10-14-11 Fatty acid oxidation Acetyl-CoA, the energy-rich molecules composed of coenzyme A and the two carbon acetyl group, plays a preeminent role in the metabolism of lipids Precursor in fatty acid biosynthesis and isoprenoid biosynthesis Fatty acids are an important and efficient energy source for many cells All of lipid metabolism focuses on Aceytl CoA. It is either needed in order to generate energy or it is in excess becaues it has been produced through glycolysis or perhaps lipid digestion. It is a focal point or decision making point of what to do with all those carbons. The two carbons from Acetyl CoA they can either be burned/used for synthesis or stored away as fat. Acetyl-CoA is a decision point. This slide is a review slide. Acetyl-CoA can either be oxidized or used for biosynthesis. Included here are fats (storage form of lipids), phospholipids (in the membrane), as well as isoprenoid synthesis (pretty much acetyl-CoA units that have been condensed together)

Digestion and Absorption of Triacylglycerols in the Small Intestine After triacylglycerol molecules are ingested, they are mixed with bile salts and digested by pancreatic lipases into fatty acids and monoacylglycerol Both are transported into the cell from the intestinal lumen Eventually reconverted to triacylglycerol, converted to chylomicrons and secreted into the lymph This is a review slide: Dietary fat is emulsified to break it down to smaller and smaller droplets that have more and more exposure to the aqueous environment where of course the enzymes are. Bile salts are used to do this, to create a fine emulsion that is “tackled” by pancreatic lipase which hydrolyzes the ester linkages to two of the fatty acids and leaves the third fatty acid attached to the glycerol. So you have monoacylglycerol. Free fatty acids that can be taken up by the enterocytes in the intestines. As soon as that happens the fat is reconstituted. Notice the fatty acids that are taken up are bound to CoA, so they become CoA (the acyl group) and they are hooked through a thioester linkage to CoA. So you have acyl CoA plus monacylglycerol and basically this high energy thioester bond serves as a donor of the acyl groups to reconstitue the triacylglycerol. The fats that are generated in the intestinal epithelium will coagulate. Because they are no longer emulsified and no longer charged into lipid droplets that are formed in structures called chylomicrons which have proteins that help organize the structure but basically are highly organized fat droplet that can be deposited into the lymph. So at the basal surface of the intestinal epithelial cell, these are secreted into a lymph duct and that way the fat from digestion get transported to the target cells. Digestion and Absorption of Triacylglycerols in the Small Intestine

Most of the triacylglycerol content in circulating Most of the triacylglycerol content in circulating chylomicrons is removed by skeletal muscle and adipose tissue cells (adipocytes) Lipoprotein lipase breaks down these triacylglycerol molecules into fatty acids and glycerol Fatty acids are taken up by the cells and glycerol is transported to the liver When lipoprotein lipase has removed 90% of the triacylglycerols in chylomicrons, the chylomicron remnants are removed from the blood by the liver The target cells for energy consumption are generally skeletal muscle and the adipocytes will pick it up and store it. So muscle cells that need energy will consume the fatty acids and the adipocytes will pick up the rest of it and store it for a later time. Lipoprotein lipases at the surface of these cells that require fatty acids break the triacylglycerols back down to fatty acids and glycerols. So they break all three ester linkages and the free fatty acids are taken up by the cell. Glycerol of course is glucogenic and can go back to the liver and be used for gluconeogenesis or even be oxidized if energy is needed. So the chylomicrons are basically these cargo trucks for the triacylglycerols to travel around the body and they gradually get unloaded at site of metabolic activity and then ultimately at the adipocytes until most of the lipid is gone from the chylomicron and at that point it is taken up by the liver and then destroyed and recycled.

