Biochemistry - Fatty Acid Oxidation and Ketone Bodies 2023 Lecture Notes PDF

Oxidation of Fatty Acids and Ketone Bodies Lecture 36 Reference: Lieberman and Peet, Chapter 30 Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD 1 Lecture Objectives a. Recall the purpose of the fatty acid β-oxidation pathway and what products are produced. b. Recall...

Oxidation of Fatty Acids and Ketone Bodies Lecture 36 Reference: Lieberman and Peet, Chapter 30 Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD 1 Lecture Objectives a. Recall the purpose of the fatty acid β-oxidation pathway and what products are produced. b. Recall the two ketone bodies made from acetyl CoA and relate why they are made from the liver. Recall which tissues can and cannot utilize these ketone bodies. c. Identify the four types of fatty acid classification, which is based on chain length. d. Recall fatty acid activation to fatty acyl CoA as a necessary step for β-oxidation. Identify the sites in the cell where fatty acids are activated. e. Relate the mechanism of long-chain fatty acid transport from the cytosol to the mitochondrial matrix. f. Using a diagram of the fatty acid oxidation pathway, relate the mechanism by which saturated fats are oxidized completely to acetyl CoA (at the level provided on the slides). g. Interpret the energy yield of ATP per 16-C saturated fatty acid oxidized. Interpret why an unsaturated 16-C fatty acid would produce slightly less ATP. h. Relate why newly synthesized fatty acids are not immediately transported into the mitochondrial matrix. i. Recall propionyl Co-A as a product of odd-chain fatty acids and relate how this product is used in the mitochondrial matrix. j. Compare and contrast the oxidation of very long fatty acids in peroxisomes to normal fatty acid oxidation in mitochondria. k. Recall the time dependence of ketone body concentration in the blood versus days of fasting. Interpret why the amount of D-β-hydroxybutyrate is much greater than acetoacetate. l. Recall ketone body synthesis and degradation in terms of starting material and products formed (synthesis) and products formed during degradation. m. Identify the metabolic conditions that regulate fatty acid oxidation and ketone body production and relate the time course (in days) of ketone body production during fasting. 2 2 Objective A Introduction • Fatty acids have several fates in the cell. • FA oxidation is the major source of energy for ATP synthesis in humans – 9 kcal / gram! • β-oxidation in the mitochondria is major pathway • generates acetyl CoA during the pathway • acetyl CoA enters TCA and produces more ATP • Many tissues, including muscle, oxidize fatty acids ß mitochondria required • Brain and nervous tissue use FAs as source of energy only very little • Generally can’t import fat across the blood-brain barrier. Small chain FA can get across the membrane. • Red blood cells lack mitochondria, so they can’t oxidize FAs 3 3 Objective A Fatty Acid Oxidation Overview • Most fatty acid oxidation occurs in the mitochondrial matrix. • Very long fatty acids are partially oxidized in peroxysomes prior to the mito matrix • Reduced coenzymes are produced (FADH2 and NADH) • Electrons donated to the electron transport chain • Acetyl CoA is produced and used by: • TCA cycle, OxPhos (most tissues) • Ketone body production (Liver) when ATP level is high 4 4 Objective B Fatty Acids and Ketone Bodies • Muscle and Liver both use β-oxidation for ATP production and energy. • During fasting, under a glucagon signal, LIVER will use excess acetyl CoA produced by β-oxidation to synthesize ketone bodies that are released into blood. • Acetoacetate and beta-hydroxybutarate are the two ketone bodies produced • Acetyl CoA is not stable in the blood. Ketone bodies are far more stable in blood and are a way to convert excess fuel for use by other tissues. • Muscle and kidney will readily use ketone bodies for energy. Brain and nervous tissue will use ketone bodies if concentration in the blood is high (i.e., after 48 hours of fasting). • Liver can not use ketone bodies since it lacks the enzyme for their activation • Prevents ketone bodies being used as they are made, which would waste energy • Red blood cells can’t use ketone bodies since they don’t have mitochondria 5 5 