Lec34 F08 Handout

8/12/2019 Lec34 F08 Handout

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Bioc 460 - Dr. Miesfeld Fall 2008

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Figure 2.

Figure 1.Lecture 34 - Fatty acid oxidation

Key Concepts- Overview of lipid metabolism- Reactions of fatty acid oxidation- Energy yield from fatty acid oxidation

- Formation of ketone bodies

Overview of lipid metabolismCarbohydrate metabolism is but onecomponent of energy production andstorage. In fact, a much larger percentageof the total energy reserves in animals islipids in the form of fat deposits consistingof energy-rich fatty acids. As shown infigure 1, there are three basic sources offatty acids in animals that can be used for

energy conversion processes, 1) fatty acidspresent in triacylglycerols (figure 2)obtained from the diet, 2) fatty acids storedas triacylglycerols in adipose tissue thatare released by hydrolysis followinghormone stimulation (glucagon orepinephrine signaling), and 3) fatty acidssynthesized in the liver from excess carbohydrates and exported as triacylglycerols. Fatty acids indietary triacylglycerols are transported from the intestines to the rest of the body by largelipoprotein particles called chylomicrons . Hormone signaling releases fatty acids from adiposetissue that bind to an abundant transport protein in serum called albumin . Lastly, fatty acids

synthesized in the liver are carried through the body as triacylglycerols by very low densitylipoprotein (VLDL) particles. Palmitateis a C 16 saturated fatty acid that can becarried through the body as a protein-fatty acid complex.

Fat is stored in fat cells(adipocytes). Obesity, especiallychildhood obesity, can be due to bothmore fat storage per cell, and to alarger number of adipocytes (figure 2).In contrast, in normal healthy adults, the

onset of old age and reduced metabolicrates leads to weight gain resultingprimarily from storing more fat per cell(although adults can also add more fatcells if they become obese).


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Olestra

Pathway Questions for Fatty Acid Metabolism1. What purpose does fatty acid metabolism serve in animals? Fatty acid oxidation in mitochondria is responsible for providing energy to cells when glucoselevels are low. Triacylglycerols stored in adipose tissue of most humans can supply energy to thebody for ~3 months during starvation. Fatty acid synthesis reactions in the cytosol of liver and adipose cells convert excess acetyl CoA

that builds up in the mitochondrial matrix when glucose levels are high into fatty acids that can bestored or exported as triacylglycerols.

2. What are the net reactions of fatty acid degradationand synthesis for the C 18 fatty acid palmitate?

Fatty acid oxidation:Palmitate + 7 NAD + + 7 FAD + 8 CoA + 7 H 2O + ATP -->

8 acetyl CoA + 7 NADH + 7 FADH 2 + AMP + 2 P i + 7 H +

Fatty acid synthesis:

8 Acetyl CoA + 7 ATP + 14 NADPH + 14 H+

-->palmitate + 8 CoA + 7 ADP + 7 P i + 14 NADP + + 6 H 2O

3. What are the key enzymes in fatty acidmetabolism? Fatty acyl CoA synthetase enzyme catalyzing the"priming" reaction in fatty acid metabolism which convertsfree fatty acids in the cytosol into fatty acyl-CoA using theenergy available from ATP and PP i hydrolysis. Whenthe energy charge in the cell is low, the fatty acyl-CoA is used for fatty acid oxidation inside themitochondria, however, when the energy charge is high, the fatty acyl-CoA is used to synthesize

triacylglycerols or membrane lipids.Carnitine acyltransferase I - catalyzes the commitment step in fatty acid oxidation which linksfatty acyl-CoA molecules to the hydroxyl group of carnitine. The activity of carnitineacyltransferase I is inhibited by malonyl CoA, the product of the acetyl-CoA carboxylase reaction,which signals that glucose levels are high and fatty acid synthesis is favored.Acetyl CoA carboxylase - catalyzes the commitment step in fatty acid synthesis using a biotin-mediated reaction mechanism that carboxylates acetyl CoA to form the C 3 compound malonylCoA. The activity of acetyl CoA carboxylase is regulated by both reversible phosphorylation (theactive conformation is dephosphorylated) and allosteric mechanisms (citrate binding stimulatesactivity, palmitoyl-CoA inhibits activity).Fatty acid synthase - this large multi-functional enzyme is responsible for catalyzing a series of

reactions that sequentially adds C 2 units to a growing fatty acid chain covalently attached to theenzyme complex. The mechanism involves the linking malonyl-CoA to an acyl carrier protein,followed by a decarboxylation and condensation reaction that extends the hydrocarbon chain.

