Lipid Metabolism II Lecture 3 - Kwame Nkrumah University of Science & Technology, Kumasi, Ghana

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana Lipid Metabolism II LECTURE 3  Learning Objectives The objectives of this lecture are to: v Examine the pathways in fatty acid oxidation (beta oxidation) v Look into the mechanisms assoc...

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana Lipid Metabolism II LECTURE 3  Learning Objectives The objectives of this lecture are to: v Examine the pathways in fatty acid oxidation (beta oxidation) v Look into the mechanisms associated with the metabolism of fatty acids. v Explore the formation of ketone bodies www.knust.edu.gh  Learning Outcomes At the end of this lecture students should be able to: v Demonstrate an understanding of the pathways in fatty acid oxidation (beta oxidation) v Describe an understanding of the mechanisms associated with the metabolism of fatty acids. v Appreciate the formation of ketone bodies www.knust.edu.gh Recap: Digestion and Absorption of fats www.knust.edu.gh www.knust.edu.gh  Mobilization of dietary Lipids v Dietary lipids must be absorbed in watery environment - in small intestine v Made soluble by emulsification with bile salts v Lipase and phospholipases A2 from pancreas release FA and Glycerol from TAG v Major source of FA which are converted to TAG on reabsorption v TAG combines with albumin and specific blood proteins to form Lipoproteins and chylomicrons which are released into the blood and transported into the liver/tissues v In tissues, lipoprotein lipase hydrolysis chylomicrons & lipoproteins for absorption into adipocytes or hepatic cells v Glycerol is converted to DHAP to enter glycolysis v FA oxidized by Beta oxidation www.knust.edu.gh  LIPOLYSIS TO β-OXIDATION Lipolysis is carried out by lipases. Once freed from glycerol, free fatty acids can enter blood and muscle fiber by diffusion. The pathway for catabolism of fatty acids is referred to as the b-oxidation pathway, because oxidation occurs at the b-carbon (C-3). Beta oxidation splits long carbon chains of the fatty acid into acetyl CoA, which can eventually enter the TCA cycle FA classified as Very long chain FA >20C Long chain FA C12-C20 Medium Chain FA C6-C12 Short Chain C4  Fatty acid oxidation v Long chain fatty acids are not soluble in water. So, in the blood, fatty acid are transported as complexed with albumin. Inside cells, they are attached to fatty acid binding protein (FABP). v They are then oxidized to produce energy. v Beta oxidation of fatty acids: Fray knoop in 1904 elucidated the principal mechanisms by which mammalian tissues oxidize fatty acids. www.knust.edu.gh  Table Fatty acids of importance to humans Numerical structure Trivial Systematic name symbol name 16:0 CH3-(CH2)14-COOH Palmitic Hexadecanoic 16:1(D9) CH3-(CH2)5-CH=CH-(CH2)7-COOH Palmitoleic cis-9-hexadecenoic 18:0 CH3-(CH2)16-COOH Stearic Octadecanoic 18:1(D 9) CH3-(CH2)7-CH=CH-(CH2)7-COOH Oleic cis-9-octadecenoic 18:2(D 9,12) CH3-(CH2)3-(CH2-CH=CH)2-(CH2)7-COOH Linoleic cis,cis-9,12- or 18:2(w6,9) octadecenoic 18:3(D 9,12,15) CH3-(CH2-CH=CH)3-(CH2)7-COOH Linolenic cis,cis,cis-9,12,15- or 18:3(w3,6,9) octadecatrienoic 20:4(D 5,8,11,14) CH3-(CH2)3-(CH2-CH=CH)4-(CH2)3-COOH arachidonic cis,cis,cis,cis-5,8,11,14- or 20:4(w6,9,12,15) eicosatetraenoic Mammals lack the enzymes to introduce double bonds at carbon atoms beyond C-9 in the fatty acid chain. Hence, mammals cannot synthesize linoleate (18:2 cis-D9, D12), linolenate (18: cis-D9, D12, D15) and arachidonate (20:4 cis-D5, D8, D11, D14). The three fatty acids are essential fatty acids.  