Lipid Metabolism - Biochemistry PDF

INTRODUCTION (slides 1-10) Lipids are a heterogeneous group of water-insoluble (hydrophobic) organic molecules. Because of their insolubility in aqueous solutions, body lipids are generally compartmentalized, as in the case of membrane-associated lipids or droplets of triacylglycerol (TG) in adipocy...

INTRODUCTION (slides 1-10) Lipids are a heterogeneous group of water-insoluble (hydrophobic) organic molecules. Because of their insolubility in aqueous solutions, body lipids are generally compartmentalized, as in the case of membrane-associated lipids or droplets of triacylglycerol (TG) in adipocytes, or transported in blood in association with protein, as in lipoprotein particles or on albumin. Lipids are a major source of energy for the body, and they also provide the hydrophobic barrier that permits partitioning of the aqueous contents of cells and subcellular structures. Deficiencies or imbalances of lipid metabolism can lead to some of the major clinical problems physicians encounter, such as atherosclerosis, diabetes, and obesity. In this chapter, we discuss the most important pathways of lipid metabolism grouped according to whether they are active under starvation or in a well-fed situation. Fasting stage (glucagon dominates): 1. Degradation of triacylglycerols 2. Degradation of fatty acids 3. Synthesis of ketone bodies Well-fed stage (insulin dominates): 4. Digestion, absorption, lipoprotein formation and circulation 5. Synthesis of fatty acids 6. Elongation of fatty acids 7. Fatty acid desaturation 8. Synthesis of triacylglycerols 1. DEGRADATION OF TRIACYLGLYCEROLS (slides 11-15) Mono-, di-, and triacylglycerols consist of one, two, or three molecules of fatty acid esterified to a molecule of glycerol. Fatty acids are esterified through their carboxyl groups, resulting in a loss of negative charge and the formation of neutral fat. Fatty acids are stored in white adipose tissue (WAT), as neutral triacylglycerol (TG), serving as the body’s major fuel storage reserve. TGs provide concentrated stores of metabolic energy because they are highly reduced. Because TGs are not soluble in water and cannot form stable micelles by themselves, they accumulate within white adipocytes to form large oily droplets that are nearly anhydrous. Specific proteins, like perilipin, cover lipid droplets, separating them from the cytoplasm. The mobilization of stored fat requires the hydrolytic release of free fatty acids (FFAs) and glycerol from their TAG form. This process of lipolysis is achieved by lipases. In humans, there are 3 lipases present that degrade the TGs of white adipocytes. Adipose TriacylGlycerol Lipase (ATGL) and Hormone-Sensitive Lipase (HSL) can cleave off the fatty acid from the first position forming diacylglycerol. From diacylglycerol, only HSL can produce monoacylglycerol. The last fatty acid in the third position is removed by MonoaclyGlycerol Lipase (MGL). 1. Glucagon binds to the receptor. The receptor activates a trimeric G-protein. 2. The G-protein activates adenylate cyclase, which produces cAMP. 3. The cAMP activates protein kinase A (PKA), which phosphorylates and acitvates HSL. 1  4. PKA also phosphorylates perilipin. CGI-58 dissociates from the phosphorylated perilipin. (CGI-58: Comparative Gene Identification-58) 5. CGI-58 binds to ATGL attached to the surface of the lipid droplet and activates it 6. ATGL cleaves off the first fatty acid from TGs. 7. Phosphorylated perilipin dissociates from the surface of the lipid droplet, and activated HSL gets access to TGs cleaving off the first and the second fatty acids from TGs. 8. MGL starts to cleave off the third fatty acid from TGs. 9. Free fatty acids are released to the blood where albumin binds them and transports them to the organs that need energy. Glycerol is also released into the blood and transported to the liver, where it can be used for ketone body synthesis or gluconeogenesis. ATGL has direct access to the TGs because it is localized on the surface of the lipid droplet. After activation by CGI-58, it starts to degrade TGs immediately, hence, it catalyzes the rate- limiting step. Activation of HSL needs more time but after activation, it does most of the task. MGL is not regulated, when it has an available substrate, it starts to cleave it. In addition to glucagon, other important hormones can activate lipolysis as well. Among them, there are Epinephrine, Norepinephrine, Adrenocorticotropic hormone (ACTH), Growth Hormone (GH), Atrial Natriuretic Factor (ANF), and cortisol. 