Objective C Fatty Acids as Fuels • Derived from dietary fat (fed state) or released into the blood by adipose tissue (fasting). • Remember…9 kcal / gram. Highest energy fuel molecule. • Fatty acids fall in to four general groups: • • • • short chain: 2-3 carbons (acetate and propionate)…gut uses these and these are the FA that can cross Blood Brain Barrier medium chain: 4-12 carbons long chain: 12-20 carbons ß most common dietary and stored fat very long chain: 20 or more carbons • Fatty acids are hydrophobic, so chains longer than 4-C are usually bound to proteins • Carried in blood by albumin, handled in cells by fatty acid binding proteins • Short chain fats are fairly soluble in water due to the polar nature of the carboxyl group and only a few CH2 groups. 6 6 Fatty Acid Oxidation Overview • Notice chain length • • • • Very long chain Long chain Medium chain Short chain • Notice location of reactions • VLC – peroxysomes • Others mitochondrial matrix • Notice the enzymes involved • Acyl-CoA Synthetase – activation • Acyltransferases – carrier molecules • Oxidation enzymes – main workers • Enzyme deficiencies can affect person’s ability to use fat for energy 7 7 Objective D Activation of Fatty Acids Words next page… 8 8 Objective D Activation of Fatty Acids • FAs are chemically unreactive, so must be activated first • goal is to get FA bound to CoA = fatty acyl CoA • Acyl CoA synthetase enzyme attaches an AMP group to the fatty acid carboxylic acid forming fatty acyl-AMP + PPi (PPi hydrolysis provides energy for reaction) • Fatty acid attached to AMP is transferred to CoA to form fatty acyl CoA • Net change is the utilization of two high energy phosphate bonds • Cleavage of AMP and PPi and hydrolysis of PPi • MAJOR MECHANISM: long chain FAs are activated by enzymes located in the ER membrane and the inner mito membrane. Activated long chain FAs are transported into the mito matrix by the carnitine shuttle (next slide) • short chain FAs are activated in the cytosol or mitochondrion • medium chain FAs cross inner mitochondrial membrane and are activated in mitochondrial matrix • Very long chain FAs are processed in peroxisomes, producing acetyl CoA and short chain fats that are complexed with carnitine in the peroxisome then exported to the mitochondria à matrix for oxidation. 9 9 Objective E and G Transport of Long Chain Fatty Acids into Mitochondria Note: malonyl CoA produced during fat synthesis will shut down CPT1 – prevents newly synthesized fats from being 10 Imported into mitochondria and immediately oxidized. 10 Transport of Long Chain Fatty Acids into Mitochondria: The Words Objective E and G • Carnitine is the carrier for long chain FAs into mito matrix • an enzyme on the outer mitochondrial membrane catalyzes the transfer of FA from CoA to carnitine • KEY CONTROL POINT: newly synthesized FAs are not immediately transported back into mitochondria • specific translocase on inner mito membrane transports fatty acyl carnitine • once inside mito matrix, second enzyme transfers FA from fatty acylcarnitine back onto CoA • long chain fatty acyl CoA in matrix is a substrate for β-oxidation 11 11 Objective F Oxidation of Fatty Acids • Fatty acids are oxidized in two carbon units, making acetyl CoA for TCA cycle. • The two carbons are the ones adjacent to the CoA, denoted α and β • first three steps (of four total) are similar to the TCA cycle between succinate and oxaloacetate. • The four steps produce FADH2, NADH, and acetyl CoA • Process repeats until all the carbons of a linear even chain FA are converted to acetyl CoA: 2 carbons at a time 12 12 Objective F β-oxidation of Even Numbered FAs 13 13 Objective F β-oxidation of Even Numbered FAs: The Words • Step 1: two H+ and 2 e- transferred to FAD forming FADH2. Results in a trans double bond between α and β carbons, called an enoyl CoA • step 2: H2O adds across double bond, forming a β-hydroxacyl CoA • step 3: β-hydroxacyl CoA is oxidized by NAD+ forming a β-ketoacyl CoA • step 4: bond between α and β carbons is cleaved by the addition of a second CoA molecule: the fatty acid chain is transferred to the new CoA • produces acetyl CoA and a fatty acyl CoA that is 2 carbons shorter • cycle repeats until all carbons of chain have been used • in final cycle, when 4 carbons remain, final product is 2 moles of acetyl CoA Enzymes for the β-oxidation pathway are very specific for tissue type and fatty acid chain length. The enzymes needed for oxidation will change as the length of the FA chain gets shorter. 