Department of Medical Biochemistry. Semmelweis University. Dr. Beáta Törőcsik. Lipid metabolism

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1 Department of Medical Biochemistry Semmelweis University Dr. Beáta Törőcsik Lipid metabolism

2 : not to learn * : structures to learn Harper s Biochemistry 30th ed. Chapter Any question regarding the lecture material: torocsik.beata@med.semmelweis-univ.hu

3 Metabolism of lipids overview. Absorption of lipids Lipoprotein metabolism Oxidation of fatty acids, ketone bodies Synthesis of fatty acids.

4 BIOMEDICAL IMPORTANCE of LIPIDS (1) relatively insoluble in water (2) soluble in nonpolar solvents They are important dietary constituents: high energy value essential fatty acids fat-soluble vitamins (A,D,E,K) and other lipophilic micronutrients Lipids are transported in the blood combined with proteins in lipoprotein particles Fat is stored in adipose tissue, Nonpolar lipids act as electrical insulators, allowing rapid propagation of depolarization waves along myelinated nerves. Lipid biochemistry is necessary for the understanding of many important biomedical conditions, including obesity, diabetes mellitus, and atherosclerosis.

5 The Cellular Compartments of Common Biological Lipids Lipidomics: New Tools and Applications, Cell 143, December 10, 2010

6 Triacylglycerols function as energy reservoirs in animals * * melting points are lower for unsaturated fatty acids melting points are higher for longer fatty acids A fat An oil

7 Molecular structures of (A) saturated, (B) monounsaturated, (C) trans fatty, and (D) polyunsaturated fatty acids (melting point: 69.6 C) (melting point: 13.4 C) (melting point: 52 C) (melting point: -9 C)

8 Nomenclature for saturated fatty acids. Nomenclature for number and position of double bonds in unsaturated fatty acids. e.g. linoleic acid (ω-3), 18:3n-3 or Δ9, Δ12, Δ15 The term omega refers to the position of double bond in relation to methyl group on the end of the fatty acid. As an example, omega-3 (ω-3) fatty acids have the last double bond 3 carbons from the terminal methyl group. n 3 is equivalent to ω3.

9 Fatty acid classification and nomenclature * * or Δ9 or Δ9, Δ12, Δ15 * * or Δ9, Δ12 or Δ5, Δ8, Δ11, Δ14

10 Essential fatty acids: Linoleate Linolenate Double bonds cannot be introduced beyond the Δ9 position Essential fatty acid deficience

11 Nomenclature for number and position of double bonds in unsaturated fatty acids. Oleic acid as an example: n 9 is equivalent to ω9. The term omega refers to the position of double bond in relation to methyl group on the end of the fatty acid. As an example, omega-3 (ω-3) fatty acids have the last double bond 3 carbons from the terminal methyl group.

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13 Adipocytes of white and brown adipose tissue

14 visceral fat versus subcutaneous fat Visceral fat has been linked to metabolic disturbances and increased risk for cardiovascular disease and type 2 diabetes. In women, it is also associated with breast cancer and the need for gallbladder surgery. Visceral fat is directly linked with higher total cholesterol and LDL (bad) cholesterol, lower HDL (good) cholesterol, and insulin resistance. Fat cells particularly visceral fat cells are biologically active. It's appropriate to think of fat as an endocrine organ or gland, producing hormones and other substances that can profoundly affect our health. (Harvard Health Publications)

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16 Absorption of lipids Fat emulsification by bile salts Pancreatic lipase

17 Structures of three common detergents. * The hydrophobic part of each molecule is shown in yellow; the hydrophilic part, in blue. The bile salt sodium deoxycholate is a natural product; the others are synthetic.

18 Cholic acid

19 Pancreatic lipase products: 2-monoacylglycerols+free fatty acids

20 When a suspension of phospholipids is mechanically dispersed in an aqueous solution, the phospholipids aggregate into one of three structures: spherical micelles or liposomes or sheet-like phospholipid bilayers

21 Structure of phospholipids * * * * * *

22 Pancreatic Phospholipase A2 also powerful detergents

23 The weight-loss drug orlistat (Xenical or Alli) blocks the action of pancreatic lipase, reducing the amount of fat that is absorbed from food.