2. UTILIZATION OF FATTY ACIDS FOR ENERGY PRODUCTION (slides 16-33) Fatty acids in the circulation are taken up by cells and used for energy production, in mitochondria, in a process integrated with energy generation from other sources. Fatty acids are broken down to acetyl-CoA in the mitochondria, with the production of NADH and FADH2. These three products are then used in the mitochondrial matrix for energy generation via the tricarboxylic acid cycle and oxidative phosphorylation. The steps of fatty acid degradation: I. Uptake and activation of fatty acids. II. Transport of fatty acids into the mitochondria. III. Systematic degradation of fatty acids: β-oxidation I. UPTAKE AND ACTIVATION OF FATTY ACIDS (slides 18-19) An FA must be converted to its activated form (bound to CoA through a thioester link) before it can participate in metabolic processes such as β-oxidation. This reaction is catalyzed by a family of fatty acyl-CoA synthetases using 2 high-energy bonds of ATP. Uptake of albumin-transported FA mobilized from adipose tissue may occur in three ways in the starving cells. 2  - Facilitated diffusion by the tranpsorter CD36: The fatty acid (FA) binds to Fatty Acid Binding Protein (FABP), which transports it to the Acyl-CoA Synthase (ACSL) present on mitochondria to form Acyl-CoA (FA-CoA). - Free diffusion through the plasma membrane. - Fatty Acid Transporter protein (FATP) binding ACSL. In the last two mechanisms, Acyl-CoA is formed immediately at the inner leaflet of the plasma membrane. Acyl-CoA will bind to Acyl-CoA Binding Protein (ACBP), and be transported in the cytosol. II. TRANSPORT OF ACYL GROUPS INTO MITOCHONDRIA (slide 20) Short-Chain Fatty Acids (SCFA, Carbon number < 5) and Medium-Chain Fatty Acids (MCFA, Carbon number < 12) can be transported into mitochondria by a simple passive diffusion. However, Long-Chain Fatty Acids (LCFA: Carbon number 12 and > 12) need carnitine- dependent transport. The acyl-group is transferred from CoA to carnitine by carnitine palmitoyltransferase I (CPT I) on the outer mitochondrial membrane. Acyl carnitine and free carnitine are then exchanged across the inner mitochondrial membrane by carnitine-acylcarnitine translocase. Finally, the fatty acyl group is transferred back to CoA by carnitine palmitoyltransferase II (CPT II) on the matrix side of the inner mitochondrial membrane. Humans can form carnitine, however, it is generated from two essential amino acids, lysine, and methionine. III. β-OXIDATION (slides 22-33) β-oxidation is a series of four reactions catalyzed by 4 different enzymes that act on a fatty acyl-CoA to produce an acetyl-CoA and a new acyl-CoA that is two carbon atoms shorter than the initial substrate: 1. Oxidation 2. Hydration 3. Oxidation 4. Cleavage Once a fatty acyl-CoA is formed at the inner surface of the inner mitochondrial membrane, it can be oxidized by acyl-CoA dehydrogenase, a flavoprotein that uses FAD as the electron acceptor (Reaction 1). The product is an enoyl-CoA with a trans-double bond between the C2 and C3 atoms and enzyme-bound FADH2. As in the tricarboxylic acid cycle, the FADH2 transfers its electrons to enzymes of oxidative phosphorylation, regenerating FAD. The second step in β-oxidation is the hydration of the trans-double bond to a β-hydroxyacyl- CoA, which is oxidized to a β-ketoacyl-CoA intermediate, with the generation of NADH in the third step. The final step is the cleavage of the chain by ketothiolase, generating acetyl-CoA and a fatty acyl-CoA that has been shortened by two carbon atoms. This shortened acyl-CoA 3 is ready for the next round of the steps mentioned before starting with acyl-CoA dehydrogenase. In most tissues, the acetyl-CoA will be used by the tricarboxylic acid cycle and the FADH2 and NADH will be reoxidized by the oxidative phosphorylation pathway with the production of ATP. Fatty acids with an odd number of carbon atoms are also oxidized by the β-oxidation pathway. However, in their case, the products of the final cleavage by thiolase are acetyl-CoA and propionyl-CoA. Propionyl-CoA is converted to succinyl-CoA, which is