14 14 Transfer of FADH2 Electrons to ETC • Acyl CoA dehydrogenase part of FA oxidation pathway. • ETF = electron transfer flavoprotein • ETF QO = electron transferring flavoprotein–coenzyme Q oxidoreductase: Flavoprotein in inner mitochondrial matrix that receives electrons and gives them to CoQ. 15 15 Objective G Energy Yield per 2 Carbon Unit Oxidized • The first two carbon unit oxidized produces 11 moles of ATP • The equivalent of 2 moles ATP are required to activate the FA • One ATP is split to AMP + PPi and PPi is hydrolyzed to 2 Pi • 1 mole FADH2 produced à1.5 moles ATP • 1 mole NADH produced à 2.5 moles ATP • oxidation of 1 mole acetyl CoA via TCA yields 9 moles ATP • subsequent 2 C units don’t need to be reactivated, so net is + 2 moles ATP = 13 moles ATP per 2 C unit utilized • Terminal two carbon unit produced is as acetyl CoA, so no FADH2 or NADH produced for the final two carbons step in beta-oxidation pathway, but this acetyl CoA will produce 9 ATP by TCA / OxPhos. • Example: palmitate (C16 FA, 7 cleavages) yields 7 FADH2; 7 NADH; 8 Acetyl CoA… 98 ATP !! • • So the 7 FADH2 and 7 NADH produce 28 ATPs just by themselves… but don’t forget that 1 ATP and 1 PPi were used 8 Acetyl CoA produce 72 ATP… (+ 8 GTP) • Compare to glucose ~ 25 mole ATP per mole glucose used. 16 16 Objective I β-oxidation of Odd Chain FAs • Same process as even chains until the final 5 carbons remain • Final reaction generates 1 acetyl CoA and one propionyl Co A (3 carbon fatty acyl CoA) • Propionyl Co A is converted to succinyl CoA and enters the TCA cycle. 17 17 Objective G Oxidation of Unsaturated FAs • Half of human fatty acids are unsaturated, and the double bonds are almost ALWAYS cis configuration. • Double bond of unsaturated C’s must be in proper configuration for βoxidation: • a trans-double bond between the α and β carbons • Mitochondrial matrix has enzymes to rearrange structure of FA double bonds to make them correct for oxidation. 18 18 Objective F Oxidation of Unsaturated FAs • extra enzymes rearrange the double bond • an isomerase and a reductase move the double bond to the α and β carbons in a trans configuration • for polyunsaturated fatty acids, double bonds are handled in the same way as they are encountered. • if two double bonds are in proximity (*within a few carbons), the isomerase and reductase move the double bonds around until a specific configuration is obtained, with the double bond between the α and β carbons: β-oxidation resumes • KEY POINT: presence of double bonds reduces amount of ATP produced because of different requirements for NADH or FADH2 production. 19 19 Oxidation of Very Long Chain FAs in Peroxysomes Reactive oxygen species H2O2. Picked up by catalase and converted to water. • process similar to β-oxidation mechanism • first step donates electrons to O2 making H2O2 rather than by the electron transport chain. Thus, no ATP is produced by this step. Objective J H2O2 is produced: bad…catalase degrades H2O2 … okay • goal of peroxysomes is to make octanoyl CoA, then move these to the mitochondrion for oxidation via the carnitine shuttle. • CAT, carnitine:acetyltransferase; COT, carnitine:octanoyltransferase, CAC: carnitine:acylcarnitine carrier; NOTE….CPTI not needed here… 20 20 Objective N Regulation of FA Oxidation • NADH and FADH2 levels are one of the main mechanisms by which FA oxidation is controlled. • If ATP levels are high, ETC/OxPhos will slow down, so NADH levels will rise, which will inhibit FA oxidation. • Fatty acid synthesis (right side of figure) will be covered later. 