Metabolism of Lipids - I

1. Metabolism of Lipids - I HP Kedilaya

2. Biological importance • Lipids are unique hydrophobic nature. • partly obtained from foods and partly derived from carbohydrates. • Fats are stored in adipocytes as highly concentrated sources of energy and oxidized for energy production.

3. Biological importance • The layer of fat as adipose tissue under the skin acts as shock absorbing cushions and also thermoinsulationagainst heat loss

4. Biological importance • Glycerophospholipids, sphingolipids and cholesterol are indispensible for cell structure and function. • Vitamin D, bile salts and steroid hormones are synthesized from cholesterol. • Prostaglandins, leukotrienes, thromboxanes and related compounds, synthesized from C20 fatty acids possess many biological actions.

6. Contents: •  Metabolism of fatty acids Fatty acid oxidation and Fatty acid synthesis • Metabolism of Ketone bodies Ketogenesis and Ketolysis • Metabolism of Triacylglycerol • Metabolism of Compound lipids • Metabolism of Cholesterol • Metabolism of lipoproteins • Fatty liver & lipotropic factors

7. Overview of Fatty Acid and Fat Metabolism VLDL (transported in blood, TAG delivered to (exported from Liver) adiposites for storage) TAG(Fat) Esterification Lipolysis (Liver) (Adipocytes ) Fatty acids TAG (Diet) Fatty acid synth (Lipogenesis) β-Oxidation (Liver, Adipocytes) (in all cells for energy) Carbohydrates, Amino acids (Diet) Acetyl CoA Ketone bodies Ketogenesis (Liver ) (for energy, especially in brain during starvation ) TCA cycle in all cells for energy

8. Metabolism of Fatty Acids- includes – • Fatty Acid Oxidation (breakdown for energy) • Fatty Acid Synthesis (Lipogenesis) – De Novo synthesis of fatty acids. (‘De Novo synthesis’ refers to synthesis of complex/large molecules from simple, small molecules.) and • Ketone body metabolism

9. Fatty acid oxidation • Fatty acids are oxidized in the body for energy mostly by β-oxidation. • Other forms of oxidation, of minor importance, are • α-oxidation and • ω-oxidation.

10. β-Oxidation of Fatty Acids • Definition β-oxidation of fatty acids is the oxidation on the β-carbonatom of the fatty acid molecule and it occurs for the energy requirement of the cell during the fastingstate. • Tissues: Most of the tissues except brain, RBC and adrenal medulla • Starting substrate: Fatty acid(Palmitic acid as example) • Pathway/Reactions of β-Oxidation

11. Pathway/Reactions of β-Oxidation β-Oxidation consists of the following three stages1. • Activation of fatty acids (in cytosol) II. Transport of fatty acids into mitochondria III. β-Oxidation proper (in the mitochondrial matrix)

12. Fatty Acid Activation (in Cytosol) Fatty acid (Palmitic acid) CoASH ATP Mg2+ Thiokinase (Acyl CoA synthetase) AMP + 2Pi Acyl CoA (Palmitoyl CoA)

13. Transport of Fatty Acyl CoA Into Mitochondria (Role of Carnitine in Oxidation of Fatty Acids) • Long chain fatty acids cannot pass through the inner mitochondrial membrane. Hence, this needs a transport mechanism involving, • carnitine, Carnitine is β-hydroxy γ-trimethyl ammonium butyrate. (CH3)3N+–CH2–CH(OH)–CH2–COO– and three proteinmolecules present in the mitochondrial membranes – • translocase, • carnitineacyltransferase (CAT I) and • carnitineacyltransferase (CAT II).