oxidized or converted to glucose through oxaloacetate (gluconeogenesis). Formation of succinyl-CoA from propionyl- CoA requires three mitochondrial enzymes and two vitamins: B7 and B12. First, propionyl-CoA is carboxylated by propionyl-CoA carboxylase forming D-methylmalonyl- CoA. This product undergoes epimerisation to form L-methylmalonyl-CoA. 3. SYNTHESIS OF KETONE BODIES (slides 34-42) I. KETOGENESIS OCCURS WHEN THERE IS A HIGH RATE OF FATTY ACID OXIDATION IN THE LIVER (slides 35-36) Under metabolic conditions associated with a high rate of fatty acid oxidation, the liver produces considerable quantities of water-soluble acetoacetate and β-hydroxybutyrate and releases them to blood for transportation. In the blood, acetoacetate continually undergoes spontaneous decarboxylation to yield acetone. These three substances are collectively known as the ketone bodies. The enzymes responsible for ketone body formation (ketogenesis) are associated mainly with the mitochondria. Acetoacetyl-CoA is formed when two acetyl-CoA molecules produced via fatty acid breakdown condense to form acetoacetyl-CoA by a reversal of the thiolase reaction. Two acetyl-CoA molecules produced via fatty acid breakdown condense to form acetoacetyl- CoA by reversing the thiolase reaction. The rate-limiting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase condenses acetoacetyl-CoA with another molecule of acetyl-CoA to form HMG-CoA. HMG-CoA lyase then causes acetyl-CoA to be released from the HMG-CoA, producing free acetoacetate. Acetoacetate can be reduced toβ-hydroxybutyrate by the mitochondrial enzyme β-hydroxybutyrate dehydrogenase, using NADH as the electron donor. The reaction is reversible under the control of the mitochondrial NADH/NAD ratio. As this ratio is high during fatty acid oxidation, β-hydroxybutyrate synthesis is favored. II. USE OF KETONE BODIES BY THE PERIPHERAL TISSUES: KETOLYSIS (slides 37-38) Ketone bodies are important sources of energy for the peripheral tissues because they (1) are soluble in aqueous solution and, therefore, do not need to be incorporated into lipoproteins or carried by albumin; 4 (2) are produced in the liver during periods when the amount of acetyl-CoA present exceeds the oxidative capacity of the liver; (3) are used in proportion to their concentration in the blood by extrahepatic tissues, such as skeletal and cardiac muscle, the intestinal mucosa, and the renal cortex. Even the brain can use ketone bodies to meet its energy needs when blood levels rise sufficiently. Thus, ketone bodies spare glucose, which is particularly important during prolonged fasting. Extrahepatic tissues utilize acetoacetate and β-hydroxybutyrate as respiratory substrates. β- Hydroxybutyrate is oxidized to acetoacetate by β-hydroxybutyrate dehydrogenase, producing NADH. Afterward, succinyl-CoA:acetoacetatyl-CoA transferase (thiophorase) provides acetoacetate with a CoA molecule from succinyl CoA. This reaction is reversible, but the product, acetoacetyl CoA, is actively removed by its cleavage to two acetyl CoA by thiolase. This facilitates the reaction forward. Extrahepatic tissues, like the brain, can efficiently oxidize acetoacetate and β-hydroxybutyrate in this way, but cells that don't have mitochondria, like red blood cells, can't. In contrast, although the liver actively produces ketone bodies, it lacks thiophorase and, therefore, cannot use them as fuel. Acetone, a waste product, is volatile and can be excreted via the lungs. III. KETOGENESIS IS REGULATED IN THREE CRUCIAL STEPS (slides 39-41) 1. Ketosis does not occur in vivo unless there is an increase in the level of circulating FFAs arising from lipolysis of triacylglycerol in adipose tissue. FFAs are the precursors of ketone bodies in the liver. Thus, the factors regulating the mobilization of FFA from adipose tissue are important in controlling ketogenesis. 