Depending on the animal’s current metabolic needs, fatty acids may be: Converted to triacylglycerols Degraded to generate energy Used for membrane synthesis Here are the alternative states of the lipids: Fatty acids themselves can be converted back to triacylglycerols and stored Degraded to Acetyl-CoA and then to CO2 for ATP synthesis They can be turned into phospholipids for membrane synthesis

Acyl-CoA molecules are fatty acid esters of CoASH In triacylglycerol (TAG) synthesis (lipogenesis), glycerol-3-phosphate or dihydroxyacetone phosphate reacts sequentially with three molecules of acyl-CoA Acyl-CoA molecules are fatty acid esters of CoASH Triacylglycerol synthesis (will talk about it later next week but he wanted to talk briefly about it here because it is one of the major outcomes of dietary fatty acids) So glycerol phosphate or dihydroxyacetone phosphate acts a skeleton on to which to deposit Acyl groups that are bound to CoA. So recognize that acyl-CoA is the high energy precursor that drives the formation of ester linkage in lipids and you form these fatty acid esters.

Triacylglycerol Synthesis So this pathway shows briefly (again will repeat this later but this is how triacylglycerol type of molecules are synthesized in an adipocyte for example) Glucose will come into the cell and be converted to dihydroxyacetone phosphate (3 carbons, so that’s just glycolysis) and then that will be reduced to glycerol-3-phosphate instead of being oxidized as glyceraldehyde-3-phosphate and then either DHAP or glycerol-3-phosphate can act as skeletons to which deposit acyl-CoA fatty acid groups. So we see glycerol phosphate being acylated to acyl-CoA, the CoA enzyme is released an ester linkage is created with the fatty acid and you now have a monoacylglycerol. Alternatively with DHAP it doesn’t get reduced first and the DHAP is used to receive a fatty acid group from acyl-CoA and these enzymes in both cases are called transferases because they transfer the acyl group and you end up with (in this case) a monoacylglycerol that is phosphorylated. Notice you already have the phosphate group on there. This is reduced then in the next step. So this is just a different sequence of events whether you start with DHAP or glycerol phosphate and that gets you to lysophosphotidic acid which can then be furher acylated to create phosphotidic acid which is a precursor for phospholipids. Phosphotidic acid just needs a head group to become a phospholipid or it can be acylated a third time to create acyl-glycerol for storage.

Lipolysis in adipocytes triggered by glucagon or epinephrine When energy reserves are low, the body’s fat stores are mobilized in a process termed lipolysis Lipolysis occurs during fasting, during vigorous exercise and in response to stress Fatty acid binding proteins are responsible for transporting the fatty acids into target organelles Lipolysis in adipocytes are triggered by the need for energy. So we see the exactly the same situation we saw for low blood glucose concentrations. Glucagon will be synthesized to increase the rate of glucose production (in this case) fatty acid production. There are receptors that act very much like the glucagon pathway that is they increase the level of cAMP, cAMP turns on Protein kinase A, and Protein kinase A phosphorylates triacylglycerol lipase to create the active form of the enzyme so it is exactly parallel to glycogen phosphorylase. Tracylglycerol lipase will then break down triacylglycerols to diacylglycerols essentially mobilizing the fat molecule, putting a charge on it making it accessible to other lipases that remove the second two acyl groups releasing glycerol and free fatty acids that are transported the adipocyte and into the plasma. Once these fatty acids get into the plasma they bind to proteins (they are amphipathic and quite hydrophobic) in the blood, fatty acid transport proteins which carry them around until they reach a zone where there is very low fatty acid concentrations, the fatty acids will come off there and be metabolized at the site of active metabolism. So this is going to occur during fasting, when blood glucose levels are low there is a need for energy (fats will suffice) or vigorous exercise or even stress and it puts the body into high gear and needing more energy will lead to glucagon and or epinephrine and break down of triacylglycerols.

They travel to target cells (tissues that are in need of energy will pick up the fatty acid and oxidize them) Glycerol that is left over will go to the liver where it will be recycled to form glucose through gluconeogenesis or alternatively oxidized to pyruvate to get a little ATP out.

