-up permitted to discriminate DEHP present in the sample to DEHP brought by the environmental contamination. In the optimized conditions, all compounds were separated in less than 10 min. The analytes were extracted from LBP samples using a liquid-liquid extraction. After optimization, recoveries were ranged from 47 to 96 %, depending on the analytes and the nature of LBP. Except for DEHP which exhibited RSD values of intermediate precision higher than 20 % at a concentration of 25 nM, all the precision results (repeatability and intermediate precision) were lower than 16 % and trueness values ranged from -16.2-19.8%. Using the validated method, the leachability of DEHP and DINCH from corresponding PVC-blood bags was investigated and the concentrations of their corresponding metabolites, MEHP and MINCH, were determined in whole blood, red cells concentrate, plasma and platelet concentrate.

Bile acids associate with glucose metabolism, but do not predict conversion from impaired fasting glucose to diabetes.

OBJECTIVE: Bile acids (BAs) are signaling molecules controlling lipid and glucose metabolism. Since BA alterations are associated with obesity and insulin resistance, plasma BAs have been considered candidates to predict type 2 diabetes (T2D) risk. We aimed to determine (1) the association of BAs with glucose homeostasis parameters and (2) their predictive association with the risk of conversion from prediabetes to new-onset diabetes (NOD) in a prospective cohort study. DESIGN: 205 patients with impaired fasting glucose (IFG) were followed each year during 5 years in the IT-DIAB cohort study. Twenty-one BA species and 7α-hydroxy-4-cholesten-3-one (C4), a marker of BA synthesis, were quantified by LC/MS-MS in plasma from fasted patients at baseline. Correlations between plasma BA species and metabolic parameters at baseline were assessed by Spearman's coefficients and the association between BAs and NOD was determined using Cox proportional-hazards models. RESULTS: Among the analyzed BA species, total hyocholic acid (HCA) and the total HCA/total chenodeoxycholic acid (CDCA) ratio, reflecting hepatic BA 6α-hydroxylation activity, negatively correlated with BMI and HOMA-IR. The total HCA/total CDCA ratio also correlated negatively with HbA1C. Conversion from IFG to NOD occurred in 33.7% of the participants during the follow-up. Plasma BA species were not independently associated with the conversion to NOD after adjustment with classical T2D risk factors. CONCLUSIONS: Fasting plasma BAs are not useful clinical biomarkers for predicting NOD in patients with IFG. However, an unexpected association between 6α-hydroxylated BAs and glucose parameters was found, suggesting a role for this specific BA pathway in metabolic homeostasis. IT-DIAB study registry number: NCT01218061.

New piperazine multi-effect drugs prevent neurofibrillary degeneration and amyloid deposition, and preserve memory in animal models of Alzheimer's disease.

Alzheimer's Disease is a devastating dementing disease involving amyloid deposits, neurofibrillary tangles, progressive and irreversible cognitive impairment. Today, only symptomatic drugs are available and therapeutic treatments, possibly acting at a multiscale level, are thus urgently needed. To that purpose, we designed multi-effects compounds by synthesizing drug candidates derived by substituting a novel N,N'-disubstituted piperazine anti-amyloid scaffold and adding acetylcholinesterase inhibition property. Two compounds were synthesized and evaluated. The most promising hybrid molecule reduces both the amyloid pathology and the Tau pathology as well as the memory impairments in a preclinical model of Alzheimer's disease. In vitro also, the compound reduces the phosphorylation of Tau and inhibits the release of Aß peptides while preserving the processing of other metabolites of the amyloid precursor protein. We synthetized and tested the first drug capable of ameliorating both the amyloid and Tau pathology in animal models of AD as well as preventing the major brain lesions and associated memory impairments. This work paves the way for future compound medicines against both Alzheimer's-related brain lesions development and the associated cognitive impairments.

Plasma asymmetric dimethylarginine concentrations are not related to differences in maximal oxygen uptake in endurance trained and untrained men.

