Synergism between SLC6A14 blockade and gemcitabine in pancreactic cancer: a 1H-NMR-based metabolomic study in pancreatic cancer cells.

Gemcitabine is the first-line chemotherapy for pancreatic cancer. To overcome the often-acquired gemcitabine resistance, other drugs are used in combination with gemcitabine. It is well-known that cancer cells reprogram cellular metabolism, coupled with the up-regulation of selective nutrient transporters to feed into the altered metabolic pathways. Our previous studies have demonstrated that the amino acid transporter SLC6A14 is markedly up-regulated in pancreatic cancer and that it is a viable therapeutic target. α-Methyltryptophan (α-MT) is a blocker of SLC6A14 and is effective against pancreatic cancer in vitro and in vivo. In the present study, we tested the hypothesis that α-MT could synergize with gemcitabine in the treatment of pancreatic cancer. We investigated the effects of combination of α-MT and gemcitabine on proliferation, migration, and apoptosis in a human pancreatic cancer cell line, and examined the underlying mechanisms using 1H-NMR-based metabolomic analysis. These studies examined the intracellular metabolite profile and the extracellular metabolite profile separately. Combination of α-MT with gemcitabine elicited marked changes in a wide variety of metabolic pathways, particularly amino acid metabolism with notable alterations in pathways involving tryptophan, branched-chain amino acids, ketone bodies, and membrane phospholipids. The metabolomic profiles of untreated control cells and cells treated with gemcitabine or α-MT were distinctly separable, and the combination regimen showed a certain extent of overlap with the individual α-MT and gemcitabine groups. This represents the first study detailing the metabolomic basis of the anticancer efficacy of gemcitabine, α-MT and their combination.

Effect of Lactic Fermentation Products on Human Epidermal Cell Differentiation, Ceramide Content, and Amino Acid Production.

INTRODUCTION: Lactic fermentation products (LFPs) are thought to affect "good" bacteria in the gut. We previously reported that oral administration of LFPs has beneficial therapeutic effects in a mouse model of atopic dermatitis. However, it is unclear how LFPs affect human epidermal cell differentiation, ceramide (Cer), and amino acid production. OBJECTIVE: The aim of this study was to determine the effects of LFPs on epidermal cell differentiation, by assessing amino acid and Cer production. METHODS: A 3-dimensional cultured human epidermis model and normal human epidermal keratinocytes were used. Cytotoxicity tests were performed using alamar Blue. Transepidermal water loss (TEWL) was used as an index to assess barrier function. Keratin 1 (K1), keratin 5 (K5), keratin 10 (K10), involucrin (INV), calpain 1, and transglutaminase (TGase) (markers of differentiation) and profilaggrin (proFLG) and bleomycin hydrolase (amino acid synthesis-related genes) expression levels were quantified by RT-PCR. In addition, TGase protein levels were measured by Western blotting. The intercellular lipid content of the stratum corneum was measured by high-performance thin-layer chromatography. Amino acids were quantified using an amino acid analyzer. Finally, bound water content in the stratum corneum was measured by differential scanning calorimetry. RESULTS: Cell viability did not change, but TEWL was significantly decreased in the cells treated with LFPs compared with the control cells. Treatment with LFPs significantly increased expression of the late-differentiation markers INV and TGase at the RNA level. Furthermore, TGase protein expression was significantly increased by treatment with LFPs. Treating a 3-dimensional cultured epidermis model with LFPs significantly increased the intercellular lipid content of the stratum corneum and production of the amino acid arginine (Arg). The amount of bound water in the stratum corneum was increased significantly in the LFP application group. CONCLUSION: Treatment with LFPs promotes human epidermal cell differentiation and increases the intercellular content of the free fatty acid, Chol, Cer [NS], Cer [AS], and Cer [AP]. This may result in improved skin barrier function. The increased amount of Arg observed in keratinocytes may help improve water retention.

A metabolomics study: Reveals the protective effect and mechanism of Terminalia chebula Retz on the cardiotoxicity induced by radix Aconiti kusnezoffii Reichb.