4. What are examples of fatty acid metabolism in real life? A variety of foods are prominently advertised as "non-fat," eventhough they can contain a high calorie count coming fromcarbohydrates. Eating too much of these high calorie non-fat foods(e.g., non-fat bagels) activates the fatty acid synthesis pathwayresulting in the conversion of acetyl CoA to fatty acids which are


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Figure 3.

Figure 4.

stored as triacylglycerols. Olestra is a fat substitute composed of a sucrose molecule with severalfatty acids attached.

Transport and storage of fatty acids and triacylglycerolsMuch of the triacylglycerol stored in adipose tissue originates from dietary lipids. Fats that enterthe small intestine from the stomach are insoluble and must be emulsified by bile acids such as

glycocholate which are secreted by the bile duct and function as detergents to promote theformation of micelles. Lipases are water soluble enzymes in the small intestine that hydrolyze theacyl ester bonds in triacylglycerols to liberate free fatty acids which then pass through themembrane on the lumenal side ofintestinal epithelial cells (figure 3 ).Pancreatic lipase cleaves the esterbond at the C-1 and C-3 carbons torelease two free fatty acids andmonoacylglyclerol, whereas, otherintestinal lipases cleave at the C-2carbon to generate glycerol and fatty

acid. The absorption and transport ofdietary triacylglycerols can be brokendown into five steps, 1) emulsificationof triacylglycerols by bile acids, 2)hydrolysis of fatty acids by intestinallipases, 3) resynthesis oftriacylglycerols inside intestinalepithelial cells, 4) packaging oftriacylglycerols into large lipoproteinparticles called chylomicrons , and 5)export of the chylomicrons to the

lymphatic system. Chylomicronstransport the triacylglycerols toadipose tissue for storage, and tomuscle cells for energy conversionprocesses.

Apolipoprotein C-II on thesurface of chylomicrons binds to andactivates lipoprotein lipase onendothelial cells which leads to therelease of fatty acids and glycerol(figure 4). Fatty acids diffuse into the

endothelial cells and then enter nearbyadipose and muscle cells where theyare stored or used for energyconversion pathways. The glycerolproduced by lipoprotein lipase returnsto the liver where it is converted todihydroxyacetone phosphate. Twoother important apolipoproteins onchylomicrons are apolipoprotein C-III


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Figure 5.

(apoC-III) and apolipoprotein B-48 (apoB-48).Dietary lipids are not the only source of triacylglycerols stored in adipocytes. The liver

synthesizes triacylglycerols fromfatty acids when glucose levels arehigh and the amount of acetyl CoAproduced exceeds the energy

requirements of the cell. Asshown in figure 5, glucoseprovides the necessary substratesfor triacylglycerol synthesis (acetylCoA for fatty acid synthesis andglycerol) using reactions in theglycolytic pathway and the citratecycle. In this metabolic scheme,glucose is converted to fructose-1,6-bisphosphate which is cleavedby aldolase to generate

dihydroxyacetone phosphate(DHAP) and glyceraldehyde-3-phosphate (GAP). The DHAP isused to make glycerol-3-phosphate, and the GAP isconverted to pyruvate which isthen oxidatively decarboxylated bythe mitochondrial enzymepyruvate dehydrogenase to formacetyl CoA. Citrate synthase , thefirst enzyme in the citrate cycle,

combines oxaloacetate and acetylCoA to generate citrate which isshuttled to the cytosol where it is cleaved by the enzyme citrate lyase to generate acetyl CoA andoxaloacetate. This process of shipping acetyl CoA out of the mitochondria using citrate andoxaloacetate is called the citrate shuttle and is described later. The cytosolic acetyl CoA isconverted to malonyl CoA by the enzyme acetyl CoA carboxylase which then serves as thebuilding block for fatty acid synthesis by fatty acid synthase . Lastly, fatty acids and glycerol arecombined to form triacylglycerols which are packaged into VLDL particles in the liver andtransported to the adipose tissue where they are stored in lipid droplets. This series of enzymaticreactions linking glycolysis, the citrate shuttle, and fatty acid synthesis, explains why eating toomany bagels or sucking on too many Jolly Ranchers , can result in an increase in total body fat