NEW FINDINGS: What is the central question of this study? Is there an association of plasma concentration of asymmetric dimethylarginine, which is related to exercise capacity in patients with cardiovascular diseases, with oxygen delivery and subsequently exercise capacity in healthy subjects in the absence of the potentially confounding influence of inflammation and oxidative stress? What is the main finding and its importance? Plasma asymmetric dimethylarginine concentrations are not related to exercise capacity in healthy subjects, while O2 delivery in the working skeletal muscle during the maximal graded-exercise test is not associated with any of the l-arginine analogues. ADMA alone does not play a crucial role in local muscle perfusion and in maintaining exercise capacity. ABSTRACT: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis that could limit oxygen (O2 ) delivery in the working skeletal muscles by altering endothelium-dependent vasodilatation. Exercise capacity is associated with plasma ADMA concentrations in patients with cardiovascular diseases, but this issue has still not been investigated in healthy subjects. We aimed to determine whether plasma ADMA concentrations were negatively associated with exercise capacity in young healthy male subjects. Ten men with maximal oxygen uptake ( V ̇ O 2 max ) > 65 mL kg-1  min-1 were included in the high exercise capacity group (HI-FIT), and 10 men with V ̇ O 2 max  < 45 mL kg-1  min-1 were included in the low exercise capacity group (LO-FIT). Plasma ADMA and other l-arginine analogue concentrations were measured before and after a maximal graded-exercise test by liquid chromatography-tandem mass spectrometry. Microvascular O2 delivery during exercise was estimated through the pattern from the sigmoid model of muscle deoxygenation in the vastus lateralis measured by near infrared spectroscopy. V ̇ O 2 max was 60% higher in the HI-FIT group (median: 70.2 mL kg-1  min-1 ; IQR: 68.0-71.9 mL kg-1  min-1 ) than in the LO-FIT group (median: 43.8 mL kg-1  min-1 ; IQR: 34.8-45.3 mL kg-1  min-1 ). Plasma ADMA concentrations did not differ between the LO-FIT and HI-FIT groups before (0.50 ± 0.06 vs. 0.54 ± 0.07 µmol L-1 , respectively) and after the maximal incremental exercise test (0.49 ± 0.08 vs. 0.55 ± 0.03 µmol L-1 , respectively). There was no significant association of plasma ADMA concentrations with the pattern of local muscle deoxygenation and exercise capacity. Exercise capacity and microvascular O2 delivery are not related to plasma ADMA concentrations in young healthy male subjects. Our findings show that ADMA does not play a crucial role in local muscle perfusion and in maintaining exercise capacity without pathological conditions.

A pharmaco-metabolomics approach in a clinical trial of ALS: Identification of predictive markers of progression.

There is an urgent and unmet need for accurate biomarkers in Amyotrophic Lateral Sclerosis. A pharmaco-metabolomics study was conducted using plasma samples from the TRO19622 (olesoxime) trial to assess the link between early metabolomic profiles and clinical outcomes. Patients included in this trial were randomized into either Group O receiving olesoxime (n = 38) or Group P receiving placebo (n = 36). The metabolomic profile was assessed at time-point one (V1) and 12 months (V12) after the initiation of the treatment. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites (Biocrates® commercial kit). Multivariate analysis based on machine learning approaches (i.e. Biosigner algorithm) was performed. Metabolomic profiles at V1 and V12 and changes in metabolomic profiles between V1 and V12 accurately discriminated between Groups O and P (p<5×10-6), and identified glycine, kynurenine and citrulline/arginine as the best predictors of group membership. Changes in metabolomic profiles were closely linked to clinical progression, and correlated with glutamine levels in Group P and amino acids, lipids and spermidine levels in Group O. Multivariate models accurately predicted disease progression and highlighted the discriminant role of sphingomyelins (SM C22:3, SM C24:1, SM OH C22:2, SM C16:1). To predict SVC from SM C24:1 in group O and SVC from SM OH C22:2 and SM C16:1 in group P+O, we noted a median sensitivity between 67% and 100%, a specificity between 66.7 and 71.4%, a positive predictive value between 66 and 75% and a negative predictive value between 70% and 100% in the test sets. This proof-of-concept study demonstrates that the metabolomics has a role in evaluating the biological effect of an investigational drug and may be a candidate biomarker as a secondary outcome measure in clinical trials.

