Endothelin receptor B-deficient mice are protected from high-fat diet-induced metabolic syndrome.

OBJECTIVE: Endothelin receptor B (ETB) together with ETA mediates cellular effects of endothelin 1 (ET-1), an autocrine and endocrine peptide produced by the endothelium and other cells. It regulates vascular tone and controls kidney function. Metabolic syndrome is due to high caloric intake and is characterized by insulin resistance, dyslipidemia, and white adipose tissue (WAT) accumulation. ETA/ETB antagonism has been demonstrated to favorably influence insulin resistance. Our study explored the role of ETB in metabolic syndrome. METHODS: Wild type (etb+/+) and rescued ETB-deficient (etb-/-) mice were fed a high-fat diet, and energy, glucose, and insulin metabolism were analyzed, and hormones and lipids measured in serum and tissues. Cell culture experiments were performed in HepG2 cells. RESULTS: Compared to etb+/+ mice, etb-/- mice exhibited better glucose tolerance and insulin sensitivity, less WAT accumulation, lower serum triglycerides, and higher energy expenditure. Protection from metabolic syndrome was paralleled by higher hepatic production of fibroblast growth factor 21 (FGF21) and higher serum levels of free thyroxine (fT4), stimulators of energy expenditure. CONCLUSIONS: ETB deficiency confers protection from metabolic syndrome by counteracting glucose intolerance, dyslipidemia, and WAT accumulation due to enhanced energy expenditure, effects at least in part dependent on enhanced production of thyroid hormone/FGF21. ETB antagonism may therefore be a novel therapeutic approach in metabolic syndrome.

A New Biomarker Profiling Strategy for Gut Microbiome Research: Valid Association of Metabolites to Metabolism of Microbiota Detected by Non-Targeted Metabolomics in Human Urine.

The gut microbiome is of tremendous relevance to human health and disease, so it is a hot topic of omics-driven biomedical research. However, a valid identification of gut microbiota-associated molecules in human blood or urine is difficult to achieve. We hypothesize that bowel evacuation is an easy-to-use approach to reveal such metabolites. A non-targeted and modifying group-assisted metabolomics approach (covering 40 types of modifications) was applied to investigate urine samples collected in two independent experiments at various time points before and after laxative use. Fasting over the same time period served as the control condition. As a result, depletion of the fecal microbiome significantly affected the levels of 331 metabolite ions in urine, including 100 modified metabolites. Dominating modifications were glucuronidations, carboxylations, sulfations, adenine conjugations, butyrylations, malonylations, and acetylations. A total of 32 compounds, including common, but also unexpected fecal microbiota-associated metabolites, were annotated. The applied strategy has potential to generate a microbiome-associated metabolite map (M3) of urine from healthy humans, and presumably also other body fluids. Comparative analyses of M3 vs. disease-related metabolite profiles, or therapy-dependent changes may open promising perspectives for human gut microbiome research and diagnostics beyond analyzing feces.

Automated LC-MS/MS: Ready for the clinical routine Laboratory?

Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a sensitive method with high specificity. However, its routine use in the clinical laboratory is hampered by its high complexity and lack of automation. Studies demonstrate excellent analytical performance using the first fully automated LC-MS/MS for 25-hydroxy vitamin D and immunosuppressant drugs (ISD) in hospital routine laboratories. Objectives: Our objectives were (1) to verify the suitability of an automated LC-MS/MS in a commercial laboratory, which differs from the needs of hospital laboratories, and (2) examine its usability among operators with various professional backgrounds. Methods: We assessed the analytical assay performance for vitamin D and the ISDs cyclosporine A and tacrolimus over five months. The assays were compared to an identical analyzer in a hospital laboratory, to in-house LC-MS/MS methods, and to chemiluminescent microparticle immunoassays (CMIA). Nine operators evaluated the usability of the fully automated LC-MS/MS system by means of a structured questionnaire. Results: The automated system exhibited a high precision (CV < 8%), accuracy (bias < 7%) and good agreement with concentrations of external quality assessment (EQA) samples. Comparable results were obtained with an identical analyzer in a hospital routine laboratory. Acceptable median deviations of results versus an in-house LC-MS/MS were observed for 25-OH vitamin D3 (-10.6%), cyclosporine A (-4.3%) and tacrolimus (-6.6%). The median bias between the automated system and immunoassays was only acceptable for 25-OH vitamin D3 (6.6%). All users stated that they had had a good experience with the fully automated LC-MS/MS system. Conclusions: A fully automated LC-MS/MS can be easily integrated for routine diagnostics in a commercial laboratory.

