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Changes in the metabolic profile within the intestine of lenok (Brachymystax lenok) when challenged to acute and lethal heat stress (HS) are studied using no-target HPLC-MS/MS metabonomic analysis. Of 51 differentially expressed metabolites identified in response to HS, 34 occurred in the positive ion mode and 17 in negative ion mode (VIP > 1, P < 0.05). Changes in metabolites (i.e. alpha-D-glucose, stachyose and L-lactate) related to carbohydrate and glycolysis are identified in HS-treated lenok. Fatty acid β-oxidation in HS-treated lenok was inhibited by accumulation of acetyl carnitine, palmitoylcarnitine, carnitine, and erucic acid. Many amino acids (L-tryptophan, D-proline, L-leucine, L-phenylalanine, L-aspartate, L-tyrosine, L-methionine, L-histidine and L-glutamine) decreased to support energy demands in HS-treated lenok. Oxidative damage in HS-treated lenok was indicated by decreased glycerophospholipid metabolites (i.e. glycerophosphocholine, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine), and increased oxylipin production (12-HETE and 9R, 10S-EpOME). Oxidative stress increased formation of eicosanoids and dicarboxylic acids, overwhelming the mitochondrial β-oxidation pathway, while minor oxidative pathways (omega-oxidation and peroxisomal beta-oxidation) were likely to be activated in HS-treated lenok.

Pozadina istraživanja: Metabolički modulator trimetazidin (TMZ) je antianginalni lijek koji se koristi u liječenju stabilne bolesti koronarnih arterija (engl. coronary artery disease, CAD). Prema pretpostavljenom mehanizmu djelovanja lijeka, povoljan klinički učinak se ostvaruje izmjenom srčanog metabolizma: preusmjeravanjem mitohondrijskog iskorištavanja supstrata ka većem korištenju ugljikohidrata, zbog čega se povećava učinkovitost proizvodnje ATP-a. Nekoliko nedavno objavljenih studija provedenih uglavnom na životinjskim modelima su preispitale točnost predloženog mehanizma djelovanja lijeka. Cilj provedenog istraživanja je bio dobiti izravan uvid u učinak TMZ na mitohondrijsku oksidaciju supstrata u lijevom vemtrikulu bolesnika oboljelih od CAD. Eksperimentalni pristup: Mitohondrijska oksidacija masnih kiselina (palmitoilkarnitin) i ugljikohidrata (piruvat) je mjerena u permeabiliziranim vlaknima lijevog ventrikula dobivenim biopsijom tijekom operativnog premoštenja koronarnih arterija (engl. coronary artery bypass grafting, CABG) kod bolesnika oboljelih od CAD koji su koristili TMZ (TMZ skupina) ili ga nisu imali u redovnoj terapiji – kontrolna skupina. Glavni rezultati: Nije zabilježena razlika između dviju skupina u oksidaciji palmitoilkarnitina i piruvata, kao niti u omjeru oksidacije ugljikohidrata i masnih kiselina. Aktivnost i ekspresija piruvat dehidrogenaze, glavnog regulirajućeg enzima ugljikohidratnog metabolizma, se nije razlikovala među skupinama. Konačno, izlaganje srčanog tkiva u in vitro uvjetima različitim koncentracijama TMZ također nije imalo značajan utjecaj na stupanj oksidacije masnih kiselina. Zaključak: koristeći srčano tkivo ciljane populacije CAD bolesnika nismo pronašli učinak TMZ primijenjenog kronično in vivo niti akutno in vitro na oksidaciju masnih kiselina i ugljikohidrata, iz čega zaključujemo kako su povoljni klinički učinci TMZ prvenstveno posljedica drugih, još nedovoljno poznatih mehanizama., Background and purpose: Trimetazidine, known as a metabolic modulator, is an anti-anginal drug used for treatment of stable coronary artery disease (CAD). It is proposed to act via modulation of cardiac metabolism: shifting the mitochondrial substrate utilization towards carbohydrates, thus increasing the efficiency of adenosine triphosphate production. This mechanism was recently challenged, however using indirect approaches and animal models, making the conclusions of these studies questionable. The goal of the current study was to directly assess the effect of trimetazidine on mitochondrial substrate oxidation in left ventricular myocardium from CAD patients. Experimental approach: Mitochondrial fatty acid (palmitoylcarnitine) and carbohydrate (pyruvate) oxidation was measured in permeabilized left ventricular fibres obtained during coronary artery bypass grafting surgery from CAD patients, which either had trimetazidine included in their therapy (TMZ group) or not (Control). Key results: There was no difference found between the two groups in either oxidation of palmitoylcarnitine or pyruvate, as well as in the ratio of carbohydrate-to-fatty acid oxidation. Activity and expression of pyruvate dehydrogenase, the key regulator of carbohydrate metabolism, was also not different. Lastly, the acute in vitro exposure of myocardial tissue to different concentrations of trimetazidine did not affect myocardial oxidation of fatty acid. Conclusion and implications: Using myocardial tissue from a target CAD patient population, we found no effect of trimetazidine (applied chronically in vivo or acutely in vitro) on cardiac fatty acid and carbohydrate oxidation, suggesting that clinical effects of trimetazidine are unlikely due to its metabolic effects, but rather to yet unidentified intracardiac mechanism.