One-week cocoa flavanol intake increases prefrontal cortex oxygenation at rest and during moderate-intensity exercise in normoxia and hypoxia.

During exercise in hypoxia, O2 delivery to brain and muscle is compromised, and oxidative stress is elicited. Cocoa flavanols (CF) have antioxidant capacities and can increase blood flow by stimulating endothelial function. We aimed to examine the effects of 7-day CF intake on oxidative stress, nitric oxide production, and tissue oxygenation in response to exercise in normobaric hypoxia (14.3% O2). In a randomized, double-blind, cross-over study, 14 well-trained male cyclists completed four trials: exercise in normoxia or hypoxia, after 7-day CF or placebo intake. Flow-mediated dilation (FMD) was measured before intake of the last dose CF or placebo. One hundred minutes later, 20-min steady-state (SS; 45% V̇o2max) and 20-min time trial (TT) (cycling) were performed. Blood samples were taken. Prefrontal and muscular oxygenation was assessed by near-infrared spectroscopy. At baseline, FMD was increased by CF. Hypoxia increased exercise-induced elevations in lipid peroxidation and antioxidant capacity. CF suppressed exercise-induced lipid peroxidation but did not influence antioxidant capacity. At rest and during SS, prefrontal and muscular oxygenation was decreased by hypoxia. CF elevated prefrontal oxygenation but did not impact muscular oxygenation. During TT, hypoxia accelerated the exercise-induced decrease in prefrontal oxygenation, but not in muscular oxygenation. During TT, CF did not alter prefrontal and muscular oxygenation. CF did not change plasma nitrite, nitrate, and arginine:citrulline. During high-intensity exercise, CF improved neither tissue oxygenation nor performance in well-trained athletes. At rest and during moderate-intensity exercise, CF reduced exercise-induced lipid peroxidation and partially restored the hypoxia-induced decline in prefrontal oxygenation. NEW & NOTEWORTHY For the first time, we showed that CF had beneficial effects on endothelial function at rest, as well as on prefrontal oxygenation at rest and during moderate-intensity exercise, both in normoxia and hypoxia. Moreover, we showed that CF intake inhibited oxidative stress during exhaustive exercise in hypoxia.

Acute cocoa Flavanols intake has minimal effects on exercise-induced oxidative stress and nitric oxide production in healthy cyclists: a randomized controlled trial.

BACKGROUND: Cocoa flavanols (CF) can stimulate vasodilation by improved nitric oxide (NO) synthesis and have antioxidant and anti-inflammatory capacities. This study aimed to examine whether acute CF intake can affect exercise-induced changes in antioxidant capacity, oxidative stress, inflammation and NO production, as well as exercise performance and recovery in well-trained cyclists. METHODS: Twelve well-trained male cyclists (mean ± SD age, VO2max: 30 ± 3 years, 63.0 ± 3.5 ml/kg/min) participated in this randomized, double-blind, cross over study. On 2 separate occasions, subjects performed two 30-min time trials 1.5 (TT1) and 3 (TT2) hours after CF (900 mg CF) or placebo (PL, 13 mg CF) intake, interposed by passive rest. Lactate, glucose, heartrate, rating of perceived exertion (RPE) and power output were measured during the TTs. Blood was drawn at baseline, before and after each TT and analyzed for epicatechin serum concentrations, trolox equivalent antioxidative capacity (TEAC), uric acid (UA), malonaldehyde (MDA), L-arginine/ADMA, citrulline, interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α plasma concentrations. Relative changes in blood markers and pacing strategy during TT were analysed by repeated measured ANOVA. TT performance was compared between PL and CF by paired t-test. RESULTS: Epicatechin concentrations were increased by CF intake. Exercise-induced increase in TEAC/UA was improved by CF intake (F(1) = 5.57; p = .038) (post-TT1: PL: 113.34 ± 3.9%, CF: 117.64 ± 3.96%, post-TT2: PL: 108.59 ± 3.95%, CF: 123.72 ± 7.4% to baseline), while exercise-induced increases in MDA, IL-1 and IL-6 were not affected by CF intake. TNF-α was unaltered by exercise and by CF. Exercise-induced decreases in L-arginine/ADMA and increases in citrulline were not affected by CF intake. TT1 and TT2 performance and exercise-induced physiological changes were unaffected by CF intake. CONCLUSION: Acute CF intake increased total antioxidant capacity in rest and during exercise, but did not affect exercise-induced lipid peroxidation, inflammation, nor NO production in healthy athletes. Acute CF intake did not improve TT performance and recovery. TRIAL REGISTRATION: ISRCTN32875, 21-11-2016, retrospectively registered.

