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LIPID MAPS Lipid Metabolomics Tutorial Fatty Acid Oxidation Professor Edward A. Dennis Department of Chemistry and Biochemistry Department of Pharmacology, School of Medicine University of California, San Diego Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or individual slides (without alteration) for academic, non-profit and non-commercial purposes. Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org E.A. DENNIS 2010 © Metabolism and Energy Overview Proteins Amino Acids Carbohydrates Simple Sugars Lipids Fatty Acids Pyruvate Acetyl CoA • Triglycerides (TG’s) are decomposed into Fatty Acids (FA’s). • FA’s are b-oxidized to release energy and create acetyl CoA fragments. • Acetyl CoA enters the power-producing Krebs cycle and electron transport chain. Energy (CO2, H2O) E.A. DENNIS 2010 © Naming Conventions: Palmitic Acid b d 16 6 e 5 4 g 3 2 1 a Carbonyl carbon e d g b a omega, always the last alkyl carbon epsilon, fifth carbon after the carbonyl delta, fourth carbon after the carbonyl gamma, third carbon after the carbonyl beta, second carbon after the carbonyl alpha, first carbon after the carbonyl E.A. DENNIS 2010 © Beta Oxidation In a Nutshell... Fatty Acid One iteration of b-Oxidation: Make fatty acyl CoA. Acyl CoA (1) Step 1: Oxidize the b-carbon (C3) Enoyl CoA (2) L-hydroxyacyl CoA (3) Ketoacyl CoA (4) Step 2: Hydrate the b-carbon Step 3: Oxidize the b-carbon, again! Step 4: Thiolyze a-b bond, releasing acetyl CoA REPEAT from step 1, w/ 2 fewer carbons Acyl CoA (shorter) Acetyl CoA TCA Cycle E.A. DENNIS 2010 © Make activated acyl CoA Triglycerides Hormone sensitive lipase Free FA’s adipocyte Albumin – carries FA’s to target tissue bloodstream Free FA’s – decomposes triglycerides – removes FA groups – free FA’s diffuse through membrane • Albumin Free FA’s Fatty acyl CoA synthase • Hormone-sensitive lipase – FA’s diffuse into tissue cells Acyl CoA • Fatty acyl CoA synthase – adds CoA to the free FA • Carnitine shuttle mitochondria Acyl CoA Carnitine shuttle – moves fatty acyl CoA into the mitochondria target tissue cell E.A. DENNIS 2010 © Regulation • Hormone Sensitive Lipase is activated by cAMP • Cyclic AMP also turns off acetyl CoA carboxylase, stopping FA synthesis • Hormones like glucagon and epinephrine increase cAMP – FA synthesis slows – Triglycerides are broken down – FA’s enter b-oxidation faster E.A. DENNIS 2010 © Location, Location, Location • Fatty Acyl CoA is oxidized inside the mitochondrial matrix • The carnitine shuttle moves it into the matrix – Free carnitine is exchanged for acyl carnitine • Carnitine acyl transferases catalyze reactions on both sides of membrane – driven by concentration gradient • CAT-1 is inhibited by high levels of malonyl CoA generated by acetyl CoA carboxylase in the pathway to Fatty Acid Synthesis. Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Lif e at the Molecular Level, 2 nd ed. Reprinted with permission of John Wiley & Sons, Inc. E.A. DENNIS 2010 © Step 1: Oxidize the b-carbon • Acyl CoA dehydrogenase oxidizes the b-carbon Fatty acyl CoA FAD Acyl CoA dehydrogenase FADH2 – 3 versions of the enzyme exist – Specific to short, medium and long chain substrates • One FADH2 is generated – makes 2 ATP’s • A trans double bond is created at the 2-carbon • Product is an Enoyl CoA D 2-trans-enoyl CoA E.A. DENNIS 2010 © Medium-chain dehydrogenase deficiency • Incidence: 1 in 10,000 live births Fatty Acid Acyl CoA (1) Enoyl CoA (2) L-hydroxyacyl CoA (3) Ketoacyl CoA (4) Acyl CoA (shorter) – More common than phenylketonuria! • Symptoms: – severe hypoglycemia >> lethargy, coma • little energy from FA’s • glucose reserves are immediately burned – contributes to sudden infant death syndrome (10% of cases) • Mechanism: – Normally, there are 3 separate fatty acyl CoA dehydrogenase enzymes for STEP 1 of b-oxidation • Specific for short, medium and long acyl chains, respectively – Autosomal recessive lack of medium chain enzyme • Treatment: Special diet and supportive care E.A. DENNIS 2010 © Step 2: Hydrate the b-carbon • Enoyl CoA hydratase adds a water