PubMed

Related Articles Cysteine depletion induces pancreatic tumor ferroptosis in mice. Science. 2020 Apr 03;368(6486):85-89 Authors: Badgley MA, Kremer DM, Maurer HC, DelGiorno KE, Lee HJ, Purohit V, Sagalovskiy IR, Ma A, Kapilian J, Firl CEM, Decker AR, Sastra SA, Palermo CF, Andrade LR, Sajjakulnukit P, Zhang L, Tolstyka ZP, Hirschhorn T, Lamb C, Liu T, Gu W, Seeley ES, Stone E, Georgiou G, Manor U, Iuga A, Wahl GM, Stockwell BR, Lyssiotis CA, Olive KP Abstract Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC - is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC - subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC. PMID: 32241947 [PubMed - in process]

Related Articles Donor sex, age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity. Haematologica. 2020 Apr 02;: Authors: D'Alessandro A, Fu X, Kanias T, Reisz JA, Culp-Hill R, Guo Y, Gladwin MT, Page G, Kleinman S, Lanteri M, Stone M, Busch MP, Zimring JC, Recipient Epidemiology and Donor Evaluation Study-III (REDS III) Abstract Red blood cell storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, the metabolic underpinnings of the capacity of RBCs to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBCs from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyze following oxidant stress. A subset of extreme hemolyzers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBCs from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBCs stored in additive solution-3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBCs stored in additive solution-1. Merging metabolomics data with results from an independent GWAS study on the same cohort, we identified metabolic markers of hemolysis and G6PD-deficiency, which were associated with extremes in oxidative hemolysis and dysregulation in NADPH and glutathione-dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and G6PD status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults. PMID: 32241843 [PubMed - as supplied by publisher]

Related Articles Research Note: Metabolic changes and physiological responses of broilers in the final stage of growth exposed to different environmental temperatures. Poult Sci. 2020 Apr;99(4):2017-2025 Authors: Wang Y, Xia L, Guo T, Heng C, Jiang L, Wang D, Wang J, Li K, Zhan X Abstract There is no information regarding the influence of heat stress (HS) on host metabolic profile. In this study, we investigated the effects of different environmental temperatures on oxidative status, hormone levels, HS indicators, and plasma metabolites in broilers. A total of 1,680 yellow-feather broilers (28 D old) were randomly allotted to 4 groups with 6 replicates. The broilers (29-57 D old) were maintained in thermostatic rooms (20°C, 25°C, 28°C, and 30°C) for 28 consecutive days. The results showed that the plasma cortisol and adrenocorticotropic hormone levels and creatine kinase and lactate dehydrogenase activities gradually increased when the temperature increased from 20°C to 30°C. However, the insulin-like growth factor-І level decreased gradually. Furthermore, heat shock protein 70 expression significantly increased in the liver and breast muscle (P < 0.01). As the temperature increased, the total anti-oxidant capacity in the plasma and liver gradually decreased, whereas the malondialdehyde level increased. The activity of plasma glutathione peroxidase and total superoxide dismutase in the liver showed a similar increasing trend (P < 0.01). In addition, 15 metabolites were identified at higher (P < 0.05) levels, whereas 2 metabolites were identified at lower (P < 0.05) levels in the 30°C treatment group than those in the 25°C treatment group. Most of these potentially diagnostic biomarkers are involved in carbohydrate, amino acid, lipid, or gut microbiome-derived metabolism, indicating that HS affected the metabolic pathways in broilers. Six candidate metabolites (tartronic acid, l-bethreine, tartaric acid, allose, glutaric acid, and neohesperidin) were selected as biomarkers, as they showed high sensitivity, specificity, and accuracy in diagnosing broilers under HS (P < 0.01). In conclusion, in the final stage of growth, we identified 6 plasma differential metabolites as potential biomarkers of HS-induced metabolic disorders in yellow-feathered broilers. This work offers new insights into the metabolic alterations of broilers exposed to HS and provides a new perspective for further study. PMID: 32241486 [PubMed - in process]

Related Articles Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis. Proc Natl Acad Sci U S A. 2019 10 08;116(41):20644-20654 Authors: Chen F, Lukat P, Iqbal AA, Saile K, Kaever V, van den Heuvel J, Blankenfeldt W, Büssow K, Pessler F Abstract cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid. PMID: 31548418 [PubMed - indexed for MEDLINE]