Ex vivo instability of lipids in whole blood: preanalytical recommendations for clinical lipidomics studies.

Reliability, robustness, and interlaboratory comparability of quantitative measurements is critical for clinical lipidomics studies. Lipids' different ex vivo stability in blood bears the risk of misinterpretation of data. Clear recommendations for the process of blood sample collection are required. We studied by UHPLC-high resolution mass spectrometry, as part of the "Preanalytics interest group" of the International Lipidomics Society, the stability of 417 lipid species in EDTA whole blood after exposure to either 4°C, 21°C, or 30°C at six different time points (0.5 h-24 h) to cover common daily routine conditions in clinical settings. In total, >800 samples were analyzed. 325 and 288 robust lipid species resisted 24 h exposure of EDTA whole blood to 21°C or 30°C, respectively. Most significant instabilities were detected for FA, LPE, and LPC. Based on our data, we recommend cooling whole blood at once and permanent. Plasma should be separated within 4 h, unless the focus is solely on robust lipids. Lists are provided to check the ex vivo (in)stability of distinct lipids and potential biomarkers of interest in whole blood. To conclude, our results contribute to the international efforts towards reliable and comparable clinical lipidomics data paving the way to the proper diagnostic application of distinct lipid patterns or lipid profiles in the future.

The Acute Cytokine Response to 30-Minute Exercise Bouts Before and After 8-Week Endurance Training in Individuals With Obesity.

CONTEXT: One acute bout of exercise leads to a rapid increase in the systemic cytokine concentration. Regular exercise might alter the cytokine response, in particular in beforehand untrained and obese individuals. OBJECTIVE: Using a proximity extension assay, we studied the effects of acute exercise as well as endurance training on a panel of 92 cytokines related to inflammation. METHODS: A total of 22 individuals (30 ± 9 years; peak oxygen uptake [VO2peak] 25.2 ± 4.2 mL/[kg × min]; body mass index [BMI] 31.7 ± 4.4) participated in an 8-week endurance exercise intervention. Blood samples were collected before and immediately after 30 minutes' ergometer exercise at 80% VO2peak. RESULTS: Before and after the training intervention, 40 and 37 cytokines, respectively, were acutely increased more than 1.2-fold (Benjamini-Hochberg [BH]-adjusted P < .05). The exercise intervention did not change the acute increase in cytokines nor the resting cytokine levels, whereas fitness was improved and adiposity reduced. The increase in fitness led to a slight increase in power output when exercising at the same heart rate, which might explain the comparable increase in cytokines before and after the intervention. The largest acute increase was found for OSM, TGFA, CXCL1 and 5, and TNFSF14 (≥ 1.9-fold, BH-adjusted P < .001). The transcript levels of these proteins in whole blood were also elevated, particularly in the trained state. Only the acute increase in IL6 (1.3-fold) was related to the increase in lactate, confirming the lactate-driven secretion of IL6. CONCLUSION: Our comprehensive proteomics approach detected several underexplored serum exerkines with up to now less understood function in the adaptation to exercise.

Exercise restores brain insulin sensitivity in sedentary adults who are overweight and obese.