To reveal the protective effect of Terminalia chebula Retz (TCR) on cardiotoxicity induced by radix of Aconitum kusnezoffii Reichb (AKR). Control, AKR, AKR-TCR 1:3, AKR-TCR 1:1, AKR-TCR 3:1 and TCR-prepared AKR groups were set up. After treatment, the heart tissues were observed by H&E staining and transmission electron microscope. Serum myoglobin (MB) and troponin (cTn) were detected by ELISA. UPLC-Q Exactive/MS analysis was performed to detect the metabolic difference among the groups. ELISA results showed that the MB and cTn values of AKR group were significantly higher than Control group (P<0.05), while those of the other groups were lower than AKR group. TCR-prepared AKR group had similar MB and cTn contents to the Control group. Histopathological examination also indicated better detoxifying effects in the TCR-prepared AKR and AKR-TCR 1:1 group. The serum metabolomics analysis showed obvious distinction between the AKR and Control groups, while AKR-TCR combination reversed the metabolomics changes induced by AKR. Through multivariate statistical analysis, 9 metabolic markers related to energy, nucleic acid and amino acid metabolism were identified. Conclusively, AKR-induced cardiotoxicity may be related to energy, nucleic acid and amino acid metabolism, and TCR can reduce the cardiotoxicity by regulating the relative metabolism pathways.

Effects of low and high doses of fenofibrate on protein, amino acid, and energy metabolism in rat.

A feared adverse effect of dyslipidaemia therapy by fibrates is myopathy. We examined the effect of fenofibrate (FF) on protein and amino acid metabolism. Rats received a low (50 mg/kg, LFFD) or high (300 mg/kg, HFFD) dose of FF or vehicle daily by oral gavage. Blood plasma, liver, and soleus and extensor digitorum longus muscles were analysed after 10 days. The FF-treated rats developed hepatomegaly associated with increased hepatic carnitine and ATP and AMP concentrations, decreased protein breakdown, and decreased concentrations of DNA and triglycerides. HFFD increased plasma ALT and AST activities. The weight and protein content of muscles in the HFFD group were lower compared with controls. In muscles of the LFFD group there were increased ATP and decreased AMP concentrations; in the HFFD group AMP was increased. In both FF-treated groups there were increased glycine, phenylalanine, and citrulline and decreased arginine and branched-chain keto acids (BCKA) in blood plasma. After HFFD there were decreased levels of branched-chain amino acids (BCAA; valine, leucine and isoleucine), methionine, and lysine and increased homocysteine. Decreased arginine and increased glycine concentrations were found in both muscles in FF-treated animals; in HFFD-treated animals lysine, methionine, and BCAA were decreased. We conclude that FF exerts protein-anabolic effects on the liver and catabolic effects on muscles. HFFD causes signs of hepatotoxicity, impairs energy and protein balance in muscles, and decreases BCAA, methionine, and lysine. It is suggested that increased glycine and decreased lysine and methionine levels are due to activated carnitine synthesis; decreased BCAA and BCKA levels are due to increased BCAA oxidation.

Serum metabonomics analysis of quercetin against the toxicity induced by cadmium in rats.

This study aimed to investigate the protective effect of quercetin against the toxicity induced by chronic exposure to low levels of cadmium in rats by an ultra performance liquid chromatography mass spectrometer. Rats were randomly divided into six groups as follows: control group (C), low dose of quercetin group (Q1: 10 mg/kg·bw), high dose of quercetin group (Q2: 50 mg/kg·bw), cadmium chloride group (D), low dose of quercetin plus cadmium chloride group (DQ1), and high dose of quercetin plus cadmium chloride group (DQ2). Cadmium chloride (CdCl2 ) was administered to rats by drinking water ad libitum in a concentration of 40 mg/L. The final amount of CdCl2 ingested was estimated from the water consumption data to be 4.85, 4.91, and 4.89 mg/kg·bw/day, for D, DQ1, and DQ2 groups, respectively. After a 12-week treatment, the serum samples of rats were collected for metabonomics analysis. Ten potential biomarkers were identified for which intensities were significantly increased or reduced as a result of the treatment. These metabolites included isorhamnetin 4'-O-glucuronide, 3-indolepropionic acid, tetracosahexaenoic acid, lysophosphatidylcholine (LysoPC) (20:5), lysoPC (18:3), lysophosphatidylethanolamine (LysoPE) (20:5/0:0), bicyclo-prostaglandin E2, sulpholithocholylglycine, lithocholyltaurine, and glycocholic acid. Results indicated that quercetin exerted a protective effect against cadmium-induced toxicity by regulating lipid and amino acid metabolism, enhancing the antioxidant defense system and protecting liver and kidney function.

