1. Fatty acid oxidation Ketone bodies Fatty acid synthesis AV. Lipid metabolism 1-2. 2011

2. Triacylglycerols Major energy reserve Oxidation: 9 kcal/g (for carbohydrates: 4 kcal/g) 11 kg of 70 kg total body weight Site of accumulation: cytoplasm of ADIPOSE CELLS Adipose tissue -specialized for synthesis, storage, mobilization of lipids Lipases

5. Number of carbons Number of double bonds Name 160Palmitate 180Stearate 200Arachidate 161Palmitoleate Cis  9 181Oleate Cis  9 182Linoleate Cis  9,  12 183Linolenate Cis  9,  12,  15 204Arachidonate Cis  5,  8,  11,  14 The most important fatty acids

6. Mobilization of triacylglycerols stored in adipose tissue Glukagon, adrenalin, ACTH ↓ receptor activation ↓ protein kinase A ↓ phosphorylation of Perilipin (when dephosphorylated it inhibits the access of lipases to TG and DG + phosphorylation of HSL (hormon-sensitive lipase) activation ↓ CGI dissociates from Perilipin then associates with ATGL (adipocyte trigliceride lipase) ↓ TG→DG + fatty acid ↓ HSL: DG→MG + fatty acid ↓ MGL (monoacylglycerol lipase): MG→G + fatty acid Lehninger, Principals of Biochemistry, 2013

7. ADIPOSE TISSUE TG Fatty acids Fatty acids - bound to albumin (10 fatty acids/albumin monomer) (free fatty acids, FFA) MUSCLE, HEART MUSCLE, RENAL CORTEX Fatty acid activation, transport into the mitochondria, β-oxidation CIRCULATION

8. Site of β-oxidation: mitochondria

9. Activation of fatty acids: Fatty acid + ATP + CoA Acyl-CoA + PPi + AMP Acyl-CoA synthetase Fast PPi hydrolysis reaction is irreversible in vivo

10. Site of fatty acid activation: cytosolic side of the mitochondrial outer membrane

11. Triacylglycerols Major energy reserve Oxidation: 9 kcal/g (for carbohydrates: 4 kcal/g) 11 kg of 70 kg total body weight Site of accumulation: cytoplasm of ADIPOSE CELLS Adipose tissue -specialized for synthesis, storage, mobilization of lipids Lipases

14. Number of carbons Number of double bonds Name 160Palmitate 180Stearate 200Arachidate 161Palmitoleate Cis  9 181Oleate Cis  9 182Linoleate Cis  9,  12 183Linolenate Cis  9,  12,  15 204Arachidonate Cis  5,  8,  11,  14 The most important fatty acids

15. Lipid droplet Mobilization of fatty acids from the adipose tissue Glukagon, adrenalin, ACTH Receptor activation Protein kináz A Phosphorylation of Perilipin (when dephosphorylated it inhibits the access of lipase to TG, phosphorylated perilipin has no such effect) + Phosphorylation of Hormon-sensitive lipase (activation) Mobilization of fatty acids from TG

16. ADIPOSE TISSUE TG Fatty acids Fatty acids - bound to albumin (10 fatty acids/albumin monomer) (free fatty acids, FFA) MUSCLE, HEART MUSCLE, RENAL CORTEX Fatty acid activation, transport into the mitochondria, β-oxidation CIRCULATION

17. Site of β-oxidation: mitochondria

18. Activation of fatty acids: Fatty acid + ATP + CoA Acyl-CoA + PPi + AMP Acyl-CoA synthetase Fast PPi hydrolysis reaction is irreversible in vivo

19. Site of fatty acid activation: cytosolic side of the mitochondrial outer membrane

20. Transport of fatty acids into the mitochondria Transport of Long-chain (12-18 C atom) fatty acids with carnitine

21. Transport of carnitine-acylcarnitine is the rate-limiting step and most important control point in fatty acid oxidation carnitine-acyltransferase I (CPT I) carnitine-acyltransferase II (CPT II) Fatty acids with <12 C enter mitochondria without carnitine and are activated in the mitochondria Transport of fatty acids into the mitochondria