BACKGROUNDInsulin resistance of the brain can unfavorably affect long-term weight maintenance and body fat distribution. Little is known if and how brain insulin sensitivity can be restored in humans. We aimed to evaluate the effects of an exercise intervention on insulin sensitivity of the brain and how this relates to exercise-induced changes in whole-body metabolism and behavior.METHODSIn this clinical trial, sedentary participants who were overweight and obese underwent an 8-week supervised aerobic training intervention. Brain insulin sensitivity was assessed in 21 participants (14 women, 7 men; age range 21-59 years; BMI range 27.5-45.5 kg/m2) using functional MRI, combined with intranasal administration of insulin, before and after the intervention.RESULTSThe exercise program resulted in enhanced brain insulin action to the level of a person of healthy weight, demonstrated by increased insulin-induced striatal activity and strengthened hippocampal functional connectivity. Improved brain insulin action correlated with increased mitochondrial respiration in skeletal muscle, reductions in visceral fat and hunger, as well as improved cognition. Mediation analyses suggest that improved brain insulin responsiveness helps mediate the peripheral exercise effects leading to healthier body fat distribution and reduced perception of hunger.CONCLUSIONOur study demonstrates that an 8-week exercise intervention in sedentary individuals can restore insulin action in the brain. Hence, the ameliorating benefits of exercise toward brain insulin resistance may provide an objective therapeutic target in humans in the challenge to reduce diabetes risk factors.TRIAL REGISTRATIONClinicalTrials.gov (NCT03151590).FUNDINGBMBF/DZD 01GI0925.

Diagnostic Performance of Sex-Specific Modified Metabolite Patterns in Urine for Screening of Prediabetes.

Aims/Hypothesis: Large-scale prediabetes screening is still a challenge since fasting blood glucose and HbA1c as the long-standing, recommended analytes have only moderate diagnostic sensitivity, and the practicability of the oral glucose tolerance test for population-based strategies is limited. To tackle this issue and to identify reliable diagnostic patterns, we developed an innovative metabolomics-based strategy deviating from common concepts by employing urine instead of blood samples, searching for sex-specific biomarkers, and focusing on modified metabolites. Methods: Non-targeted, modification group-assisted metabolomics by liquid chromatography-mass spectrometry (LC-MS) was applied to second morning urine samples of 340 individuals from a prediabetes cohort. Normal (n = 208) and impaired glucose-tolerant (IGT; n = 132) individuals, matched for age and BMI, were randomly divided in discovery and validation cohorts. ReliefF, a feature selection algorithm, was used to extract sex-specific diagnostic patterns of modified metabolites for the detection of IGT. The diagnostic performance was compared with conventional screening parameters fasting plasma glucose (FPG), HbA1c, and fasting insulin. Results: Female- and male-specific diagnostic patterns were identified in urine. Only three biomarkers were identical in both. The patterns showed better AUC and diagnostic sensitivity for prediabetes screening of IGT than FPG, HbA1c, insulin, or a combination of FPG and HbA1c. The AUC of the male-specific pattern in the validation cohort was 0.889 with a diagnostic sensitivity of 92.6% and increased to an AUC of 0.977 in combination with HbA1c. In comparison, the AUCs of FPG, HbA1c, and insulin alone reached 0.573, 0.668, and 0.571, respectively. Validation of the diagnostic pattern of female subjects showed an AUC of 0.722, which still exceeded the AUCs of FPG, HbA1c, and insulin (0.595, 0.604, and 0.634, respectively). Modified metabolites in the urinary patterns include advanced glycation end products (pentosidine-glucuronide and glutamyl-lysine-sulfate) and microbiota-associated compounds (indoxyl sulfate and dihydroxyphenyl-gamma-valerolactone-glucuronide). Conclusions/Interpretation: Our results demonstrate that the sex-specific search for diagnostic metabolite biomarkers can be superior to common metabolomics strategies. The diagnostic performance for IGT detection was significantly better than routinely applied blood parameters. Together with recently developed fully automatic LC-MS systems, this opens up future perspectives for the application of sex-specific diagnostic patterns for prediabetes screening in urine.