2. After uptake by the liver, FFAs are either oxidized to CO2 or ketone bodies or esterified to triacylglycerol and phospholipid. There is the regulation of entry of fatty acids into the oxidative pathway by carnitine palmitoyltransferase-I (CPT-I), and the remainder of the fatty acid taken up is esterified. Malonyl-CoA, the initial intermediate in fatty acid biosynthesis is a potent inhibitor of CPT-I. CPT-I activity is low in the fed state, leading to depression of fatty acid oxidation, and high in starvation, allowing fatty acid oxidation to increase. In the fed state, therefore, FFAs enter the liver cell in low concentrations and are nearly all esterified to acylglycerols and transported out of the liver in very-low-density lipoprotein (VLDL). However, as the concentration of FFA increases with the onset of starvation, acetyl-CoA carboxylase is inhibited, and malonyl-CoA decreases, releasing the inhibition of CPT-I and allowing more acyl- CoA to be β-oxidized. 3. In turn, the acetyl-CoA formed in β-oxidation is oxidized in the citric acid cycle, or it enters the pathway of ketogenesis via acetoacetyl-CoA to form ketone bodies. A fall in the concentration of oxaloacetate, particularly within the mitochondria can impair the ability of the citric acid cycle to metabolize acetyl-CoA and divert fatty acid oxidation toward ketogenesis. Such a fall may occur because of an increase in the [NADH]/[NAD+]ratio caused when increased β-oxidation alters the equilibrium between oxaloacetate and malate so that the concentration of oxaloacetate is decreased. Malate is transported out of mitochondria 5 into the cytosol for gluconeogenesis elevated due to low blood glucose levels. The activation by acetyl-CoA of pyruvate carboxylase, which catalyzes the conversion of pyruvate to oxaloacetate, partially alleviates this problem, but in conditions such as starvation and untreated diabetes mellitus, ketone bodies are produced. (Note: During starvation, pyruvate can be formed from amino acids, not from glucose.) The decreased availability of OAA for condensation with acetyl CoA results in the increased use of acetyl CoA for ketone body synthesis. The partitioning of acetyl-CoA between the ketogenic pathway and the pathway of oxidation to CO2 is regulated by the energy charge of the liver. The total free energy captured in ATP which results from the β-oxidation of FFA remains constant and is sufficient for the hepatocyte. As serum levels rise, a large proportion of the acetyl-CoA produced from their breakdown is converted to ketone bodies and less is oxidized via the citric acid cycle to CO2. Thus, ketogenesis may be regarded as a mechanism that allows the liver to oxidize increasing quantities of fatty acids within the constraints of a tightly coupled system of oxidative phosphorylation. IV. KETOACIDOSIS RESULTS FROM PROLONGED KETONE BODY FORMATION (slide 42) Higher than normal quantities of ketone bodies present in the blood or urine constitute ketonemia (hyperketonemia) or ketonuria, respectively. The overall condition is called ketoacidosis. An exaggeration of this general pattern of metabolism produces the pathologic states found in diabetes mellitus type I. Ketoacidosis may also be seen in cases of excessive ethanol consumption. Nonpathologic forms of increased ketone body synthesis are also found under conditions of high-fat feeding and after severe exercise in the postabsorptive state. Acetoacetic and 3-hydroxybutyric acids are purely strong acids normally buffered in blood or other tissues. However, their continual excretion in quantity progressively depletes the alkali reserve, causing ketoacidosis. Each ketone body loses a proton [H+] as it circulates in the blood, which lowers the pH. Also, in uncontrolled diabetes mellitus type-1, urinary loss of glucose and ketone bodies results in dehydration. Therefore, the increased number of H+ circulating in a decreased volume of plasma can cause a severe acidosis known as diabetic ketoacidosis (DKA). A frequent symptom of DKA is a fruity odor on the breath, resulting from increased production of acetone. To balance the pH in the blood, “Kussmaul breathing” can be observed, which is deep, labored breathing to exhale more CO2. The exchange of H+ and K+ between the plasma/tissue fluid and cells leads to increased K+ urination which may cause arrhythmia, cardiac arrest, fatigue, and vomiting. DKA may result in unconsciousness from the combination of a severely increased blood sugar level, dehydration, shock, and exhaustion. This condition may be fatal in uncontrolled diabetes mellitus, the patient even may fall into a coma. If it is proved that unconsciousness is caused by uncontrolled diabetes mellitus type-1, the immediate intervention needs the addition of insulin, potassium replacement, and intravenous fluid. 