despite consumption of these non-fat, carbohydrate-rich, foods.The fatty acid oxidation pathway in mitochondriaThe degradation of fatty acids in animal cells requires enzymatic reactions that occur insidemitochondria as first described by Eugene Kennedy and Albert Lehninger in the late 1940s.Subsequent work showed that fatty acids need to be activated by coenzyme A on the cytosolicside of the outer mitochondrial membrane and then transported into the mitochondrial matrix by aspecific carrier system. Fatty acids stored in adipose cells are released into the blood in responseto hormone signaling by activation of cellular lipases which cleave stored triacylglycerides. The


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Figure 6.

Figure 7.

fatty acids provide a rich source ofenergy for tissues throughout thebody when glycogen stores havebeen depleted, especially duringendurance exercise and dieting.

Figure 6 shows the two step

fatty acyl-CoA reaction catalyzed bymedium chain fatty acyl CoAsynthetase. In the first step, thecarboxylate ion of the fatty acidattacks a phosphate in ATP to forman acyl-adenylate intermediate andrelease pyrophosphate (PP i) whichis quickly hydrolyzed by the enzymeinorganic pyrophosphatase toform 2 P i. In the second step of thefatty acyl CoA synthetase reaction,

the palmitoyl-adenylate intermediateis attacked by the thiol group of CoAto form the thioester palmitoyl-CoAproduct and release AMP. As with other reactions we have seen involving PP i, the rapid removalof PP i as a product of the fatty acyl-adenylation reaction serves to pull the reaction to the right,making it even more favorable. Remember to count this reaction as requiring 2 ATP since itrequires two rounds of nucleoside kinase (1 ATP each) to convert AMP back into ATP.

The fatty acyl-CoA products of the fatty acyl CoA synthetase reaction have two fates. If theenergy charge of the cell is low, then they will be imported into the mitochondrial matrix by thecarnitine transport cycle and degraded by the fatty acid oxidation reactions to yield acetyl CoA,FADH 2 and NADH. However, if the energy charge is high, and fatty acid synthesis is favored, then

mitochondrial import of fatty acyl-CoA is inhibited and the fatty acyl-CoA molecule is used insteadfor triacylglycerol or membrane lipid synthesis in the cytosol. Figure 7 illustrates the carnitinetransport cycle which involves the function of three proteins and a molecular tag called carnitine .In the first reaction, carnitine acyltransferase I , which is located in the outer mitochondrialmembrane, replaces the CoA moiety with carnitine to form fatty acyl carnitine which is translocated


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Figure 8.

Figure 9.

across the inner mitochondrial membrane. The carnitinetranslocating protein is an antiporter that exchanges afatty acyl carnitine molecule for a carnitine. Once inside themitochondrial matrix, fatty acyl carnitine is converted backto fatty acyl CoA in a reaction catalyzed by carnitineacyltransferase II releasing the carnitine so that it can be

shuttled back across the inner mitochondrial membrane.Once the electron-rich carbons of fatty acids aremoved into the mitochondrial matrix, their high energy redoxpotential is traded in for a substantial payout of ATP (figure8). This energy conversion process of fatty acid --> ATPinvolves oxidation of fatty acids by sequential degradationof C 2 units leading to the generation FADH 2, NADH, andacetyl CoA. The subsequent oxidation of these reactionproducts by the citrate cycle and oxidative phosphorylationgenerates large amounts of ATP. Figure 9 illustrates the -oxidation pathway used to degrade the C 16 fatty acid

