Lipid Metabolism Synthesis and Degradation PDF

LIPID Metabolism Biosynthesis and Degradation Amos M Sakwe, Ph. D., MSCI School of Graduate Studies West Basic Sciences Bldg. Rm.# 2141B (Office), #3108 (Lab) Phone: 615-327-6064 Email: [email protected] Lecture Outline • • • • • Fatty Acid biosynthesis and Lipogenesis Regulation of Fatty acids syn...

LIPID Metabolism Biosynthesis and Degradation Amos M Sakwe, Ph. D., MSCI School of Graduate Studies West Basic Sciences Bldg. Rm.# 2141B (Office), #3108 (Lab) Phone: 615-327-6064 Email: [email protected] Lecture Outline • • • • • Fatty Acid biosynthesis and Lipogenesis Regulation of Fatty acids synthesis Elongation and Desaturation of fatty acids Synthesis of polyunsaturated fatty acids Lipolysis and Degradation of fatty acids – – – – – – Mobilization of fatty acids from fats Degradation of fatty acids – β-Oxidation Energy yield from β-Oxidation of fatty acids Other forms of lipid oxidation Regulation of lipid metabolism Ketogenesis Fatty Acid Biosynthesis • The main pathway for synthesis of fatty acids occurs in the cytosol • Fatty acid synthesis is a stepwise assembly of two carbon units to form the 16-carbon palmitate (C16, saturated) • Fatty acid biosynthesis is an important part of lipogenesis and together with glycolysis, it generates fats from blood sugar. Fatty acid biosynthesis takes place in three stages: 1) Transport of Mitochondrial Acetyl CoA into the cytosol via the Citrate Transport System (Citrate Shuttle) 2) Formation of Malonyl CoA from Acetyl CoA by Acetyl CoA carboxylase (ACC) 3) Assembly of the fatty acid chain on the Acyl Carrier Protein, catalyzed by Fatty Acid Synthase (FAS) The building block Necessary for initiation Summary of Fatty Acids Biosynthesis During the Fed State Stage 3 Stage 2 Stage 1 Adapted from N. Miesfeld, Univ. of Arizona Step 1: Transport of Mitochondrial Acetyl CoA into the cytosol via the Citrate Transport System 3. Citrate shuttle not only transports Acetyl CoA but also produces NADPH FA 1. Acetyl CoA condenses with oxaloacetate to form Citrate ---> antiported out with inward movement of anion 2. Citrate cleaved by cytosolic Citrate Lyase to oxaloacetate + acetyl CoA Two Sources of NADPH for Fatty Acid Biosynthesis The reaction catalyzed by malic enzyme (Decarboxylating Malate dehydrogenase) to generates 1 NADPH for each Acetyl-CoA equivalent that is transported. Most of the NADPH needed for fatty acid biosynthesis comes from the Pentose Phosphate Pathway (PPP). Step 2: Formation of Malonyl CoA from Acetyl CoA • The reaction is catalyzed by Acetyl-CoA Carboxylase (ACC), • ACC is the major regulatory enzyme in fatty acid biosynthesis • The formation of Malonyl CoA from Acetyl CoA is the committed step in fatty acid synthesis. • Enzyme has three functional domains: • Biotin carboxylase, and • Transcarboxylase (acetyl CoA carboxytransferase) and • Biotin carboxyl-carrier The swinging arm mechanism of Acetyl-CoA Carboxylase Overall Reaction Acetyl CoA + CO2 + ATP --> Malonyl CoA + ADP + Pi Step 3: Synthesis of fatty acid chain • This is catalyzed by Fatty Acid Synthase (FAS) • Mammalian Fatty Acid Synthase is a homodimer, with two identical subunits, each containing 6 enzyme activities, and an Acyl Carrier Protein (ACP) Fatty Acyl Synthase (FAS) is a multifunctional enzyme