Fatty acids are linked to Coenzyme A before oxidation An acyl-adenylate intermediate is formed acyl CoA synthetase Glucagon is doing two things here: it is activating glycogen breakdown and activating lipid breakdown. Which would it do first? What is the priority here? Remember that glycogen breakdown is an immediate response that it occurs inside metabolically active cells (particularly muscles) it immediately leads to glucose that can be oxidized in generating ATP without any other conversion or transport. When you breakdown lipids in an adipocyte all you do is get the fatty acid into the blood then they need to travel to the tissues (this can take several minutes to hours for the fuel to find its target by that time chances are glucose needs have been met by glycogen metabolism and glucagon will no longer be present and you will stop hydrolyzing lipids. So the lipid response to glucagon is much slower and persistent and will only really have an effect with glycogen stores are depleted and there is not an immediate replenishment of glucose and glycogen. Now lets take a look at the beta oxidation pathway (the way fatty acids are oxidized). It is called beta oxidation because it breaks the fatty acid at the beta bond. The first step is the formation of acyl-CoA from the fatty acid so you need to the activate the fatty acid by binding it to CoA and forming a thioester bond in the acyl-CoA. This is a very high energy bond that can be used both in the synthesis and degradation. In order to make it you need to hydrolyze ATP to AMP and this both releases the energy of two phosphate bonds as well as creates an unstable intermediate called acyl adenolate. In other words, the acyl group initially gets attached to ATP and the pyrophosphate gets released. Fatty acid plus ATP creates an acyl adenolate intermediate where the acyl group is attached to AMP and the two phosphates from ATP are released. That pyrophosphate needs to be cleaved to inorganic phosphate in order to drive the reaction forward. So the phosphatases will do that and that’s why the two phosphates on the ATP were used initially so you can drive this reaction forward by hydrolizing pyrophosphate. Same thing we saw before. As I said we are looking at this reaction total at the top by breaking it down into two steps. Once the acyl adenolate intermediate is formed there is a high energy bond there that can be used to drive the synthesis of the thioester bond to connect acyl group to CoA to get acyl-CoA and the end product is the AMP plus two inorganic phosphates (which are not shown) So we now have the fatty acid attached to CoA which makes it activated and ready for degradation for beta oxidation.

1. Acyl-CoA converted into acylcarnitine Carnitine is used to transfer acyl groups into the mitochondrion where most b-oxidation occurs 1. Acyl-CoA converted into acylcarnitine 2. Translocase transfers acylcarnitine into matrix 3. Acyl-CoA regenerated 4. Carnitine recycled to intermembrane space Beta oxidation of fatty acids occurs in the mitochondrial matrix. Everything so far we have seen was out in the cytoplasm (the fatty acids were taken in the cytoplasm, the amino adenolate intermediate was created and acyl-CoA was created there) In order to oxidize this stuff you need to get it into the matrix of the mitochondria and that’s done using a deveice called the carnitine shuttle. Carnitine is connected to the acyl group in order to make it transportable. So we see translocase enzyme taking acyl-CoA and moving the acyl group on to carnitine (is an amino acid and a amino alcohol, commonly referred to as an amino alcohol because its alcohol group is the business end of things). There is an ester linkage now with the carnitine molecule so the acyl group is covalently bond to the carnitine and the carnitine is basically a handle where this translocase can grab and transport it across the innermitochondrial membrane. In the picture on the bottom right: Here we see carnitine combined with acyl-CoA. To release free CoA and now we have the acyl group bound to carnitine instead of CoA. The acyl carnitine gets transloacted into the matrix from the cytoplasmic side inot the matrix, then another carnitine trasnferase (carnitine acyltransferase II, the one inside the mitochondrial matrix) transfers the acyl group back to CoA, so you now have acyl CoA in the mitochondrial matrix you regenerate the carnitine, it gets transported by the translocase outside to the cytoplasmic side where carnitine acyltransferase I can recharge it.

Fatty Acid Transport into the Mitochondrion This is a picture that puts it more into perspective. Remember the mitochondrion has an outer membrane and an inner membrane and ultimately the matrix so there is this intermembrane space. The synthetase that forms the fatty acyl CoA is located in the outer membrane so it takes from the cytosol these free fatty acids and combines them with CoA. Notice it’s a synthetase enzyme because the reaction requires ATP and now you have acyl-CoA (the adenolate intermediate). Acyl-CoA is transferred to carnitine by carnitine acyltransferase I which happens to be also located in the outer mitochondrial membrane and you have acylcarnitine generated in the innermembrane space which is suitable substrate for the carnitine transloacator and it moves acylcarnitine into the matrix where CoA is used to pick up the acyl group and carnitine is regenerated by carnitine acyltransferase II (second transfer of the acyl group) the carnitine transfers back out and you end up with a net input of acyl-CoA into the matrix where this fatty acid can now be burned. Fatty Acid Transport into the Mitochondrion

Fatty Acid Degradation Most fatty acids are degraded by the sequential removal of two carbon fragments from the carboxyl end as acetyl-CoA; this is known as b-oxidation Once formed, acetyl-CoA and other short chain products are used for energy production or as metabolic intermediates So lets look at the beta oxidation process now. We got our acyl-CoA groups in the matrix and we need to start breakding down that fatty acid chain which can be 20 or so carbons long. This is the process of beta oxidation because the cleavage occurs between the alpha and beta bonds (remember those are the carbons that are closest to the carboxyl group on the fatty acid so its kinda chewed from the inside out. As a result you get acetyl-CoA formed and this stuff can be used for energy production or for synthesis of cholesterol, isoprenoids, etc..

The b-oxidation of saturated fatty acids is a series of The b-oxidation of saturated fatty acids is a series of four reactions that constitute one cycle of b-oxidation During each later cycle, a two carbon fragment is removed (b-oxidation spiral) Acetyl-CoA molecules produced are used in the citric acid cycle (or for isoprenoid synthesis) acyl CoA dehydrogenase enoyl CoA hydratase L-3-hydroxyacyl CoA dehydrogenase Here is beta oxidation. Basically is three steps: oxidation, acyl-CoA is oxidized first. FAD is the oxidizing agent and is reduced to FADH2 and the enzyme is acyl CoA dehydrogenase converting the saturated bond in acyl-CoA. You manuver that bond so you can break it. You take the saturated bond and put it into a enoyl bond so you have enoyl-CoA where you have this double bond next door to the thioester linkage. They enoyl-CoA is hydrated (water is added across the bond, 2nd step) by enoyl CoA hydratase and now you have a hydroxyl group and a saturated carbon next door in other words hydroxyacyl CoA. That hydroxy group makes its hydroxyacyl. So you got now a one oxidation step (hydration step) this is followed by a second oxidation step in which NAD+ is used as the oxidant to oxidize the hydroxyacyl CoA to ketoacyl CoA in other words there is a keto group instead of an alcohol group. NADH is produced and 3-ketoacyl CoA can be cleaved by thiolase to create a free acetyl CoA and a shorter Acyl CoA (a fatty acid that is attached to CoA that is two carbons shorter) Notice where the acetyl CoA came from it came from the original CoA and the new CoA has come in and has done the beta oxidation and now carries the rest of the acyl chain on it. The old CoA becomes the Acetyl CoA. ketothiolase

This chart lists the enzyme that are important (which all of them are important to understand the process) First we form acyl-CoA that is ATP requiring process forms the acyl adenolate intermediate Then the acyl carnitine needs to be formed in order to transport into the mitochondrial matrix. In the matrix the acyl-CoA is regenerated and oxidized first by FAD Then it is hydrated as water across the double bond resulted from the oxidation. Then is is oxidized a seoncd time using NAD to create NADH and you end up with 3-ketoacyl CoA the beta ketohiolase breaks that into the acetyl CoA and the shorter acyl CoA.

The Complete Oxidation of a Fatty Acid The aerobic oxidation of a fatty acid generates a large number of ATP molecules The yield of ATP from the oxidation of palmitoyl- CoA is 108 ATP (106 net) It is possible to take a fatty acid of ~20 carbons long and do the math in other words for every 2 carbons that comes off you generate a NADH and a FADH2 you also use an ATP to activate the fatty acid in the first place to make acyl CoA Oxidation of a single fatty acid chain you get a lot of ATP out (108 ATP) since you have to use ATP to activate it you get 106 ATP net. In other words fatty acids are a very dense source of ATP

