GLUT1-mediated glucose import in B cells is critical for anaplerotic balance and humoral immunity.

Glucose uptake increases during B cell activation and antibody-secreting cell (ASC) differentiation, but conflicting findings prevent a clear metabolic profile at different stages of B cell activation. Deletion of the glucose transporter type 1 (GLUT1) gene in mature B cells (GLUT1-cKO) results in normal B cell development, but it reduces germinal center B cells and ASCs. GLUT1-cKO mice show decreased antigen-specific antibody titers after vaccination. In vitro, GLUT1-deficient B cells show impaired activation, whereas established plasmablasts abolish glycolysis, relying on mitochondrial activity and fatty acids. Transcriptomics and metabolomics reveal an altered anaplerotic balance in GLUT1-deficient ASCs. Despite attempts to compensate for glucose deprivation by increasing mitochondrial mass and gene expression associated with glycolysis, the tricarboxylic acid cycle, and hexosamine synthesis, GLUT1-deficient ASCs lack the metabolites for energy production and mitochondrial respiration, limiting protein synthesis. We identify GLUT1 as a critical metabolic player defining the germinal center response and humoral immunity.

ATP citrate lyase (ACLY)-dependent immunometabolism in mucosal T cells drives experimental colitis in vivo.

OBJECTIVE: Mucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory immune cell function, we here focus on the enzyme ATP citrate lyase (ACLY) in mucosal T cell immunometabolism and its relevance for IBD. DESIGN: ACLY expression and its immunometabolic impact on colitogenic T cell function were analysed in mucosal T cells from patients with IBD and in two experimental colitis models. RESULTS: ACLY was markedly expressed in colon tissue under steady-state conditions but was significantly downregulated in lamina propria mononuclear cells in experimental dextran sodium sulfate-induced colitis and in CD4+ and to a lesser extent in CD8+ T cells infiltrating the inflamed gut in patients with IBD. ACLY-deficient CD4+ T cells showed an impaired capacity to induce intestinal inflammation in a transfer colitis model as compared with wild-type T cells. Assessment of T cell immunometabolism revealed that ACLY deficiency dampened the production of IBD-relevant cytokines and impaired glycolytic ATP production but enriched metabolites involved in the biosynthesis of phospholipids and phosphatidylcholine. Interestingly, the short-chain fatty acid butyrate was identified as a potent suppressor of ACLY expression in T cells, while IL-36α and resolvin E1 induced ACLY levels. In a translational approach, in vivo administration of the butyrate prodrug tributyrin downregulated mucosal infiltration of ACLYhigh CD4+ T cells and ameliorated chronic colitis. CONCLUSION: ACLY controls mucosal T cell immunometabolism and experimental colitis. Therapeutic modulation of ACLY expression in T cells emerges as a novel strategy to promote the resolution of intestinal inflammation.

A supervised machine-learning approach for the efficient development of a multi method (LC-MS) for a large number of drugs and subsets thereof: focus on oral antitumor agents.

OBJECTIVES: Accumulating evidence argues for a more widespread use of therapeutic drug monitoring (TDM) to support individualized medicine, especially for therapies where toxicity and efficacy are critical issues, such as in oncology. However, development of TDM assays struggles to keep pace with the rapid introduction of new drugs. Therefore, novel approaches for faster assay development are needed that also allow effortless inclusion of newly approved drugs as well as customization to smaller subsets if scientific or clinical situations require. METHODS: We applied and evaluated two machine-learning approaches i.e., a regression-based approach and an artificial neural network (ANN) to retention time (RT) prediction for efficient development of a liquid chromatography mass spectrometry (LC-MS) method quantifying 73 oral antitumor drugs (OADs) and five active metabolites. Individual steps included training, evaluation, comparison, and application of the superior approach to RT prediction, followed by stipulation of the optimal gradient. RESULTS: Both approaches showed excellent results for RT prediction (mean difference ± standard deviation: 2.08 % ± 9.44 % ANN; 1.78 % ± 1.93 % regression-based approach). Using the regression-based approach, the optimum gradient (4.91 % MeOH/min) was predicted with a total run time of 17.92 min. The associated method was fully validated following FDA and EMA guidelines. Exemplary modification and application of the regression-based approach to a subset of 14 uro-oncological agents resulted in a considerably shortened run time of 9.29 min. CONCLUSIONS: Using a regression-based approach, a multi drug LC-MS assay for RT prediction was efficiently developed, which can be easily expanded to newly approved OADs and customized to smaller subsets if required.

L-arginine metabolism inhibits arthritis and inflammatory bone loss.

OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.

A Metabolomic Analysis of Sensitivity and Specificity of 23 Previously Proposed Biomarkers for Renal Transporter-Mediated Drug-Drug Interactions.

Endogenous biomarkers are discussed as tools for detection of drug-drug interactions (DDIs) mediated by renal transport proteins, such as organic cation transporter 2 (OCT2), multidrug and toxin extrusion proteins (MATE1 and MATE2-K) and organic anion transporters (OAT1 and OAT3). Whereas sensitivity of some endogenous biomarkers against at least one clinical transporter inhibitor has frequently been shown, intra-study comparisons of the extent of effects of inhibitors on different biomarkers are frequently lacking. Moreover, in vivo specificity of such discussed biomarkers has frequently not been studied. We therefore investigated changes of 10 previously described putative biomarkers for inhibition of OCT2/MATEs, as well as 15 previously described putative biomarkers for OATs in human plasma and urine samples of healthy volunteers in response to treatment with 4 inhibitors of transport proteins [verapamil (P-glycoprotein), rifampin (organic anion transporting polypeptides), cimetidine (OCT2/MATEs), and probenecid (OATs)]. Two of the putative biomarkers had been suggested for both OCT2/MATEs and OATs. All substances were unequivocally identified in an untargeted metabolomics assay. The OCT2/MATE biomarkers choline and trimethylamine N-oxide were both sensitive and specific (median log2-fold changes -1.18 in estimated renal elimination and -0.85 in urinary excretion, respectively). For renal OATs, indoleacetyl glutamine and indoleacetic acid (median log2-fold changes -3.77 and -2.85 in estimated renal elimination, respectively) were the candidates for sensitive and specific biomarkers with the most extensive change, followed by taurine, indolelactic acid, and hypoxanthine. This comprehensive study adds further knowledge on sensitivity and specificity of 23 previously described biomarkers of renal OCT2/MATE- and OAT-mediated DDIs.

N1 -Methylnicotinamide as Biomarker for MATE-Mediated Renal Drug-Drug Interactions: Impact of Cimetidine, Rifampin, Verapamil, and Probenecid.

N1 -methylnicotinamide (NMN) has been proposed as endogenous biomarker for drug-drug interactions mediated by inhibition of multidrug and toxin extrusion proteins (MATEs) at the renal proximal tubule. We analyzed NMN in plasma and urine samples of two clinical trials investigating a new probe drug cocktail (consisting of digoxin, metformin, furosemide, and rosuvastatin) dedicated to clinically relevant drug transporters. In trial 1, NMN was investigated after single-dose treatment with individual cocktail components or after cocktail treatment. In trial 2, NMN was investigated after treatment with cocktail alone or with cocktail + inhibitor (cimetidine, a MATE inhibitor; or rifampin, verapamil, or probenecid, inhibitors of other transporters). In trial 1, NMN kinetics in plasma and urine were essentially not affected by individual cocktail components or after cocktail treatment. In trial 2, NMN renal clearance from 0 to 12 hours (CLR,0-12 ) geometric mean ratio (GMR) after cocktail + cimetidine vs. cocktail alone was 75% (90% confidence interval (CI): 65-87%). NMN CLR GMR after cocktail + verapamil, + rifampin, or + probenecid vs. cocktail alone was 99% (90% CI: 81-121%), 91% (90% CI: 75-111%), and 107% (90% CI: 91-126%), respectively. Compared with creatinine CLR and creatinine area under the plasma-concentration time curve, NMN CLR was more specific and more sensitive for renal MATE inhibition. Absence of impact of the cocktail on NMN in trial 1 allows for utilization of NMN in studies using this transporter cocktail. Trial 2 data support that NMN CLR is a specific and sensitive marker for MATE-mediated renal drug-drug interactions.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) is localized in lysosomes and mediates resistance against kinase inhibitors.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3), a splice variant of the hepatic uptake transporter OATP1B3 (liver-type; Lt-OATP1B3), is expressed in several tumor entities including colorectal carcinoma (CRC) and breast cancer. In CRC, high OATP1B3 expression has been associated with reduced progression-free and overall survival. Several kinase inhibitors used for antitumor treatment are substrates and/or inhibitors of OATP1B3 (e.g. encorafenib, vemurafenib). The functional importance of Ct-OATP1B3 has not been elucidated so far. HEK293 cells stably overexpressing Ct-OATP1B3 protein were established and compared with control cells. Confocal laser scanning microscopy, immunoblot, and proteomics-based expression analysis demonstrated that Ct-OATP1B3 protein is intracellularly localized in lysosomes of stably-transfetced cells. Cytotoxicity experiments showed that cells recombinantly expressing the Ct-OATP1B3 protein were more resistant against the kinase inhibitor encorafenib compared to control cells [e.g. encorafenib (100 µM) survival rates: 89.5% vs. 52.8%]. In line with these findings, colorectal cancer DLD1 cells endogenously expressing Ct-OATP1B3 protein had poorer survival rates when the OATP1B3 substrate bromosulfophthalein (BSP) was coincubated with encorafenib or vemurafenib compared to the incubation with the kinase inhibitor alone. This indicates a competitive inhibition of Ct-OATP1B3-mediated uptake into lysosomes by BSP. Accordingly, mass spectrometry-based drug analysis of lysosomes showed a reduced lysosomal accumulation of encorafenib in DLD1 cells additionally exposed to BSP. These results demonstrate that Ct-OATP1B3 protein is localized in the lysosomal membrane and can mediate transport of certain kinase inhibitors into lysosomes revealing a new mechanism of resistance. Significance Statement We describe the characterization of a splice variant of the liver-type uptake transporter OATP1B3 expressed in several tumor entities. This variant is localized in lysosomes mediating resistance against kinase inhibitors which are substrates of this transport protein by transporting them into lysosomes and thereby reducing the cytoplasmic concentration of these antitumor agents. Therefore, the expression of the Ct-OATP1B3 protein is associated with a better survival of cells revealing a new mechanism of drug resistance.