Omics to Explore Amyotrophic Lateral Sclerosis Evolution: the Central Role of Arginine and Proline Metabolism.

In amyotrophic lateral sclerosis (ALS), motor neuron degeneration is associated with systemic metabolic impairment. However, the evolution of metabolism alteration is partially unknown and its link with disease progression has never been described. For the first time, we ran a study focused on (1) the evolution of metabolism disturbance during disease progression through omics approaches and (2) the relation between metabolome profile and clinical evolution. SOD1-G93A (mSOD1) transgenic mice (n = 11) and wild-type (WT) littermates (n = 17) were studied during 20 weeks. Metabolomic profile of muscle and cerebral cortex was analysed at week 20, and plasma samples were assessed at four time points over 20 weeks. The relevant metabolic pathways highlighted by metabolomic analysis were explored by a targeted transcriptomic approach in mice. Plasma metabolomics were also performed in 24 ALS patients and 24 gender- and age-matched controls. Metabolomic analysis of muscle and cerebral cortex enabled an excellent discrimination between mSOD1 and WT mice (p < 0.001). These alterations included especially tryptophan, arginine, and proline metabolism pathways (including polyamines) as also revealed by transcriptomic analysis and findings in ALS patients. Multivariate models performed to explain clinical findings in ALS mice, and patients were excellent (p < 0.01) and highlighted three main metabolic pathways: arginine and proline, tryptophan, and branched amino acid metabolism. This work is the first longitudinal study that evaluates metabolism alteration in ALS, including the analysis of different tissues and using a combination of omics methods. We particularly identified arginine and proline metabolism. This pathway is also associated with disease progression and may open new perspectives of therapeutic targets.

Combined Metabolomics and Transcriptomics Approaches to Assess the IL-6 Blockade as a Therapeutic of ALS: Deleterious Alteration of Lipid Metabolism.

In amyotrophic lateral sclerosis (ALS), motor neuron degeneration occurs simultaneously with systemic metabolic impairment and neuroinflammation. Playing an important role in the regulation of both phenomena, interleukin (IL)-6, a major cytokine of the inflammatory response has been proposed as a target for management of ALS. Although a pilot clinical trial provided promising results in humans, another recent preclinical study showed that knocking out the IL-6 gene in mice carrying ALS did not improve clinical outcome. In this study, we aimed to determine the relevance of the IL-6 pathway blockade in a mouse model of ALS by using a pharmacological antagonist of IL-6, a murine surrogate of tocilizumab, namely MR16-1. We analyzed the immunological and metabolic effects of IL-6 blockade by cytokine measurement, blood cell immunophenotyping, targeted metabolomics, and transcriptomics. A deleterious clinical effect of MR16-1 was revealed, with a speeding up of weight loss (p = 0.0041) and decreasing body weight (p < 0.05). A significant increase in regulatory T-cell count (p = 0.0268) and a decrease in C-X-C ligand-1 concentrations in plasma (p = 0.0479) were observed. Metabolomic and transcriptomic analyses revealed that MR16-1 mainly affected branched-chain amino acid, lipid, arginine, and proline metabolism. IL-6 blockade negatively affected body weight, despite a moderated anti-inflammatory effect. Metabolic effects of IL-6 were mild compared with metabolic disturbances observed in ALS, but a modification of lipid metabolism by therapy was identified. These results indicate that IL-6 blockade did not improve clinical outcome of a mutant superoxide dismutase 1 mouse model of ALS.