Heart-type fatty-acid binding protein (FABP3) is an essential cytosolic lipid transport protein found in cardiomyocytes. FABP3 binds fatty acids (FAs) reversibly and with high affinity. Acylcarnitines (ACs) are an esterified form of FAs that play an important role in cellular energy metabolism. However, an increased concentration of ACs can exert detrimental effects on cardiac mitochondria and lead to severe cardiac damage. In the present study, we evaluated the ability of FABP3 to bind long-chain ACs (LCACs) and protect cells from their harmful effects. We characterized the novel binding mechanism between FABP3 and LCACs by a cytotoxicity assay, nuclear magnetic resonance, and isothermal titration calorimetry. Our data demonstrate that FABP3 is capable of binding both FAs and LCACs as well as decreasing the cytotoxicity of LCACs. Our findings reveal that LCACs and FAs compete for the binding site of FABP3. Thus, the protective mechanism of FABP3 is found to be concentration dependent.

BACKGROUND Sepsis-induced cardiomyopathy (SIC) is a major contributing factor for morbidity and mortality in sepsis. Accumulative evidence has suggested that cardiac mitochondrial oxidative phosphorylation is attenuated in sepsis, but the underlying molecular mechanisms remain incompletely understood. METHODS Adult male mice of 9-12 weeks old were subjected to sham or cecal ligation and puncture procedure. Echocardiography in vivo and Langendorff-perfused hearts were used to assess cardiac function 24 hours after the procedures. Unbiased proteomics analysis was performed to profile mitochondrial proteins in the hearts of both sham and SIC mice. Seahorse respirator technology was used to evaluate oxygen consumption in purified mitochondria. RESULTS Of the 665 mitochondrial proteins identified in the proteomics assay, 35 were altered in septic mice. The mitochondrial remodeling involved various energy metabolism pathways including subunits of the electron transport chain, fatty acid catabolism, and carbohydrate oxidative metabolism. We also identified a significant increase of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) and inhibition of PDH activity in septic hearts. Furthermore, compared to sham mice, mitochondrial oxygen consumption of septic mice was significantly reduced when pyruvate was provided as a substrate. However, it was unchanged when PDH was bypassed by directly supplying the Complex I substrate NADH, or by using the Complex II substrate succinate, or using Complex IV substrate, or by providing the beta-oxidation substrate palmitoylcarnitine, neither of which require PDH for mitochondrial oxygen consumption. CONCLUSIONS These data demonstrate a broad mitochondrial protein remodeling, PDH inactivation and impaired pyruvate-fueled oxidative phosphorylation during SIC, and provide a molecular framework for further exploration.

Scientists have long established that fatty acids are the primary substrates for kidney mitochondria. However, to date we still do not know how long-chain and middle-chain fatty acids are oxidized at the mitochondrial level. Our previous research has shown that mitochondria from the heart, brain, and kidney oxidize palmitoylcarnitine at a high rate only in the presence of succinate, glutamate, or pyruvate. In this paper, we report properties of the isolated kidney mitochondria and how malate and succinate affect the oxidation of C16 and C8 acylcarnitines. The isolated kidney mitochondria contain very few endogenous substrates and require malate to oxidize pyruvate, glutamate, and C16 or C8 acylcarnitines. We discovered that with 10 µM of C16 or C8 acylcarnitines, low concentrations of malate (0.2 mM) or succinate (0.5 mM) enhance the States 4 and 3 respiratory rates several times. The highest respiration rates were observed with C16 or C8 acylcarnitines and 5 mM succinate mixtures. Results show that kidney mitochondria, unlike the heart and brain mitochondria, lack the intrinsic inhibition of succinate dehydrogenase. Additionally, results show that the oxidation of fatty acid by the small respirasome’s supercomplex generates a high level of CoQH2, and this makes SDH in the presence of succinate reverse the flow of electrons from CoQH2 to reduce fumarate to succinate. Finally, we report evidence that succinate dehydrogenase is a key mitochondrial enzyme that allows fast oxidation of fatty acids and turns the TCA cycle function from the catabolic to the anabolic and anaplerotic metabolic pathways.

BackgroundKeloids are a fibroproliferative disease characterized by unsatisfactory therapeutic effects and a high recurrence rate.ObjectiveThis study aimed to investigate keloid-related circulating metabolic signatures.MethodsUntargeted metabolomic analysis was performed to compare the metabolic features of 15 keloid patients with those of paired healthy volunteers in the discovery cohort. The circulating metabolic signatures were selected using the least absolute shrinkage. Furthermore, the selection operators were quantified using multiple reaction monitoring-based target metabolite detection methods in the training and test cohorts.ResultsMore than ten thousand metabolic features were consistently observed in all the plasma samples from the discovery cohort, and 30 significantly different metabolites were identified. Four differentially expressed metabolites including palmitoylcarnitine, sphingosine, phosphocholine, and phenylalanylisoleucine, were discovered to be related to keloid risk in the training and test cohorts. In addition, using linear and logistic regression models, the respective risk scores for keloids based on a 4-metabolite fingerprint classifier were established to distinguish keloids from healthy volunteers.ConclusionsIn summary, our findings show that the characteristics of circulating metabolic fingerprinting manifest phenotypic variation in keloid onset.