Effects of treatment with SGLT-2 inhibitors on arginine-related cardiovascular and renal biomarkers.

BACKGROUND: In patients with type 2 diabetes (T2D) sodium-glucose cotransporter 2 (SGLT-2) inhibitors improve glycaemic control as well as cardiovascular and renal outcomes. Their effects on L-arginine (Arg) related risk markers asymmetric and symmetric dimethylarginine (ADMA and SDMA) and the protective biomarker L-homoarginine (hArg) linking T2D to cardiovascular and renal disease have not yet been reported. METHODS: Plasma and 24-h urine samples taken before and after 6 weeks of treatment were available from two prospective, randomized, double-blind, placebo-controlled, cross-over trials with empagliflozin (71 patients analyzed, NCT02471963) and dapagliflozin (59 patients analyzed, NCT02383238). In these samples, concentrations of hArg, Arg, ADMA, SDMA, and creatinine were determined by liquid-chromatography coupled to tandem mass-spectrometry. Additionally, intraindividual changes of the biomarkers in plasma were correlated with intraindividual changes of clinical parameters. RESULTS: Treatment with empagliflozin and dapagliflozin was associated with a reduction of plasma hArg by 17.5% and 13.7% (both p < 0.001), respectively, and increase in plasma SDMA concentration of 6.7% and 3.6%, respectively (p < 0.001 and p < 0.05), while plasma Arg and ADMA concentrations were not significantly altered. 24-h urinary excretion of ADMA was reduced by 15.2% after treatment with empagliflozin (p < 0.001) but not after dapagliflozin treatment, while excretion of the other markers was not significantly altered. Renal clearance of SDMA was reduced by 9.1% and 3.9% for both drugs (both p < 0.05). A reduction in ADMA clearance was observable after empagliflozin treatment only (- 15.5%, p < 0.001), but not after dapagliflozin. Renal clearance of hArg and Arg was not significantly altered. Treatment effects on L-arginine related biomarkers were not constantly correlated with effects on glycated hemoglobin, fasting plasma glucose, body mass index, and systolic blood pressure. CONCLUSIONS: Treatment with SGLT-2 inhibitors has divergent effects on Arg-related biomarkers and could affect risk estimates associated with these markers. The observed effects are unlikely to explain the known cardiovascular and renal benefits of treatment with empagliflozin or dapagliflozin but still may indicate new therapeutic approaches in patients treated with SGLT-2 inhibitors. Trial registration http://www.clinicaltrials.gov : NCT02471963 (registered 15th June 2015, retrospectively registered) and NCT02383238.

An Easily Expandable Multi-Drug LC-MS Assay for the Simultaneous Quantification of 57 Oral Antitumor Drugs in Human Plasma.

