1. Lipid and Ketone Metabolism Week 12

2. 2 Lipids Lipids: structure & function Transport of lipids: albumin-binding & lipoprotein Storage & release of lipids – adipose tissue Catabolism: energy release from fatty acid Activation of FA & carnitine shuttle  oxidation of palmitic acid Ketone bodies

3. 3 What are lipids? Not soluble in water – man (or animal) is ~80% water Soluble in organic solvents – acetone, ether Saponifiable (can be split by hyrolysis) Triglyceride (fats), waxes Phospholipids, sphingolipids, glycolipids, lipoproteins Non-saponifiable Carotenoids, cholesterol, fat soluble vitamins (D, K, E, A)

4. 4 Functions of lipids Fuel for energy metabolism Membrane structure Phospholipid, cholesterol Hormone action Steroids, prostaglandins Electron transfer Ubiquinone Antioxidant Vitamin E

5. 5 Importance of Lipid Metabolism In health –Provision of energy in fasting/starvation –Provision of energy in pregnancy & for lactation In diseases –Endocrine disease Diabetes mellitus, Cushing’s syndrome –Production/metabolic diseases Ketosis, pregnancy toxaemia, peri-parturient syndrome, fatty liver disease, hyperlipidaemia

6. 6 Linoleic, linolenic and arachidonic are essential fatty acids with >1 double bond Important for membrane function Cis double bond not trans

7. 7 Lipid of di-acyl glycerol provides the hydrophobic nature of phospholipid membrane

8. 8 Transport of lipid Problem of solubility From intestine – lipoprotein (chylomicrons) From liver – very low density lipoprotein From tissues to liver – high density lipoprotein From adipose tissue – fatty acid binds to albumin

9. 9 Lipoproteins Chylomicrons: from intestine to adipose & other tissues Very low density lipoprotein (VLDL) TG, cholesterol ester & phospholipid to tissues Low density lipoprotein (LDL) Remnant of VLDL after release of lipid High density lipoprotein (HDL) Collects lipid, especially cholesterol for return to liver

10. 10 Lipoproteins: Central hydrophobic core of triglyceride, cholesterol ester Monolayer of phospholipid in outer surface, hydrophilic head to outer- face Apolipoprotein in phospholipid monolayer

11. 11 Lipoprotein composition (%) ChyloVLDLLDLHDL Protein1102550 Triglyceride9060103 Cholesterol ester & cholesterol 5154520 Phopholipid4152027 Size (  m) 1000502010

12. 12 Apolipoproteins Apolipoprotein: –Have many hydrophobic amino acids –Hydrophilic amino acid on surface –Provide structure –Some have activity in lipid metabolism Apo A: predominantly in HDL, Cofactor for lecithin cholestrol acyl transferase (LCAT) Apo B :Predominantly in HDL & VLDL Apo C:Predominantly in HDL VLDL; Activates lipoprotein lipase in transfer of TG to adipose tissue Apo D:Predominantly in HDL Apo E: Predominantly in HDL & VLDL

13. 13 Fats in diet carried by chylomicron to tissues eg adipose Liver exports TG, CE PL in VLDL to tissues VLDL release lipids and become LDL LDL remnants to tissues and liver HDL is collecting lipid from tissues & from LDL to return to liver (reverse transport)

14. 14 HDL released from liver Collects excess cholesterol from tissues Produces cholesterol ester and lyso-lecithin from cholesterol and lecithin (phosphotidyl choline) Cholesterol ester to VLDL and TG from VLDL HDL absorbed by liver

15. 15 Transport from Adipose tissue – When energy needed Hormones (adrenaline, glucagon, cortisol) activate hormone sensitive lipase in Adipose Tissue Triglyceride  3 Fatty acid + Glycerol Released from adipose to blood Fatty acid disrupts membranes Fatty acid bound to albumin – protects membranes Fatty acid goes to tissues (eg muscle & liver) for energy Glycerol goes to liver for gluconeogenesis

16. 16 In cell (eg liver, muscle), FA linked to coenzyme A by high energy bond Required ATP, releases AMP and PPi 2 x high energy ~phos bonds used

17. 17 Carnitine Shuttle CoA can not enter mitochondria Outer membrane: After activation FA-CoA transfers FA to carnitine to produce fatty acyl-carnitine- enzyme inhibited by malonyl CoA Inner membrane: FA transferred back to CoA, carnitine recycles