Exercise-Induced N-Lactoylphenylalanine Predicts Adipose Tissue Loss during Endurance Training in Overweight and Obese Humans.

Physical exercise is a powerful measure to prevent cardiometabolic diseases. However, the individual response to lifestyle interventions is variable and cannot, to date, be predicted. N-Lactoylphenylalanine (Lac-Phe) produced during exercise has recently been shown to mediate weight loss in obese mice. Lac-Phe could also contribute to, and potentially explain differences in, the effectiveness of exercise interventions in humans. Sedentary overweight and obese subjects completed an 8-week supervised endurance exercise intervention (n = 22). Before and after the intervention, plasma levels of Lac-Phe were determined by UHPLC-MS in the resting state and immediately after an acute bout of endurance exercise. Adipose tissue volume was quantified using MRI. Acute exercise caused a pronounced increase in Lac-Phe, both before and after the intervention. Higher levels of Lac-Phe after acute exercise were associated with a greater reduction in abdominal subcutaneous and, to a lower degree, visceral adipose tissue during the intervention. Lac-Phe produced during physical activity could contribute to weight loss by acting as a signaling molecule that regulates food intake, as previously shown in mice. Quantification of Lac-Phe during an exercise test could be employed as a tool to predict and potentially improve the individual response to exercise-based lifestyle interventions in overweight humans and those with obesity.

Exercise prevents fatty liver by modifying the compensatory response of mitochondrial metabolism to excess substrate availability.

OBJECTIVE: Liver mitochondria adapt to high-calorie intake. We investigated how exercise alters the early compensatory response of mitochondria, thus preventing fatty liver disease as a long-term consequence of overnutrition. METHODS: We compared the effects of a steatogenic high-energy diet (HED) for six weeks on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome, and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high-resolution respirometry. RESULTS: HED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism, and lipogenesis. Training also counteracted the HED-induced glucose intolerance, the increase in fasting insulin, and in liver fat by lowering diacylglycerol species and c-Jun N-terminal kinase (JNK) phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content. CONCLUSION: We provide a comprehensive insight into the early adaptations of mitochondria in the liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity.

Signals from the Circle: Tricarboxylic Acid Cycle Intermediates as Myometabokines.

Regular physical activity is an effective strategy to prevent and ameliorate aging-associated diseases. In particular, training increases muscle performance and improves whole-body metabolism. Since exercise affects the whole organism, it has countless health benefits. The systemic effects of exercise can, in part, be explained by communication between the contracting skeletal muscle and other organs and cell types. While small proteins and peptides known as myokines are the most prominent candidates to mediate this tissue cross-talk, recent investigations have paid increasing attention to metabolites. The purpose of this review is to highlight the potential role of tricarboxylic acid (TCA) metabolites as humoral mediators of exercise adaptation processes. We focus on TCA metabolites that are released from human skeletal muscle in response to exercise and provide an overview of their potential auto-, para- or endocrine health-promoting effects.

Comparison of the metabolome in urine prior and eight weeks after radical prostatectomy uncovers pathologic and molecular features of prostate cancer.

Prostate cancer (PCa) is associated with cellular metabolism alterations leading to changes of the metabolome. So far, studies investigating these alterations mainly focused on comparisons of metabolite profiles of PCa patients and healthy controls. In the present study we compared for the first time metabolite profiles in a significant number of paired urine samples collected before and eight weeks after radical prostatectomy (rPX) in 34 patients with PCa. Our comprehensive non-targeted liquid chromatographic-mass spectrometric metabolomics approach covered > 3000 metabolite ion masses. We annotated 23 metabolites showing significant changes eight weeks after rPX. While the levels of uridine and six acylcarnitines in urine were increased before surgery, lower levels were detected for 16 metabolites, like e.g. citrate, phenyl-lactic acid, choline, myo-inositol, emphasizing a relevant pathophysiological role of these biomarkers and the associated metabolic pathways. These results have important implications for potential use of metabolome analyses for detection of prostate cancer and related pathologic and molecular features.