Oral anticancer drugs have led to significant improvements in the treatment of multiple tumor entities. However, in patients undergoing oral antitumor therapy, plasma concentrations are highly variable, resulting in risks of reduced therapeutic effects or an increase in side effects. One important tool to reduce this variability is therapeutic drug monitoring. In this work we describe a method to simultaneously quantify the plasma concentrations of 57 oral antitumor agents. Quantification of these drugs was achieved using liquid chromatography coupled to an Orbitrap mass spectrometer. The method was fully validated according to the FDA guidelines and constitutes a simple and robust way for exposure monitoring of a wide variety of oral anticancer drugs. Applicability to clinical routine was demonstrated by the analysis of 71 plasma samples taken from 39 patients. In summary, this new multi-drug method allows simultaneous quantification of 57 oral antitumor drugs, which can be applied to exposure monitoring in clinical studies, taking into account the broad variety of oral antitumor drugs prescribed in clinical routine.

Vectorial transport of the arginine derivatives asymmetric dimethylarginine (ADMA) and L-homoarginine by OATP4C1 and P-glycoprotein studied in double-transfected MDCK cells.

Elevated plasma concentrations of the uremic toxin asymmetric dimethylarginine (ADMA) and low plasma concentrations of L-homoarginine are independently associated with cardiovascular events and mortality. Key enzymes involved in the homeostasis of both arginine derivatives are expressed in proximal tubule cells of the kidney. To get access to these enzymes, transport proteins are important. One of the transporters mediating the transport of ADMA and L-homoarginine is the solute carrier superfamily (SLC) member OATP4C1, located in the basolateral membrane of proximal tubule cells. To gain insights into the role of export pumps in the transport of both substances, we established a double-transfected MDCK cell line expressing OATP4C1 and the export pump P-glycoprotein (P-gp). Using MDCK cell monolayers, we demonstrated in time-dependent and concentration-dependent vectorial transport experiments that ADMA and L-homoarginine are transported from the basolateral to the apical compartment of MDCK-OATP4C1-P-gp cells with significantly higher transport rates compared to single-transfected MDCK-OATP4C1, MDCK-P-gp and MDCK-VC (control) cells (e.g. transport ratio MDCK-OATP4C1-P-gp/MDCK-VC: for 50 µM ADMA = 2.0-fold, for 50 µM L-homoarginine = 3.4-fold). These results indicate that both OATP4C1 and P-gp transport the arginine derivatives ADMA and L-homoarginine and are, therefore, important for the homoeostasis of both substances.

Trimethylamine-N-oxide (TMAO) determined by LC-MS/MS: distribution and correlates in the population-based PopGen cohort.

Background Accumulating evidence indicates that trimethylamine-N-oxide (TMAO) may play a causal role in cardiovascular disease (CVD), chronic kidney disease (CKD) and type 2 diabetes (T2D). TMAO plasma concentrations show considerable intra- and inter-individual variation, underscoring the need for a reference interval in the general population to identify elevated TMAO concentrations. Methods TMAO concentrations were determined using an LC-MS/MS assay in a community-based sample of the PopGen control cohort consisting of 694 participants (54% men; aged 25-82 years) free of clinical CVD, CKD and T2D. We defined reference intervals for TMAO concentrations in human plasma using the 2.5th and 97.5th percentiles. Using multivariable regression analysis we analyzed the association of estimated glomerular filtration rate (eGFR), sex, and dietary intake and TMAO plasma concentrations. Results TMAO plasma concentrations were positively skewed and differed by sex. The median TMAO plasma concentration in men was 3.91 (Q1-Q3: 2.87-6.10) µmol/L and the reference interval 1.28-19.67 µmol/L (2.5th-97.5th percentile). In women median TMAO plasma concentration was 3.56 (Q1-Q3: 2.41-5.15) µmol/L and the reference interval 1.08-17.12 µmol/L. In multivariable regression analysis plasma TMAO was associated with sex, renal function and diet. The association of TMAO and diet was significant for intake of fish and shellfish in men only. Conclusions In a community-based sample free of apparent CVD and renal disease, we report the distribution of TMAO plasma concentrations with sex, renal function and diet as factors associated with plasma TMAO, and suggest reference intervals. These data may facilitate standardized comparisons of TMAO across populations.

Establishment of reference values for the lysine acetylation marker Nɛ-acetyllysine in small volume human plasma samples by a multi-target LC-MS/MS method.