Beta oxidation of fatty acids Beta oxidation of fatty acid is Occurs in the mitochondria the catabolic process by which Produces Acetyl-CoA is fed fatty acids are broken down to directly into the Krebs Cycle generate acetyl-CoA  All Reactions Occur Between α and β Carbons Beta oxidation β-carbon α-carbon The oxidation of the hydrocarbon chain occurs by a sequential cleavage of two carbon atoms, occurring at the ᵦ-carbon atom. α β www.knust.edu.gh  Where & How Does Beta Oxidation Occur v Fatty acids are degraded in the mitochondrial matrix via the Beta-Oxidation Pathway. v Fatty acyl-CoA formed outside can pass through the outer mitochondrial membrane, but cannot penetrate the inner membrane. v Transfer of the fatty acid moiety across the mitochondrial inner membrane involves carnitine. www.knust.edu.gh  Beta oxidation of fatty Acids Involves three stages – ACTIVATION OF FATTY ACIDS IN CYTOSOL – TRANSPORTATION OF ACTIVATED FATTY ACIDS INTO MITOCHODRIA(CARTININE SHUTTLE) – BETA OXIDATION IN THE MITOCHONDRIA MATRIX  Beta oxidation of fatty Acids Preparation for Oxidation v Before Oxidation, Fatty Acids Must be Activated and Transported to the Mitochondrion. v Activation Begins in the Cytoplasm (FA THIOKINASE, ACYL COA SYNTHETASE, ACYL COA LIGASE) Long Chain Fatty Acyl-CoA Ligase Fatty Acid Fatty Acyl-CoA www.knust.edu.gh  FATTY ACID ACTIVATION BY ESTERIFICATION WITH CoASH Acyl CoA Synthetase CoASH + RCO2H + ATP RCOSCoA + AMP + PP i Pyrophosphatase 2 Pi DG0’(KJ/mole) ATP AMP + PPi -32.3 CoASH + RCO2H RCOSCoA +31.5 PPi 2 Pi -33.6 -34.4 Fatty acids must be esterified to Coenzyme A before they can undergo oxidative degradation, be utilized for synthesis of complex lipids, or be attached to proteins as lipid anchors. Acyl-CoA Synthases (Thiokinases) of ER & outer mitochondrial membranes catalyze activation of long chain fatty acids, esterifying them to coenzyme A. This process is ATP-dependent, & occurs in 2 steps. There are different Acyl-CoA Synthases for fatty acids of different chain lengths. 22  FOUR ENZYMES AND REACTIONS IN β −OXIDATION: 1)DEHYDROGENATION: Dehydrogenation occurs between the α and β carbons (C2 and C3) in an FAD-linked reaction catalyzed by Acyl CoA dehydrogenase. The oxidative power of FAD is required to oxidize the alkyl chain. The product contains a trans- double bond. Involvement of the beta-carbon in this and subsequent steps gives the pathway its name. There are three fatty acyl CoA dehydrogenases, each specific for a different acyl chain length: 1) Long chain fatty acyl CoA dehydrogenase (LCAD) acts on chains greater than C12. 2) Medium chain fatty acyl CoA dehydrogenase (MCAD) acts on chains of C6 to C12. 3) Short chain fatty acyl CoA dehydrogenase (SCAD) acts on chains of C4 to C6. MCAD deficiency is thought to be one of the most common inborn errors of metabolism. 2)HYDRATION : Hydration of the double bond is catalyzed by Enoyl CoA hydratase. The product is an L-3-hydroxyacyl CoA; yielding 1 NADH  FOUR ENZYMES AND REACTIONS IN β −OXIDATION 3)2ND DEHYDROGENATION : A second dehydrogenation, of the alcohol, occurs in a NAD-linked reaction catalyzed by β-hydroxyacyl CoA dehydrogenase. The product is a ketone. 4)THIOLYTIC CLEAVAGE : Thiolytic cleavage of the thioester is catalyzed by β- ketoacyl CoA thiolase, yielding 1 acetyl-CoA and a fatty acid that has now been shortened by 2 carbons (acyl-CoA.) The shortened fatty acyl group is now ready for another round of β-oxidation. This cycle repeats until the FFA has been completely reduced to acetyl-CoA or, in the case of fatty acids with odd numbers of carbon atoms, acetyl-CoA and 1 mol of propionyl-CoA per mol of fatty acid 1. Oxidation: The fatty acyl Beta oxidation of fatty Acids CoA is dehydrogenated to 4. The B-keto acyl-CoA undergoes thiolytic trans-enoyl CoA by FAD cleavage, splitting off a molecule of acyl which becomes FADH2. CoA and a fatty acid (2-carbon less). FADH2 when oxidized in the electron transport Further repeated cycles produces acetyl chain will produce 2 ATPs. CoA for the TCA cycle. 