Free fatty acids, glicentin and glucose-dependent insulinotropic polypeptide as potential major determinants of fasting substrate oxidation.

The selection of carbohydrates or fat to generate intracellular energy is thought to be crucial for long-term metabolic health. While most studies assess fuel selection after a metabolic challenge, the determinants of substrate oxidation in the fasted state remain largely unexplored. We therefore assessed the respiratory quotient by indirect calorimetry as a read-out for substrate oxidation following an overnight fast. This cross-sectional analysis consisted of 192 (92 women, 100 men) either lean or obese participants. Following an overnight fast, the respiratory quotient (RQ) was assessed, after which a 5-point 75-g oral glucose tolerance test was performed. Unlike glucose and insulin, fasting free fatty acids (FFA) correlated negatively with fasting RQ (p < 0.0001). Participants with high levels of the ketone body ß-hydroxybutyric acid had significantly lower RQ values. Fasting levels of glucose-dependent insulinotropic polypeptide (GIP) and glicentin were positively associated with fasting RQ (all p ≤ 0.03), whereas GLP-1 showed no significant association. Neither BMI, nor total body fat, nor body fat distribution correlated with fasting RQ. No relationship between the RQ and diabetes or the metabolic syndrome could be observed. In the fasting state, FFA concentrations were strongly linked to the preferentially oxidized substrate. Our data did not indicate any relationship between fasting substrate oxidation and metabolic diseases, including obesity, diabetes, and the metabolic syndrome. Since glicentin and GIP are linked to fuel selection in the fasting state, novel therapeutic approaches that target these hormones may have the potential to modulate substrate oxidation.

Systematic, Modifying Group-Assisted Strategy Expanding Coverage of Metabolite Annotation in Liquid Chromatography-Mass Spectrometry-Based Nontargeted Metabolomics Studies.

From microbes to human beings, nontargeted metabolic profiling by liquid chromatography (LC)-mass spectrometry (MS) has been commonly used to investigate metabolic alterations. Still, a major challenge is the annotation of metabolites from thousands of detected features. The aim of our research was to go beyond coverage of metabolite annotation in common nontargeted metabolomics studies by an integrated multistep strategy applying data-dependent acquisition (DDA)-based ultrahigh-performance liquid chromatography (UHPLC)-high-resolution mass spectrometry (HRMS) analysis followed by comprehensive neutral loss matches for characteristic metabolite modifications and database searches in a successive manner. Using pooled human urine as a model sample for method establishment, we found 22% of the detected compounds having modifying structures. Major types of metabolite modifications in urine were glucuronidation (33%), sulfation (20%), and acetylation (6%). Among the 383 annotated metabolites, 100 were confirmed by standard compounds and 50 modified metabolites not present in common databases such as human metabolite database (HMDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were structurally elucidated. Practicability was tested by the investigation of urines from pregnant women diagnosed with gestational diabetes mellitus vs healthy controls. Overall, 83 differential metabolites were annotated and 67% of them were modified metabolites including five previously unreported compounds. To conclude, the systematic modifying group-assisted strategy can be taken as a useful tool to extend the number of annotated metabolites in biological and biomedical nontargeted studies.

Evaluation of the first immunosuppressive drug assay available on a fully automated LC-MS/MS-based clinical analyzer suggests a new era in laboratory medicine.