For ectothermic species, adaptation to thermal changes is of critical importance. Mitochondrial oxidative phosphorylation (OXPHOS), which leverages multiple electron pathways to produce energy needed for survival, is among the crucial metabolic processes impacted by temperature. Our aim in this study was to identify how changes in temperature affect the less-studied electron transferring flavoprotein pathway, fed by fatty acid substrates. We used the planarian Dugesia tigrina, acclimated for 4 weeks at 10°C (cold acclimated) or 20°C (normothermic). Respirometry experiments were conducted at an assay temperature of either 10 or 20°C to study specific states of the OXPHOS process using the fatty acid substrates palmitoylcarnitine (long chain), octanoylcarnitine (medium chain) or acetylcarnitine (short chain). Following cold acclimation, octanoylcarnitine exhibited increases in both the OXPHOS and electron transfer (ET, non-coupled) states, indicating that the pathway involved in medium-chain length fatty acids adjusts to cold temperatures. Acetylcarnitine only showed an increase in the OXPHOS state as a result of cold acclimation, but not in the ET state, indicative of a change in phosphorylation system capacity rather than fatty acid β-oxidation. Palmitoylcarnitine oxidation was unaffected. Our results show that cold acclimation in D. tigrina caused a specific adjustment in the capacity to metabolize medium-chain fatty acids rather than an adjustment in the activity of the enzymes carnitine-acylcarnitine translocase, carnitine acyltransferase and carnitine palmitoyltransferase-2. Here, we provide novel evidence of the alterations in fatty acid β-oxidation during cold acclimation in D. tigrina.

When stable and near-normoglycemic, patients with “A−β+” ketosis-prone diabetes (KPD) manifest accelerated leucine catabolism and blunted ketone oxidation, which may underlie their proclivity to develop diabetic ketoacidosis (DKA). To understand metabolic derangements in A−β+ KPD patients during DKA, we compared serum metabolomics profiles of adults during acute hyperglycemic crises, without (n = 21) or with (n = 74) DKA, and healthy control subjects (n = 17). Based on 65 kDa GAD islet autoantibody status, C-peptide, and clinical features, 53 DKA patients were categorized as having KPD and 21 type 1 diabetes (T1D); 21 nonketotic patients were categorized as having type 2 diabetes (T2D). Patients with KPD and patients with T1D had higher counterregulatory hormones and lower insulin-to-glucagon ratio than patients with T2D and control subjects. Compared with patients withT2D and control subjects, patients with KPD and patients with T1D had lower free carnitine and higher long-chain acylcarnitines and acetylcarnitine (C2) but lower palmitoylcarnitine (C16)-to-C2 ratio; a positive relationship between C16 and C2 but negative relationship between carnitine and β-hydroxybutyrate (BOHB); higher branched-chain amino acids (BCAAs) and their ketoacids but lower ketoisocaproate (KIC)-to-Leu, ketomethylvalerate (KMV)-to-Ile, ketoisovalerate (KIV)-to-Val, isovalerylcarnitine-to-KIC+KMV, propionylcarnitine-to-KIV+KMV, KIC+KMV-to-C2, and KIC-to-BOHB ratios; and lower glutamate and 3-methylhistidine. These data suggest that during DKA, patients with KPD resemble patients with T1D in having impaired BCAA catabolism and accelerated fatty acid flux to ketones—a reversal of their distinctive BCAA metabolic defect when stable. The natural history of A−β+ KPD is marked by chronic but varying dysregulation of BCAA metabolism.

This study was focused on the mechanisms of the protective effects of L-arginine and the nitric oxide donor SNAP under the induction of cyclosporine A-dependent mitochondrial permeability transition pore (MPTP) by excess of Ca2+ or D,L-palmitoylcarnitine (PC). The possible involvement of a signaling system that included mitochondrial enzymes NO-synthase (mtNOS), guanylate cyclase (GC), and cGMP-dependent protein kinase G (PKG) (mtNOS/GC/PKG-SS) in the regulation of MPTP was investigated. The experiments were performed on isolated liver mitochondria using inhibitory analysis. The results of the study have shown that L-arginine and SNAP caused a dose-dependent effects on the opening of MPTP. L‑Arginine and SNAP at low concentrations (5–100 μM) activated protective mechanisms, providing an increase in the mitochondrial calcium buffer capacity (CRC) and the critical (threshold) concentration of palmitoylcarnitine (PC*) required for MPTP induction. At higher concentrations, L-arginine (more than 500 μM) and SNAP (more than 100 μM) caused the opposite effect, that is, they suppressed respiration and promoted pore opening. Inhibitors of NOS, GC, and PKG eliminated the protective effects observed at low concentrations of L‑arginine and SNAP. The experiments conducted with a specific inhibitor of inducible NOS (W1400) showed that this calcium-independent enzyme did not participate in the regulation of MPTP in our experiments. Based on the results obtained, it can be assumed that the calcium-dependent mitochondrial signaling system mtNOS/GC/PKG-SS is involved in the regulation of MPTP, providing an increase in CRC and PC*. At high concentrations of L-arginine or SNAP, excess of NO overcame the protection provided by mtPKG and promoted pore opening.

Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine accumulation and limited FAO associate with lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation, and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at 200 and 500 µM did not trigger cell stress responses (phospho-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50–96% compared with WT. Growing cells in carnitine-free media abolished differences between WT and CPT2 KO, but this did not fully rescue PA-induced insulin resistance. The results suggest that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, further supporting the hypothesis that select acylcarnitines participate in cell signaling and, when in excess, can compromise cell function. Since carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced “lipotoxicity” in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.