24 Harper figure 43 2 Digestion and absorption of triacylglycerols

25 Long-chain fatty acids are esterified to yield to triacylglycerol in the mucosal cells and together with the other products of lipid digestion, secreted as chylomicrons into the lymphatics, entering the bloodstream via the thoracic duct. Short- and medium-chain fatty acids are mainly absorbed into the hepatic portal vein as free fatty acids.

26 Lipid transport

27 lipoprotein metabolic pathways

28 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 Lipid droplet

29 ATGL: Adipose triglyceride lipase HSL: hormone sensitive lipase CGI-58: comparative gene identification 58 PKA: protein kinase A triacylglycerol lipolysis

30 triacylglycerol lipolysis Triacylglycerol ATGL MAGL: Monoacylglycerol lipase MAGL HSL Diacylglycerol Monoacylglycerol

31 Three-dimensional structure of HSA complexed with endogenous and exogenous ligands bound to the FA sites

32 Putative functions of fatty acid binding protein (FABP) in the cell fatty acid transport protein gene expression

33 X-ray structure of rat intestinal fatty acid binding protein in complex with palmitate

34 lipoprotein metabolic pathways LPL: LIPOPROTEIN LIPASE in heart: K m for TG low in adipose tissue : K m for TG high (10X) During starvation heart enzyme, lactating mammary gland enzyme remains saturated HL. Hepatic lipase Harrison's Principles of Internal Medicine, 19e

35 Generalized structure of a plasma lipoprotein chylomicron

36 Esterification of cholesterol in tissues by acyl-coa cholesterol acyltransferase (ACAT) Synthesis of cholesteryl esters. Esterification converts cholesterol to an even more hydrophobic form for storage and transport.

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39 RNA editing of APOB pre-mrna

40 A helical wheel projection of the amphipathic helix constituting residues 148 to 164 of apolipoprotein A-I lipoprotein helices float on phospholipid surfaces, much like logs on water

41 TABLE 25 1 Composition of the Lipoproteins in Plasma of Humans

42 The formation and secretion of (A) chylomicrons by an intestinal cell and (B) very low density lipoproteins by a hepatic cell.

43 Metabolic fate of chylomicrons

44 Metabolic fate of very low density lipoproteins (VLDL) and production of low density lipoproteins (LDL)

45 Michael S. Brown Joseph L. Goldstein The Nobel Prize in Physiology or Medicine 1985 was awarded jointly to Michael S. Brown and Joseph L. Goldstein "for their discoveries concerning the regulation of cholesterol metabolism" Studies on homozygous familial hypercholesterolemia (FH)

46 LDL receptor recycling Schematic representation of the formation of a clathrin-coated vesicle

47 LDL - LDL receptor binding is ph dependent

48 Metabolism of high-density lipoprotein (HDL) in reverse cholesterol transport Discussed in detail in Prof. Kolev s lecture

49 Esterification of cholesterol in plasma by LCAT Reaction catalyzed by lecithin-cholesterol acyl transferase (LCAT). This enzyme is present on the surface of HDL and is stimulated by the HDL component apoa-i. Cholesteryl esters accumulate within nascent HDLs, converting them to mature HDLs.

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52 * * * Conversion of glycerol to the glycolytic intermediate dihydroxyacetone phosphate.

53 β-oxidation, ketogenesis

54 Overview of fatty acid metabolism showing the major pathways and end products

55 ADIPOSE TISSUE TG Fatty acids CIRCULATION 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

56 Site of β-oxidation: mitochondria

57 Fatty Acid Activation by acyl-coa synthetases (also called thiokinases) Site of fatty acid activation: cytosolic side of the mitochondrial outer membrane

58 Role of carnitine in the transport of long chain fatty acids through the inner mitochondrial membrane Transport of carnitine-acylcarnitine is the rate-limiting step and most important control point in fatty acid oxidation CPT-I is inhibited by malonyl-coa the key precursor of fatty acid synthesis