6 4. DIGESTION, ABSORPTION, LIPOPROTEIN FORMATION AND CIRCULATION (slides 44-57) SEE MORE DETAILS IN LIPID METABOLISM II Water-insoluble lipids are packed together with water-soluble proteins (apoproteins) and transported in lipoprotein particles in the hydrophilic phase of blood plasma and other extracellular fluids. These lipoprotein particles make the plasma opalic and unsuitable for most of the laboratory tests. Lipoproteins are complex particles with a central core containing mainly hydrophobic cholesterol esters and triacylglycerol surrounded by a single surface layer of amphipathic phospholipid and cholesterol molecules. These are oriented so that their polar groups face outward to the aqueous medium, as in the cell membrane. The protein moiety of a lipoprotein is known as an apolipoprotein or apoprotein, constituting nearly 70% of some HDL and as little as 1% of chylomicrons. Plasma lipoproteins can be divided into different classes based on size, lipid composition, and apolipoproteins. Since fat is less dense than water, the density of a lipoprotein decreases as the protein-lipid ratio increases. Five major groups of lipoproteins have been identified that are important physiologically and in clinical diagnosis: (1) chylomicrons, derived from intestinal absorption of triacylglycerol and other lipids; (2) very-low-density lipoproteins (VLDL), derived from the liver for the export of triacylglycerol band cholesterol; (3) intermediate-density lipoprotein (IDL) formed in the circulation from VLDL (4) low-density lipoproteins (LDL), formed from IDL in the circulation, and responsible for the transport of cholesterol in humans and representing a final stage in the catabolism of VLDL; (5) high-density lipoproteins, (HDL), involved in reverse cholesterol transport. Chylomicron, remnant Chylomicron, VLDL, and IDL are too large to exit the circulation. Hence, the fat content of Chylomicron, VLDL, and IDL can be utilized only using lipoprotein lipase (LpL) anchored to the surface of endothelial cells and cleaving out fatty acids from TGs. The TG content gradually decreases converting Chylomicron to remnant Chylomicron, VLDL to IDL, and IDL to LDL in the circulation. LDL and HDL are small enough to penetrate through the walls of capillaries. Tissue cells can uptake them using specific cell surface receptors. However, only hepatocytes can take up Remnant Chylomicron and IDL directly from the Disse space using specific cell surface receptors. One or more apolipoproteins are present in each lipoprotein. They are usually abbreviated as apo followed by the letters A, B, C, etc. Some apolipoproteins are integral and cannot be removed (eg, apo B), whereas others are bound to the surface and are free to transfer to other lipoproteins (eg, apos C and E). The major apolipoproteins of HDL are apo As. The main apolipoprotein of LDL is apo B (B-100), which is found also in VLDL. Chylomicrons contain a truncated form of apo B-100, termed apoB-48 as it constitutes 48% of the full-sized molecule. Apo B-48 is synthesized in the intestine, while B-100 is synthesized in the liver. To produce Apo B-48, a stop signal is introduced into the mRNA transcript for Apo B-100 by an RNA editing 7 enzyme. Apos C and Apo E, found in VLDL, HDL, chylomicrons, and chylomicron remnants, are also on the surface of the particles. Apolipoproteins carry out several roles: (1) they can be part of the structure of the lipoprotein, for example, apo B; (2) they are enzyme cofactors, for example, C-II for lipoprotein lipase, A for lecithin:cholesterol acyltransferase (LCAT) (3) they act as ligands for interaction with lipoprotein receptors in tissues, for example, apo B- 100 for the LDL receptor, apo E for the LDL-receptor–related protein (LRP), which recognizes remnant chylomicron, and apo A for the HDL receptor (SR-B1). Circulation of lipoproteins involves: - EXOGENOUS PATHWAY transporting absorbed lipids TO THE LIVER from the intestine - ENDOGENOUS PATHWAY transporting lipids FROM THE LIVER to extrahepatic tissues and cholesterol back to the liver I. EXOGENOUS PATHWAY The particle released by the intestinal mucosal cell is called a nascent chylomicron. When it reaches the plasma, the particle is rapidly modified, and lipoprotein lipase (LPL) starts to degrade its TG content mainly