In this work, the effect of thyroxine on energy and oxidative metabolism in the mitochondria of the rat heart was studied. Hyperthyroidism was observed in experimental animals after chronic administration of T4, which was accompanied by an increase in serum concentrations of free triiodothyronine (T3) and thyroxine (T4) by 1.8 and 3.4 times, respectively. The hyperthyroid rats (HR) had hypertrophy of the heart. In HR, there was a change in the oxygen consumption in the mitochondria of the heart, especially when using palmitoylcarnitine. The assay of respiratory chain enzymes revealed that the activities of complexes I, I + III, III, IV increased, whereas the activities of complexes II, II + III decreased in heart mitochondria of the experimental animals. It was shown that the level of respiratory complexes of the electron transport chain in hyperthyroid rats increased, except for complex V, the quantity of which was reduced. The development of oxidative stress in HR was observed: an increase in the hydrogen peroxide production rate, increase in lipid peroxidation and reduced glutathione. The activity of superoxide dismutase in the heart of HR was higher than in the control. At the same time, the activity of glutathione peroxidase decreased. The obtained data indicate that increased concentrations of thyroid hormones lead to changes in energy metabolism and the development of oxidative stress in the heart of rats, which in turn contributes to heart dysfunction.

Previous evidence suggests that palmitoylcarnitine incubations trigger mitochondrial-mediated apoptosis in HT29 colorectal adenocarcinoma cells, yet nontransformed cells appear insensitive. The mechanism by which palmitoylcarnitine induces cancer cell death is unclear. The purpose of this investigation was to examine the relationship between mitochondrial kinetics and glutathione buffering in determining the effect of palmitoylcarnitine on cell survival. HT29 and HCT 116 colorectal adenocarcinoma cells, CCD 841 nontransformed colon cells, and MCF7 breast adenocarcinoma cells were exposed to 0 μM, 50 μM, and 100 μM palmitoylcarnitine for 24–48 h. HCT 116 and HT29 cells showed decreased cell survival following palmitoylcarnitine compared with CCD 841 cells. Palmitoylcarnitine stimulated H2O2emission in HT29 and CCD 841 cells but increased it to a greater level in HT29 cells due largely to a higher basal H2O2emission. This greater H2O2emission was associated with lower glutathione buffering capacity and caspase-3 activation in HT29 cells. The glutathione-depleting agent buthionine sulfoximine sensitized CCD 841 cells and further sensitized HT29 cells to palmitoylcarnitine-induced decreases in cell survival. MCF7 cells did not produce H2O2when exposed to palmitoylcarnitine and were able to maintain glutathione levels. Furthermore, HT29 cells demonstrated the lowest mitochondrial oxidative kinetics vs. CCD 841 and MCF7 cells. The results demonstrate that colorectal cancer is sensitive to palmitoylcarnitine due in part to an inability to prevent oxidative stress through glutathione-redox coupling, thereby rendering the cells sensitive to elevations in H2O2. These findings suggest that the relationship between inherent metabolic capacities and redox regulation is altered early in response to palmitoylcarnitine.

Sleep fragmentation (SF) commonly occurs in several pathologic conditions and is especially associated with impairments of hippocampus-dependent neurocognitive functions. Although the effects of SF on hippocampus in terms of protein or gene levels were examined in several studies, the impact of SF at the metabolite level has not been investigated. Thus, in this study, the differentially expressed large-scale metabolite profiles of hippocampus in a rat model of SF were investigated using untargeted metabolomics approaches. Forty-eight rats were divided into the following 4 groups: 4-day SF group, 4-day exercise control (EC) group, 15-day SF group, and 15-day EC group (n = 12, each). SF was accomplished by forced exercise using a walking wheel system with 30-s on/90-s off cycles, and EC condition was set at 10-min on/30-min off. The metabolite profiles of rat hippocampi in the SF and EC groups were analyzed using liquid chromatography/mass spectrometry. Multivariate analysis revealed distinctive metabolic profiles and marker signals between the SF and corresponding EC groups. Metabolic changes were significant only in the 15-day SF group. In the 15-day SF group, L-tryptophan, myristoylcarnitine, and palmitoylcarnitine were significantly increased, while adenosine monophosphate, hypoxanthine, L-glutamate, L-aspartate, L-methionine, and glycerophosphocholine were decreased compared to the EC group. The alanine, aspartate, and glutamate metabolism pathway was observed as the common key pathway in the 15-day SF groups. The results from this untargeted metabolomics study provide a perspective on metabolic impact of SF on the hippocampus.

Neurosyphilis is a chronic central nervous system infectious disease caused by Treponema pallidum. Our aim was to study the metabolic profiling in the cerebrospinal fluid of neurosyphilis patients and identify specific potential biomarkers. Fifteen cerebrospinal fluid samples from neurosyphilis patients and 14 non-neurosyphilis samples were analyzed by liquid chromatography-mass spectrometer (LC-MS). The LC-MS data were preprocessed by supervised pattern recognition to obtain diagnostic models. Both orthogonal projections to a latent structures discriminant analysis (OPLS-DA) and a t test were used to obtain specific metabolites for neurosyphilis. LC-MS data showed that the metabolites in cerebrospinal fluid (CSF) from neurosyphilis are different from the non-neurosyphilis group. The OPLS-DA model parameters R2Y and Q2Y are both more than 0.7 and indicated a satisfactory diagnostic performance. Bilirubin, L-histidine, prostaglandin E2, alpha-kamlolenic acid, and butyryl-L-carnitine and palmitoyl-L-carnitine were identified as novel potential biomarkers for neurosyphilis. The metabolic study of CSF may provide a new way to explore the pathogenesis of neurosyphilis.