Identification of a crucial amino acid responsible for the loss of specifying FGFR1-KLB affinity of the iodinated FGF21.

Previous studies suggest that specific binding to the complex consisting of fibroblast growth factor receptor-1 (FGFR1) and the coreceptor beta-Klotho (KLB) is the premise for human FGF19 and FGF21 activating the downstream signaling cascades, and regulating the metabolic homeostasis. However, it was found that human FGF21 loses its ability to bind to FGFR1-KLB after iodination with Na125 I and chloramine T, whereas human FGF19 retained its affinity for FGFR1-KLB even after iodination. The molecular mechanisms underlying these differences remained elusive. In this study, we first demonstrated that an intramolecular disulfide bond was formed between cysteine-102 and cysteine-121 in FGF21, implying that the oxidation of the cysteine to cysteic acid, which may interfere with the active conformation of FGF21, did not occur during the iodination procedures, and thus ruled out the possibility of the two conserved cysteine residues mediating the loss of FGF21 binding affinity to FGFR1-KLB upon iodination. Site-directed mutagenesis and molecular modeling were further applied to determine the residue(s) responsible for the loss of FGFR1-KLB affinity. The results showed that mutation of a single tyrosine-207, but not the other five tyrosine residues in FGF21, to a phenylalanine retained the FGFR1-KLB affinity of FGF21 even after iodination, whereas replacing the corresponding phenylalanine residue with tyrosine in FGF19 did not alter its binding affinity to FGFR1-KLB, but decreased the receptor binding ability of the iodinated protein, suggesting that tyrosine-207 is the crucial amino acid responsible for the loss of specifying FGFR1-KLB affinity of the iodinated FGF21.

Glucose and amino acid metabolism in mice depend mutually on glucagon and insulin receptor signaling.

Glucagon and insulin are important regulators of blood glucose. The importance of insulin receptor signaling for alpha-cell secretion and of glucagon receptor signaling for beta-cell secretion is widely discussed and of clinical interest. Amino acids are powerful secretagogues for both hormones, and glucagon controls amino acid metabolism through ureagenesis. The role of insulin in amino acid metabolism is less clear. Female C57BL/6JRj mice received an insulin receptor antagonist (IRA) (S961; 30 nmol/kg), a glucagon receptor antagonist (GRA) (25-2648; 100 mg/kg), or both GRA and IRA (GRA + IRA) 3 h before intravenous administration of similar volumes of saline, glucose (0.5 g/kg), or amino acids (1 µmol/g) while anesthetized with isoflurane. IRA caused basal hyperglycemia, hyperinsulinemia, and hyperglucagonemia. Unexpectedly, IRA lowered basal plasma concentrations of amino acids, whereas GRA increased amino acids, lowered glycemia, and increased glucagon but did not influence insulin concentrations. After administration of GRA + IRA, insulin secretion was significantly reduced compared with IRA administration alone. Blood glucose responses to a glucose and amino acid challenge were similar after vehicle and GRA + IRA administration but greater after IRA and lower after GRA. Anesthesia may have influenced the results, which otherwise strongly suggest that both hormones are essential for the maintenance of glucose homeostasis and that the secretion of both is regulated by powerful negative feedback mechanisms. In addition, insulin limits glucagon secretion, while endogenous glucagon stimulates insulin secretion, revealed during lack of insulin autocrine feedback. Finally, glucagon receptor signaling seems to be of greater importance for amino acid metabolism than insulin receptor signaling.