Related Articles Inhibition of Δ24-dehydrocholesterol reductase activates pro-resolving lipid mediator biosynthesis and inflammation resolution. Proc Natl Acad Sci U S A. 2019 10 08;116(41):20623-20634 Authors: Körner A, Zhou E, Müller C, Mohammed Y, Herceg S, Bracher F, Rensen PCN, Wang Y, Mirakaj V, Giera M Abstract Targeting metabolism through bioactive key metabolites is an upcoming future therapeutic strategy. We questioned how modifying intracellular lipid metabolism could be a possible means for alleviating inflammation. Using a recently developed chemical probe (SH42), we inhibited distal cholesterol biosynthesis through selective inhibition of Δ24-dehydrocholesterol reductase (DHCR24). Inhibition of DHCR24 led to an antiinflammatory/proresolving phenotype in a murine peritonitis model. Subsequently, we investigated several omics layers in order to link our phenotypic observations with key metabolic alterations. Lipidomic analysis revealed a significant increase in endogenous polyunsaturated fatty acid (PUFA) biosynthesis. These data integrated with gene expression analysis, revealing increased expression of the desaturase Fads6 and the key proresolving enzyme Alox-12/15 Protein array analysis, as well as immune cell phenotype and functional analysis, substantiated these results confirming the antiinflammatory/proresolving phenotype. Ultimately, lipid mediator (LM) analysis revealed the increased production of bioactive lipids, channeling the observed metabolic alterations into a key class of metabolites known for their capacity to change the inflammatory phenotype. PMID: 31548397 [PubMed - indexed for MEDLINE]

Related Articles Expression and gene regulation network of RBM8A in hepatocellular carcinoma based on data mining. Aging (Albany NY). 2019 01 22;11(2):423-447 Authors: Lin Y, Liang R, Qiu Y, Lv Y, Zhang J, Qin G, Yuan C, Liu Z, Li Y, Zou D, Mao Y Abstract RNA binding motif protein 8A (RBM8A) is an RNA binding protein in a core component of the exon junction complex. Abnormal RBM8A expression is associated with carcinogenesis. We used sequencing data from the Cancer Genome Atlas database and Gene Expression Omnibus, analyzed RBM8A expression and gene regulation networks in hepatocellular carcinoma (HCC). Expression was analyzed using OncomineTM and Gene Expression Profiling Interactive Analysis tools, while RBM8A alterations and related functional networks were identified using cBioPortal. LinkedOmics was used to identify differential gene expression with RBM8A and to analyze Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Gene enrichment analysis examined target networks of kinases, miRNAs and transcription factors. We found that RBM8A is overexpressed and the RBM8A gene often amplified in HCC. Expression of this gene is linked to functional networks involving the ribosome and RNA metabolic signaling pathways. Functional network analysis suggested that RBM8A regulates the spliceosome, ribosome, DNA replication and cell cycle signaling via pathways involving several cancer-related kinases, miRNAs and E2F Transcription Factor 1. Our results demonstrate that data mining efficiently reveals information about RBM8A expression and potential regulatory networks in HCC, laying a foundation for further study of the role of RBM8A in carcinogenesis. PMID: 30670676 [PubMed - indexed for MEDLINE]

37 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2020/04/03PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

17 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2020/04/02PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

17 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2020/04/01PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

17 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2020/03/31PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

17 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2020/03/31PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

Related Articles Integrated omics analysis of sweat reveals an aberrant amino acid metabolism pathway in Vogt-Koyanagi-Harada disease. Clin Exp Immunol. 2020 Mar 28;: Authors: Cui X, Su G, Zhang L, Yi S, Cao Q, Zhou C, Kijlstra A, Yang P Abstract Vogt-Koyanagi-Harada (VKH) disease is an autoimmune disease leading to visual impairment. Its pathogenic mechanisms remain poorly understood. Our purpose was to investigate the distinctive protein and metabolic profiles of sweat in patients with VKH disease. In the present study, proteomics and metabolomics analysis was performed on 60 sweat samples (30 VKH patients and 30 normal controls) using liquid chromatography tandem mass spectrometry. Parallel reaction monitoring (PRM) analysis was used to validate the results of our omics analysis. In total, we were able to detect 716 proteins and 175 metabolites. Among them, 116 proteins (99 decreased and 17 increased) were observed to be significantly different in VKH patients when compared to controls. Twenty-one differentially expressed metabolites were identified in VKH patients, of which eighteen included Choline, L-Tryptophan, Betaine and L-Serine were reduced, while the rest were increased. Our multi-omics strategy reveals an important role for the amino acid metabolic pathway in the pathogenesis of VKH disease. Significant differences in proteins and metabolites were identified in the sweat of VKH patients, and to some extent, an aberrant amino acid metabolism pathway may be a pathogenic factor in the pathogenesis of VKH disease. PMID: 32222072 [PubMed - as supplied by publisher]