The decline in fat oxidation at higher power outputs of exercise is a complex interaction between several mechanisms; however, the influence of mitochondrial bioenergetics in this process remains elusive. Therefore, using permeabilized muscle fibers from mouse skeletal muscle, we aimed to determine if acute exercise altered mitochondrial sensitivity to (1) adenosine diphosphate (ADP) and inorganic phosphate (Pi), or (2) carnitine palmitoyltransferase-I (CPT-I) independent (palmitoylcarnitine, PC) and dependent [palmitoyl-CoA (P-CoA), malonyl-CoA (M-CoA), and l-carnitine] substrates, in an intensity-dependent manner. As the apparent ADP Km increased to a similar extent following low (LI) and high (HI) intensity exercise compared with sedentary (SED) animals, and Pi sensitivity was unaltered by exercise, regulation of phosphate provision likely does not contribute to the well-established intensity-dependent shift in substrate utilization. Mitochondrial sensitivity to PC and P-CoA was not influenced by exercise, while M-CoA sensitivity was attenuated similarly following LI and HI. In contrast, CPT-I sensitivity to l-carnitine was only altered following HI, as HI exercise attenuated l-carnitine sensitivity by ∼40%. Moreover, modeling the in vivo concentrations of l-carnitine and P-CoA during exercise suggests that CPT-I flux is ∼25% lower following HI, attributed equally to reductions in l-carnitine content and l-carnitine sensitivity. Altogether, these data further implicate CPT-I flux as a key event influencing metabolic interactions during exercise, as a decline in l-carnitine sensitivity in addition to availability at higher power outputs could impair mitochondrial fatty acid oxidation.

BackgroundIntrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and is associated with increased risks in fetal complications. Currently, the exact mechanism of this disease is unknown. The purpose of this study was to develop potential biomarkers for the diagnosis and prediction of ICP.MethodsWe enrolled 40 pregnant women diagnosed with ICP and 40 healthy pregnant controls. The number of placental samples and serum samples between the two groups was 10 and 40 respectively. Ultra-performance liquid chromatography tandem high-resolution mass spectrometry was used to analyze placental metabolomics. Then, we verified the differentially expressed proteins and metabolites, both placental and blood serum, in the first, second, and third trimesters.ResultsMetabolomic analysis of placental tissue revealed that fatty acid metabolism and primary bile acid biosynthesis were enriched. In the integrated proteomic and metabolomic analysis of placental tissue, peroxisomal acyl-CoA oxidase 1 (ACOX1), L-palmitoylcarnitine, and glycocholic acid were found to be three potential biomarkers. In a follow–up analysis, expression levels of both placental and serum ACOX1, L-palmitoylcarnitine, and glycocholic acid in both placenta and serum were found to be significantly higher in third-trimester ICP patients; the areas under the ROC curves were 0.823, 0.896, and 0.985, respectively. Expression levels of serum ACOX1, L-palmitoylcarnitine, and glycocholic acid were also significantly higher in first- and second-trimester ICP patients; the areas under the ROC curves were 0.726, 0.657, and 0.686 in the first trimester and 0.718, 0.727, and 0.670 in the second trimester, respectively. Together, levels of the three aforementioned biomarkers increased the value for diagnosing and predicting ICP (AUC: 0.993 for the third, 0.891 for the second, and 0.932 for the first trimesters).ConclusionsL-palmitoylcarnitine, ACOX1, and glycocholic acid levels taken together may serve as a new biomarker set for the diagnosis and prediction of ICP.

Introduction Skeletal muscle mitochondria have dynamic shifts in oxidative metabolism to meet energy demands of aerobic exercise. Specific complexes oxidize lipid and nonlipid substrates. It is unclear if aerobic exercise stimulates intrinsic oxidative metabolism of mitochondria or varies between substrates. Methods We studied mitochondrial metabolism in sedentary male and female adults (n = 11F/4M) who were free of major medical conditions with mean ± SD age of 28 ± 7 yr, peak aerobic capacity of 2.0 ± 0.4 L·min-1, and body mass index of 22.2 ± 2 kg·m-2. Biopsies were collected from the vastus lateralis muscle on separate study days at rest or 15 min after exercise (1 h cycling at 65% peak aerobic capacity). Isolated mitochondria were analyzed using high-resolution respirometry of separate titration protocols for lipid (palmitoylcarnitine, F-linked) and nonlipid substrates (glutamate-malate, N-linked; succinate S-linked). Titration protocols distinguished between oxidative phosphorylation and leak respiration and included the measurement of reactive oxygen species emission (H2O2). Western blotting determined the protein abundance of electron transfer flavoprotein (ETF) subunits, including inhibitory methylation site on ETF-β. Results Aerobic exercise induced modest increases in mitochondrial respiration because of increased coupled respiration across F-linked (+13%, P = 0.08), N(S)-linked (+14%, P = 0.09), and N-linked substrates (+17%, P = 0.08). Prior exercise did not change P:O ratio. Electron leak to H2O2 increased 6% increased after exercise (P = 0.06) for lipid substrates but not for nonlipid. The protein abundance of ETF-α or ETF-β subunit or inhibitory methylation on ETF-β was not different between rest and after exercise. Conclusion In sedentary adults, the single bout of moderate-intensity cycling induced modest increases for intrinsic mitochondrial oxidative phosphorylation that was consistent across multiple substrates.