palmitate. The ! -oxidation pathway is so named becausethe sequential C 2 cleavage reaction (thiolysis) occurs at the! carbon of the fatty acid, thereby releasing the C-1carboxyl carbon and " carbon as the acetate component ofacetyl CoA. In the first of four reactions, the enzyme acylCoA dehydrogenase catalyzes a dehydrogenation reaction(oxidation ) that introduces a trans C=C bond between the " and ! carbons of the fatty acyl-CoA molecule using amechanism that reduces an enzyme bound FAD to formFADH 2. Mitochondria actually contain three isozymes ofacyl CoA dehydrogenase which differ in their specificity forhydrocarbon chains of different lengths. These are referredto as long chain (C 12 to C 18 ), medium chain (C 4 to C 14 ) andshort chain (C 4 to C 8 ) acyl CoA dehydrogenases. Thesecond reaction in the ! oxidation pathway is a hydration step catalyzed by the enzyme enoyl CoA hydratase thatadds H 2O across the C=C bond to convert trans- # 2-enoyl-CoA to 3-L-hydroxyacyl-CoA. The third reaction is anotherdehydrogenation ( oxidation ) step in which the enzyme -hydroxyacyl-CoA dehydrogenase removes an electronpair from the substrate and donates it to NAD + to formNADH. Finally, coenzyme A is used in thiolysis reactioncatalyzed by the enzyme acyl CoA acetyltransferase (alsocalled thiolase ) that releases a molecule of acetyl CoA andin the process, results in the formation of an fatty acyl CoAproduct that is two carbons shorter than the startingsubstrate. These four reactions together convert palmitoyl-CoA (C 16 ) into myristoyl-CoA (C 14 ), and in the process,generate 1 FADH 2, 1 NADH and 1 acetyl CoA. Themyristoyl CoA product becomes the substrate for another


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Figure 10.

round of ! oxidation resulting in the production of one more molecule of FADH 2, NADH and acetylCoA.

The complete oxidation of palmitoyl-CoA (C 16 ) requires seven rounds of the ! oxidationpathway to convert one molecule of palmitoyl CoA into eight molecules of acetyl CoA in a netreaction that can be written as:

Palmitoyl-CoA + 7 CoA + 7 FAD + 7 NAD+ + 7 H 2O -->

8 acetyl CoA + 7 FADH 2 + 7 NADH + 7 H +

We are now ready to tally up the net ATP yield from cashing in the redox potential ofpalmitate using the ! oxidation pathway. As shown in figure 10, after seven rounds of ! oxidation,palmitoyl-CoA yields 8 acetyl CoA, 7NADH and 7 FADH 2. The oxidation ofacetyl CoA by the citrate cycle thengenerates 24 NADH, 8 FADH 2 and 8GTP (ATP). Finally, the combinedreactions of the electron transport

system and oxidative phosphorylationconverts these 31 NADH into ~77.5

ATP (31 x ~2.5 ATP), and the 15FADH 2 are converted into ~22.5 ATP(15 x ~1.5 ATP), to give a subtotal of100 ATP. After subtracting the 2 ATPrequired for fatty acyl CoA activation(AMP --> PP i), and adding the 8 ATPobtained from eight turns of the citratecycle, you receive a total payout of 106 ATP .

To appreciate the increased energy yield from fatty acid oxidation as compared to that of

glucose oxidation, consider that the oxidation of stearate in muscle cells, a fully saturated C 18 fattyacid, yields 120 ATP using the same bookkeeping methods for ATP energy exchange. In contrast,the complete oxidation of three molecules of glucose (3 x C 6 = C 18 ) generates only 90 ATP (or 96

ATP in liver cells). The increased energy yield of 33% (120/90 = 133%) is due to the increasednumber of electrons available in stearate for donation to the electron transport system (stearate isa saturated fatty acid and more highly reduced than glucose). When you take into account the factthat the fatty acids in triacylglycerols are hydrophobic and not as hydrated as glucose in glycogen,it is easy to see why evolution exploited the energy storage properties of lipids over carbohydrates.

Besides the payout of ATP that comes from fatty acid oxidation, another benefit is thegeneration of H 2O that occurs when O 2 is reduced by the finalreaction in the electron transport system, as well as, the formation of

H2O in the ATP synthesis reaction of oxidative phosphorylation asshown in the three reactions below:

2 NADH + 2 H + + O 2 --> 2 H 2O2 FADH 2 + O 2 --> 2 H 2O

ADP + PO 42- --> ATP + H 2O

The water production that accompanies fatty oxidation benefits animals that live in dry climateswhere liquid water is scarce, for example, the desert kangaroo rat and Arabian camel. Large


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Figure 12.