Cardiovascular disease (CVD) and chronic kidney disease (CKD) constitute substantial burdens for public health. The identification and validation of risk markers for CVD and CKD in epidemiological studies requires frequent adaption of existing analytical methods as well as development of new methods. In this study, an analytical procedure to simultaneously quantify ten endogenous biomarkers for CVD and CKD is described. An easy-to-handle sample preparation requiring only 20 µL of human plasma is followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was successfully validated according to established guidelines meeting required criteria for accuracy, precision, recovery, linearity, selectivity, and limits of quantification. The scalability of the method for application in larger cohorts was assessed using a set of plasma samples from healthy volunteers (n = 391) providing first reference values for the recently established biomarker Nɛ-acetyllysine (Nɛ-AcLys). Other biomarkers analyzed were creatinine, ß-aminoisobutyric acid (ß-AIB), carnitine, 1-methylnicotinamide (1-MNA), citrulline, symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), homoarginine (hArg), and ornithine. All obtained results are within reference values specified elsewhere. Overall, these results demonstrate the suitability of the method for simultaneous quantification of ten endogenous biomarkers for CVD and CKD in plasma samples from larger cohorts and allow validation of Nɛ-AcLys as a biomarker in large cohorts.

Clinical Aspects of Transporter-Mediated Drug-Drug Interactions.

Drug transporters play an essential role in disposition and effects of multiple drugs. Plasma concentrations of the victim drug can be modified by drug-drug interactions occurring in enterocytes (e.g., P-glycoprotein), hepatocytes (e.g., organic anion-transporting polypeptide 1B1 (OATP1B1)), and/or renal proximal tubular cells (e.g., organic cation transporter 2 (OCT2)/multidrug and toxin extrusion 1 and 2-K (MATE1/MATE2-K)). In addition, transporter-mediated drug-drug interactions can cause altered local tissue concentrations and possibly altered effects/toxicity (e.g., in liver and kidneys). During drug development, there is now an intensive in vitro screening of new molecular entities as transporter substrates and inhibitors, followed if necessary by drug-drug interaction studies in healthy volunteers. Nevertheless, there are still unresolved issues, which will also be discussed in this review article (e.g., the clinical significance of transporter-mediated drug-drug interactions of particular relevance to the elderly who are prescribed multiple drugs, with additional impaired liver or kidney function, and the extent to which medication safety in real life could be improved by a reduction of those interactions).

Changing Biosynthetic Profiles by Expressing bldA in Streptomyces Strains.

Recently we described an unusual way of activating a cryptic gene cluster when we explored the origin of the bald phenotype of Streptomyces calvus. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a new natural products. In this study we report on the expression of bldA in several Streptomyces strains that have been described as "poorly sporulating" strains. In seven out of 15 cases, HPLC profiling revealed the production of new compounds, and in two cases the overproduction of known compounds. Two compounds were isolated and their structures were determined.

A cryptic polyene biosynthetic gene cluster in Streptomyces calvus is expressed upon complementation with a functional bldA gene.

Streptomyces calvus is best known as the producer of the fluorinated natural product nucleocidin. This strain of Streptomycetes is also unusual for displaying a "bald" phenotype that is deficient in the formation of aerial mycelium and spores. Genome sequencing of this organism revealed a point mutation in the bldA gene that is predicted to encode a misfolded Leu-tRNA(UUA) molecule. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a polyeneoic acid amide, 4-Z-annimycin, and a minor amount of the isomer, 4-E-annimycin. Bioassays reveal that these compounds inhibit morphological differentiation in other Actinobacteria. The annimycin gene cluster encoding a type 1 polyketide synthase was identified and verified through disruption studies. This study underscores the importance of the bldA gene in regulating the expression of cryptic biosynthetic genes.

Complete genome sequence of Saccharothrix espanaensis DSM 44229(T) and comparison to the other completely sequenced Pseudonocardiaceae.

BACKGROUND: The genus Saccharothrix is a representative of the family Pseudonocardiaceae, known to include producer strains of a wide variety of potent antibiotics. Saccharothrix espanaensis produces both saccharomicins A and B of the promising new class of heptadecaglycoside antibiotics, active against both bacteria and yeast. RESULTS: To better assess its capabilities, the complete genome sequence of S. espanaensis was established. With a size of 9,360,653 bp, coding for 8,501 genes, it stands alongside other Pseudonocardiaceae with large genomes. Besides a predicted core genome of 810 genes shared in the family, S. espanaensis has a large number of accessory genes: 2,967 singletons when compared to the family, of which 1,292 have no clear orthologs in the RefSeq database. The genome analysis revealed the presence of 26 biosynthetic gene clusters potentially encoding secondary metabolites. Among them, the cluster coding for the saccharomicins could be identified. CONCLUSION: S. espanaensis is the first completely sequenced species of the genus Saccharothrix. The genome discloses the cluster responsible for the biosynthesis of the saccharomicins, the largest oligosaccharide antibiotic currently identified. Moreover, the genome revealed 25 additional putative secondary metabolite gene clusters further suggesting the strain's potential for natural product synthesis.