21 21 Objective B Metabolism of Ketone Bodies: Acetoacetate, β-hydroxybutyrate • Most tissues can use ketone bodies at any time, especially muscle and kidney • Brain and nerve tissue will only use ketone bodies when the amount is high in the blood (long fasts) 22 22 Objective K Ketone Bodies and Fasting 23 23 Objective L Synthesis of Ketone Bodies • two acetyl CoA react to form acetoacyl CoA • This reacts with a third acetyl CoA to produce 3-hydroxyl-3-methyl glutaryl CoA (HMG CoA) • HMG CoA is cleaved to acetoacetate and acetyl CoA 24 24 Objective L Utilization of Ketone Bodies • Ketone bodies travel in the blood to tissues where they are oxidized as fuel • muscle, kidney primarily • not liver, because it lacks key enzyme (thiotransferase) for oxidation (make them, don’t use them!) • brain : during prolonged fasting / starvation, ketone body concentration gets high enough that they enter brain cells and can be utilized. • Acetoacetate enters cells, or it is produced in cells by the oxidation of β-hydroxybutyrate • reverse reaction of β-hydroxybutyrate formation, produces NADH • NADH used to generate ATP from oxidative phosphorylation • thus more energy is obtained from β-hydroxybutyrate 25 25 Objective L Fates of Acetoacetate • Can enter blood stream to be utilized by other tissues, but acetoacetate will spontaneously decarboxylate, forming acetone • acetone cannot be utilized, it is expired by lungs • To protect against acetone formation, acetoacetate can be reduced to beta-hydroxybutarate: more stable form. • This reaction is readily reversible, so the two ketone bodies are interconverted easily • The NADH / NAD+ ratio is the key determinant for the relative amount of each species • Humans tend to favor β-hydroxybutyrate • Tissues importing β-hydroxybutarate get one additional NADH from the conversion to acetoacetate, in addition to the two acetyl CoA molecules from acetoacetate. 26 26 Objective L Oxidation of Ketone Bodies • succinyl CoA combines with the acetoacetate to form succinate and acetoacetyl CoA • acetoacetyl CoA combines with a new CoA resulting in the formation of two molecules of acetyl coA, which enter the TCA cycle • two moles acetyl coA enter TCA cycle should = 18 moles ATP produced • energy produced using ketone bodies derived from fatty acids is nearly the same as taking fatty acids all the way to CO2 and H2O. Advantages? • • • • liver only partially oxidizes FAs to ketone bodies Other tissues can use the ketone bodies as fuel brain can use ketone bodies during starvation, decreasing need for glucose decreased use of muscle protein aas as carbon source for glucose production 27 27 Objective M Metabolic Conditions that Favor Formation of Ketone Bodies • Stimulated when there is high blood levels of fatty acids • fasting, starvation or due to a high fat, low carbohydrate diet • High fatty acid oxidation activity leads to accumulation of acetyl CoA, high NADH and high ATP • glucagon / insulin ratio is high (due to low glucose) • liver is synthesizing glucose by gluconeogenesis pyruvate produced from lacate or alanine • pyruvate is converted to OAA, which is transported out of mito by aspartate / malate shuttle to make glucose in cytosol • high NADH helps drive OAA to malate • less OAA is available for conversion to citrate, so acetyl CoA accumulates • High acetyl CoA signals the formation of ketone bodies 28 28 Question Why are fatty acids a better source of fuel than glucose for tissues with high energy demands? a. Fatty acid oxidation can be used by more tissue types than glycolysis. b. The compartmentalization of fatty acid oxidation in mitochondria is more efficient for the electron transport chain and ATP synthase. c. A typical fatty acid produces about 50 ATP molecules, whereas a glucose molecule yields about 25 ATP molecules. d. A typical fatty acid yields about 100 ATP molecules whereas a glucose molecule yields only about 25 ATP molecules. 29 29 Question A patient has an enzyme imbalance, such that her cells have a chronically high level of malonyl CoA. Which of the following would be a likely consequence of this imbalance? a. Her acetylCoA levels would be vanishingly small. b. Her cells will make excess ketone bodies c. Fatty acid oxidation will be inhibited in her cells d. Fatty acid oxidation will be increased in her cells 30 30 Question A patient has a defect in his carnitine-palmatoyl transferase I enzyme that makes this enzyme inactive. Which of the following would be a likely consequence of this defect? A. B. C. D. His mitochondria will oxidize newly synthesized fatty acids His fatty acid oxidation will take place in the cell cytoplasm He will generate significantly less ketone bodies during fasting. His fatty acid oxidation will be upregulated to compensate. X 31 31 Thank You! 32 32