Related Articles Salivary metabolites to detect patients with cancer: a systematic review. Int J Clin Oncol. 2020 Mar 27;: Authors: Assad DX, Mascarenhas ECP, de Lima CL, de Toledo IP, Chardin H, Combes A, Acevedo AC, Guerra ENS Abstract Novel adjunctive screening aids are needed to reduce the morbidity and mortality related to cancer, and every effort should be made for early diagnosis. This systematic review aimed to evaluate salivary metabolites and their diagnostic value in patients with cancer.The systematic review was performed in two phases and included studies that focused on the diagnostic value of salivary metabolites in humans with solid malignant neoplasms. Five electronic databases were searched, and the risk of bias in individual studies was evaluated using the revised Quality Assessment of Diagnostic Accuracy Studies criteria (QUADAS-2). All procedures were performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Of the 1151 studies retrieved, 25 were included; 13 studies used targeted and 12 untargeted metabolomics approaches. Most studies included patients with breast and oral cancer. Except for one, all studies had case-control designs, and none fulfilled all quality assessments. Overall, 140 salivary metabolites were described. The most frequently reported metabolites were alanine, valine, and leucine. Among the 11 studies that reported diagnostic test accuracy (DTA) values, proline, threonine, and histidine in combination and monoacylglycerol alone demonstrated the highest DTA for breast cancer. Combined choline, betaine, pipecolinic acid, and L-carnitine showed better discriminatory performance for early oral cancer.This systematic review highlights the current evidence on salivary metabolites that may be used as a future strategy to diagnose cancer. Further studies including larger sample sizes with confirmation of the results by untargeted analysis are warranted. PMID: 32221803 [PubMed - as supplied by publisher]

Related Articles Nuclear Magnetic Resonance to Detect Rumen Metabolites Associated with Enteric Methane Emissions from Beef Cattle. Sci Rep. 2020 Mar 27;10(1):5578 Authors: Bica R, Palarea-Albaladejo J, Kew W, Uhrin D, Pacheco D, Macrae A, Dewhurst RJ Abstract This study presents the application of metabolomics to evaluate changes in the rumen metabolites of beef cattle fed with three different diet types: forage-rich, mixed and concentrate-rich. Rumen fluid samples were analysed by 1H-NMR spectroscopy and the resulting spectra were used to characterise and compare metabolomic profiles between diet types and assess the potential for NMR metabolite signals to be used as proxies of methane emissions (CH4 in g/kg DMI). The dataset available consisted of 128 measurements taken from 4 experiments with CH4 measurements taken in respiration chambers. Predictive modelling of CH4 was conducted by partial least squares (PLS) regression, fitting calibration models either using metabolite signals only as predictors or using metabolite signals as well as other diet and animal covariates (DMI, ME, weight, BW0.75, DMI/BW0.75). Cross-validated R2 were 0.57 and 0.70 for the two models respectively. The cattle offered the concentrate-rich diet showed increases in alanine, valerate, propionate, glucose, tyrosine, proline and isoleucine. Lower methane yield was associated with the concentrate-rich diet (p < 0.001). The results provided new insight into the relationship between rumen metabolites, CH4 production and diets, as well as showing that metabolites alone have an acceptable association with the variation in CH4 production from beef cattle. PMID: 32221381 [PubMed - as supplied by publisher]

Related Articles Serum Metabolites in Hand-Arm Vibration Exposed Workers. J Occup Environ Med. 2020 Mar 20;: Authors: Vihlborg P, Graff P, Hagenbjörk A, Hadrévi J, Bryngelsson IL, Eriksson K Abstract OBJECTIVE: To investigate whether low molecular organic biomarkers could be identified in blood samples from vibration exposed workers using a metabolomics. METHODS: The study population consisted of 38 metalworkers. All participants underwent a standardized medical examination. Blood samples were collected before and after work shift and analyzed with GC-TOFMS. Multivariate modeling (orthogonal partial least-squares analysis with discriminant analysis [OPLS-DA]) were used to verify differences in metabolic profiles. RESULTS: Twenty-two study participants reported vascular symptoms judged as vibration-related. The metabolic profile from participants with vibration-induced white fingers (VWF) was distinctly separated from participants without VWF, both before and after vibration exposure. CONCLUSION: Metabolites that differed between the groups were identified both before and after exposure. Some of these metabolites might be indicators of health effects from exposure to vibrations. This is the first time that a metabolomic approach has been used in workers exposed to vibrations. PMID: 32221116 [PubMed - as supplied by publisher]