14. Figure: Transport of fatty acyl CoA from cytosol to mitochondrial matrix CoASH Acyl CoA CAT I Acyl carnitine Carnitine CYTOSOL Translocase Inner Mitochondrial Membrane MITOCHONDRIAL MATRIX CAT II Acyl carnitine Carnitine Acyl CoA CoASH β-Oxidation

15. III . β-Oxidation Proper(Palmitic acid/PalmitoylCoA as example) Subcellular site: Mitochondrial matrix Starting substrate:AcylCoA/PalmitoylCoA During β-oxidation, fatty acids undergo oxidative removal of successive two-carbon units as acetyl CoA, starting from carboxyl end of fatty acyl chain.

16. III. β-Oxidation Proper Fatty Acyl CoA Mitochondrial matrix β-Oxidation ETC Acetyl CoA NADH, FADH2 ATP TCA Cycle

17. III . β-Oxidation Proper Each cycle of β-oxidation, accompanied by removal of a two-carbon unit as acetyl CoA, has four steps/reactions in the following order. • Dehydrogenation • Hydration • Dehydrogenation • Thiolytic cleavage

18. III . β-Oxidation Proper • Palmitic acid (C16) requires 7 cycles of β-oxidation, each cycle producing one molecule each of acetyl CoA, NADH and FADH2.

19. Reactions of β-Oxidation of Acyl CoA (Palmitoyl CoA as Example) Acyl CoA (Palmitoyl CoA) FAD Acyl CoA dehydrogenase ETC 1.5 ATP FADH2 Δ2 – Transenoyl CoA H2O NADH Enoyl CoA hydratase FADH2 β-Hydroxyacyl CoA NAD+ β-Hydroxyacyl CoA dehydrogenase ETC 2.5 ATP NADH + H+ β-Ketoacyl CoA ATP CoASH Thiolase 6 more cycles ETC Acyl CoA + Acetyl CoA (2 carbon less) TCA cycle

20. III . β-Oxidation Proper • Overall reaction of β-oxidation of palmitoyl CoA • Palmitoyl CoA+7 CoASH+ 7 FAD+7NAD++7 H2O • 8 Acetyl CoA+ 7FADH2 +7NADH + 7H+

21. Energetics of β-Oxidation of Palmitic acid • MechanismATP Yield • I. β-Oxidation proper (7 cycles): • 7 NADH (by oxidative phosphorylation, • each NADH gives 2.5 ATP) 17.5 • 7 FADH2(by oxidative phosphorylation, • each FADH2 gives 1.5 ATP) 10.5 • II. From 8 acetyl CoA oxidized by TCA Cycle, • Each acetyl CoA provides 10 ATP 80 •  Total energy from one mole of palmitoyl CoA 108 • Energy utilized for activation (formation of palmitoyl CoA) –2 Net ATP yield of β-oxidation of one molecule of palmitate = 106

22. Regulation of β-Oxidation • by availability of the substrate, namely fatty acids. • During fasting state rate of β-oxidation is increased due to high level of plasma free fatty acid, which is released from adipose tissue because of low insulin/glucagon ratio found during fasting. (See chapter ‘Lipolysis in Adipose Tissue’)

23. Regulation of β Oxidation • by availability of the substrate, namely fatty acids. • During fed state rate of β-oxidation is decreased as there is an increased level of malonylCoA, a substrate of fatty acid synthesis. CAT I is inhibited by malonylCoA decreasing the entry of fatty acids into mitochondrial matrix.

24. Regulation of β Oxidation fasting state ↓insulin/glucagon ratio ↑Lipolysis adipose tissue ↑plasma free fatty acid ↑β-Oxidation

25. Oxidation of Odd Chain Fatty acids Odd chain fatty acid Several rounds of β-oxidation Propionyl CoA Succinyl CoA Acetyl CoA (Intermediate of TCA cycle)

26. Fatty Acid Biosynthesis(De Novo Synthesis of Fatty Acid) • Synthesis is not the reversal of β-oxidation. • dietary carbohydrates and amino acids, when consumed in excess, converted to fatty acids and stored as triacylglycerols.