hydroxymethylglutaryl * mitochondrial matrix Ketone Body Formation * D-3-hydroxybutyrate dehydrogenase 3-ketothiolase HMG CoA cleavage enzyme hydroxymethylglutaryl CoA synthase * mitochondrial matrix Another consequence of using lipids for fuel is the formation of ketone bodies. Ketone bodies arise when there is a lot of acetyl-CoA concentrations are high. Why would acetyl-CoA concentrations get high and it is basically when acetyl-CoA is coming from the lipid oxidation rather than the glucose oxidation. When substrates are being taken out of the TCA cycle for anabolic purposes. When you deplete that carbon in the TCA cycle you no longer have oxaloacetate to condense acetyl-CoA to run the cycle so acetyl-CoA will pile up. This is primarily what's happening when lipids are oxidized when glucose is being oxidized the TCA cycle can be largely replenished through gluconeogenesis but glucose is not oxidized there is no supply of pyruvate for gluconeogenesis so under these conditions lipids are oxidized and energy supplies are met but because the TCA cycle isnt running at full speed because it has been depleted acetyl-CoA concentrations pile up. So when acetyl-CoA is in excess, ketothiolase combines these two together to make what is called acetoacetyl CoA which is then converted to HMG CoA by HMG CoA synthase. What it is doing here it is adding a third acetyl group in the form of CoA, combining three acetyl groups together and that’s the hydroxy-methyl-glutaryl. This is all happening in the mitochondrial matrix. Acetyl-CoA is coming from two sources pyruvate dehydrogenase and from acyl CoA oxidation/fatty acid oxidation both of which are in the mitochondrial matrix. In the mitochondrial matrix where the TCA cycle has been compromised because a lot of the carbon in the cycle has been drawn off for other purposes either gluconeogenesis (primarily) or protein synthesis which reduces the amount of oxaloacetate so the acetyl-CoA can’t be condensed and used up so it piles up. So as the acetyl-CoA supply piles up it drives these first two reactions the formation of HMG CoA (which is basically 3 acetyl-CoA units condensed together) The thioester bond can be cleaved to liberate acetoacetate as well as one of the orginal acetyl-CoA molecules. So this is our first ketone body (acetoacetic acid) it’s a small metabolite that can diffuse out and go into the blood and it’s a ketone body. They are common in individual that cannot metabolize glucose properly (diabetics) because they can’t oxidize glucose to create acetyl-CoA int hat way so they oxidize fats instead. Acetoacetate is just one of the ketone bodies and can be further metabolized to 3-hydroxy-butyrate by reduction. So if the mitochondrion has a lot of NADH present then it can drive this back to 3-hydroxy-butyrate or the acetoacetate can spontaneously decarboxylate to form acetone (third ketone body) these 3 substance are in very high concentration during the process we call ketogenesis or ketoses (is the condition in the patient) Ketone Bodies - excess acetyl-CoA is converted to ketone bodies (ketogenesis) Forms acetoacetate, b-hydroxybutyrate and acetone

In a case of diabetics they will have a lot of blood glucose but one form of diabetes is the inability to take up glucose because insulin is absent to tell the cells to take it up or glucose transporters are compromised so there is a lot of blood glucose but it is not available for metabolism. So instead in order to supply the energy needs of the cell, fatty acids are oxidized the problem is that the liver (TCA cycle) is being depleted of its intermediates for gluconeogenesis because the liver is in the business of maintaining blood glucose and also protein synthesis. So if the glucose can’t be oxidized the fatty acids will instead and there is no oxaloacetate to take up all that acetyl-CoA. So acetyl-CoA builds up and ketone bodies are produced. Ketone bodies are acids that go inot the blood, pH drops you get acidosis eventually you get coma and death.

Under certain conditions ketone bodies are the preferred fuel Under certain conditions ketone bodies are the preferred fuel. Under starvation conditions of course glucose is not available and the TCA cycle is being tapped very heavily for gluconeogenesis because the blood glucose needs to be maintained in order to sustain the brain and minimal activity in the tissues so blood glucose drops really quickly in these conditions and there is can only be maintained at a level that gluconeogenesis can provide so that leads to the formation of ketone bodies because fats are being oxidized to acetyl-CoA and acetyl-CoA can’t be oxidized via the TCA cycle instead it combines to form acetoacetate which gets secreted by the liver which becomes fuel. And ketone bodies can actually feed the brain after glucose levels have depleted to a certain level. The brain prefers glucose as an energy source however at some point in starvation (week or so) it will switch to ketone bodies to obtain energy that it needs because glucose is limiting. So during starvation ketone bodies allow the fats that couldn’t be oxidized in the tissues be converted to a substance that can be used by the brain.