Chronic maternal cortisol excess during late gestation leads to metabolic alterations in the newborn heart.

Our laboratory has previously shown in an ovine model of pregnancy that abnormal elevations in maternal cortisol during late gestation lead to increased fetal cardiac arrhythmias and mortality during peripartum. Furthermore, transcriptomic analysis of the fetal heart suggested alterations in TCA cycle intermediates and lipid metabolites in animals exposed to excess cortisol in utero. Therefore, we utilized a sheep model of pregnancy to determine how chronic increases in maternal cortisol alter maternal and fetal serum before birth and neonatal cardiac metabolites and lipids at term. Ewes were either infused with 1 mg·kg-1·day-1 of cortisol starting at gestational day 115 ( n = 9) or untreated ( n = 6). Serum was collected from the mother and fetus (gestational day 125), and hearts were collected following birth. Proton nuclear magnetic resonance (1H-NMR) spectroscopy was conducted to measure metabolic profiles of newborn heart specimens as well as fetal and maternal serum specimens. Mass spectrometry was conducted to measure lipid profiles of newborn heart specimens. We observed alterations in amino acid and TCA cycle metabolism as well as lipid and glycerophospholipid metabolism in newborn hearts after excess maternal cortisol in late gestation. In addition, we observed alterations in amino acid and TCA cycle metabolites in fetal but not in maternal serum during late gestation. These results suggest that fetal exposure to excess maternal cortisol alters placental and fetal metabolism before birth and limits normal cardiac metabolic maturation, which may contribute to increased risk of peripartum cardiac arrhythmias observed in these animals or later life cardiomyopathies.

No evidence of attenuation of placental insulin-stimulated Akt phosphorylation and amino acid transport in maternal obesity and gestational diabetes mellitus.

Pregnancies complicated by obesity and/or gestational diabetes (GDM) are associated with peripheral insulin resistance; however, the insulin responsiveness of the placenta in these pregnancy complications remains largely unknown. We tested the hypothesis that primary human trophoblast cells and placental villous explants will be insulin responsive, characterized by amino acid transport, Akt and Erk activity with maternal obesity, and/or GDM. We evaluated term placentas from women with normal body mass index (BMI) (normal; n = 15), obesity (OB; n = 11), normal BMI with GDM (N-GDM; n = 11), and obesity with GDM (OB-GDM; n = 11). In a subgroup, primary human trophoblast cells (PHT) were isolated, and in an independent subgroup placental villous explants were exposed to varying concentrations of insulin. Amino acid transport capacity and insulin signaling activity were determined. Insulin significantly increased amino acid transport activity to a similar degree in PHT cells isolated from normal (+21%), N-GDM (+38%), OB (+37%), and OB-GDM (+35%) pregnancies. Insulin increased Akt and Erk phosphorylation in PHT cells (3-fold) and in villous explants (2-fold) in all groups to a similar degree. In contrast to the peripheral maternal insulin resistance commonly associated with obesity and/or GDM, we found that the placenta is insulin sensitive in these pregnancy complications. We suggest that elevated maternal insulin levels in pregnancies complicated by obesity and/or GDM promote critical placental functions, including amino acid transport. Insulin-stimulated placental nutrient delivery may contribute to the increased risk of fetal overgrowth and adiposity in these pregnancies. Moreover, our findings may inform efforts to optimize insulin regimens for women with GDM.

Effects of a casein hydrolysate versus intact casein on gastric emptying and amino acid responses.