The KS subunit has a Cys-SH group, while the ACP subunit contains a Phosphopantetheine prosthetic group Acyl Carrier Protein-ACP • Contains a long and flexible Pantothenic acid (4’-phospho-pantethiene) group • Intermediates in fatty acid synthesis are attached to ACP • ACP shuttles the growing chain from one active site to another during fatty acid biosynthesis • At the end of each cycle, the ACP transfers the growing FA chain to the KS subunit to allow another Malonyl CoA to be attached to the ACP. Stage 3: Synthesis of fatty acid chain Consists of three separate steps: ➢ 1. Initiation - Acetyl CoA is attached to Ketoacyl Synthase (AT) and Malonyl CoA is attached to the acyl carrier protein (MT) ➢ 2. Fatty acid synthesis ▪ Condensation of Acetyl CoA and Malonyl CoA-ACP to form Acetoacetyl-ACP (KS) ▪ Reduction - NADPH is oxidized to form Hydroxybutyryl ACP (KR) ▪ Dehydration - formation of double bond (HD) ▪ Reduction - NADPH is source of e- and H+ to form butyryl-ACP (ER) The product, is then translocated from the ACP subunit to the KS subunit to allow the ACP subunit to accept the next Malonyl CoA. ➢ 3. Release of product (Palmitate) from FAS catalyzed by Thioesterase 1. Initiation of Fatty Acid Synthesis Acetyl CoA Malonyl CoA ACP KS Keto-acyl synthase (KS) FAS Initiation Condensation (KS) Next three (3) steps for each cycle reduce the β-carbonyl group of the product (Acetoacetyl-ACP) to CH2. 2. Assembly of Fatty acid Chain 1st Reduction (KR) Dehydration (HD) 2nd Reduction (ER) Condensation (KS) These reactions result in the net addition of 2Cs to the growing fatty acid chain/cycle. Fatty acid Synthesis - Summary Initiation Cycle 1 = 4C Cycle 2 = 6C Cycle 3 = 8C Fatty acid biosynthesis is the sequential addition of 2Cs/cycle from Malonyl CoA to a growing chain. Cycle 7 = 16C 3. Release of final product - Palmitate In the final step, the enzyme Palmitoyl Thioesterase (PT) catalyzes the hydrolysis reaction that releases palmitate from the FAS Synthesis of C16 palmitate: • 1 molecule of Acetyl CoA for initiation • Seven turns of the cycle (i.e. 7 Malonyl CoA) • Uses 14 NADPH. Net fatty acid synthesis reaction Acetyl-CoA + 7 Malonyl-CoA + 7ATP + 14NADPH + 14H+ Palmitate + 8 CoA + 7ADP + 7Pi + 14NADP+ + 7CO2 + 6H2O Regulation of Fatty Acid Synthesis • Acetyl-CoA Carboxylase is the Most Important Enzyme in the Regulation of Fatty Acid Synthesis Allosteric Regulation by metabolites: • Citrate • Palmitoyl-CoA = polymerization/ depolymerization Regulation by Hormones: • Glucagon/epi nephrine • Insulin = Phosphorylation/ dephosphorylation 1. Metabolite or Allosteric regulation of Acetyl-CoA Carboxylase •Acetyl CoA carboxylase is most active when it is in a homopolymeric form. •Citrate is an allosteric activator; abundant supply of citrate promotes ACC polymerization/activity. •Palmitoyl CoA acts as an allosteric inhibitor, and abundant supply of fatty acids favors depolymerization and inactivation of ACC (enzyme remains monomeric) 2. Hormonal regulation of Acetyl CoA Carboxylase e.g. HSL e.g. ACC Regulation of Fatty Acid Synthesis Regulation of Acetyl CoA Carboxylase by phosphorylation/dephosphorylation Malonyl CoA  Insulin Malonyl CoA  Glucagon Epinephrine Norepinephrine Hormonal regulation of acetyl-CoA carboxylase •Insulin stimulates the activity of protein