Acyl-Coenzyme A Thioesters for Pesticides, Parkinson s, and Metabolism Nathaniel W Snyder, PhD, MPH Blair Lab August 11, 214 1
Biological Importance of Acyl-CoAs Krebs Cycle Fatty Acid Metabolism Acyl- Coenzyme A Posttranslational Modifications Xenobiotic Metabolism 2
MS/MS of Acyl-Coenzyme A Thioesters Neutral Loss 57.2 Da Snyder, et al. RCM. 214, Basu, et al. Anal. Chem. 211, Snyder, et al. In Preparation 3
Rotenone and Parkinson s Disease Rotenone Widely used pesticide (before 27) Decades of laboratory/animal studies on toxicity Hypothesized similarity to 1-methyl-4-phenylpyridinium (MPP+) and precursor MPTP Exposure associated with Parkinson s disease Agricultural Health Study Farming and Movement Evaluation 11 PD cases (confirmed by two neurologist consensus) 358 age, sex, state matched controls PD associated with rotenone exposure R = 2.5 (1.3-4.7) Tanner et al. EHP. 211 4
Rotenone and Metabolism H H H 3 C rotenone CH 3 5
Rotenone Alters Short Chain Acyl-CoA Levels Acetyl-CoA Acetyl-CoA (%) 12 1 8 6 4 2 Succinyl-CoA 1-1 -9-8 -7-6 log[rotenone] M 1 βhb-coa Succinyl-CoA (%) 8 6 4 2-1 -9-8 -7-6 log[rotenone] M BHB-CoA (%) 8 6 4 2-1 -9-8 -7-6 log[rotenone] M Basu, et al. Chemical Research in Toxicology. 211 6
Rotenone Alters Fatty Acid Metabolism [ 13 C16]- Palmitate % labeling in Acetyl CoA M+2 2 15 1 5 DMS Rotenone [ 13 C 6 ]- Glucose Pyruvate Acetyl-CoA Malonyl-CoA Fatty Acyl- CoAs BHB-CoA [ 13 C 16 ]- Palmitate Acetoacetyl-CoA [ 13 C 4 ]- ctanoate % labeling in Acetyl CoA M+2 2 15 1 5 xaloacetate Malate Fumarate Succinate Succinyl-CoA Citrate cis-aconitate Isocitrate α-keto-glutarate HMG-CoA Propionyl-CoA Worth, et al. In Review JBC. 214 (see poster TXI:58. Tu Evening) 7
Rotenone Reduces Medium Chain Acyl-CoAs 1.5 1.25 DMS Rotenone relative abundance 1..75.5.25 * * *. C2: C4: C6: C8: C1: acyl-coa C12: C14: C16: C18: 8
Palmitoylcarnitine Increases with Rotenone Relative abundance of palmitoyl carnitine 3 2 1 DMS Rotenone 9
Many Roads to Acetyl-CoA unlabeled carbon 13 C from reduc1ve metabolism 13 C from oxida1ve metabolism H H oxaloacetate H H MD H L-malate FH H H fumarate SD H H succinate SCS H SCoA Succinyl-CoA SCoA acetyl-coa H CS ACL H H H H H H citrate H H aconitase α-ketoglutarate αkgd H D-isocitrate H IDH H GDH H lipids SCoA acetyl-coa NH 2 H L-glutamate H H H L-malate H MD H H H H oxaloacetate H H glutaminase H FH H 2 N H fumarate NH 2 H C-1 L-glutamine H ASS ASL H H AAT NH 2 L-aspartate 1
Reductive Metabolism of Glutamine [ 13 15 C 5 N2 ]- Glutamine labeling in Citrate (% of pool) [ 13 15 C 5 N2 ]- Glutamine labeling in Isocitrate (% of pool) [ 13 15 C 5 N2 ]- Glutamine labeling in α KG (% of pool) 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 * * DMS Rotenone * DMS Rotenone * Alpha-Ketoglutarate DMS Rotenone Isocitrate * Citrate M+ M+1 M+2 M+3 M+4 M+5 M+6 M+ M+1 M+2 M+3 M+4 M+5 M+6 * * M+ M+1 M+2 M+3 M+4 M+5 Isotopomer * * H citrate H aconitase α-ketoglutarate H H H GDH H D-isocitrate H IDH H lipids H H [ 13 15 C 5 N2 ]- Glutamine labeling in Acetyl CoA (% of pool) SCoA acetyl-coa H NH 2 H L-glutamate H H H 1 8 6 4 2 ACL H H H H glutaminase Acetyl CoA DMS Rotenone M+ M+1 M+2 H H 2 N Isotopomer NH 2 H C-1 L-glutamine * 11
Glutamine Support Palmitoyl-CoA Synthesis [ 13 15 C 5 N2 ]- Glutamine % labeling in Palmitoyl CoA 6 4 2 * M+2 M+4 M+6 * Isotopomer DMS Rotenone 12
Wider Metabolic Perturbations Rotenone Metabolites D-Panthethine 13
Moving to New Models- Platelets Basu, et al. Bioanalysis. 213. Worth, et al. In Preparation 14
Conclusions Rotenone Association with Parkinson s Metabolic compensation from multiple sources revealed through acyl- CoA analysis Absolute quantitation has failings Tracer studies to show relative contributions from different carbon sources Lipid synthesis and β-oxidation? 15
Summary Future Directions Toxicological/pharmacological mechanisms of action Connect to wider metabolism Better model systems for disease relevance Cause or consequence? ther mitochondrial complex inhibitors xidative stress inducing exposures Metabolic rescue 16
Acknowledgements Dr. Ian A. Blair Andrew Worth Dr. Clementina Mesaros Dr. Qingqing Wang Dr. Sankha Bobby Basu Christine Shwed Dr. Xiaojing Liu Dr. Stacy Gelhaus Alejandro, Linda, Sonia, Zinan Dr. Suhong Zhang CEET Lynch Lab Brass Lab Dr. Peisong Ma Funding Wharton Innovation Fund NIH Grants R1-CA-158328 to IAB T32-GM-876 P42-ES-2372 Superfund 17