Post-translational regulation of metabolism in fumarate hydratase deficient cancer cells.

Deregulated signal transduction and energy metabolism are hallmarks of cancer and both play a fundamental role in tumorigenesis. While it is increasingly recognised that signalling and metabolism are highly interconnected, the underpinning mechanisms of their co-regulation are still largely unknown. Here we designed and acquired proteomics, phosphoproteomics, and metabolomics experiments in fumarate hydratase (FH) deficient cells and developed a computational modelling approach to identify putative regulatory phosphorylation-sites of metabolic enzymes. We identified previously reported functionally relevant phosphosites and potentially novel regulatory residues in enzymes of the central carbon metabolism. In particular, we showed that pyruvate dehydrogenase (PDHA1) enzymatic activity is inhibited by increased phosphorylation in FH-deficient cells, restricting carbon entry from glucose to the tricarboxylic acid cycle. Moreover, we confirmed PDHA1 phosphorylation in human FH-deficient tumours. Our work provides a novel approach to investigate how post-translational modifications of enzymes regulate metabolism and could have important implications for understanding the metabolic transformation of FH-deficient cancers with potential clinical applications

Ischemic preconditioning protects against cardiac ischemia reperfusion injury without affecting succinate accumulation or oxidation.

Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted and then restored, and underlies many disorders, notably myocardial infarction and stroke. While reperfusion of ischemic tissue is essential for survival, it also initiates cell death through generation of mitochondrial reactive oxygen species (ROS). Recent work has revealed a novel pathway underlying ROS production at reperfusion in vivo in which the accumulation of succinate during ischemia and its subsequent rapid oxidation at reperfusion drives ROS production at complex I by reverse electron transport (RET). Pharmacologically inhibiting ischemic succinate accumulation, or slowing succinate metabolism at reperfusion, have been shown to be cardioprotective against IR injury. Here, we determined whether ischemic preconditioning (IPC) contributes to cardioprotection by altering kinetics of succinate accumulation and oxidation during IR. Mice were subjected to a 30-minute occlusion of the left anterior descending coronary artery followed by reperfusion, with or without a protective IPC protocol prior to sustained ischemia. We found that IPC had no effect on ischemic succinate accumulation with both control and IPC mice having profound increases in succinate compared to normoxia. Furthermore, after only 1-minute reperfusion succinate was rapidly metabolised returning to near pre-ischemic levels in both groups. We conclude that IPC does not affect ischemic succinate accumulation, or its oxidation at reperfusion

Mitochondrial Protein Lipoylation and the 2-Oxoglutarate Dehydrogenase Complex Controls HIF1α Stability in Aerobic Conditions.

Hypoxia-inducible transcription factors (HIFs) control adaptation to low oxygen environments by activating genes involved in metabolism, angiogenesis, and redox homeostasis. The finding that HIFs are also regulated by small molecule metabolites highlights the need to understand the complexity of their cellular regulation. Here we use a forward genetic screen in near-haploid human cells to identify genes that stabilize HIFs under aerobic conditions. We identify two mitochondrial genes, oxoglutarate dehydrogenase (OGDH) and lipoic acid synthase (LIAS), which when mutated stabilize HIF1α in a non-hydroxylated form. Disruption of OGDH complex activity in OGDH or LIAS mutants promotes L-2-hydroxyglutarate formation, which inhibits the activity of the HIFα prolyl hydroxylases (PHDs) and TET 2-oxoglutarate dependent dioxygenases. We also find that PHD activity is decreased in patients with homozygous germline mutations in lipoic acid synthesis, leading to HIF1 activation. Thus, mutations affecting OGDHC activity may have broad implications for epigenetic regulation and tumorigenesis.This work was supported by a Wellcome Trust Senior Clinical Research Fellowship to J.A.N. (102770/Z/13/Z), Wellcome Trust Principal Research Fellowship to P.J.L. (084957/Z/08/Z), and the Medical Research Council (A.S.H.C. and C.F.). The Cambridge Institute for Medical Research is in receipt of a Wellcome Trust Strategic Award (100140).This is the final version of the article. It first appeared from Elsevier (Cell Press) via https://doi.org/10.1016/j.cmet.2016.09.01