PURPOSE: Milk proteins and/or their hydrolysates have been reported to have beneficial effects for improving postprandial glycaemia. Gastric emptying is a major determinant of postprandial glycaemia, yet limited studies have examined the effects of intact milk proteins compared to hydrolysates on gastric emptying. We investigated gastric emptying of a casein hydrolysate compared to intact casein. METHODS: Nine overweight and obese adults (mean ± SD age: 59.5 ± 6.5 years and BMI 28.4 ± 2.6 kg/m2) were studied in a randomised crossover design. Gastric emptying was assessed by paracetamol absorption test, with HPLC-MS being used for determining paracetamol and its primary metabolites in plasma. Glucose, insulin and amino acid responses were also assessed. RESULTS: Linear mixed model analysis showed no effect of treatment [F(1, 55) = 2.1, P = 0.16] or treatment × time interactions [F(6, 54) = 1.5, P = 0.21] for paracetamol concentrations. In addition, there were no significant differences between the intact casein and hydrolysate for any of the gastric emptying outcome measures (Cmax, AUC0-30min, AUC0-60min; AUC0-240min). However, insulin was increased in the early postprandial period (iAUC0-15min, iAUC0-30min;P < 0.05) and there was a treatment effect for glucose [F(1, 53) = 5.3, P = 0.03] following the casein hydrolysate compared to intact casein. No significant differences in amino acids were found between the two conditions. CONCLUSIONS: Gastric emptying of a casein hydrolysate compared to intact casein does not differ. Mechanisms other than gastric emptying, for example the presence of a bioactive peptide sequence, may contribute to the glycaemic management effects of certain milk protein hydrolysates and warrant further investigation.

Effects of maternal alpha-ketoglutarate supplementation during lactation on the performance of lactating sows and suckling piglets.

The aim of the study was to investigate if dietary alpha-ketoglutarate (AKG) supplementation may improve the performance of lactating sows and their suckling piglets. After farrowing, 24 lactating sows (Large White × Landrace) with similar body weight (BW) were assigned to the control and AKG groups based on parity, and their lactation diets were supplemented with 0.00 or 0.25% AKG, respectively. It was found that supplementing the diet of lactating sows with 0.25% AKG enhanced growth performance of the suckling piglets from d 7 to d 21 of the lactation period, improved villus height of ileum and tended (p = 0.085) to increase mean volumetric bone mineral density of femur in the weanling piglets. In the lactating sows, dietary supplementation of AKG decreased plasma urea level on d 14 of lactation, decreased plasma calcium (Ca) concentrations from d 7 to d 21 of lactation and increased lactose and Ca levels in ordinary milk. Thus, it was proposed that AKG supplementation stimulates the capacity for lactose synthesis and Ca uptake in the mammary gland, thereby altering the composition of the ordinary milk which might be associated with the enhanced performance of piglets during the suckling period. These findings could lead to a better application of AKG in lactating nutrition, and therefore, promoting pork production.

Anti-Cancer Effects of Emodin on HepG2 Cells as Revealed by 1H NMR Based Metabolic Profiling.

Hepatic carcinoma is one of the most common cancers in the world, with a high incidence. Emodin is an anthraquinone derived from Polygonum multiflorum Thunb, possessing anti-cancer activity. The purpose of this study is to investigate the anti-cancer effect of different dosages of emodin on HepG2 cells using a 1H NMR based metabolic approach complemented with qRT-PCR and flow cytometry to identify potential markers and discover the targets to explore the underlying mechanism. Emodin can dose-dependently inhibit the growth of HepG2 cells, perturb cell cycle progression, down-regulate the expression of genes and proteins related to glycolysis, and trigger intracellular ROS generation. Orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA) and correlation network analysis of the 1H NMR data showed significant changes in many endogenous metabolites after emodin exposure concerning oxidative stress and disturbances in amino acid and energy metabolism. These findings are helpful to understand the anti-cancer mechanism of emodin and provide a theoretical basis for its future application and development.

Progesterone and plasma metabolites in women with and in those without premenstrual dysphoric disorder.