phosphatase 2A. • stimulates fatty acid synthesis via stimulation of dephosphorylation of ACC •Inhibits hydrolysis of stored triacylglycerides by dephosphorylation of Hormone Sensitive Lipase (HSL). •Phosphorylation of ACC inhibits its activity, while dephosphorylation activates the enzyme. Synthesis of longer Fatty Acid Chains Condensation • Occurs in the ER or Microsomes and in mitochondria • Catalyzed by enzymes other than FAS and does not require ACP • Precursors are • Acetyl CoA in Mitochondria • Malonyl CoA in Microsomes Reduction Dehydration Reduction Desaturation of Fatty Acids Synthesis of nonessential monounsaturated fatty acids from saturated fatty acids Microsomal 9 Desaturase enzyme system 10 9 -3 And -6 Fatty Acids Must Be Derived From Diet 6 18  12 9  9  1  ω- nomenclature; Systematic nomenclature • In mammals, double bonds can be introduced only up to the Δ9 position but never beyond • Δ4, Δ5, Δ6, and Δ9 desaturases are present in most animals • In contrast, plants are able to synthesize fatty acids with double bonds at the Δ12 and Δ15 positions. • Double bonds introduced into fatty acids are always separated from each other by a methylene group Two Essential Fatty Acids • Unsaturated FA with double bonds after C9 cannot be synthesized in animals and must be obtained from plant sources. • Such fatty acids are denoted essential fatty acids (EFA) • Linoleic acid (LA): omega-6 fatty acid • -linolenic acid (LNA or ALA): omega-3 fatty acid Deficiency of EFAs leads to poor growth, poor wound healing and dermatitis Formation of long chain Polyunsaturated Fatty Acids in Eukaryotes Requires Desaturase & Elongase Enzyme Systems Desaturation (reduction) does not yield ATP In mammals, complete desaturation requires two other proteins Cytochrome b5 and NADH-Cytochrome b5 reductase Although FAD is used as the cofactor, desaturation reactions do not yield ATP The fate of Palmitate Palmitate ATP + CoA-SH Palmitoyl CoA synthetase AMP + PPi Palmitoyl CoA Chain Elongation Chain desaturation Longer chain FAs 2 Esterification Storage lipids: Unsaturated FA 3 Acylglycerols (TAGs) 1 Membrane/structural lipids: Glycerophospholipids Cholesterol esters 4 Summary of Fatty Acid Synthesis Lipid droplets • The newly synthesized fatty acids have mainly two fates: • Incorporation into triacylglycerols for storage (Lipogenesis) • Incorporation into membranes as phospholipids (during rapid growth) Filipin = Cholesterol staining LAMP-1: Late endosomal marker Triacylglycerol –Stored Lipids • Tri-fatty acid esters of glycerol • Nonpolar, neutral lipids • Stored in adipocytes CH2OH H C OH CH2OH Glycerol G l y c e r o l Fatty acid Fatty acid Fatty acid Triacylglycerol Triacylglycerol Lipogenesis • Lipogenesis is the process of synthesis of fatty acids from acetyl-CoA and the esterification of the fatty acids to produce Triacylglycerol (Storage Fat). • The nutritional state of the organism is the main factor regulating the rate of lipogenesis. • Excess glucose is diverted into synthesis of fatty acids (Glucose is the primary substrate for lipogenesis) • During starvation or in diabetes patients, fats are broken down into fatty acids (lipolysis) and are mostly mobilized for energy production. Degradation of Triacylglycerol - Lipolysis • Triacylglycerols are hydrolyzed by lipases to their