Dissection of metabolic reprogramming in polycystic kidney disease reveals coordinated rewiring of bioenergetic pathways.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder caused by loss-of-function mutations in PKD1 or PKD2. Increased glycolysis is a prominent feature of the disease, but how it impacts on other metabolic pathways is unknown. Here, we present an analysis of mouse Pkd1 mutant cells and kidneys to investigate the metabolic reprogramming of this pathology. We show that loss of Pkd1 leads to profound metabolic changes that affect glycolysis, mitochondrial metabolism, and fatty acid synthesis (FAS). We find that Pkd1-mutant cells preferentially use glutamine to fuel the TCA cycle and to sustain FAS. Interfering with either glutamine uptake or FAS retards cell growth and survival. We also find that glutamine is diverted to asparagine via asparagine synthetase (ASNS). Transcriptional profiling of PKD1-mutant human kidneys confirmed these alterations. We find that silencing of Asns is lethal in Pkd1-mutant cells when combined with glucose deprivation, suggesting therapeutic approaches for ADPKD

Two parallel pathways connect glutamine metabolism and mTORC1 activity to regulate glutamoptosis.

Glutamoptosis is the induction of apoptotic cell death as a consequence of the aberrant activation of glutaminolysis and mTORC1 signaling during nutritional imbalance in proliferating cells. The role of the bioenergetic sensor AMPK during glutamoptosis is not defined yet. Here, we show that AMPK reactivation blocks both the glutamine-dependent activation of mTORC1 and glutamoptosis in vitro and in vivo. We also show that glutamine is used for asparagine synthesis and the GABA shunt to produce ATP and to inhibit AMPK, independently of glutaminolysis. Overall, our results indicate that glutamine metabolism is connected with mTORC1 activation through two parallel pathways: an acute alpha-ketoglutarate-dependent pathway; and a secondary ATP/AMPK-dependent pathway. This dual metabolic connection between glutamine and mTORC1 must be considered for the future design of therapeutic strategies to prevent cell growth in diseases such as cancer.This work was supported by funds from the following institutions: Agencia Estatal de Investigación/European Regional Development Fund, European Union (PGC2018-096244- B-I00, SAF2016-75442-R), Ministry of Science, Innovation and Universities of Spain, Spanish National Research Council—CSIC, Institut National de la Santé et de la Recherche Médicale —INSERM, Université de Bordeaux, Fondation pour la Recherche Médicale, the Conseil Régional d’Aquitaine, SIRIC-BRIO, Fondation ARC, and Institut Européen de Chimie et Biologie. C.B. was recipient of fellowships from the Minister of Higher Education, Research and Innovation (France) and the Fondation ARC (France). We thank Prof. Patricia Boya (Centro de Investigaciones Biologicas, Madrid, Spain) for kindly providing with the ATG5+/+ and ATG5−/− MEFs. We thank Prof. Benoit Viollet (Institute Cochin, Paris, France) for kindly providing with the AMPK+/+ and AMPK−/− MEFs, and the CA-AMPK plasmid

Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition.

FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.P.G. is funded by the Wellcome Trust (109967/Z/15/Z) and was previously supported by the Academy of medical Sciences and Lady Tata Memorial Trust. The Huntly lab is funded by European Research Council, MRC, Bloodwise, the Kay Kendall Leukaemia Fund, the Cambridge NIHR Biomedical Research Centre, and core support grants to the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute. C.F. and A.S.H.C are funded by the Medical Research Council, Core Grant to the Cancer Unit. P.M-P. is supported by a grant from Cancer Research UK (C56179/A21617). D.S. is a Postdoctoral Fellow of the Mildred-Scheel Organisation, German Cancer Aid. This research was supported by the CIMR Flow Cytometry Core Facility. We would like to thank the Welcome Trust Sanger Institute facility for the MiSeq run