18. 18  -Oxidation of saturated fatty acid a)Dehydrogenase (oxidation) at  - carbon with FAD reduced and trans == formed b)Hydratase, hydrates double bond c)Dehydrogenase with NAD reduced d)Thiolase cleavage with release of acetyl CoA and CoA on shortened fatty acid

19. 19  -Oxidation of saturated fatty acid a)Dehydrogenase (oxidation) at b- carbon with FAD reduced and trans == formed b)Hydratase, hydrates double bond c)Dehydrogenase with NAD reduced d)Thiolase cleavage with release of acetyl CoA and CoA on shortened fatty acid e)Note similarity to steps of TCA cycle

20. 20  -oxidation of palmitic acid 16 carbon fatty acid 7 cycles yields 8 acetyl CoA 7 FADH2 7 NADH + H + Which gives 131 ATP Equivalent of 2 ATP used in activation of FA:- net 129 ATP

21. 21 Palmitic acid yields >2x ATP per gram compared to glucose Fatty acids have no water of hydration

22. 22 If FA has odd number of carbons,  -oxidation proceeds till C 3 fatty acyl CoA (propionyl CoA) Carboxylase requiring biotin Racemase (isomerase) converts between sterioisomers Mutase – internal transfer of carboxyl group- required Vit B 12 Succinyl CoA to TCA cycle and gluconeogenesis THIS IS VERY IMPORTANT FOR RUMINANTS

23. 23  -Oxidation of poly unsaturated fatty acid  -oxidation operates for saturated FA (no double bonds) When unsaturated other enzymes are involved for oxidation of double bonds Reaction requires 2 x isomerases and NADPH dependent reductase as well as the usual enzymes Altered reaction costs 5 ATP per double bond

24. 24 Catabolism of lipid Stimulated by hormone, (glucagon, adrenalin) Adipose tissue releases FA & glycerol In liver & muscle FA is activated & enters mitochondria Oxidised to acetyl CoA & enters TCA cycle In liver, excess acetyl CoA forms ketone bodies, secreted to blood & used in other tissues (not brain)

25. 25 Control of lipid catabolism Whole animal level –Hormone sensitive lipase Triglyceride  fatty acid + glycerol –Activated by glucagon, adrenaline Cellular regulation –Carnitine shuttle inhibited by malonyl CoA –Malonyl CoA is a reactant in FA synthesis –Malonyl CoA elevated when cell is energy rich

26. 26 Formation of Ketone Bodies In Liver (only organ with enzymes): 3 x acetyl CoA form 3-OH, 3-methyl glutaryl CoA 1 acetyl CoA released Acetoacetate* formed Reduction forms  -hydroxybutyrate* Decarboxylation forms acetone* Exported to blood Ketone bodies are more soluble that fatty acid & help to make energy available for tissues in fasting & starvation * These are the ketone bodies

27. 27 Ketosis of acetyl Co-A

28. 28 Use of Ketone Bodies In tissues (not brain in most species):  OH butyrate & acetoacetate to acetyl CoA Enters TCA cycle for ATP production Excess production of ketone bodies occurs in metabolic diseases – diabetes mellitus, bovine ketosis, ovine toxaemia

29. 29 Ketones in Ruminants Carbohydrate in diet of ruminants forms ‘volatile fatty acids’ in rumen These are acetate C 2, propionate C 3 and butyrate C 4 fatty acids Butyrate converted to  -OH butyrate (ie ketone body) in rumen wall before entry to blood Much energy for ruminant tissue metabolism come from this ketone body

30. 30 Lipid Anabolism – FA Synthesis When body/cell is energy rich Liver, adipose tissue & mammary gland In cytoplasm, separate from  -oxidation Substrate is acetyl CoA (2 carbons) From glycolysis or amino acid catabolism Exported from mitochondria by citrate shuttle Primary product – palmitoyl CoA, (16 carbons) Then modified - elongated, desaturated, conjugated

31. 31 Lipid Anabolism- FA Synthesis Co-factors: NADPH + H +, CoA Multi-enzyme complex Fatty Acid Synthase complex Control point - Acetyl CoA carboxylase Activated by citrate; inhibited by palmitoyl CoA Hormone activation (insulin); inhibition (glucagon) Nutrition: cabrohydrate activates; lipid inhibits

32. 32 FA Synthesis in Ruminants Fatty acid synthesis primarily in adipose tissue and mammary gland Little synthesis in liver Substrate: –Acetyl CoA from acetate in blood –Acetate in blood is rumen product ‘volatile fatty acid’ –Activated by acetyl CoA synthase Acetate + CoASH + ATP  Acetyl CoA + AMP + PPi