Research question Which metabolites are altered in the peritoneal cavity of women with endometriosis? Could the mouse endometriosis model simulate these alterations? Design Thirteen women with endometriosis and seven women with other benign gynaecological diseases, who underwent laparoscopic surgery, were included in this study. None had received hormonal therapy for 3 months before surgery. For the animal experiments, six and five mice were included in the endometriosis and control groups, respectively. Peritoneal fluid from the patients and peritoneal lavage fluid from the mice was collected and analysed. Non-targeted metabolomics via liquid chromatography with tandem mass spectrometry was used to identify the altered metabolites in the peritoneal fluid of endometriosis patients and mouse models. MetaboAnalyst 4.0 was used to visualize the data. Results Several metabolites in the peritoneal cavity were significantly altered in both humans and mice with endometriosis. Concentrations of lysophosphatidylcholine (LysopC) (P=0.017 in patients and P=0.041 in the mouse model) and derivatives of phosphoethanolamine (1-arachidonoyl-sn-glycero-3-phosphoethanolamine in patients, P=0.027; 1-oleoyl-sn-glycero-3-phosphoethanolamine in patients, P=0.0086; and phosphorylethanolamine in the mouse model, P=0.0027) were significantly up-regulated in both, whereas concentrations of acylcarnitines ( l -palmitoylcarnitine, P=0.047; and stearoylcarnitine, P=0.029) and kynurenine (P=0.045) were significantly increased only in humans. The human and mouse samples shared three altered enriched metabolite sets. Conclusions Women with endometriosis show an altered metabolic state in the abdominal cavity. The endometriosis mouse model shared half of the significantly altered metabolite sets found in the abdominal cavity of humans.

Recent studies in alcohol use disorders (AUDs) have demonstrated some connections between carnitine metabolism and the pathophysiology of the disease. In this scoping review, we aimed to collate and examine existing research available on carnitine metabolism and AUDs and develop hypotheses surrounding the role carnitine may play in AUD. A scoping review method was used to search electronic databases in September 2019. The database search terms used included "alcohol, alcoholism, alcohol abuse, alcohol consumption, alcohol drinking patterns, alcohol-induced disorders, alcoholic intoxication, alcohol-related disorders, binge drinking, Wernicke encephalopathy, acylcarnitine, acetyl-l-carnitine, acetylcarnitine, carnitine and palmitoylcarnitine." The inclusion criteria included English language, human-based, AUD diagnosis and measured blood or tissue carnitine or used carnitine as a treatment. Of 586 studies that were identified and screened, 65 underwent abstract review, and 41 were fully reviewed. Eighteen studies were ultimately included for analysis. Data were summarized in an electronic data extraction form. We found that there is limited literature available. Alcohol use appears to impact carnitine metabolism, most clearly in the setting of alcoholic cirrhosis. Six studies found carnitine to be increased in AUD, of which 5 were conducted in patients with alcoholic cirrhosis. Only 3 placebo-controlled trials were identified and provide some support for the use of carnitine in AUD to decrease cravings, anhedonia, and withdrawal and improve cognition. The increase in plasma carnitine in alcoholic cirrhosis may be related to disordered fatty acid metabolism and oxidative stress that occurs in AUD. The multiple possible therapeutic effects carnitine could have on ethanol metabolism and the early evidence available for carnitine supplementation as a treatment for AUD provide a foundation for future randomized control trials of carnitine for treating AUD.

Mitochondria produce and scavenge reactive oxygen species (ROS); however, whether oxidative distress due to exogenous stress arises from excessive production or impaired scavenging remains unclear. We assessed the effect of copper (Cu) and thermal stress on kinetics of ROS (H2O2) consumption in mitochondria isolated from fish heart. Mitochondria were energized with succinate, glutamate-malate or palmitoylcarnitine (PC) and incubated with 1–25 μM Cu at 11 (control) and 23 °C. We found that H2O2 consumption capacity of heart mitochondria varies with substrate and is additively reduced by temperature rise and Cu. While Cu is a potent inhibitor of H2O2 consumption in mitochondria oxidizing glutamate-malate and succinate, mitochondria oxidizing PC are resistant to the inhibitory effect of the metal. Moreover, the sensitivity of H2O2 consumption pathways to Cu depend on the substrate and are greatly impaired during oxidation of glutamate-malate. Pharmacological manipulation of mitochondrial antioxidant systems revealed that NADPH-dependent peroxidase systems are the centerpieces of ROS scavenging in heart mitochondria, with the glutathione-dependent pathway being the most prominent while catalase played a minimal role. Surprisingly, Cu is as efficacious in inhibiting thioredoxin-dependent peroxidase pathway as auranofin, a selective inhibitor of thioredoxin reductase. Taken together, our study uncovered unique mechanisms by which Cu alters mitochondrial H2O2 homeostasis including its ability to inhibit specific mitochondrial ROS scavenging pathways on a par with conventional inhibitors. Importantly, because of additive inhibitory effect on mitochondrial ROS removal mechanisms, hearts of organisms jointly exposed to Cu and thermal stress are likely at increased risk of oxidative distress.