OBJECTIVES: Due to its high specificity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the gold standard in diagnostic areas such as therapeutic monitoring of immunosuppressive drugs (ISDs). However, many laboratories still rely on immunoassays for ISD quantification in a tradeoff between analytical performance and the advantages of fully automated analyzers - shorter turnaround times, greater ease of use, and 24/7 availability. METHODS: The LC-MS/MS-based Thermo Scientific™ Cascadion™ SM Immunosuppressant Panel was evaluated for >6 months in the routine laboratory of a university hospital. We assessed the analytical performance of the panel and compared it to conventional LC-MS/MS as well as to immunoassays (cyclosporine A, sirolimus, tacrolimus (Siemens) and everolimus (Thermo Fisher)). In addition, both ISD panel and Cascadion analyzer were scrutinized with regards to, e.g., turnaround time, usability, and robustness. RESULTS: All ISDs showed high linearity and precision (CV≤6%) and a good correlation with conventional LC-MS/MS. The mean deviation to the immunoassays was 17-19% and negative for all ISDs except everolimus with a positive 19% bias. No weak points were revealed when challenging assay and system with, e.g., high haematocrit, sedimented whole blood or priority samples. The Cascadion integrated well into our 24/7 routine and could easily be operated simultaneously with several other analyzers by technical staff without LC-MS experience. CONCLUSIONS: The ISD panel showed excellent analytical performance and demonstrated that a fully automated LC-MS-based analysis starting from primary samples is feasible, suggesting that LC-MS could become an integral part of 24/7 diagnostics in the near future.

Human Prostate Cancer is Characterized by an Increase in Urea Cycle Metabolites.

Despite it being the most common incident of cancer among men, the pathophysiological mechanisms contributing to prostate cancer (PCa) are still poorly understood. Altered mitochondrial metabolism is postulated to play a role in the development of PCa. To determine the key metabolites (which included mitochondrial oncometabolites), benign prostatic and cancer tissues of patients with PCa were analyzed using capillary electrophoresis and liquid chromatography coupled with mass spectrometry. Gene expression was studied using real-time PCR. In PCa tissues, we found reduced levels of early tricarboxylic acid cycle metabolites, whereas the contents of urea cycle metabolites including aspartate, argininosuccinate, arginine, proline, and the oncometabolite fumarate were higher than that in benign controls. Fumarate content correlated positively with the gene expression of oncogenic HIF1α and NFκB pathways, which were significantly higher in the PCa samples than in the benign controls. Furthermore, data from the TCGA database demonstrated that prostate cancer patients with activated NFκB pathway had a lower survival rate. In summary, our data showed that fumarate content was positively associated with carcinogenic genes.

Identification and regulation of the xenometabolite derivatives cis- and trans-3,4-methylene-heptanoylcarnitine in plasma and skeletal muscle of exercising humans.

Little is known about xenometabolites in human metabolism, particularly under exercising conditions. Previously, an exercise-modifiable, likely xenometabolite derivative, cis-3,4-methylene-heptanoylcarnitine, was reported in human plasma. Here, we identified trans-3,4-methylene-heptanoylcarnitine, and its cis-isomer, in plasma and skeletal muscle by liquid chromatography-mass spectrometry. We analyzed the regulation by exercise and the arterial-to-venous differences of these cyclopropane ring-containing carnitine esters over the hepatosplanchnic bed and the exercising leg in plasma samples obtained in three separate studies from young, lean and healthy males. Compared with other medium-chain acylcarnitines, the plasma concentrations of the 3,4-methylene-heptanoylcarnitine isomers only marginally increased with exercise. Both isomers showed a more than twofold increase in the skeletal muscle tissue of the exercising leg; this may have been due to the net effect of fatty acid oxidation in the exercising muscle and uptake from blood. The latter idea is supported by a more than twofold increased net uptake in the exercising leg only. Both isomers showed a constant release from the hepatosplanchnic bed, with an increased release of the trans-isomer after exercise. The isomers differ in their plasma concentration, with a four times higher concentration of the cis-isomer regardless of the exercise state. This is the first approach studying kinetics and fluxes of xenolipid isomers from tissues under exercise conditions, supporting the hypothesis that hepatic metabolism of cyclopropane ring-containing fatty acids is one source of these acylcarnitines in plasma. The data also provide clear evidence for an exercise-dependent regulation of xenometabolites, opening perspectives for future studies about the physiological role of this largely unknown class of metabolites.

Muscle-Liver Substrate Fluxes in Exercising Humans and Potential Effects on Hepatic Metabolism.