for storage in adipose tissue and energy generation in the muscle. The remnant chylomicron transfers the lipids to the liver, where it is taken up using LRP receptors. II. ENDOGENOUS PATHWAY The liver converts and sorts the lipids originating from the exogenous pathway. The liver also produces triacylglycerols from glucose and introduces them into VLDL. Note: The liver does not store lipids. Similarly to chylomicron, when it reaches the plasma, the particle is rapidly modified, and lipoprotein lipase (LPL) starts to degrade its TG content mainly for storage in adipose tissue and energy generation in the muscle. Loosing some of the TG content, the composition and density of the particle change, and VLDL is converted first to IDL and then LDL in the circulation. LDL containing mainly cholesterol and cholesterol ester is small enough to leave the circulation, and the tissue cells can take it up directly using their LDL receptor. The discoidal nascent HDL is produced by the liver. It collects excess cholesterol directly from cells pumped out by ABCA1. Lecithin cholesterol acyltransferase (LCAT) enzyme present in the body fluid produces cholesterol ester from cholesterol. The water-insoluble cholesterol ester is embedded in the core of the HDL altering its shape of HDL. The spherical HDL transports cholesterol back to the liver safely. HDL also delivers a high amount of cholesterol to endocrine glands for steroid hormone synthesis. 8 5. SYNTHESIS OF FATTY ACIDS IN THE CYTOSOL (slides 58-78) I. TRANSPORT OF ACETYL GROUPS FROM MITOCHONDRIA TO THE CYTOSOL IN THE FORM OF CITRATE (slides 59-61) Glucose is the major precursor for fatty acid synthesis mainly occurring in the liver. At high energy supply, the activity of the Citric Acid Cycle is inhibited by ATP and NADH. The acetyl groups generated from glucose through pyruvate or from ketogenic amino acids cannot be degraded but will be used for fatty acid synthesis. In this case, acetly-CoA formed in mitochondria should be transported to the cytosol, where fatty acid synthesis occurs. However, there is no transporter for acetyl-CoA in the mitochondrial membranes. The citrate cleavage pathway overcomes this problem. Citrate, formed by citrate synthase from acetyl group and oxaloacetate in the tricarboxylic acid cycle, is transported across the mitochondrial inner membrane via the tricarboxylate transporter. The citrate is then cleaved in the cytosol by ATP citrate lyase (also called citrate cleavage enzyme) to form acetyl-CoA and oxaloacetate. This reaction is not a reversal of citrate synthase, since it requires the hydrolysis of ATP. The appearance of citric acid in the cytosol indicates a high energy level of the cell and acts as an allosteric activator of acetyl-CoA carboxylase, the rate-limiting enzyme in lipogenesis (see in the next paragraph). The oxaloacetate generated by citrate cleavage in the cytosol is not readily transported back into the mitochondria. It is instead reduced to malate by the cytosolic isoform of malate dehydrogenase. The malate then undergoes oxidative decarboxylation to pyruvate catalyzed by malic enzyme. The pyruvate enters the mitochondria for further metabolism. The removal of citrate from the mitochondria must be accompanied by its replacement (anaplerosis) to maintain tricarboxylic acid cycle flux. This is achieved by the conversion of pyruvate to oxaloacetate by pyruvate carboxylase, the major anaplerotic enzyme in mitochondria. II. THE RATE-LIMITING STEP OF THE SYNTHESIS: FORMATION OF MALONYL-CoA FROM ACETYL-CoA BY ACETYL-CoA CARBOXYLASE (slides 61-67) Synthesis of fatty acids from acetyl-CoA requires the activated intermediate, malonyl CoA, made by carboxylation of acetyl-CoA by acetyl-CoA carboxylase. The reaction needs biotin, ATP, and bicarbonate as the source of CO2. This reaction is similar to the carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase. Acetyl-CoA carboxylase catalyzes the rate-limiting step in fatty acid synthesis. The mammalian enzyme is activated by citrate or isocitrate. This represents feed-forward activation of fatty acid synthesis because citrate is exported from the mitochondria to generate cytosolic acetyl- CoA for fatty acid synthesis. Acetyl-CoA carboxylase is also inhibited by long-chain acyl-CoAs, resulting in feedback inhibition of fatty acid synthesis