BACKGROUND: The molecular mechanisms underpinning the progesterone-triggering mood symptoms in women with premenstrual dysphoric disorder (PMDD) are unknown. Cell metabolism is a potential source of variability. Very little is known about the effect of progesterone sensitivity on the metabolome. In this study, we aimed to characterize the effects of progesterone on the global metabolic profile and explore the differences between women with PMDD and controls. METHODS: Plasma was obtained from 12 women with prospectively confirmed PMDD and 25 controls under two hormone conditions: (1) gonadal suppression induced by leuprolide acetate (3.75 mg IM monthly) and (2) add-back phase with leuprolide and progesterone (200 mg twice daily by vaginal suppository). The global metabolic profile was obtained using liquid and gas chromatography followed by mass spectrometry. Differences between groups and time points were tested using repeated measures analysis of variance. The false discovery rate was calculated to account for multiple testing. RESULTS: Amino acids and their derivatives represented 78% (28/36) of the known compounds that were found in significantly lower plasma concentrations after progesterone administration than during gonadal suppression. The concentration of tyrosine was nominally significantly decreased after progesterone add-back in controls, but not in cases (P = 0.02). CONCLUSION: Plasma levels of some amino acids are decreased in response to progesterone. Albeit preliminary, evidence further suggests that progesterone has a different effect on the metabolic profiles of women with PMDD compared to controls. Further research is needed to replicate our findings in a larger sample and to identify the unknown compounds, especially those differentially expressed.

Effects of Histidine Supplementation on Global Serum and Urine 1H NMR-based Metabolomics and Serum Amino Acid Profiles in Obese Women from a Randomized Controlled Study.

The aim of current study was to investigate the metabolic changes associated with histidine supplementation in serum and urine metabolic signatures and serum amino acid (AA) profiles. Serum and urine 1H NMR-based metabolomics and serum AA profiles were employed in 32 and 37 obese women with metabolic syndrome (MetS) intervened with placebo or histidine for 12 weeks. Multivariable statistical analysis were conducted to define characteristic metabolites. In serum 1H NMR metabolic profiles, increases in histidine, glutamine, aspartate, glycine, choline, and trimethylamine-N-oxide (TMAO) were observed; meanwhile, decreases in cholesterol, triglycerides, fatty acids and unsaturated lipids, acetone, and α/ß-glucose were exhibited after histidine supplement. In urine 1H NMR metabolic profiles, citrate, creatinine/creatine, methylguanidine, and betaine + TMAO were higher, while hippurate was lower in histidine supplement group. In serum AA profiles, 10 AAs changed after histidine supplementation, including increased histidine, glycine, alanine, lysine, asparagine, and tyrosine and decreased leucine, isoleucine, ornithine, and citrulline. The study showed a systemic metabolic response in serum and urine metabolomics and AA profiles to histidine supplementation, showing significantly changed metabolism in AAs, lipid, and glucose in obese women with MetS.

Glucagon's effect on liver protein metabolism in vivo.

The postprandial state is characterized by a storage of nutrients in the liver, muscle, and adipose tissue for later utilization. In the case of a protein-rich meal, amino acids (AA) stimulate glucagon secretion by the α-cell. The aim of the present study was to determine the impact of the rise in glucagon on AA metabolism, particularly in the liver. We used a conscious catheterized dog model to recreate a postprandial condition using a pancreatic clamp. Portal infusions of glucose, AA, and insulin were used to achieve postprandial levels, while portal glucagon infusion was either maintained at the basal level or increased by three-fold. The high glucagon infusion reduced the increase in arterial AA concentrations compared with the basal glucagon level (-23%, P < 0.05). In the presence of high glucagon, liver AA metabolism shifted toward a more catabolic state with less protein synthesis (-36%) and increased urea production (+52%). Net hepatic glucose uptake was reduced modestly (-35%), and AA were preferentially used in gluconeogenesis, leading to lower glycogen synthesis (-54%). The phosphorylation of AMPK was increased by the high glucagon infusion (+40%), and this could be responsible for increasing the expression of genes related to pathways producing energy and lowering those involved in energy consumption. In conclusion, the rise in glucagon associated with a protein-rich meal promotes a catabolic utilization of AA in the liver, thereby, opposing the storage of AA in proteins.

Ciprofloxacin triggered glutamate production by Corynebacterium glutamicum.