22. Three isoenzymes: -long-chain fatty acids (C 12-18) -medium chain fatty acids (MCAD, 4-14) -short-chain fatty acids (4-8) MCAD deficiency is relatively frequent

23. Specific for L-stereoisomere

24. Oxidation of fatty acids with >12 C is carried out by a multienzyme bound to the mitochondrial inner membrane, in which the last three enzymes are tightly associated (trifunctional protein), when the chain is < 12 C soluble enzymes in the matrix continue the oxidation β-oxidation of fatty acids

27. Conversion of glycerol to glycolysis intermediate – in the liver glycerol kinaseGlycerol-P dehydrogenase

28. hormones (adrenaline, glucagon) Malonyl-CoA Increased level of free fatty acids Oxidation Inhibiton of Perilipin Activation of hormone-sensitive lipase NADH Inhibition of carnitine acyltransferase I Entry of fatty acids into mitochondria is inhibited Oxidation Inhibition of 3-hydroxyacyl CoA dehydrogenase thiolase Inhibition of ß-oxidation High energy state Regulation of fatty acid oxidation Acetyl-CoA

29. Long-term regulation: PPAR (peroxisome proliferator/activated receptors) nuclear receptor – transcription factors PPARα – muscle, adipose tissue, liver regulate the transcription of fatty acid transporters, CPT I és CPT II, and acyl-CoA dehydrogenase - energy need (fasting, between-meal periods) PPARα activation transcription of enzymes of fatty acid oxidation - fetus - principal fuels for heart: glucose and lactate -neonatal – fatty acid Regulation of metabolic transition by PPARα - sustained exercise – PPARα expression in muscles

30. The most common genetic defect in fatty acid oxidation Acyl-CoA dehydrogenase deficiency For the medium length acyl-CoA dehydrogenase Prevalence: 1:40 – mutation in one of the chromosomes 1:10000 – two mutant copies – disease manifestation symptoms in the first years -hypoglycemia – with decreased ketone body formation (decreased fatty acid oxidation and gluconeogenesis in the liver) -accumulation of lipids in the liver -vomiting, drowsiness Therapy: frequent carbohydrate-rich meals + carnitine supply

31. Deficiency of carnitine transport into the mitochondria Long-chain fatty acid transport Carnitine deficiency -high-affinity plasma membrane transporter (heart, kidney, muscle – but not liver) muscle cramps – weakness – death addition of carnitine -secondary carnitine deficiency due to deficiency on β-oxidation acyl-carnitine in the urine Carnititne acyltransferase deficiency most common - CPT II gene mutation – partial loss of enzyme activity muscle weakness when more serious – hypoglycemia with decreased ketone body formation

32. GLUCOSE PYRUVATE OXALOACETATE CITRATE GLUCONEOGENESIS ACETYL-CoA FATTY ACID ß-oxidation Ketone bodies FORMATION OF KETONE BODIES ANAPLEROTIKUS REAKCIÓ Fatty acid oxidation + lack of oxaloacetate fasting untreated diabetes

34. Synthesis of ketone bodies in the liver

35. Ketone bodies as fuels

36. Oxidation of ketone bodies in the extrahepatic tissues heart muscle striatal muscle kidney brain

37. Ketone bodies can be regarded as a transport form of acetyl groups Important sources of energy: heart muscle, renal cortex (preference to glucose, 1/3 of the energy) brain - glucose is the major fuel but in starvation and diabetes brain uses acetoacetate

38. Ketone bodies Fasting Diabetes high level of ketone bodies in the blood KETOSIS Formation in the liver exceeds the use in the periphery. Level of ketone bodies after an overnight fast: ~0.05 mM 2 days starvation: 2 mM (40-fold increase!) 40 days: 7 mM

40. Fatty acid synthesis - repeated cycles – in each cycle the chain is extended by two carbons – four steps in each cycle Enzyme: fatty acid synthase Seven active site for different reactions in separate domains of a single large polypeptide