Related Articles Autophagy induction by thiostrepton improves the efficacy of immunogenic chemotherapy. J Immunother Cancer. 2020 Mar;8(1): Authors: Wang Y, Xie W, Humeau J, Chen G, Liu P, Pol J, Zhang Z, Kepp O, Kroemer G Abstract BACKGROUND: Immunogenic cell death (ICD) is a peculiar modality of cellular demise that elicits adaptive immune responses and triggers T cell-dependent immunity. METHODS: Fluorescent biosensors were employed for an unbiased drug screen approach aiming at the identification of ICD enhancers. RESULTS: Here, we discovered thiostrepton as an enhancer of ICD able to boost chemotherapy-induced ATP release, calreticulin exposure and high-mobility group box 1 exodus. Moreover, thiostrepton enhanced anticancer immune responses of oxaliplatin (OXA) in vivo in immunocompetent mice, yet failed to do so in immunodeficient animals. Consistently, thiostrepton combined with OXA altered the ratio of cytotoxic T lymphocytes to regulatory T cells, thus overcoming immunosuppression and reinstating anticancer immunosurveillance. CONCLUSION: Altogether, these results indicate that thiostrepton can be advantageously combined with chemotherapy to enhance anticancer immunogenicity. PMID: 32221018 [PubMed - as supplied by publisher]

Related Articles Metabolic profiling by reversed-phase/ion-exchange mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Mar 16;1143:122072 Authors: Le A, Mak J, Cowan TM Abstract Metabolic profiling is commonly achieved by mass spectrometry (MS) following reversed-phase (RP) and hydrophilic interaction chromatography (HILIC) either performed independently, leading to overlapping datasets, or in a coupled configuration, requiring multiple liquid chromatography (LC) systems. To overcome these limitations, we developed a single, 20-minute chromatographic method using an in-line RP-ion-exchange (IEX) column arrangement and a single LC system. This configuration separates clinically significant polar and non-polar compounds without derivatization or ion-pairing reagents, allowing ionization in both polarities. An in-house library was created with 397 authentic standards, including acylcarnitines, amino acids, bile acids, nucleosides, organic acids, steroid hormones, and vitamins. Analysis of pooled plasma and urine samples revealed 5445 and 4111 ion features, leading to 88 and 82 confirmed metabolite identifications, respectively. Metabolites were detected at clinically relevant concentrations with good precision, and good chromatographic separation was demonstrated for clinically significant isomers including methylmalonic acid and succinic acid, as well as alloisoleucine and isoleucine/leucine. Evaluation of the samples by unsupervised principal component analysis showed excellent analytical quality. PMID: 32220802 [PubMed - as supplied by publisher]

Related Articles Multi-omics: Differential expression of IFN-γ results in distinctive mechanistic features linking chronic inflammation, gut dysbiosis, and autoimmune diseases. J Autoimmun. 2020 Mar 24;:102436 Authors: Bae HR, Leung PSC, Hodge DL, Fenimore JM, Jeon SM, Thovarai V, Dzutsev A, Welcher AA, Boedigheimer M, Damore MA, Choi MS, Fravell RA, Trinchieri G, Gershwin ME, Young HA Abstract Low grade, chronic inflammation is a critical risk factor for immunologic dysfunction including autoimmune diseases. However, the multiplicity of complex mechanisms and lack of relevant murine models limit our understanding of the precise role of chronic inflammation. To address these hurdles, we took advantage of multi-omics data and a unique murine model with a low but chronic expression of IFN-γ, generated by replacement of the AU-rich element (ARE) in the 3' UTR region of IFN-γ mRNA with random nucleotides. Herein, we demonstrate that low but differential expression of IFN-γ in mice by homozygous or heterozygous ARE replacement triggers distinctive gut microbial alterations, of which alteration is female-biased with autoimmune-associated microbiota. Metabolomics data indicates that gut microbiota-dependent metabolites have more robust sex-differences than microbiome profiling, particularly those involved in fatty acid oxidation and nuclear receptor signaling. More importantly, homozygous ARE-Del mice have dramatic changes in tryptophan metabolism, bile acid and long-chain lipid metabolism, which interact with gut microbiota and nuclear receptor signaling similarly with sex-dependent metabolites. Consistent with these findings, nuclear receptor signaling, encompassing molecules such as PPARs, FXR, and LXRs, was detectable as a top canonical pathway in comparison of blood and tissue-specific gene expression between female homozygous vs heterozygous ARE-Del mice. Further analysis implies that dysregulated autophagy in macrophages is critical for breaking self-tolerance and gut homeostasis, while pathways interact with nuclear receptor signaling to regulate inflammatory responses. Overall, pathway-based integration of multi-omics data provides systemic and cellular insights about how chronic inflammation driven by IFN-γ results in the development of autoimmune diseases with specific etiopathological features. PMID: 32220507 [PubMed - as supplied by publisher]