27. Fatty Acid Biosynthesis(De Novo Synthesis of Fatty Acid) • Tissues: Liver, adipose tissue, kidney, brain and mammary glands • Intracellular site: Cytosol • Substrates required: Acetyl CoA, ATP and NADPH (provides reducing equivalents; main source: HMP shunt pathway)

28. Fatty Acid Biosynthesis(De Novo Synthesis of Fatty Acid) Steps of fatty acid synthesis include: • Transport of acetyl CoA from mitochondrial matrix to cytosol • Formation of malonylCoA from acetyl CoA • Synthesis of fatty acid involving fatty acid synthase complex

29. Steps 1. Transport of Acetyl CoA to Mitochondrial Matrix Acetyl CoA (C2) Oxaloacetate TCA cycle Citrate MITOCHONDRIA ATP ADP + Pi CYTOSOL Acetyl CoA (C2) Citrate ATP citrate lyase Oxaloacetate

30. Steps 2 -- Formation of Malonyl CoA Acetyl CoA (C2) 1. Transport of acetyl CoA MITOCHONDRIA CYTOSOL Acetyl CoA (C2) CO2 ATP Acetyl CoA carboxylase Biotin 2. Formation of Malonyl CoA ADP + Pi Malonyl CoA (C3)

31. Step 3 – Synthesis of Fatty Acid Involving Fatty Acid Synthase Complex Fatty Acid Synthase (FAS) Complex1 • FAS complex is a multienzyme complex and enzymes form a dimer with identical subunits. • The multienzyme complex facilitates easy interaction of substrates with the active sites of the enzymes.

32. Figure: Fatty Acid Synthase Complex – A Diagrammatic Representation Functional subdivisiona Functional division Reducing Unit Acetyl transferase Ketoacyl reductase Dehydratase Substrate Entry and Condensing Unit Releasing Unit Malonyl transferase Enoyl reductase Thioesterase Ketoacyl synthase Acyl carrier protein 4´-phosphopantethein Cys Subunit SH SH division SH SH 4´-phosphopantethein Cys Ketoacyl synthase Acyl carrier protein Thioesterase Malonyl transferase Enoyl reductase Acetyl transferase Dehydratase Ketoacyl reductase

33. Reactions of Fatty Acid Synthase Complex SH -Pan- Enz 1-Cys-SH Enzyme (FAS) SH -Cys-Enz 2-Pan-SH Malonyl CoA Acetyl/acyl CoA Malonyl transferase Acyl transferase CoASH CoASH • Enz 1-Cys-S-acyl • Enz 2-Pan-S-malonyl Acyl-malonyl-enzyme Ketoacyl synthase CO2 - Enz 1-Cys-SH - Enz 2 -Pan-S-ketoacyl Ketoacyl enzyme

34. Enzyme (FAS) Malonyl CoA Acetyl/acyl CoA Malonyl transferase Acyl transferase CoASH CoASH • Enz 1-Cys-S-acyl • Enz 2-Pan-S-malonyl Acyl-malonyl-enzyme Ketoacyl synthase CO2 - Enz 1-Cys-SH - Enz 2 -Pan-S-ketoacyl Ketoacyl enzyme

35. CoASH CoASH • Enz 1-Cys-S-acyl • Enz 2-Pan-S-malonyl Acyl-malonyl-enzyme Ketoacyl synthase CO2 - Enz 1-Cys-SH - Enz 2 -Pan-S-ketoacyl Ketoacyl enzyme

36. Acyl-malonyl-enzyme Ketoacyl synthase CO2 - Enz 1-Cys-SH - Enz 2 -Pan-S-ketoacyl Ketoacyl enzyme

37. - Enz 1-Cys-SH - Enz 2 -Pan-S-ketoacyl Ketoacyl enzyme NADPH + H+ Ketoacyl reductase NADP+ - Enz 1-Cys-SH - Enz 2 -Pan-S-hydroxyacyl Hydroxyacyl-enzyme Dehydratase H2O - Enz 1-Cys-SH - Enz 2 -Pan-S-enoyl Enoyl-enzyme