constituent fatty acids and glycerol. • Free fatty acids are released into the plasma. • Fatty acids are degraded by -oxidation in Mitochondria Sources of Fatty acids for degradation • Degradation of free fatty acids (FFAs) is the major source of energy during fasting (starvation), Intense exercise (exhaustion) and metabolic conditions with poor glucose utilization e.g. type 2 Diabetes Two major sources of FFAs: • Dietary Lipids • Stored (intracellular) Lipids 36 Mobilization of Dietary Fats for energy 37 Mobilization of Fats from Adipose tissues • Low energy/blood glucose triggers the secretion of Epinephrine and Glucagon which activate Adenylate Cyclase • Elevated cAMP activates cAMP dependent Protein kinase • Phosphorylation of Hormone sensitive lipase (HSL) catalyzes the release fatty acids from TAGs • The released fatty acids are transported through the blood stream by binding to albumin and transported to other tissues e.g. skeletal muscles for β-oxidation and energy production. 38 Mobilization of fatty acids from Triacylglycerols • • • • Adipose triacylglycerol lipase (ATGL) Hormone-sensitive lipase (HSL) Perilipin (lipid droplet-associated protein) Monoacylglycerol lipase (MGL) Fatty Acid Oxidation ▪ FAs are stored in more reduced state than Carbohydrates Very closely packed in storage tissues Stored lipids are the primary source of energy in most organisms. ▪ Fatty acids are oxidized (or degraded) mainly by oxidation in Mitochondria ▪ The Beta Carbon is oxidized through a series of reactions. 40 Oxidation of Fatty Acids Major Fatty acid oxidation pathways • Mitochondrial -oxidation: 2 carbon units (Acetyl CoA) are sequentially removed from the carboxyl terminal • Peroxisomal -oxidation: Oxidation of very long (17-26 carbon) fatty acids begins in peroxisomes Minor Fatty acid oxidation pathways • α-oxidation: the carboxyl terminal carbon is oxidized and released as CO2 • ω-Oxidation: the ω-carbon is oxidized to COOH to form a dicarboxylic acid 41 Cellular sites of Fatty Acid oxidation • Short- (<C6) and medium-chain (C6-C12) fatty acids are oxidized exclusively in Mitochondria. • Long-chain fatty acids (C14–C16) are oxidized in both Mitochondria and Peroxisomes. • Very-long-chain fatty acids (C17–C26) are preferentially oxidized in Peroxisomes. • Peroxisomes are small, single membrane-bound organelles (microbodies) and lack genetic material but contain several metabolic enzymes 42 Mitochondrial β-oxidation Three stages: ▪ Activation of fatty acids in the cytosol by Acyl CoA synthetase ▪ Transport of fatty acids into mitochondria by Carnitine acyltransferases (CAT I and CAT II) ▪ Oxidation of the β Carbon and thiolysis to form 2carbon fragments (acetyl CoA) in the mitochondrial matrix 43 Stage 1: The fatty acid Activation + ATP 2ADP 44 The Fatty Acid Activation Reaction….. PPi + H2O 2 Pi AMP + ATP 2ADP Fatty acid + HS-CoA + 2ATP + H2O Acyl-CoA + 2ADP + 2Pi A net 2 ATP equivalents are used to activate one fatty acid to fatty acyl CoA. 45 Stage 2: Transport of Fatty acids into mitochondria: The Carnitine Shuttle System 3 main components: Carnitine, Carnitine Acyltransferases: (CAT I and CAT II) Acylcarnitine translocase Carnitine It is an amino acid derivative synthesized from L-Lysine and L-Methionine in the liver and kidneys. It is stored in skeletal muscles, heart, brain, and sperm It plays a major role in the conversion of fat into energy by shuttling activated long chain Fatty Acids into mitochondria. 