Related Articles Systems pharmacology reveals the mechanism of activity of Physalis alkekengi L. var. franchetii against lipopolysaccharide-induced acute lung injury. J Cell Mol Med. 2020 Mar 27;: Authors: Yang Y, Ding Z, Wang Y, Zhong R, Feng Y, Xia T, Xie Y, Yang B, Sun X, Shu Z Abstract Acute lung injury (ALI) is an important cause of mortality of patients with sepsis, shock, trauma, pneumonia, multiple transfusions and pancreatitis. Physalis alkekengi L. var. franchetii (Mast.) Makino (PAF) has been extensively used in Chinese folk medicine because of a good therapeutic effect in respiratory diseases. Here, an integrated approach combining network pharmacology, proton nuclear magnetic resonance-based metabolomics, histopathological analysis and biochemical assays was used to elucidate the mechanism of PAF against ALI induced by lipopolysaccharide (LPS) in a mouse model. We found that the compounds present in PAF interact with 32 targets to effectively improve the damage in the lung undergoing ALI. We predicted the putative signalling pathway involved by using the network pharmacology and then used the orthogonal signal correction partial least-squares discriminant analysis to analyse the disturbances in the serum metabolome in mouse. We also used ELISA, RT-qPCR, Western blotting, immunohistochemistry and TUNEL assay to confirm the potential signalling pathways involved. We found that PAF reduced the release of cytokines, such as TNF-α, and the accumulation of oxidation products; decreased the levels of NF-κB, p-p38, ERK, JNK, p53, caspase-3 and COX-2; and enhanced the translocation of Nrf2 from the cytoplasm to the nucleus. Collectively, PAF significantly reduced oxidative stress injury and inflammation, at the same time correcting the energy metabolism imbalance caused by ALI, increasing the amount of antioxidant-related metabolites and reducing the apoptosis of lung cells. These observations suggest that PAF may be an effective candidate preparation alleviating ALI. PMID: 32220053 [PubMed - as supplied by publisher]

Related Articles PKA-independent vasopressin signaling in renal collecting duct. FASEB J. 2020 Mar 26;: Authors: Datta A, Yang CR, Limbutara K, Chou CL, Rinschen MM, Raghuram V, Knepper MA Abstract Vasopressin regulates renal water excretion by binding to a Gα s-coupled receptor (V2R) in collecting duct cells, resulting in increased water permeability through regulation of the aquaporin-2 (AQP2) water channel. This action is widely accepted to be associated with cAMP-mediated activation of protein kinase A (PKA). Here, we use phosphoproteomics in collecting duct cells in which PKA has been deleted (CRISPR-Cas9) to identify PKA-independent responses to vasopressin. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Upregulated sites in PKA-null cells include Ser256 of AQP2, which is critical to regulation of AQP2 trafficking. In addition, phosphorylation changes in the protein kinases Stk39 (SPAK) and Prkci (an atypical PKC) are consistent with PKA-independent regulation of these protein kinases. Target motif analysis of the phosphopeptides increased in PKA-null cells indicates that vasopressin activates one or more members of the AMPK/SNF1-subfamily of basophilic protein kinases. In vitro phosphorylation assays using recombinant, purified SNF1-subfamily kinases confirmed postulated target specificities. Of interest, measured IBMX-dependent cAMP levels were an order of magnitude higher in PKA-null than in PKA-intact cells, indicative of a PKA-dependent feedback mechanism. Overall, the findings support the conclusion that V2-receptor mediated signaling in collecting duct cells is in part PKA-independent. PMID: 32219907 [PubMed - as supplied by publisher]