2. Hydration: Trans-enoyl CoA is then hydrated to the L- somer which is L-B-hydroxy acyl CoA by trans-enoyl CoA is hydratase. 3. NAD+ dependent hydrogenase converts L-B- hydroxy acyl CoA to B-keto acyl-CoA producing NADH which can be oxidized by the electron transport chain will produce 3 ATPs. www.knust.edu.gh  Clinical application of carnitine deficiency v Deficiency of the translocase have been reported in a few cases where the transport of long chain fatty acids was defective. v In this condition muscle cramps are precipitated by fasting, exercise and high fat diet. v Carnitine deficiency is reported in preterm infants, in whom impaired fatty acid oxidation is noticed. So more glucose is utilized resulting in episodes of hypoglycemia. www.knust.edu.gh  Calculations Carbons in Fatty Acetyl CoA β-oxidation cycles Acid C/2 (C/2) -1 12 6 5 14 7 6 16 8 7 18 9 8 Note: In each round of β-oxidation one molecule of FADH2 and one molecule of NADH+H+ are produced which generates 2 and 3 ATP molecules, respectively  BETA OXIDATION OF ODD CARBON FATTY ACIDS Odd chain fatty acids are less common. Formed by some bacteria in the stomachs of ruminants, plants, marine organisms & human colon. β-oxidation occurs pretty same much as with even chain FA until the final thiolase cleavage which results in a 3 carbon acyl-CoA (Propionyl-CoA) Special set of 3 enzymes are required to further oxidize propionyl-CoA Ø Propionyl-CoA carboxylase, Ø Methylmalonyl-CoA epimerase and Ø Methymalonyl-CoA mutase.  BETA OXIDATION OF ODD CARBON FATTY ACIDS The first enzyme, having the coenzyme biotin attached, catalyzes the carboxylation of the propionyl-CoA to the D-stereoisomer of methylmalonyl-CoA. The epimerase then catalyzes the conversion of the D-stereoisomer to its L form, L-methylmalonyl-CoA. The last enzyme, the mutase then catalyzes the rearrangement of the molecules of L-methylmalonyl-CoA to succinyl-CoA. This enzyme requires the coenzyme B12, derived from vitamin B12. Succinyl CoA enters the TCA cycle  β-OXIDATION OF UNSATURATED FATTY ACIDS In β-Oxidation of unsaturated FA the location of a cis bond prevents the formation of a trans-Δ2 bond. These situations are handled by an additional two enzymes: Enoyl CoA Isomerase or 2,4 Dienoyl CoA Reductase. If the acyl CoA contains a cis-Δ3 bond, then cis-Δ3-Enoyl CoA isomerase will convert the bond to a trans-Δ2 bond, which is a regular substrate. If the acyl CoA contains a cis-Δ4 double bond, then its dehydrogenation yields a 2,4-dienoyl intermediate, which is not a substrate for enoyl CoA hydratase. However, the enzyme 2,4 Dienoyl CoA reductase reduces the intermediate, using NADPH, into trans-Δ3-enoyl CoA. As in the above case, this compound is converted into a suitable intermediate by 3,2-Enoyl CoA isomerase.  KETONE BODIES During fasting or carbohydrate starvation, oxaloacetate is depleted in liver due to gluconeogenesis. This impedes entry of A-CoA into Krebs cycle. A-CoA in liver mitochondria is converted then to ketone bodies, acetoacetate & b- hydroxybutyrate. Made up of two molecules – Acetoacetate and β-hdyroxybutyrate Only Acetoacetate is the true ketone body Brain uses nearly 70% of body's glucose. Fat – major energy store releases Free Fatty acids under increased demand. FFA cannot cross the blood brain barrier + low capacity for FA oxidation Ketones are used under increased demand and starvation conditions by the brain  Significance of Ketogenesis 1) Ketone bodies are water soluble, they are convenient to transport in blood, and readily taken up by non-hepatic tissues. In the early stages of fasting, the use of ketone bodies by heart, skeletal muscle conserves glucose for support of central nervous system. With prolonged starvation, brain can up take more ketone bodies to spare glucose consumption. (2) High concentration of ketone bodies can induce ketonemia and ketonuria, and even ketosacidosis. When carbohydrate catabolism is blocked by a disease e.g diabetes mellitus, inadequate sugar supply, the blood concentration of ketone bodies may increase, the patient may suffer from ketosis and acidosis – ketoacidosis.  