High-throughput metabolite profiling provides the opportunity to reveal metabolic mechanisms and identify biomarkers. Psoriasis is an immune-mediated chronic inflammatory disease. However, the role of metabolism in psoriasis pathogenesis remains unclear. Methods: Plasma samples of individuals (45 psoriasis and 45 sex-, age-, and BMI-matched healthy controls) were collected. Non-targeted metabolomics and amino acid- or carnitine-targeted metabolomics were conducted, then, plasma samples of mice induced by imiquimod (IMQ) were subjected to the amino acid- and carnitine-targeted metabolomic profiling. Flow cytometry was used to study the effect of L-carnitine (LC(C0)) on IMQ-induced psoriatic inflammation. Results: Through the non-targeted metabolomics approach, we detected significantly altered amino acids and carnitines in psoriasis patients. Amino acid-targeted metabolomic profiling identified 37 amino acids altered in psoriasis, of these 23 were markedly upregulated, including essential amino acids (EAAs), and branched-chain amino acids (BCAAs), whereas glutamine, cysteine, and asparagine were significantly down-regulated. Carnitine-targeted metabolomic profiling identified 40 significantly altered carnitines, 14 of which included palmitoylcarnitine (C16) and were markedly downregulated in psoriasis, whereas hexanoylcarnitine (C6) and 3-OH-octadecenoylcarnitine (C18:1-OH) were significantly upregulated. Interestingly, glutamine, asparagine, and C16 levels were negatively correlated with the PASI score. Moreover, a higher abundance of LC(C0) was associated with markedly reduced IMQ-induced epidermal thickening and infiltration of Th17 cells in skin lesions, indicating LC(C0) supplementation as a potential therapy for psoriasis treatment. Conclusion: Our results suggested the metabolism of amino acids and carnitines are significantly altered in psoriasis, especially the metabolism of EAAs, BCAAs, and LC(C0), which may play key roles in the pathogenesis of psoriasis.

Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine accumulation and limited FAO associate with lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation, and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at 200 and 500 µM did not trigger cell stress responses (phospho-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50-96% compared with WT. Growing cells in carnitine-free media abolished differences between WT and CPT2 KO, but this did not fully rescue PA-induced insulin resistance. The results suggest that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, further supporting the hypothesis that select acylcarnitines participate in cell signaling and, when in excess, can compromise cell function. Since carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced "lipotoxicity" in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.

Aim: Insulin-resistant skeletal muscle is characterized by metabolic inflexibility with associated alterations in substrate selection, mediated by peroxisome-proliferator activated receptor delta (PPARdelta. Although it is established that PPARdelta contributes to the alteration of energy metabolism, it is not clear whether it plays a role in mitochondrial fuel competition. While nutrient overload may impair metabolic flexibility by fuel congestion within mitochondria, in absence of obesity defects at a mitochondrial level have not yet been excluded. We sought to determine whether reduced PPAR content in insulin-resistant rat skeletal muscle of a non-obese rat model of T2DM (Goto-Kakizaki, GK) ameliorate the inhibitory effect of fatty acid (i.e. palmitoylcarnitine) on mitochondrial carbohydrate oxidization (i.e. pyruvate) in muscle fibers. Methods: Bioenergetic function was characterized in oxidative soleus (S) and glycolytic white gastrocnemius (WG) muscles with measurement of respiration rates in permeabilized fibers in the presence of complex I, II, IV and fatty acid substrates. Mitochondrial content was measured by citrate synthase (CS) and succinate dehydrogenase activity (SDH). Western blot was used to determine protein expression of PPARdelta, PDK isoform 2 and 4. Results: CS and SDH activity, key markers of mitochondrial content, were reduced by ~10-30% in diabetic vs control, and the effect was evident in both oxidative and glycolytic muscles. PPARdelta(p

The interaction of lipids (entry mechanism) with respect to both oxy- and deoxy-myoglobin was explored using unrestrained Molecular Dynamics simulations. The results indicated a spontaneous entry of both palmitic and palmitoylcarnitine molecules into the oxy-Mb structure at the main binding site, whereas in deoxy-Mb, both the lipid ligands move away from the protein surface. For the alternative binding locations, entry of the ligands was independent of the oxygenation state. Presented here are the tables with the myoglobin binding energies for palmitic acid and palmitoylcarnitine estimated using Alchemical Free Energy Perturbation approach for the key structures obtained in unrestrained Molecular Dynamics simulations. These data are referenced in the original article "Exploring the entry route of palmitic acid and palmitoylcarnitine into myoglobin", reference number YABBI7787.

Background Metabolomics is one of the “omics” technologies, and is a comprehensive analysis of small molecule metabolites which include amino acid, nucleotides, carbohydrates and fatty acid. The purpose of the present study was to compare the differences of metabolite profiling between patients with ossification of the posterior longitudinal ligament (OPLL) and control subjects. Methods We analyzed plasma metabolites in patients with cervical OPLL (n = 10) and in control subjects (n = 10). Ionic metabolites were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and lipophilic metabolites were analyzed using liquid chromatograph time-of-flight mass spectrometry (LC-TOFMS). Results A total of 259 metabolites (144 metabolites in CE-TOFMS and 115 metabolites in LC-TOFMS) were detected. Among the 259 metabolites, six metabolites, namely acylcarnitine (AC) (14:0), palmitoylcarnitine, AC (18:2), fatty acid (FA) (24:2), thyroxine, thiaproline were significantly larger in OPLL group, even in analyzes excluding patients with diabetes mellitus and hyperlipidemia. Conclusions We examined the metabolite profiling in patients with OPLL for the first time and detected six metabolites showing suggestive association with disease. These results of the present study could lead to new insights into clarifying the molecular pathomechanisms of OPLL.