38. Ketoacyl reductase NADP+ - Enz 1-Cys-SH - Enz 2 -Pan-S-hydroxyacyl Hydroxyacyl-enzyme Dehydratase H2O - Enz 1-Cys-SH - Enz 2 -Pan-S-enoyl Enoyl-enzyme

39. Hydroxyacyl-enzyme Dehydratase H2O - Enz 1-Cys-SH - Enz 2 -Pan-S-enoyl Enoyl-enzyme

40. - Enz 1-Cys-SH - Enz 2 -Pan-S-enoyl Enoyl-enzyme NADPH + H+ Enoyl reductase NADP+ - Enz 1-Cys-SH - Enz 2 -Pan-S-acyl Acyl-enzyme

41. Enzyme (FAS) Malonyl CoA Acyl group transferred from • Enz 1-Cys-S-acyl • Enz 2-Pan-S- Enz 2 to Enz 1 malonyl - Enz 1-Cys-SH - Enz 2 -Pan-S-acyl After 7 cycles Thioesterase Acyl-enzyme Palmitate

42. Regulation of Fatty Acid Synthesis Fed State ↑Insulin/glucagon ratio Activation Inhibiton, Malonyl CoA, Fatty acyl CoA Acetyl CoA Carboxylase (End products, High fat diet) Activation ↑Glucose (Intracellular ) ↑Citrate (Intracellular )

43. Ketone Body Metabolism and Ketosis ketone bodies – A collective name for • Acetone, • Acetoacetate (acetoacetic acid) and • β-hydroxybutyrate (β-hydroxy butyric acid) • synthesized in the liver from fatty acids metabolic fuel molecules during prolonged fasting and Starvationespecially for brain

44. Ketone Body Metabolism and Ketosis O CH3 – C – CH3 Acetone O O C – CH2 – C – CH3 Acetoacetate -O O OH β-hydroxy butyrate C – CH2 – CH – CH3 -O

45. Ketone Body Metabolism and Ketosis • Ketosis, accumulation of ketone bodies in the body, a metabolic disorder seen in starvation and uncontrolled diabetes mellitus (Type 1)

46. Ketone Body Metabolism and Ketosis Ketone body metabolism includes – • 1) Ketogenesis (synthesis of ketone bodies), • 2)Ketolysis (breakdown of ketone bodies for energy) and

47. 1) Ketogenesis(Synthesis of Ketone Bodies) • Acetyl CoA immediate precursor However, since acetyl CoA is formed from fatty acids by β-oxidation, fatty acids are the ultimateprecursors • Site:Liver • Sub-cellularsite: Mitochondria

48. Pathway/Reactions (Liver) Fatty acid β-oxidation Acetyl CoA + Acetyl CoA Thiolase CoASH Acetoacetyl CoA Acetyl CoA HMG CoA Synthase CoASH HMG CoA (β-Hydroxy β-Methyl Glutaryl CoA)

49. Pathway/Reactions (Liver) Fatty acid Acetyl CoA β-oxidation HMG CoA Synthase Acetyl CoA + Acetyl CoA Thiolase CoASH Acetoacetyl CoA Acetyl CoA HMG CoA Synthase CoASH HMG CoA (β-Hydroxy β-Methyl Glutaryl CoA)

50. Pathway/Reactions (Liver) Fatty acid Acetyl CoA β-oxidation HMG CoA Synthase Acetyl CoA + Acetyl CoA Thiolase CoASH Acetoacetyl CoA Acetyl CoA HMG CoA Synthase CoASH HMG CoA (β-Hydroxy β-Methyl Glutaryl CoA) HMG CoA Lyase Acetyl CoA Acetoacetate NADH + H+ Spontaneous CO2 β-hydroxy butyrate dehydrogenase Acetone NAD+ Exhaled by lungs (acetone is volatile) β-hydroxy butyrate