CLINICAL SIGNIFICANCE OF KETOGENESIS Ketone bodies -relatively low rate during normal physiological status. Normal physiological responses to carbohydrate shortages cause the liver to increase the production of ketone bodies from the acetyl-CoA generated from fatty acid oxidation primarily allowing the heart and skeletal muscles to use ketone bodies for energy, thereby preserving the limited glucose for use by the brain. Ketone bodies - strong acids (≥ 3.5), and their increase lowers the pH of the blood –Keto acidosis.  CLINICAL SIGNIFICANCE OF KETOGENESIS Acidification of the blood impairs the ability of hemoglobin to bind oxygen. Accumulation of ketone bodies is abnormal (but not necessarily harmful) is called ketosis/keto acidosis and can be quantified by sampling the patient's exhaled air, and testing for acetone by gas chromatography. Ketosis, leads to profound clinical manifestations in untreated insulin- dependent diabetes mellitus- Diabetic Ketoacidosis (DKA) This is the result of reduced supply of glucose and a concomitant increase in fatty acid oxidation. The increased production of A-CoA leads to ketosis that exceeds the ability of peripheral tissues to oxidize them.  Key points vFatty acid oxidation is a regulated and coordinated process affected by the state of the body. vThe body compensates for the absence of energy using diverse means to ensure brain and cellular function. vAccumulation of ketone bodies in the blood can lead to ketoacidosis. vDeficiency of vital enzymes in the cascade of fatty acid metabolism has medical significance. www.knust.edu.gh Kwame Nkrumah University of Science & Technology, Kumasi, Ghana www.knust.edu.gh  It is not a reversal of oxidation. Requirements The major fatty acid synthesised is Acetyl coA palmitic acid. A 16c saturated FA NADPH ATP SITE- cytosol – Liver – Lactating mammary glands – Adipose tissues – lungs  Transport of Acetyl CoA to Cytoplasm starting material for de novo synthesis is acetyl CoA. formed inside the mitochondria from pyruvate. inner membrane is not freely permeable to acetyl CoA acetyl CoA units are delivered to the cytoplasm as citrate www.knust.edu.gh www.knust.edu.gh www.knust.edu.gh www.knust.edu.gh  Fatty Acid Synthase (FAS) Complex multi-enzyme complex enzymes form a dimer with identical subunits Each subunit of the complex is organized into 3 (condensing unit, reduction unit , releasing unit) domains with 7 enzymes Advantages of Multi-enzyme Complex – Intermediates of the reaction can easily interact with the active sites of the enzymes – One gene codes all the enzymes; so all the enzymes are in equimolecular concentrations – So the efficiency of the process is enhanced.  Fatty Acid Synthase (FAS) Complex 1st Domain or Condensing Unit initial substrate binding site enzymes involved are beta-keto acyl synthase or condensing enzyme (CE); acetyl transferase (AT) and malonyl trans acylase (MT) 2nd Domain or Reduction Unit contains the dehydratase (DH); enoyl reductase (ER); beta-keto acyl reductase (KR) and acyl carrier protein (ACP) The acyl carrier protein is a polypeptide chain having a phospho-pantotheine group, to which the acyl groups are attached in thioester linkage. So ACP acts like the CoA carrying fatty acyl groups Fatty acid synthase complex. Upper and lower units are two monomers of the complex. Dotted line represents functional division  Fatty Acid Synthase (FAS) Complex Fatty Acid Synthase (FAS) Complex thio-esterase (TE)- It is involved in the