by the end products of the pathway. Phosphorylation of acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase A (PKA) or AMP-dependent protein kinase (AMPK) inhibits this enzyme. Glucagon or epinephrine activates PKA through signal transduction, hence, activation of PKA provides information to the cell about the low energy supply in the whole body. However, activation of AMPK gives information about the low energy level within the individual cell. When it is low, ATP 9 concentration decreases (it is converted to ADP), and AMP concentration increases, due to the activity of adenylate kinase producing ATP from ADP molecules: ADP+ADP=ATP+AMP. Insulin signaling activates phosphoprotein phosphatase resulting in activation of acetyl-CoA carboxylase. IMPORTANT CONNECTION TO THE REGULATION OF FATTY ACID DEGRADATION (slides 66-67). Both fatty acid synthesis and degradation must not be active at the same time. The rate of fatty acid oxidation in mitochondria is controlled by regulating the entry of substrate into this organelle. The key enzyme is carnitine palmitoyltransferase I (CPT I), which synthesizes acyl-carnitine from cytosolic acyl-CoA. In the liver, acetyl-CoA carboxylase is activated in the fed state, because cyclic-AMP-dependent phosphorylation is low, and the enzyme is activated by citrate. The resulting high concentration of malonyl-CoA blocks fatty acid oxidation by inhibiting CPT I. Conversely, in the fasted state, the activity of acetyl-CoA carboxylase in the liver is low because the enzyme is phosphorylated, and cytosolic citrate is also decreased. Hence, malonyl-CoA is not produced, so CPT1 remains active and the transport of fatty acids into mitochondria is not inhibited. In this case, the cell can degrade fatty acids to acetyl-CoAs to produce energy (ATP) and ketone bodies. III. FATTY ACID SYNTHASE IS A MULTIFUNCTIONAL PROTEIN WITH DIMERIC STRUCTURE (slides 68-78) The synthesis of fatty acids occurs in the cytosol using acetyl-CoA and malonyl-CoA. The saturated C16 fatty acid, palmitic acid, is synthesized by the fatty acid synthase (FAS), and many other fatty acids are derived from it. FAS is composed of two identical subunits each of which is a multienzyme polypeptide that contains all of the necessary catalytic activities. The two subunits are oriented in opposite directions under each other and cooperate to form two palmitic acid molecules simultaneously. The level of FAS synthase is controlled by the rate of its synthesis and degradation. Insulin increases the rate of fatty acid synthesis by stimulating transcription of the FAS gene, thereby increasing enzyme levels. Palmitic acids are synthesized by sequential addition of two-carbon units to the activated carboxyl end of a growing chain by FAS. The chemical steps of the synthesis are the reverse of fatty acid degradation occurring in the opposite sequence: 1. Condensation, 2. Reduction, 3. Dehydration, 4. Reduction. In the beginning, an acetyl group from CoA is transferred to the sulfhydryl group of a cysteine residue of the condensing enzyme (CE) by acetyltransferase activity (AT). Next, the malonyl group from CoA is transferred to the sulfhydryl group containing moiety on acyl carrier protein (ACP) of the other subunit, opposite the CE, by the malonyl transferase (MT). 1. Condensation: Then the CE links the acetyl chain to C2 of the malonyl group, with the loss of CO2. This is the same CO2 that was added by acetyl-CoA carboxylase, so the carbon atoms in the palmitate are derived entirely from acetyl-CoA. The acyl product is β-ketoacyl-ACP. 10  2. Reduction: β-ketoacyl-ACP is reduced by β-ketoacyl-ACP reductase (KR) to β- hydroxyacyl-ACP using NADPH. 3. Dehydration: β-hydroxyacyl-ACP dehydratase (DH) eliminates a water molecule and forms β-enoyl-ACP having a double bond. 4. Reduction: The double bond of β-enoyl-ACP is saturated by β-enoyl-ACP reductase (ER) using another NADPH molecule. The domains containing KR, DH, and ER activities are in the same subunits next to ACP, and ACP, like a robotic arm, serves the growing chain for these enzyme activities. After that, the condensing enzyme (CE) takes the four carbon-containing acyl group from the ACP. ACP is free again and can bind the next incoming malonyl group transferred by the malonyl transferase (MT), and the cycle is repeated six more