Context Carnitine and its metabolites are centrally involved in fatty acid metabolism. Although elevated circulating concentrations have been observed in obesity and insulin resistance, prospective studies examining whether these metabolites are associated with incident type 2 diabetes mellitus (T2D) are sparse. Objective We performed a comprehensive evaluation of metabolites along the carnitine pathway relative to incident T2D. Design A total of 2519 patients (73.1% men) with coronary artery disease, but without T2D, were followed for median 7.7 years until the end of 2009, during which 173 (6.9%) new cases of T2D were identified. Serum levels of free carnitine, its precursors trimethyllysine (TML) and γ-butyrobetaine, and the esters acetyl-, propionyl-, (iso)valeryl-, octanoyl-, and palmitoylcarnitine were measured by liquid chromatography/tandem mass spectrometry. Risk associations were explored by logistic regression and reported per (log-transformed) standard deviation increment. Results Median age at inclusion was 62 years and median body mass index (BMI) 26.0 kg/m2. In models adjusted for age, sex, fasting status, BMI, estimated glomerular filtration rate, glycated hemoglobin A1c, triglyceride and high-density lipoprotein cholesterol levels, and study center, serum levels of TML and palmitoylcarnitine associated positively [odds ratio (95% confidence interval), 1.22 (1.04 to 1.43) and 1.24 (1.04 to 1.49), respectively], whereas γ-butyrobetaine associated negatively [odds ratio (95% confidence interval) 0.81 (0.66 to 0.98)] with T2D risk. Conclusion Serum levels of TML, γ-butyrobetaine, and the long-chained palmitoylcarnitine predict long-term risk of T2D independently of traditional risk factors, possibly reflecting dysfunctional fatty acid metabolism in patients susceptible to T2D development.

Mitochondrial reactive oxygen species (ROS) have been implicated in organ damage caused by environmental stressors, prompting studies on the effect of oxygen deprivation and metal exposure on ROS metabolism. However, how anoxia and copper (Cu) jointly influence heart mitochondrial ROS metabolism is not understood. We used rainbow trout heart mitochondria to probe the effects of anoxia-reoxygenation and Cu on hydrogen peroxide (H2O2) emission during oxidation of palmitoylcarnitine (PC), succinate, or glutamate-malate. In addition, we examined the influence of anoxia-reoxygenation and Cu on site-specific H2O2 emission capacities and key antioxidant enzymes, glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Results showed that anoxia-reoxygenation suppressed H2O2 emission regardless of substrate type or duration of anoxia. Anoxia-reoxygenation reduced mitochondrial sensitivity to Cu during oxidation of succinate or glutamate-malate whereas high Cu concentration additively stimulated H2O2 emission in mitochondria oxidizing PC. Prolonged anoxia-reoxygenation stimulated H2O2 emission from sites OF and IF, inhibited emission from sites IQ, IIF and IIIQo, and disparately altered the sensitivity of the sites to Cu. Interestingly, anoxia-reoxygenation increased GPx and TrxR activities, more prominently when reoxygenation followed a short duration of anoxia. Cu did not alter GPx but reduced TrxR activity in normoxic and anoxic-reoxygenated mitochondria. Overall, our study revealed potential mechanisms that may reduce oxidative damage associated with anoxia-reoxygenation and Cu exposure in heart mitochondria. The increased and decreased H2O2 emission from NADH/NAD+ and QH2/Q isopotential sites, respectively, may represent a balance between H2O2 required for oxygen deprivation-induced signaling and prevention of ROS burst associated with anoxia-reoxygenation.

H2O2 is endogenously generated and its removal in the matrix of skeletal muscle mitochondria (SMM) is dependent on NADPH likely provided by NAD(P)+ transhydrogenase (NNT) and isocitrate dehydrogenase (IDH2). Importantly, NNT activity is linked to mitochondrial protonmotive force. Here, we demonstrate the presence of NNT function in detergent-solubilized and intact functional SMM isolated from rats and wild type (Nnt+/+) mice, but not in SMM from congenic mice carrying a mutated NNT gene (Nnt−/-). Further comparisons between SMM from both Nnt mouse genotypes revealed that the NADPH supplied by NNT supports up to 600 pmol/mg/min of H2O2 removal under selected conditions. Surprisingly, SMM from Nnt−/- mice removed exogenous H2O2 at wild-type levels and exhibited a maintained or even decreased net emission of endogenous H2O2 when substrates that support Krebs cycle reactions were present (e.g., pyruvate plus malate or palmitoylcarnitine plus malate). These results may be explained by a compensation for the lack of NNT, since the total activities of concurrent NADP+-reducing enzymes (IDH2, malic enzymes and glutamate dehydrogenase) were ~70% elevated in Nnt−/- mice. Importantly, respiratory rates were similar between SMM from both Nnt genotypes despite differing NNT contributions to H2O2 removal and their implications for an evolving concept in the literature are discussed. We concluded that NNT is capable of meaningfully sustaining NADPH-dependent H2O2 removal in intact SMM. Nonetheless, if the available substrates favor non-NNT sources of NADPH, the H2O2 removal by SMM is maintained in Nnt−/- mice SMM.