release of the palmitate synthesised. (RELEASING UNIT)  STEPS IN F.A SYNTHESIS Step 1: Carboxylation of Acetyl CoA first step in the fatty acid synthesis is the carboxylation of acetyl CoA to form malonyl CoA acetyl CoA carboxylase it is the rate-limiting enzyme enzyme is allosterically regulated, the major effectors being citrate (positive) and palmitoyl CoA (negative)  STEP 2 2A- The acetyl transacylase catalyses the transfer of the acetyl group (2 carbons) to the cysteinyl SH group of the condensing enzyme (CE) of the other monomer of the fatty acid synthase complex 2B- One molecule of acetyl CoA (2 carbon) and one molecule of malonyl CoA (3 carbon) bind to the multienzyme complex. Malonyl transacylase transfers the malonyl group to the SH group of the ACP of one monomer of the enzyme  Four-step repeating cycle, extension by 2-carbons /cycle Condensation – Loss of the CO2 used in the formation of Malonyl CoA Reduction – NADPH used as the reducing agent (from PPP) Dehydration Reduction  STEP 3 Step 3: Condensation The acetyl (2C) and malonyl (3C) units are condensed to form beta- keto acyl ACP or acetoacetyl ACP During this process one carbon is lost as CO2 enzyme is called condensing enzyme or keto acyl synthase  Step 4: Reduction The acetoacetyl ACP is reduced by NADPH dependent beta-keto acyl reductase to form beta-hydroxy fatty acyl ACP Step 5: Dehydration – beta-hydroxy fatty acyl ACP is then dehydrated by a dehydratase (DH) to form enoyl ACP – Step 6: Second Reduction – The enoyl ACP is again reduced by enoyl reductase (ER) utilizing a 2nd molecule of NADPH to form butyryl ACP  Cycling of Reactions The butyryl group (4C) is now transferred to the SH group of the condensing enzyme on the other monomer and a 2nd malonyl CoA molecule binds to the phospho-pantothenyl SH group. The sequence of reactions, namely condensation, reduction, dehydration and reduction (steps 3,4,5,6) are repeated seven times, till the 16-carbon palmitic acid is formed  Step 7: Palmitic Acid is Release Thio-esterase activity (TE) releases palmitate from the multienzyme complex.  Regulation of Fatty Acid Synthesis 1. Availability of Substrates Fatty acid synthesis occurs when carbohydrate is abundant and the level of fatty acids is low. availability of citrate in the cytoplasm is the most important regulatory factor producing a short-term effect. 2. Acetyl CoA Carboxylase key enzyme Phosphorylation inactivates acetyl CoA carboxylase The enzyme is inhibited by palmitoyl CoA, the end product 3. Insulin Favors Lipogenesis Insulin enhances the uptake of glucose by cells and increases the activity of pyruvate dehydrogenase. acetyl CoA carboxylase. Insulin also depresses the hormone sensitive lipase 4. Glucagon inhibits Lipogenesis Glucagon and epinephrine inactivate the acetyl CoA carboxylase by phosphorylating the enzyme Kwame Nkrumah University of Science & Technology, Kumasi, Ghana  SYNTHESIS OF TRIGLYCERIDES (TAG) Liver and adipose tissue are the major sites of triacylglycerol (TAG) synthesis. The TAG synthesis in adipose tissue is for storage of energy whereas in liver it is mainly secreted as VLDL and is transported to peripheral tissues. TAG is synthesised by esterification of fatty acyl CoA with either glycerol-3- phosphate or dihydroxy acetone phosphate (DHAP)  Key points Lipid metabolism – an integral part in survival of living organism Derangement in metabolism leads to metabolic disorders which may be life threatening. Pathways are well regulated and cordinated by series of enzymes and formation of intermediates which may be fed into other pathways.  READING Assignment v What is alpha oxidation of fatty acids? Where does it occur? What mechanisms are involved? v What is omega oxidation? Where does it occur? What mechanisms are involved? www.knust.edu.gh  Thank you 1/26/24