molecule across the double bond • Product is b-hydroxyacyl CoA – S- stereoisomer E.A. DENNIS 2010 © Step 3: Oxidize the b-carbon, Again! • Beta-hydroxyacyl CoA dehydrogenase oxidizes the b-carbon again • One NADH is created – makes 3 ATP’s • A ketoacyl CoA is produced E.A. DENNIS 2010 © Step 4: Thiolyze off acetyl CoA CoA-SH Acyl CoA: Acyltransferase (“thiolase”) • Thiolase (aka: Acyl CoA acyltranferase) splits the ketoacyl • A new CoASH is consumed • Acetyl CoA is released • A new, shorter Acyl CoA remains and reenters the cycle E.A. DENNIS 2010 © REPEAT with a Shorter Acyl CoA • Palmitoyl CoA (16 carbons) becomes myristoyl CoA (14 carbons). • Each iteration releases another acetyl CoA. • 7 iterations will release 8 acetyl CoA fragments from the original palmitoyl CoA. • Net equation: Palmitoyl CoA + 7CoASH + 7FAD + + 7NAD+ + 7H20 yields: 8 Acetyl CoA + 7 FADH 2 + 7NADH + 7H+ E.A. DENNIS 2010 © b-Oxidation Cycle Removal of 2 carbons from acyl chain Fatty acyl-CoA [1] Acyl CoA dehydrogenase [4] Acyl CoA: acyltransferase D 2-trans-enoyl CoA b-ketoacyl CoA Acetyl-CoA [3] b-hydroxyacyl CoA dehydrogenase enoyl CoA hydratase [2] TCA cycle b-hydroxyacyl CoA E.A. DENNIS 2010 © How Much Energy? Each palmitoyl CoA group released from a triglyceride: – directly produces 7 NADH & 7 FADH2 • which generate 21 and 14 ATP’s, respectively – releases 8 acetyl CoA molecules for TCA Cycle • which each generate 12 ATP Grand total is 131 ATP per fully oxidized palmitoyl CoA Efficiency of Energy Recovery = 40% ** Recently, some have calculated energetically less ATP equivalents per NADH/FADH2 [2.5 and 1.5 instead of 3 and 2] which lowers the numbers somewhat (Berg). E.A. DENNIS 2010 © Efficiency of Fat Storage Fat has 9 kcal/gram = 9 kcal/cc Both Carbohydrate and Protein have 4 kcal/gram = 4 kcal/3cc or 1.33 kcal/cc Fat/(Carbohydrate or Protein) = 6x/cc!!! E.A. DENNIS 2010 © Problem: Oxidizing Unsaturated FA’s Linoleic Acid C12 (Even # Unsat) C9 (Odd # Unsat) 3 NAD+ + 3FAD + CoA-SH 3 rounds of b- oxidation 3 NADH + 3FADH2 + 3Acetyl-CoA Problem: b-g cis double bond E.A. DENNIS 2010 © Special Case 1: Odd Unsaturations The GOOD News: • Enzyme enoyl-CoA isomerase moves the double bond over 1 position. • Oxidation process resumes The BAD News: • The first oxidation step is skipped • One less FADH2 is made – 2 fewer ATPs E.A. DENNIS 2010 © Special Case 2: Even Unsaturations 4 2 The GOOD News: • Enzymes 2,4 dienoyl-CoA reductase and 3,2 dienoyl-CoA isomerase convert the g-d and a-b double bonds into a single a-b unsaturation. • Oxidation process resumes 3 H+ + NADPH NADP+ 3 4 2 3 4 The BAD News: • The conversion costs one NADPH directly. • Net effect is the loss of one NADH – 3 fewer ATPs 2 E.A. DENNIS 2010 © FA Synthesis vs. FA Oxidation Synthesis Oxidation Cytosol Mitochondria ACP CoA Malonyl CoA Acetyl CoA b-hydroxyl acyl step R-config S-config Electron carriers NADPH NADH, FADH Liver Muscle, liver Cell Location Acyl carrier 2-Carbon Piece Primary tissue site E.A. DENNIS 2010 © FA Synthesis vs. FA Oxidation (cont) 1. They are not the reverse of one another. – Different subcellular locations – R and S isomers of b-hydroxyacyl intermediates cannot easily jump to the other pathway – Electrons donated from oxidation (NADH, FADH2) cannot directly enter synthesis, which uses NADPH. 2. Separate, semi-independent pathways allow more sophisticated regulation. – Accelerating one pathway does not mean slowing the other. E.A. DENNIS 2010 © Acknowledgement This tutorial is based on an evolving subset of lectures and accompanying slides presented to medical students in the Cell Biology and Biochemistry course at the School of Medicine of the University of California, San Diego. I wish to thank Dr. Bridget Quinn and Dr. Keith Cross for aid in developing many of the original slides, Dr. Eoin Fahy for advice in applying the LIPID MAPS nomenclature and structural drawing conventions [Fahy et al (2005) J Lipid Res, 46, 839-61; Fahy et al (2009) J Lipid Res, 50, S9-14] and Masada Disenhouse for help in adopting to the tutorial format. Edward A. Dennis September, 2010 La Jolla, California E.A. DENNIS 2010 ©