Figure 11.animals that hibernate over the winter, like the Alaskan brownbear, also take advantage of fatty acid oxidation in order toreplace H 2O that is lost by respiration. Fatty acid oxidation ofpalmitate yields 46 H 2O molecules generated by theoxidation of 31 NADH and 15 FADH 2 in the electron transportsystem, and 106 H 2O molecules formed by the synthesis of 106

ATP by the ATP synthase reaction. By subtracting the 16 H 2Omolecules required by the citrate cycle (2 H 2O for every acetylCoA oxidized), and the 7 H 2O molecules used during ! oxidation, the oxidation of 1 mole of palmitate yields 129 molesof H 2O (152 - 23 = 129). This works out to be an amazing ~9milliliters of H 2O that are generated from the complete oxidationof just 1 gram of palmitate.

Ketogenesis is a salvage pathway for acetyl CoAWhen carbohydrate sources are limited due to starvation, or when glucose homeostasis isdefective, as is the case in diabetes, ongoing ! oxidation in liver cell mitochondria results in the

build-up of excess acetyl CoA. This occurs because flux through the citrate cycle is diminisheddue to the depletion of oxaloacetate which issiphoned away from the citrate cycle to producepyruvate for gluconeogenesis. Ketogenesis is aprocess in liver cell mitochondria that takes theexcess acetyl CoA and converts it to acetoacetate and D- -hydroxybutyrate , two energy-richcompounds that are sometimes called " ketonebodies " for historical reasons (figure 11).

As shown in figure 12, three mitochondrialreactions are required to convert two acetyl CoA

molecules into acetoacetate which is then reduced toform D- ! -hydroxybutyrate. Acyl-CoAacetyltransferase (thiolase) is the same enzymethat releases one molecule of acetyl CoA in reaction4 of the ! oxidation pathway, however in this case,the reaction is driven toward condensation by thehigh concentration of acetyl CoA in the mitochondriaunder ketogenic conditions. In the next step, theenzyme HMG-CoA synthase adds another acetylCoA group to form the intermediate ! -hydroxy- ! -methylglutaryl-CoA, abbreviated as HMG-CoA , andthen the enzyme HMG-CoA lyase removes one ofthe original acetyl CoA groups to yield acetoacetate.

Acetoacetate and D- ! -hydroxybutyrate are exportedfrom the liver and used by other tissues such asskeletal and heart muscle to generate acetyl CoAfor energy conversion reactions (figure 13 ). Eventhe brain which prefers glucose as an energy source,can adapt to using ketone bodies as chemical energy


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Figure 14.

Figure 13.

during times of extreme starvation as described in lecture 40.While ketogenesis is an important survival mechanism that maintains high rates of fatty acid

oxidation when carbohydrates stores are depleted, it can also lead to pathological conditions ifacetoacetate and D- ! -hydroxybutyrate levels in the blood get too high. Acidosis is a conditionreferring to low blood pH which can occur when ketogenesis produces more acetoacetate and D- ! -hydroxybutyrate than what can be utilized by the peripheral tissues (these are both carboxylic

acids). In patients with undiagnosed diabetes, which is a metabolic form of carbohydrate"starvation," elevated concentrations of acetoacetate and D- ! -hydroxybutyrate in the blood andurine can be several orders of magnitude higher than normal causing nausea, vomiting andstomach pain. Moreover, these individuals also have high levels of acetone in their blood whichcan be detected on their breath as a fruity odor. Acetone is a spontaneous breakdown product ofacetoacetate (decarboxylation), or is formed by enzymatic cleavage of acetoacetate by theenzyme acetoacetate decarboxylase (figure 12).

The smell of acetone, along with the other debilitating symptoms, have been known totragically mislead law officers into thinking these severe cases of diabetes were the result ofexcess alcohol consumption. Ketogenesis is also a hallmark of fasting in which glycogen storesare depleted and energy is derived almost entirely from fatty acid degradation (figure 14). This is

also true of many low carbohydrate diets that include higherthan normal levels of fats as a substitute for bread, fruit andstarchy vegetables (rice, beans, potatoes). Although thereare numerous drawbacks to the extreme versions of theselow carbohydrate/high fat diets, there is metabolic logic to whya carbohydrate-deficient diet will result in a significantdecrease in body fat composition due to increased rates offatty acid oxidation and ketogenesis.

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Lec34 F08 Handout