BACKGROUND: Corynebacterium glutamicum is a well-studied bacterium which naturally overproduces glutamate when induced by an elicitor. Glutamate production is accompanied by decreased 2-oxoglutatate dehydrogenase activity. Elicitors of glutamate production by C. glutamicum analyzed to molecular detail target the cell envelope. RESULTS: Ciprofloxacin, an inhibitor of bacterial DNA gyrase and topoisomerase IV, was shown to inhibit growth of C. glutamicum wild type with concomitant excretion of glutamate. Enzyme assays showed that 2-oxoglutarate dehydrogenase activity was decreased due to ciprofloxacin addition. Transcriptome analysis revealed that this inhibitor of DNA gyrase increased RNA levels of genes involved in DNA synthesis, repair and modification. Glutamate production triggered by ciprofloxacin led to glutamate titers of up to 37 ± 1 mM and a substrate specific glutamate yield of 0.13 g/g. Even in the absence of the putative glutamate exporter gene yggB, ciprofloxacin effectively triggered glutamate production. When C. glutamicum wild type was cultivated under nitrogen-limiting conditions, 2-oxoglutarate rather than glutamate was produced as consequence of exposure to ciprofloxacin. Recombinant C. glutamicum strains overproducing lysine, arginine, ornithine, and putrescine, respectively, secreted glutamate instead of the desired amino acid when exposed to ciprofloxacin. CONCLUSIONS: Ciprofloxacin induced DNA synthesis and repair genes, reduced 2-oxoglutarate dehydrogenase activity and elicited glutamate production by C. glutamicum. Production of 2-oxoglutarate could be triggered by ciprofloxacin under nitrogen-limiting conditions.

LC-MS metabolomics of psoriasis patients reveals disease severity-dependent increases in circulating amino acids that are ameliorated by anti-TNFα treatment.

Psoriasis is an immune-mediated highly heterogeneous skin disease in which genetic as well as environmental factors play important roles. In spite of the local manifestations of the disease, psoriasis may progress to affect organs deeper than the skin. These effects are documented by epidemiological studies, but they are not yet mechanistically understood. In order to provide insight into the systemic effects of psoriasis, we performed a nontargeted high-resolution LC-MS metabolomics analysis to measure plasma metabolites from individuals with mild or severe psoriasis as well as healthy controls. Additionally, the effects of the anti-TNFα drug Etanercept on metabolic profiles were investigated in patients with severe psoriasis. Our analyses identified significant psoriasis-associated perturbations in three metabolic pathways: (1) arginine and proline, (2) glycine, serine and threonine, and (3) alanine, aspartate, and glutamate. Etanercept treatment reversed the majority of psoriasis-associated trends in circulating metabolites, shifting the metabolic phenotypes of severe psoriasis toward that of healthy controls. Circulating metabolite levels pre- and post-Etanercept treatment correlated with psoriasis area and severity index (PASI) clinical scoring (R(2) = 0.80; p < 0.0001). Although the responsible mechanism(s) are unclear, these results suggest that psoriasis severity-associated metabolic perturbations may stem from increased demand for collagen synthesis and keratinocyte hyperproliferation or potentially the incidence of cachexia. Data suggest that levels of circulating amino acids are useful for monitoring both the severity of disease as well as therapeutic response to anti-TNFα treatment.

Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model.

Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we tested the hypothesis that prolonged systemic pyruvate supplementation activates pyruvate oxidation in an immature swine model in vivo. Twelve male mixed-breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (group C) or pyruvate (group P) during the final 6 h of 8 h of ECMO. Over the final hour, piglets received [2-(13)C] pyruvate, as a reference substrate for oxidation, and [(13)C6]-l-leucine, as an indicator for amino acid oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of the citric acid cycle intermediates. An increase in anaplerotic flux through pyruvate carboxylation in group P occurred compared with no change in pyruvate oxidation. Additionally, pyruvate promoted an increase in the phosphorylation state of several nutrient-sensitive enzymes, like AMP-activated protein kinase and acetyl CoA carboxylase, suggesting activation for fatty acid oxidation. Pyruvate also promoted O-GlcNAcylation through the hexosamine biosynthetic pathway. In conclusion, although prolonged pyruvate supplementation did not alter pyruvate oxidation, it did elicit changes in nutrient- and energy-sensitive pathways. Therefore, the observed results support the further study of pyruvate and its downstream effect on cardiac function.