33. 33 Acetyl CoA + Oxaloacetate ADP, Pi Citrate + CoA + ATP Oxaloacetate + Acetyl CoA + ADP + Pi Malate Pyruvate Mitochondria Cytoplasm COO -, ATP carboxylase Citrate synthase (TCA cycle) FATTY ACID SYNTHESIS Citrate lyase NAD + NADH + + H + Malate DH Malate enzyme NADPH + + H + NADP + CITRATE SHUTTLE (Malate Shuttle also operates)

34. 34 Acetyl CoA carboxylase, activated by citrate, inhibited by palmitoyl CoA (product) Activated by insulin, inhibited by glucagon Upregulated by hi carbon/low fat diet Downregulated by hi fat/low carbon diet Control site for FA synthesis

35. 35

36. 36 Fatty acid synthase complex: 7 enzymes and acyl carrier protein (ACP) in dimer formation ACP has active –SH and binds growing FA chain Cys with –SH also present Each enzyme required for FA synthesis

37. 37 Transacylase: acetyl CoA to cys-SH, malonyl CoA to ACP-SH Condensation by ketoacyl synthase: acetyl replaces carboxyl grp of malonyl, becomes C 4 Reductase: C 3 keto group becomes hydroxyl (NADPH) Dehydratase: hydroxyl grp lost, double bond in place Reductase: double bond is hydrogenated (NADPH) Acetyl Transacylase: C4 FA transferred to cys-SH, new malonyl to ACP-SH Reactions catalysed by fatty acid synthase

38. 38

39. 39 Fatty Acid Synthesis Continues for 6 cycles C 16 palmitoyl Co A is released by thioesterase Released palmitoyl CoA further modified –Elongated by addition of acetyl groups –Desaturated to give unsaturated FA –Conjugated to triglyceride and phospholipid

40. 40 In endoplasmic reticulum C 2 groups from malonyl CoA with loss of CO 2 Multi-enzyme complex needed – FA elongase Very long chain FA (22-24 carbons) needed for central nervous system In Mitochnodria Elongation by reversal of  -oxidation

41. 41 Example: palmitoyl CoA (C16)  Palmitoleioyl CoA C 16 C 16, cis  9 In endoplasmic reticulum, NADH & cytochrome b 5 required

42. 42 In endoplasmic reticulum In Liver: glycerol-3-phos from glycerol, product (TG) goes to VLDL synthesis In adipose: glycerol-3 phos from glucose via glycolysis (no glycerol kinase), therefore only when animal has high glucose * Note error in fig: Pi released from phosphatidic acid not taken up *

43. 43 Control of FA synthesis In fed state with excess of energy –Increased insulin from pancreas Activates acetyl CoA carboxylase Activates lipoprotein lipase in adipose tissue, increasing TG breakdown in VLDL & transfer of Fatty acid to adipocyte –Decreased glucagon – (insulin antagonist) –High glucose Enters adipose tissue, provides glycerol, essential for TG re- synthesis

44. 44 Synthesis of Phospholipid In endoplasmic reticulum A) diacyl glycerol (as in TG formation) reacts with CDP-choline giving phosphatidyl choline and CMP Same with CDP-serine & CDP-ethanolamine B) phosphatidic acid (TG formation), react with CTP to form CDP-diacyl glycerol then with inositol to form phosphatidyl inositol From endoplasmic reticulum to membranes

45. 45 Synthesis of cholesterol Liver predominant site Acetyl CoA to mevalonate 3 x acetyl CoA forms HMG CoA (as in ketone formation) Reduction to mevalonate (C 6 ) Converted to isopentenyl pyro phosphate (C 5 ; diphosphate) 5 x isopent pyrophos condensed to give squalene

46. 46 Natural conformation of squalene encourages ring formation by cyclase

47. 47 Cholesterol Forms cholesterol ester for transport in VLDL (enzyme is LCAT) Forms bile acids with glycine, taurine for digestion Modifications lead to hormones No degradation in body (ie no enzyme to break ring structure)

48. 48 Regulation of cholesterol synthesis Committing enzyme:- 3-hydroxy 3-methyl glutaryl reductase (formation of mevalonic acid) Control by cholesterol concentration in cell –Hi chol inhibits: low chol activates Control by hormones –Insulin & thyroid hormone activates: glucagon & cortisol inhibit Control by diet –Enzyme synthesis increases in fasting –Enzyme synthesis decreases when dietary cholesterol is high Target for ‘statin’ drugs