59 carnitine transport into the mitochondria Yes No No

60 Acylation of carnitine catalyzed by carnitine palmitoyltransferase * *

61 Stages of fatty acid oxidation

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64 The β-oxidation pathway Acyl-CoA dehydrogenase: 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

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66 Palmitoyl-CoA: C16:0

67 GLUCONEOGENESIS FORMATION OF KETONE BODIES FATTY ACID GLUCOSE ß-oxidation PYRUVATE ACETYL-CoA ANAPLEROTIIC REACTION OXALOACETATE CITRATE Ketone bodies Ketogenesis occurs primarily in liver mitochondria, acetyl-coa is converted to acetoacetate or D-- hydroxybutyrate. Fatty acid oxidation + lack of oxaloacetate fasting untreated diabetes chronic alcoholism

68 Ketone bodies are water-soluble equivalents of fatty acids * * *

69 Synthesis of ketone bodies I working in the reverse direction from the way it does in the final step of β-oxidation HMG-CoA is also a precursor in cholesterol biosynthesis

70 Synthesis of ketone bodies II

71 Ketone bodies as fuels

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

73 Ketone bodies can be regarded as a transport form of acetyl groups Important sources of energy: heart muscle, renal cortex brain - glucose is the major fuel but in starvation and diabetes brain uses acetoacetate

74 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

75 The well-fed state: the lipogenic liver

76 The fasting state: the glucogenic liver

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78 carnitine acyltransferase I CAT1, carnitine palmitoyltransferase, CPTI

79 * Elevated level of propionyl-coa causes enzyme inhibition, e.g. pyruvate carboxylase inhibition * * *

80 Causes of carnitine deficiency: Systemic: 1. Primary: OCTN2 defect hypoglycemia without associated acidosis or ketosis 2. Secondary: methylmalonyl-coa defect, B12 vitamin deficiency hypoglycemia metabolic acidosis Myopathic: Impaired uptake of increased loss (defect of mucle transporters)

81 Regulation of β-oxidation

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

83 Lipid synthesis

84 Overview of fatty acid metabolism showing the major pathways and end products

85 acetyl-coa carboxylase bicarbonate ACC reaction, like those of the other biotin-dependent carboxylases, occurs in two steps, a CO2 activation and a carboxylation ACC is a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis and one of its rate controlling steps. IRREVERSIBLE Biotin dependent carboxylases in human: ACC propionyl-coa carboxylase pyruvate carboxylase β-methylcrotonyl-coa carboxylase (which participates in the degradation of leucine). BUT: glutamate carboxylase Vitamine K dependent

86 The structure of Pyruvate Carboxylase Sarawut Jitrapakdee et al. Biochem. J. 2008;413:

87 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

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

89 2. transfer of the activated carboxil group from biotin to acetyl-coa

90 Biological tethers

91 Critical SH-groups carry the intermediates during the synthesis of fatty acids 1. Acyl carrier protein The prosthetic group is 4 -phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP Phosphopantetheine contains the B5 vitamin pantothenic acid, also found in the coenzyme A molecule:

92 Critical SH-groups carry the intermediates during the synthesis of fatty acid foszfopantetein (attached to acil carrier protein [ACP]) 2. β-ketoacyl-acp-synthase

93 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

94 Structural overview of the mammalian (porcine) fatty acid synthase 5 SEPTEMBER 2008 VOL 321 SCIENCE

95 Substrate shuttling by the ACP in mfas. 5 SEPTEMBER 2008 VOL 321 SCIENCE

96 B. A. Sequence of events during synthesis of a fatty acid the enzyme complex must be charged with the correct acyl groups: A. the acetyl group of acetyl-coa is transferred to the Cys-SH group of the β- ketoacyl-acp synthase. This reaction is catalyzed by acetyl-coa ACP transacetylase (AT) B. transfer of the malonyl group from malonyl-coa to the -SH group of ACP, is catalyzed by malonyl-coa ACP transferase (MT)

97 Step 1 Condensation condensation of the activated acetyl and malonyl groups to form acetoacetyl-acp, an acetoacetyl group bound to ACP through the phosphopantetheine-sh group; simultaneously, a molecule of CO2 is produced. Enzyme: β-ketoacyl-acp synthase (KS) 1.

98 Differences between fatty acid synthesis and β-oxidation 2. reduction of the carbonyl group citosol mitochondria (KR) D-β-Hydroxybutyryl-ACP (D- β-hydroxyacyl-acp) 3. dehydration (HD) 4. reduction of the double bond (ER) in β-oxidation: L- β-hydroxybutyryl-coa (L- β-hydroxyacyl-coa) NADPH NADH/FADH 2 Single polypeptide chain Enzymes are not associated

99 A. The butyryl group is transferred from the phosphopantetheine-sh group of ACP to the Cys -SH Következő group of β--ketoacyl-acp synthase (like in A reaction) To start the next cycle of four reactions that lengthens the chain by two more carbons, another malonyl group is linked to the now unoccupied phosphopantetheine-sh group of ACP (like in B reaction). B

100 Beginning of the second round of the fatty acid synthesis cycle

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

102 STOICHIOMETRY

103 Shuttle for transfer of acetyl groups from mitochondria to the cytosol

104 Main sources of NADPH for fatty acid synthesis

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

106 -regulation of Acetyl-CoA carboxylase- Phosphorylation inactivates the enzyme Dephosphorylated form active -Polymerizes into long filaments insulin (activates phosphoprotein phosphatase

107 Parallel Regulation of fatty acid synthesis and oxidation

108 Sites of regulation of fatty acid metabolism.

109 Subcellular localization of lipid metabolism

110 Routes of synthesis of other fatty acids essential essential

111 Electron transfer in the desaturation of fatty acids in vertebrates Mammalian systems contain four terminal desaturases of broad chain-length specificities designated Δ9-, Δ6-, Δ5-, and Δ4-fatty acyl-coa desaturases. site: ER

112 Biosynthesis of phosphatidic acid a b adipocytes lack glycerol kinase

113 Phosphatidic acid in lipid biosynthesis.

114 Glyceroneogenesis Is Important for Triacylglycerol Biosynthesis The dihydroxyacetone phosphate used to make glycerol-3-phosphate for triacylglycerol synthesis comes either from glucose via the glycolytic pathway or from oxaloacetate via an abbreviated version of gluconeogenesis termed glyceroneogenesis. Glyceroneogenesis is necessary in times of starvation, (when glycolysis is inhibited) since approximately 30% of the fatty acids that enter the liver during a fast are reesterified to triacylglycerol and exported as VLDL. Adipocytes also carry out glyceroneogenesis in times of starvation. They do not carry out gluconeogenesis but contain the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), which is upregulated when glucose concentration is low, and participates in the glyceroneogenesis required for triacylglycerol biosynthesis.

115 Flux through the triacylglycerol cycle between liver and adipose tissue is controlled to a large degree by the activity of PEPCK, which limits the rate of both gluconeogenesis (in liver) and glyceroneogenesis (in liver and adipose tissue). Glucocorticoid hormones such as cortisol regulate the levels of PEPCK reciprocally in the liver and adipose tissue. Acting through the glucocorticoid receptor, these steroid hormones increase the expression of the gene encoding PEP carboxykinase in the liver, thus increasing gluconeogenesis and glyceroneogenesis. In adipose tissue, they inhibit PEPCK expression thus increasing fatty acid release.

116 A class of drugs called thiazolidinediones are now used to treat type 2 diabetes. In this disease, high levels of free fatty acids in the blood interfere with glucose utilization in muscle and promote insulin resistance. Thiazolidinediones activate a nuclear receptor called peroxisome proliferator-activated receptor γ (PPARγ), which induces the activity of PEP carboxykinase. Therapeutically, thiazolidinediones increase the rate of glyceroneogenesis, thus increasing the resynthesis of triacylglycerol in adipose tissue and reducing the amount of free fatty acid in the blood.

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118 Pyridoxal phosphate is an essential cofactor in the glycogen phosphorylase reaction as well; involved in acid catalysis