46 Fatty acids are transported into mitochondria by carnitine acyl transferases (CATI/CATII) Malonyl CoA The net effect is the transport of Acyl CoA from the cytosol into the mitochodria matrix 47 Stage 3: -oxidation reactions In fatty acid degradation, 2 carbon units (acetyl CoA) are sequentially removed from the carboxyl terminal by oxidation of the β (C3) carbon The cleavage occurs between the α (C2) and the β (C3) carbon in four reactions 1. Formation of a trans double bond by Acyl CoA dehydrogenase 2. Hydration of the double bond by Enoyl-CoA hydratase 3. NAD+ dependent dehydrogenation of β-hydroxyacylCoA 4. Cleavage of Cα-Cβ bond by thiolase 48 Sequence of Reactions: 1. Oxidation (FAD) 2. Hydration 3. Oxidation (NAD+) 1 OXIDATION 4. Cleavage or Thiolysis Deficiency or inhibition of Acyl CoA dehydrogenase is fatal Symptoms include; severe hypoglycemia, vomiting, comma and death. e.g. in Sudden Infant Death syndrome due to mutation of Lys304 to Glu. Enzyme cannot bind Fatty-acyl CoA or FAD. 2 HYDRATION 49 3 OXIDATION 4 THIOLYSIS The net effect of these reactions is to Oxidize the β carbon and to shorten the fatty acid chain by 2 carbons 50 -oxidation Net reaction for palmitate (C16) Palmitoyl-CoA + 7 CoA + 7 FAD + 7 NAD+ + 7 H2O 8 acetyl CoA + 7 FADH2 + 7 NADH + 7 H+ 51 ENERGY GENRATION FROM FATTY ACID OXIDATION ▪ Each β-oxidation cycle of fatty acids yields 1 Acetyl CoA 1 FADH2 1 NADH + H+ Palmitate (C16) ▪ Acetyl CoA is coupled to the citric acid cycle that generates 1 GTP, 1 FADH2, 3 NADH + H+ ▪ The reducing equivalents are coupled to the Electron transport chain to yield ATP 52 Energy Yield From Palmitate Assuming: Source ATP 1FADH2 = 1.5 ATP 1 NADH + H+ = 2.5 ATP 1 FADH2 x 1.5 ATP = 1.5 ATP 1 NADH x 2.5 ATP = 2.5 ATP 1 Acetyl CoA 1GTP, 1 FADH2, 3 NADH x 10 ATP The ATP yield for every β-oxidation cycle is 14 ATP TOTAL Total = 10 ATP = 14 ATP β-Oxidation of Palmitate (C16) yields: Source ATP Total 7 FADH2 x 1.5 ATP = 10.5 ATP 7 NADH x 2.5 ATP = 17.5 ATP 8 acetyl CoA x 10 ATP = 80 ATP FA Activation = - 2 ATP NET 53 = 106 ATP For an even-numbered saturated fatty the number of oxidation cycles and number of FADH2 and NADH produced can be calculated as: • Even-numbered saturated fatty acid: No. of carbons in fatty acid = (X) Number of acetyl CoA 2 produced X-1= Number of - oxidation cycles =Number of FADH2 =Number of NADH • For example - oxidation of palmitate (C-16): 16/2 = 8 (Number of acetyl CoA produced) 8-1 = 7 - oxidation cycles = 7 FADH2 = 7NADH For an even-numbered saturated fatty acid (C2n) the total ATP yield can be calculated as: (n - 1) * 14 + 10 - 2 = total ATP For palmitate (C16, n = 8), the ATP yield is: (8 - 1) * 14 + 10 - 2 = 106 ATP For stearate (C18, n = 9), the ATP yield is: (9 - 1) * 14 + 10 - 2 = 120 ATP Oxidation of Monounsaturated Fatty Acids Oxidation of Polyunsaturated Fatty Acids Oxidation of Polyunsaturated Fatty Acids Oxidation of a Fatty Acids with Odd Number of Carbon Atoms additionally yields Propionyl-CoA Propionyl-CoA is converted to Succinyl CoA Propionyl-CoA is converted to succinyl-CoA, a citric acid cycle intermediate Propionyl residue from an oddchain fatty acid is the