Metabolomic Profiling in Acute ST-Segment-Elevation Myocardial Infarction Identifies Succinate as an Early Marker of Human Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury following ST-segment-elevation myocardial infarction (STEMI) is a leading determinant of clinical outcome. In experimental models of myocardial ischemia, succinate accumulation leading to mitochondrial dysfunction is a major cause of ischemia-reperfusion injury; however, the potential importance and specificity of myocardial succinate accumulation in human STEMI is unknown. We sought to identify the metabolites released from the heart in patients undergoing primary percutaneous coronary intervention for emergency treatment of STEMI. METHODS AND RESULTS: Blood samples were obtained from the coronary artery, coronary sinus, and peripheral vein in patients undergoing primary percutaneous coronary intervention for acute STEMI and in control patients undergoing nonemergency coronary angiography or percutaneous coronary intervention for stable angina or non-STEMI. Plasma metabolites were analyzed by targeted liquid chromatography and mass spectrometry. Metabolite levels for coronary artery, coronary sinus, and peripheral vein were compared to derive cardiac and systemic release ratios. In STEMI patients, cardiac magnetic resonance imaging was performed 2 days and 6 months after primary percutaneous coronary intervention to quantify acute myocardial edema and final infarct size, respectively. In total, 115 patients undergoing acute STEMI and 26 control patients were included. Succinate was the only metabolite significantly increased in coronary sinus blood compared with venous blood in STEMI patients, indicating cardiac release of succinate. STEMI patients had higher succinate concentrations in arterial, coronary sinus, and peripheral venous blood than patients with non-STEMI or stable angina. Furthermore, cardiac succinate release in STEMI correlated with the extent of acute myocardial injury, quantified by cardiac magnetic resonance imaging. CONCLUSION: Succinate release by the myocardium correlates with the extent of ischemia

Causal integration of multi-omics data with prior knowledge to generate mechanistic hypotheses.

Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies

Prevalensi Labioschisis Di Rsup. Prof. Dr. R. D. Kandou Manado Periode Januari 2011 – Oktober 2012

: Cleft lip or labioschisis is an inherited disorder that can occur on the lips to the ceiling. Cleft lip is a disruption in the face of growth since the fourth week of embryonic life. Method: This research in retrospection description research for knowning prevalence cleft lip or labioschisis in surgical department RSUP. Prof. Dr. R. D. Kandou Manado, period of Januari 2011 – October 2012. Output: Prevalence of Labioschisis and Labiopalatochisis on Januari 2011 – October 2012 is 57% and 43%. Presentation for each of kind harelipped are : unilateral labioschisis 47%, bilateral labioschisis 5%, unilateral palatum of labioshisis 28%, and bilateral palatum of labioschisis 12%, submucosa 1%, and cleft palate lips 7%. Presentation according to the place of defect : right 18%, left 57%, bilateral 25%, and status not complete 54%. Presentation according to age for doing operation : 0-4 years 73%, 5-9 years 10%, 10-14 years 7%, and >15 years 10%. Presentation labioschisis according to sex : Man 58%, and women 42%. Presentation labioschisis according to etiology : genetic factor 25%, environment factor 62%, and unknown factor 13%. Presentation of labioschisis that be surgery 93%, and not surgery 7%. Presentation of labioschisis according to complication surgery : bleeding post surgery 1%, secunder infection 4%, dehisensi/establish scar 4%, and not complication 91%. Conclusion: Prevalence labioschisis still decrease in each year, kind of labioschisis that large is unilateral labioschisis and localization defect is often on left edge. Labioschisis is happen more to man. Factor that to cause labioschisis between : genetic factor, environment factor and unknown factor. Labioschisis is often more to surgery 0-4 years old

Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation.

Neural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain