BIO 361 Lecture 36: Fatty Acid Metabolism PDF

BIO 361 Final (Lectures 31-41) Lecture 36: ● ● ● ● Let’s review a bit Metabolism of dietary triglycerides We break it down into component fatty acids and monoglycerides in the small intestine, absorbed in the intestinal cells, converted to triglycerides, packaged into chylomicrons, shipped out in...

BIO 361 Final (Lectures 31-41) Lecture 36: ● ● ● ● Let’s review a bit Metabolism of dietary triglycerides We break it down into component fatty acids and monoglycerides in the small intestine, absorbed in the intestinal cells, converted to triglycerides, packaged into chylomicrons, shipped out into circulation, then stored in lipid droplets, and how the triglycerides in lipid droplets can be liberated and cleaved into their component fatty acids through a series of enzymes Today we’re going to focus on how the target tissues that take up fatty acids can convert them into energy ● ● We will discuss how fatty acids, in this case palmitate, will be converted into acetyl-CoA WIth each cycle, we will be cleaving two carbons from the fatty acid and converting it into an acetyl molecule of coA ● ● We’re now shifting gears away from blood and circulation and more into the muscle tissues We’ll focus on how fatty acids are imported into the mitochondria for fatty acid oxidation pathways ● ● ● ● ● ● Imagine you are a muscle cell and you need fatty acids for energy One way to get fatty acids is cleave triglycerides by lipid protein lipase and that would be taken up by the muscle tissue One other approach is during times of starvation or fasting, fatty acids can be released by adipose tissues into the bloodstream and taken by muscle cells These fatty acids have to be converted into acetyl-CoA What this means that a molecule of coenzyme A will be attached to the fatty acid molecule The enzymes that do this are called Acyl-CoA synthetase ● These enzymes are membrane enzymes that are cytoplasmic orientated and they will convert fatty acids into acyl-CoA by attaching a co-enzyme molecule to a fatty acid ● ● This is the mechanism We have a fatty acid with an acyl chain , and the enzyme COA synthetase requires a molecule of ATP to activate the fatty acid, which means to convert the molecule of fatty acid to acyl-CoA Fatty acid comes in and attacks the alpha-phosphate of the ATP, and the phosphodiester bond is broken Now the fatty acid is attached to the alpha phosphate and you have pyrophosphate that gets hydrolyzed Now an enzyme CoA, which has a sulfur on it, will attack the fatty acid carbon of the acyl adenylate intermediate Now the fatty acid gets transferred to a coenzyme A to yield our Acyl-CoA A byproduct is AMP What drives the reaction forward is the cleavage of the high energy bond between the beta and gamma phosphate During one cycle of this reaction we break down two high energy bonds ○ First we break down the bond between the alpha and beta phosphate when the fatty acid attacks, and then between the beta and gamma phosphate when the pyrophosphate is hydrolyzed ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● So in essence it’s thought we’re using 2 ATP equivalents because we’re breaking two high energy bonds We’re using two energy, two molecules of ATP to convert it to an acyl-CoA Now we have a problem The problem is the fatty acid attached to acyl-CoA is in the cytoplasm, but the enzymes that will convert the fatty acids via the beta oxidation pathway are actually in the matrix of the mitochondria Acyl-CoA isn’t permeable, so it can’t just diffuse from the cytosol into the matrix of the mitochondria Cells use a series of enzymes called Carnitine Palmitoyltransferase (CPTs) There’s two: CPT I and CPT II CPT I is on the outer mitochondrial membrane and faces the cytosol, CPT II is found on the inner mitochondrial matrix and faces the matrix So CPT I takes the acyl-CoA, and transfers the fatty acid group to a molecule of carnitine in the cytoplasm What this does is converts the acyl-CoA into acyl-carnitine The enzyme CoA is liberated and returns back to the cytosol Now the acyl-carnitine is permeable to the mitochondrial membrane ● ● ● ● ● ● There’s also a transporter that recognizes acyl-carnitine that will transport it to the matrix of the mitochondria So fatty acids are