only part of a fatty acid that is glucogenic. ATP yield from 1 propionate: 1 GTP 1 FADH2 1 NADH ~5 ATP Peroxisomes Oxidize Very Long Chain Fatty Acids (C20- C26) -oxidation sequence ends at octanoyl-CoA Regulation of Fatty acid oxidation 62 Regulation of Fatty Acid Metabolism -Oxidation of Fatty Acids Synthesis • Cytosol • Requires NADPH • Acyl carrier protein Beta Oxidation • Mitochondria • NAD, FAD • CoA Clinical Aspects Impaired Oxidation of Fatty Acids Gives Rise to Diseases Associated With Hypoglycemia Carnitine deficiency CPT-I and II deficiency Medium-chain acyl-CoA dehydrogenase deficiency Ketogenesis ▪ Ketogenesis is the formation of ketone bodies as a result of increased fatty acid breakdown. ▪ Ketogenesis occurs in the liver when glycogen stores are depleted e.g. during fasting, starvation, exhaustion and in undiagnosed diabetes ▪ Ketone bodies are produced mainly in the mitochondria of liver cells and are used by other tissues including the brain ▪ 3 Types of Ketone Bodies: • Acetoacetate; • D-β-Hydroxybutyrate; • Acetone Brain, Heart, Kidneys Other peripheral tissues 67 Ketogenesis - Reactions 1 1. Acetoacetyl CoA is formed from 2 molecules of Acetyl CoA (Thiolase) 2. Acetoacetyl CoA condenses with another Acetyl CoA to form 3-HMG CoA (HMG CoA synthase) 3. 3-HMG CoA is cleaved to Acetyl CoA and Acetoacetate (HMG CoA lyase) ▪Acetoacetate and D-βHydroxybutyrate are interconvertible. 2 3 ▪ Acetoacetate can also spontaneously undergo decarboxylation to yield Acetone 68 Ketone bodies are an energy source for tissues other than the Liver • Ratio of [β-hydroxybutyrate] to [Acetoacetate] varies between 1:1 and 10:1 depending on the mitochondrial redox state (NADH/NAD+) • Liver lacks β-ketoacyl-CoA transferase and cannot use ketone bodies as an energy source. 69 Clinical Aspects-Ketogenesis • Ketone bodies are created at moderate levels in our bodies, at times when carbohydrates are not readily available. • Excessive production of ketone bodies occurs during prolonged starvation or diabetes mellitus. • Higher than normal levels of ketogenesis leading to accumulation of ketone bodies in blood or urine is called hyperketonemia or hyperketonuria. The condition is called ketosis. • Abnormally high concentration of acetoacetate and D-hydroxybutyrate lower blood pH. This state is called ketoacidosis Regulation of Ketogenesis Three major Control points 1) Control of release of free fatty acids from TAGs by lipolysis Control pt. Fed state versus starvation or diabetes (Insulin deficiency). 2) Fate of Acyl CoA: ▪Entry of acyl CoA into liver mitochondria for β-oxidation or ▪ Esterification to form TAGs Control Point: Carnitine and carnitine acyltranesferase I 3) Fate of Acetyl CoA: ▪Oxidation to CO2 in the citric acid cycle or ▪Ketogenesis Control point: Level of Oxaloacetate and energy needs Summary of lipid metabolism Fasted state, Starvation Triacylglycerols & Phospholipids Fed state Glycerol DHAP Glucose Fatty Acids Stage 1 Glyceraldehyde-3-Phosphate Long Chain FAs Unsaturated FAs Palmitoyl CoA Acyl CoA Stage 2 Palmitate Stage 3 FA synthesis Acyl CoA Glycolysis Stage 3 β-Oxidation CAT I Pyruvate Pyruvate Malonyl CoA Acetyl CoA Acetyl CoA carboxylase Stage 2 Acetyl CoA Citric acid cycle Citrate ATP Mitochondrial matrix Cytosol Stage 1 Citrate Citrate Shuttle 72