CONTEXT: The liver is crucial to maintain energy homeostasis during exercise. Skeletal muscle-derived metabolites can contribute to the regulation of hepatic metabolism. OBJECTIVE: We aim to elucidate which metabolites are released from the working muscles and taken up by the liver in exercising humans and their potential influence on hepatic function. METHODS: In two separate studies, young healthy men fasted overnight and then performed an acute bout of exercise. Arterial-to-venous differences of metabolites over the hepato-splanchnic bed and over the exercising and resting leg were investigated by capillary electrophoresis- and liquid chromatography-mass spectrometry metabolomics platforms. Liver transcriptome data of exercising mice were analyzed by pathway analysis to find a potential overlap between exercise-regulated metabolites and activators of hepatic transcription. RESULTS: During exercise, hepatic O2 uptake and CO2 delivery were increased two-fold. In contrast to all other free fatty acids (FFA), those FFA with 18 or more carbon atoms and a high degree of saturation showed a constant release in the liver vein and only minor changes by exercise. FFA 6:0 and 8:0 were released from the working leg and taken up by the hepato-splanchnic bed. Succinate and malate showed a pronounced hepatic uptake during exercise and were also released from the exercising leg. The transcriptional response in the liver of exercising mice indicates the activation of HIF-, NRF2-, and cAMP-dependent gene transcription. These pathways can also be activated by succinate. CONCLUSION: Metabolites circulate between working muscles and the liver and may support the metabolic adaption to exercise by acting both as substrates and as signaling molecules.

Linking bioenergetic function of mitochondria to tissue-specific molecular fingerprints.

Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxidoreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosynthetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function.

Hepatokines-a novel group of exercise factors.

Regular physical activity not only improves the exercise capacity of the skeletal muscle performing the contractions, but it is beneficial for the whole body. An extensive search for "exercise factors" mediating these beneficial effects has been going on for decades. Particularly skeletal muscle tissue has been investigated as a source of circulating exercise factors, and several myokines have been identified. However, exercise also has an impact on other tissues. The liver is interposed between energy storing and energy utilising tissues and is highly active during exercise, maintaining energy homeostasis. Recently, a novel group of exercise factors-termed hepatokines-has emerged. These proteins (fibroblast growth factor 21, follistatin, angiopoietin-like protein 4, heat shock protein 72, insulin-like growth factor binding protein 1) are released from the liver and increased in the bloodstream during or in the recovery after an exercise bout. In this narrative review, we evaluate this new group of exercise factors focusing on the regulation and potential function in exercise metabolism and adaptations. These hepatokines may convey some of the beneficial whole-body effects of exercise that could ameliorate metabolic diseases, such as obesity or type 2 diabetes.

Serum or plasma, what is the difference? Investigations to facilitate the sample material selection decision making process for metabolomics studies and beyond.

In analytical chemistry serum as well as plasma are recommended as sample material of choice. However, blood processing for the generation of serum or plasma is rather different. Whether plasma or serum is the preferable sample material is still controversial discussed. We performed in paired samples three UHPLC-mass spectrometry-driven metabolomics studies. In study 1 metabolite profiles of serum vs plasma were compared. 46% out of 216 identified metabolites showed significant different levels (paired Wilcoxon signed-rank test, p < 0.05, FDR <0.01) with only three metabolites (methionine, C2:0- and C3:0-carnitine) showing lower levels in serum. In study 2 comparison of three different serum blood collection tubes revealed that coagulation and associated processes distinctly alter metabolite levels depending on the tube-specific clotting process. Most pronounced differences were found for the dipeptide phenylalanine-phenylalanine (highest levels in silicate containing serum blood collection tubes). In study 3 possible adverse effects of platelets, which still remain in standard plasma even after correct processing, were investigated. No differences in a pattern of 216 metabolites were detected in the comparison of standard and platelet-free plasma (PFP). Our results give novel insights in fundamental differences between serum and plasma, thereby providing valuable information for analytical chemists for decision making to either use serum or plasma before starting complex and time-consuming analytical investigations.