only transported into the mitochondria if they are attached to a molecule of carnitine, or acyl-carnitine Once it's in the mitochondria, CP II catalyzes the reverse reaction, where it transfers the fatty acid from the acyl-carnitine back to a coenzyme A molecule in the matrix, and we will re-generate an acyl-CoA in the matrix This is the only way we can transport a long chain fatty acid into the mitochondrial matrix They’re the primary fatty acids we consume in our diet To summarize again, we have an acyl-CoA in the cytosol, CPT I transfers the acyl group to a carnitine to generate acyl-carnitine, and then we transfer this using a special transporter into the matrix of the mitochondria, and then CPT II will catalyze the reverse reaction by transferring the fatty acid from an acyl-carnitine back onto an acyl-CoA, and the carnitine can be transported back to the cytosol to begin another cycle ● Now let’s discuss how the acyl-CoA, or fatty acid attached to an enzyme CoA, how that will be converted sequentially into acetyl-CoA and NADH and FADH2, and how those carbon bonds will be used for energy ● So beta oxidation or fatty acid oxidation will convert a acyl-CoA into acetyl-CoA ● We’re going to cleave two carbons at a time each cycle ● ● Four enzymes used in this process We’re going to generate an FADH2, an NADH, and an acetyl-CoA with each cycle ● The fatty acid will then go back and repeat this cycle until we generate acetyl-CoA ● ● ● First enzyme: Acyl-CoA dehydrogenase (AD) Beta oxidation takes place on the beta carbon This enzyme create an alpha-beta trans double bond between the alpha and beta double bond As we remove these two hydrogens, we end up generating an alpha-beta trans double bond The two electrons are transferred to FAD to generate FADH2, which is used for energy ● ● ● ● ● Second reaction, we have an enzyme called enoyl-Coa Hydratase (EH) We take the alpha-beta trans double bond and there will be added a water onto it EH adds a water ● ● ● ● The next enzyme is an HAD Enzyme creates a ketone on the beta carbon Hydroxyl is converted and electrons onto NAD and is converted to NADH Again, HAD oxidizes the bond and generates a ketone on the beta carbon, which generates NADH ● ● ● ● Fourth enzyme is KT KT generates an acetyl-CoA and Acyl-CoA two carbons shorter So the six carbon fatty acid is converted into the four carbon fatty acid, which is then re-used in the first step The longer the fatty acid, the longer the beta oxidation ● The whole point is that now the products of beta oxidation, FADH2, NADH, and acetyl-CoA can now enter the citric acid cycle via oxidative phosphorylation and can generate energy ● ● ● ● ● ● ● Let’s see how much energy is made from beta-oxidation Here we have palmitate, which is a 16 carbon fatty acid, so we have 7 rounds of beta oxidation Each cycle we generate an NADH, an FADH2, and an acetyl-CoA With 7 rounds of beta oxidation, and 7 FADH2 are generated, each FADH2 generating approximately 1.5 ATPs, end up with 10.5 ATPs Here we’re generating 8 acetyl-CoA, because the last round of beta oxidation we have 4 carbons, so we get two acetyl-CoA We get 108 ATP molecules We can generate a lot of energy using beta oxidation and fatty acids ● ● ● There are a lot of disorders of beta oxidation, and they affect that Acyl-CoA dehydrogenase (AH) and HAD These are the first and third enzymes A mutation in HAD leads to lethal cardiomyopathy for babies ○ The baby suckles on the mother’s milk which contains fatty acids, if the baby can’t undergo beta oxidation, the heart will never absorb the fatty acids, and the heart dies ● Screen for these disorders ● Now let’s switch gears and talk about the B-oxidation of the unsaturated fatty acids (Oils in diet) ● ● ● ● ● ● ● ● ● ● ● ● So our cells have evolved mechanisms to use fatty acids with double bonds for energy Cells adapted to double bonds found in fatty acids So we have the four enzymes, AD, EH, HAD, KT Imagine we had a fatty acid and it has two double bonds So we go through everything, we cleave the bond between the alpha and beta carbon, and we generate acetyl-CoA, and we have a fatty acid attached to acetyl-CoA Everything goes smoothly, but the problem lies where we have a fatty acid with a double bond on the beta and gamma position Enzyme called enoyl-CoA Isomerase will shift double bond on gamma position onto the alpha-beta position Isomerase converts beta-gamma double bond into alpha beta double bond, and this is the substrate for EH, and we can continue with beta-oxidation Now we’re good with double bond for EH, but there’s another problem Although it uses alpha-beta trans double bond containing substrates, it can’t do so if there’s a double bond at the 4-5 position Dienoyl-CoA reductase converts the double bond into the beta gamma position, but that’s again a problem But we have Enoyl-CoA isomerase, which takes hte bond and puts it back between alpha and beta carbon as trans double bond, and now that is a substrate for the EH enzyme ● ● ● ● ● ● ● ● ● ● ● ● ● So in summary, cells evolved mechanisms for the issue of EH inability to process the double bond in unsaturated fatty acids Two enzymes, enoyl-CoA isomerase and di-enoyl CoA reductase Cells have elaborate mechanisms to extract energy out of fatty acids no matter the structure Summary of Beta Oxidation Started in cytoplasm, taken up fatty acids Fatty acids converted to fatty acyl-CoA through the acyl-CoA synthetase enzymes With the help of CPT 1, acyl-CoA converted into acyl-carnitine, and transported into the matrix CPT 2 converts acyl-carnitine back into acyl-CoA Once in the matrix, the first enzyme, AD, generates an alpha-beta trans double bond → FADH2 generated EH hydrates the alpha-beta trans double bond HAD then converts the hydroxyl into a ketone, electrons transferred on NAD+ → NADH generated KT then cleaves the alpha-beta carbon and generates acetyl-CoA Then a new enzyme of coenzyme A will attack the beta carbon, generating a new molecule of acyl-CoA where the fatty acid is attached, and that fatty acid is two carbons shorter ● ● ● ● ● ● ● ● ● ● ● That’s great, but now we have to discuss a major physiological problem Let’s see we haven’t eaten in a few days (or even overnight) Glucagon levels rise, fatty acids are liberated from adipose tissue and shipped out to muscle tissue, and in muscle tissue they’re converted to acetyl-CoA The problem is that fatty acids, if there in the bloodstream, cannot be taken up by the brain effectively Only a very small amount of fatty acids can be taken up by the brain This presents a major problem Now let’s look at the capillary in the brain The brain has a blood-brain barrier ○ This means that cells line the blood vessels and form tight junctions, and this means many molecules cannot enter the brain from the bloodstream. These molecules are not permeable, including fatty acids The blood-brain barrier helps to act as a protective barrier against harmful compounds (bacterial toxins, etc.) However, what this means is in times of starvation, adipose tissue generates fatty acids, these fatty acids cannot be used by the brain for energy There must be alternate mechanisms that supply the brain with energy during times of fasting/starvation ● ● ● ● These set of molecules are called ketone bodies Ketone bodies are four carbon molecules that will be generated form acetyl-CoA from the liver, and only in the liver mitochondria Only liver mitochondria can generate ketone bodies During times of starvation, as we havel lipolysis and adipose tissues release fatty acids, the liver will take up a significant portion of these fatty acids in the bloodstream and convert them into acetyl-CoA, and this build up of acetyl-CoA will be funneled into the biosynthesis of these ketone bodies by the liver, which will then be released into circulation, taken up by the brain, and then converted back into acetyl-CoA and used for energy ● ● ● ● ● ● ● ● ● ● ● ● ● There are two ketone bodies Acetoacetate and Hydroxybutyrate They’re both four carbon units Result in condensation from two acetyl-CoA Each acetyl-CoA has two carbons, when fused together, have four carbon units On beta carbon we have a ketone, similarity between beta oxidation pathway and acetoacetate Only synthesized in the liver and released by the liver Used for energy during times of fasting and times of starvation Used generally in people who have low blood sugar Ketogenic diet is based on the generation of these ketones If you have low blood sugar, the body will be generating and releasing large amounts of fatty acids, and converting those fatty acids into ketones What’s important about ketones is that they can diffuse into the brain Ketones, unlike fatty acids, can be taken up by the brain and permeable in blood-brain barrier ● ● ● Produced from acetyl-CoA in the liver and only in matrix of mitochondria Although we make these under normal conditions, we’re always making them, since if we have acetyl-CoA in the mitochondria, some of that acetyl-CoA will be funneled into generation of ketones Ketone levels rise if: ○ We have a high fat diet → high amounts of fat, liver is generating large amounts of acetyl-CoA ○ Fasted for long time → Adipose tissue releases fatty acids, liver takes fatty acids and generates acetyl-CoA and convert acetyl-CoA into ketones ○ Untreated diabetics → Insulin is not effective or efficient (depending on type 1 or type 2), and so glucose is not being catalyzed and being taken up by the cells, so in essence your cells are starving ■ Blood sugar highly elevated, but cells are not seeing that sugar and so they cannot see that sugar because it is not taken up by the cell effectively, so fatty acids are mobilized and will be converted into ketones in the liver ● This is a way to diagnose a diabetic that is still used today ● ● ● ● ● ● ● ● One important concept to also discuss is why the brain is a special organ Brain compromises only 2% of bodyweight mass wise, but it uses a large part of glucose throughout the day, about 20% of glucose If you run out of glucose, you will die Brain (i.e neurons) is heavily reliant on glucose metabolism ○ Cannot survive in the absence of glucose Highly reduced glucose levels also lead to brain seizures and abnormalities, demonstrating how reliant neurons are on glucose and cannot survive without them During times of starvation, our bodies have very fine mechanisms to ensure glucose levels in blood remain steady and do not drop too much ○ If they drop too much, brain cannot take up glucose, and you would die During times of starvation and fasting, muscle tissues and livers will shift their metabolism from glucose to fatty acids Fatty acids released by adipose tissues, and muscle tissue and liver will take up fatty acid and shift metabolism to fatty acids and away from glucose ○ This happens to ensure glucose is preserved for the brain ● So now let’s discuss the biosynthesis of ketones from acetyl-CoA ● ● ● ● ● ● So again, liver shifts metabolism away from sugar towards fats (glucose to fatty acids) Fats are taken up from circulation from adipose tissues which releases them, liver converts them to acetyl-CoA ○ This is a source of energy since we have beta oxidation and NADH, FADH2, etc. However, when there’s a buildup of NADH or acetyl-CoA, these feedback inhibit pyruvate dehydrogenase What this means is, as we develop a higher energy state in the liver from fatty acid metabolism, this can feedback and inhibit the breakdown of glucose ○ So we’re reducing glucose glycolysis/metabolism in the liver and shifting to fatty acids Acetyl-CoA stimulates pyruvate carboxylase to stimulate and increase gluconeogenesis As we metabolize fatty acids in liver, we inhibit glycolysis, stimulate gluconeogenesis, and now that excess sugar can be released into bloodstream and sent to the brain for energy since brain is highly dependent on glucose ● ● ● ● ● ● ● ● ● Ketogenesis pathway and mechanism We have two acetyl-CoA here Steps involved in generation in ketones have resemble to beta oxidation, except in reverse KT is first enzyme and conjugates from one acetyl-CoA to another acetyl-CoA to generate four carbon acetoacetyl-CoA, and we have a beta carbon ketone (similar to beta oxidation) Now we have another acetyl-CoA that attacks a carbon on acetoacetyl-CoA, and we get HMG-CoA Now what’s important is in the liver mitochondria we have a liver specific enzyme called HMG-CoA lyase ○ Liver specific ○ Expressed in liver mitochondrial matrix Only in the matrix of mitochondria we have this enzyme that will cleave the HMG-CoA and generate a four carbon ketone body called Acetoacetate, and an acetyl-CoA Two acetyl-CoA conjugated → acetoacetyl-CoA → acetoacetyl-CoA + acetyl-CoA → HMG-acetyl-CoA, and then HMG-CoA lyase → acetoacetate + acetyl-CoA This is the process of ketogenesis ● These ketones will be released into the bloodstream and be taken up by other tissues, principally the brain but also the heart and even the muscle, and these ketones will be converted back to acetyl-CoA for energy ● Again, the point here is ketone bodies are converted back to acetyl-CoA in the target tissue ● Advantage of ketone bodies is that it provides a way in essence to transport acetyl-CoA to tissues ○ This is because acetyl-CoA is not permeable to membranes So you can take two carbons from acetyl-CoA and transport them to a target tissues through the formation of ketone Ketone bodies also helps to lower the demand by the brain for glucose during starvation conditions since we can adequately supply the brain with energy so neurons won’t die It also helps to reduce the amount of protein that has to be broken down for gluconeogenesis because the liver is shifting metabolism away from glucose towards fat so it can save glucose for brain, and this reduces the amount of muscle broken down for energy ● ● ● ● ● ● ● ● ● So there are complications with ketones as with anything Now if we have a patient with diabetic ketoacidosis or a patient with high levels of ketones (indicating untreated diabetic) or a severe alcoholic, both patients will have elevated levels of ketones in blood The problem with this is ketones are acids, and they will reduce the blood pH If the blood is too acidic, you will suffer complications and eventually die Diabetic Ketoacidosis ○ Tissues unable to take up and utilize glucose ○ Excess ketone body production ○ Acetone can be meleed in blood ○ Blood pH drops ○ Common to see in diabetic patients Alcoholic ketoacidosis ○ Found in alcoholics ○ High levels of NADH, depletion of oxaloacetate required for gluconeogenesis ○ Elevated ketone body production ○ Blood pH drops ○ This is more rare to see in alcoholics, because this requires prolonged alcoholism without eating anything ● ● ● ● ● ● ● ● ● ● ● ● ● ● Ketogenic diet Ketogenic diet is a fad, and the idea is to consume low levels of sugar and high levels of fat, and shift metabolism from glucose metabolism towards fat metabolism into ketones Has pros and cons Now ketogenic diet could be therapeutic An example where the ketogenic can help is if there isa glut1 deficiency Glut1 is a transporter expressed in the blood-brain barrier ○ This is how glucose enters the brain and can be used for energy There are people who have mutations in glut 1 Mutations in glut1 reduce glucose uptake by brain If you have two defective copies of the gene, then you wouldn’t even be born because you would die right away However if someone is born with one defective copy, they will survive and be born Now the problem with those with glut1 deficiency is they will have seizures The reason for these seizures is because the amount of glucose getting into the brain is highly reduced, because there is one defective copy of the transporter Now because the brain is highly reliant on glucose, these children don’t get the glucose in the brain, and so they have seizures Children with Glut1 deficiency (heterozygous) frequently develop seizures that are poorly controlled by anti-epileptic medications ● ● ● ● Now what’s interesting and good is that if you take these children and you put them on a ketogenic diet/high fat diet, the amount of ketones produced are sufficient to supplement the reduced levels of glucose in these patients that can’t get to the brain So if the patient isn’t getting 50% of the glucose you need, but ketones can provide that excess energy because ketones will be converted to acetyl-CoA in the brain, then that is sufficient to reduce seizures in patients and restore relatively normal function of neurons This is how the ketogenic diet can be therapeutic Fatty acid oxidation is an incredibly important way to generate energy, and during times of starvation, liver and muscle will shift towards fatty acid oxidation because adipose tissues releases large amounts of fatty acids, and in doing so, the products of oxidation (NADH/FADH2/acetyl-CoA) will inhibit glycolysis and save that extra sugar that can be used for the brain, and it can stimulate gluconeogenesis by the liver to “stretch that glucose currency” for as many days as possible so you can survive for as long as you can if you are starving