The amino acid response to a mixed meal in patients with type 2 diabetes: effect of sitagliptin treatment.

AIMS: Amino acid (AA) metabolism is altered in type 2 diabetes (T2D), and fasting levels of α-hydroxybutyrate (α-HB), a biomarker for insulin resistance, have been suggested to track AA metabolism. We investigated the changes in AA and α-HB induced by a mixed-meal tolerance test (MTT) and the effects of sitagliptin treatment. METHODS: Forty-seven T2D patients [56 ± 7 years, body mass index (BMI) 29.9 ± 4.2 kg/m(2) ] were randomized to sitagliptin (100 mg/day, 6 weeks) or placebo. Seven age- and BMI-matched non-diabetic subjects served as control (CT). RESULTS: During a 5-h MTT, branched-chain AA (BCAA) peaked earlier in T2D than CT [75(25) vs. 62(3) mmol/l · h over 2 h, median(interquartile range), p = 0.05], and rose higher [5-h increment: 31(23) vs. 19(24) mmol/l · h, p = 0.05]. Fasting α-HB was higher [7.5(2.7) vs. 5.9(1.3) µg/ml, p = 0.04 T2D vs. CT], and its meal-induced increments were larger [24(99) vs. -41(86) µg/ml · h, p = 0.006]. Plasma non-esterified fatty acids (NEFA) declined during MTT, but their increments were greater in patients (53 ± 16 vs. 35 ± 10 mEq/l · h, p = 0.005). Compared to placebo, both BCAA [-6.4(21.1) vs. 0.0(48.0) mmol/l · h, p = 0.01] and α-HB increments [-114(250) vs. 114(428) µg/ml · h, p = 0.002] decreased with sitagliptin, and meal-induced NEFA suppression was improved. Changes in BCAA and α-HB were reciprocally related to changes in insulin sensitivity (ρ = -0.37 and -0.43, p ≤ 0.01). CONCLUSIONS: T2D is associated with a hyperaminoacidaemic response to MTT, which circulating α-HB levels track. Sitagliptin-induced glycaemic improvement was associated with reductions in BCAA and α-HB excursions and better NEFA suppression, in parallel with improved insulin sensitivity, confirming that α-HB is a readout of metabolic overload.

Amino acid concentrations in critically ill children following cardiac surgery*.

OBJECTIVE: Guidelines for administering amino acids to critically ill children are largely based on uncontrolled observational studies and expert opinion, without support from rigorous outcome studies. Also, data on circulating amino acid concentrations during critical illness are scarce. We thoroughly studied the time profiles of circulating amino acid concentrations in critically ill children who received standard nutritional care according to international guidelines. DESIGN: This is a subanalysis of pediatric critically ill patients included in a large (n = 700) randomized controlled study on intensive insulin therapy. SETTING: The study was conducted at a university hospital PICU. PATIENTS: We studied 100 patients in PICU for at least 3 days following cardiac surgery. INTERVENTIONS: Patients were assigned to intensive insulin therapy targeting normal-for-age fasting blood glucose concentrations or insulin infusion only to prevent excessive hyperglycemia. MEASUREMENTS AND MAIN RESULTS: Plasma amino acid concentrations were measured at admission, day 3, and day 7 in PICU. At admission, the concentrations of most amino acids were comparable to those reported for healthy children. Total amino acid concentrations remained stable during ICU stay, but individual amino acids showed different time profiles with eight of them showing an increase and five a decrease. Nonsurviving children had higher total amino acid concentrations and individual amino acids compared with survivors at admission and/or during ICU stay. Intensive insulin therapy lowered the concentrations of total amino acids and several individual amino acids. Neonates showed somewhat different amino acid profiles with rather increased concentrations from baseline with time in ICU for total amino acids and several individual amino acids as compared with older infants and children. CONCLUSIONS: Circulating amino acid concentrations in critically ill children after cardiac surgery differ according to survival status, blood glucose control with intensive insulin therapy, and age.