41. Fatty acid synthesis –Lipogenesis- not a reversal of degradation SYNTHESISBREAKDOWN SiteCytosolMitochondrial matrix Intermediates bound toAcyl-carrier proteinCoA EnzymesJoined in a single polypeptide chain (fatty acid synthase) Not associated Reducing equivalentsNADPHNAD, FAD UnitsMalonyl-CoAAcetyl-CoA 3-hydroxyacyl-derivativeD-enantiomerL-enantiomer

42. Fatty acid synthesis: *Liver *Adipose tissue *Lactating mammary gland

43. Committed step in fatty acid synthesis: formation of malonyl-CoA from acetyl- CoA acetyl-CoA carboxylase - prosthetic group: biotin

44. Acetyl-CoA carboxylase has three activities in a single polypeptide Biotin – covalently bound to Lys έ-amino group 1. transfer of carboxyl group to biotin ATP-dependent

45. move of activated CO 2 from the biotin carboxylase region to the transcarboxylase active site

46. 2. transfer of the activated carboxil group from biotin to acetyl-CoA

47. Acyl carrier protein SH group is the site of ently of malonyl group during fatty acid synthesis Critical SH-groups carry the intermediates during the synthesis of fatty acids β-ketoacyl-ACP synthase

48. Fatty acid synthase Acetyl group from Acetyl-CoA is transferred to Cys-SH of β-ketoacyl-ACP synthase (KS) by MAT

49. Malonyl group is transferred to ACP-SH by MAT

50. Acetyl group (from Acetyl- CoA) is transferred to the malonyl group on ACP (methyl terminal) Acetoacetyl-ACP

51. by β-ketoacyl-ACP reductase Reduction

52. Dehydration by β-hydroxyacyl-ACP dehydratase

53. Reduction by enoyl-ACP reductase

55. ACP is recharged with another malonyl group by MAT Second round of fatty acid synthesis cycle

56. The overall process of palmitate synthesis Seven cycles for the synthesis of palmitate Palmitate is released from ACP by thioesterase (TE)

57. STOICHIOMETRY Seven cycles for the synthesis of palmitate Ac-CoA + 7 malonyl-CoA + 14 NADPH + H +  Palmitate + 7 CO 2 + 14 NADP + + 8 CoA + 6 H 2 O 7 Ac-CoA + 7 CO 2 + 7 ATP  7 malonyl-CoA + 7 ADP + 7 P i Overall: 8 Ac-CoA + 7 ATP + 14 NADPH + H +   palmitate + 14 NADP + + 8 CoA + 6 H 2 O + 7 ADP + 7 P i

58. Fatty acid synthase is present exclusively in the cytosol In adipocytes and hepatocytes cytosolic [NADPH]/[NADP] ratio is high (~75) – strongly reducing environment In hepatocytes and lactating mammary gland cytosolic NADPH is generated largely by pentose phosphate pathway but malic enzyme is also significant Citrate carries Acetyl-CoA from mitochondria to the cytosol for fatty acid synthesis ATP:citrate lyase malic enzyme

60. Source of NADPH for fatty acid synthesis

61. Regulation of fatty acid synthesis -regulation of Acetyl-CoA carboxylase- - allosteric stimulation by citrate -regulation by covalent modification Dephosphorylated form – active -Polymerizes into long filaments insulin Active dephosphorylated ACC Phosphorylation inactivates the enzyme -negative feed-back inhibition by palmitate

62. Control of fatty acid synthesis Short term regulation Ac-CoA ATP isocitrate dehydrogenase inhibited citrate – stimulates Ac-CoA carboxylase +carries the substrate (Ac-CoA) (indicates that two-carbon units & ATP are available for synthesis)

63. Glucagon cAMP  activation of protein kinases phosphorylation of Ac-CoA carboxylase - switch off Palmitoyl-CoA *Inhibits Ac-CoA carboxylase *Inhibits translocation of citrate from mitochondria to cytosol *Inhibits glucose 6-P dehydrogenase  NADPH  Control of fatty acid synthesis

64. Long term regulation – low fat, high carbohydrate diet the amount of Acetyl-CoA carboxylase & fatty acid synthase is increased - fasting and high fat diet the amount of Acetyl-CoA carboxylase is decreased Control of fatty acid synthesis High-fat low carbohydrate diet – Atkins diet fatty acid mobilization - ketone body formation (loss in the urine)