times. After completing the last cycle, the condensing enzyme cannot take the C16 acyl group from ACP, instead, thioesterase cleaves off it forming palmitic acid. Note that at this stage, the sulfhydryl groups of ACP and condensing enzyme (CE) are free again, so other cycles of palmitic acid synthesis can begin. The released product is converted to palmitoyl-CoA, preparing it for modification or incorporation into complex lipids. The synthesis of one palmitate requires 14 NADPH. The pentose phosphate pathway is a major supplier of the NADPH. Two NADPH are produced for each molecule of glucose 6-phosphate that enters this pathway. The cytosolic conversion of malate to pyruvate, in which malate is oxidized and decarboxylated by cytosolic malic enzyme, also produces cytosolic NADPH for fatty acid synthase. 6. ELONGATION OF FATTY ACIDS MAY OCCUR IN ENDOPLASMIC RETICULUM AND MITOCHONDRIA (slides 79-80) Although palmitate, a 16-carbon, fully saturated fatty acid, is the end product of FAS activity, it can be further elongated by the addition of two-carbon units to the carboxylate end primarily in the smooth endoplasmic reticulum (SER). Elongation requires a system of separate enzymes rather than a multifunctional enzyme, but the chemical steps are the same as the steps carried out by FAS. Malonyl-CoA is the two-carbon donor (CO2 is released), and NADPH supplies the electrons. The brain has additional elongation capabilities, allowing it to produce the very–long-chain fatty acids (over 22 carbons) that are required for the synthesis of brain lipids. Fatty acyl-CoA substrates shorter than 16 carbons can be elongated in mitochondria by the addition of two-carbon units. The process is essentially a reversal of beta-oxidation, except that one NADPH and one NADH are required (beta-oxidation yields two NADH) and no CO2 is formed. The two-carbon donor is acetyl-CoA produced in mitochondria mainly by pyruvate dehydrogenase (PDH). 11 7. DESATURATION OF FATTY ACIDS MAY OCCUR IN CARBON POSITIONS 4, 5, 6, AND 9 (slides 81-83) The synthesis of unsaturated fatty acids is important for regulating the fluidity of triacylglycerols and membrane phospholipids. It is also required for the synthesis of cholesterol esters in the liver and waxy secretions in the skin, which preferentially use newly Synthesized fatty acids, rather than dietary ones. Fatty acid desaturation occurs in the smooth endoplasmic reticulum (SER), and the reactions and enzymes that introduce cis double bonds are significantly different from the acyl-CoA dehydrogenases of mitochondrial β-oxidation. The desaturase is a “mixed-function oxidase” as it oxidases two substrates at the same time: acyl-CoA and the Cytb5 reductase. The three components of the whole system are the desaturase enzyme, cytochrome b5, and NADPH- cytochrome b5 reductase. First, electrons are transferred from NADPH to the FAD moiety of NADPH-cytochrome b5 reductase. The heme Fe3+ of cytochrome b5 is then reduced to the Fe2+ state. The electron transfer to the nonheme Fe3+ of the desaturase subsequently converts it into the Fe2+ state, which enables the enzyme to interact with O2 and the saturated fatty acyl-CoA substrate. A double bond is formed and two molecules of H2O are released. Two electrons come from NADPH and two from the single bond of the fatty acyl substrate. The driving force of the reaction is the formation of water molecules from respiratory oxygen activated by the transferred electrons which needs 4 hydrogen ions, 2 from the surrounding solution and 2 from the carbons forming the saturated bond. Insulin increases gene transcription and thus the levels of desaturases in the liver, whereas dietary polyunsaturated fatty acids have the opposite effect. Humans have the enzymes to produce double bonds at carbon atoms C-4, C-5, C-6, and C-9, but not beyond. Hence, mammals cannot synthesize linoleate and linolenate which are essential for us and must be supplied in the diet. Linoleate and linolenate furnished by the diet are the starting points for synthesizing other unsaturated fatty acids through elongation and desaturation processes. The insulin signaling pathway activates gene expression of desaturases. 8. SYNTHESIS AND STORAGE OF TRIACYLGLYREOLS (TGs, slides 84-95) LIPOPROTEIN LIPASE AND CD36 TRANSPORTER ARE REQUIRED FOR THE UPTAKE OF FATTY ACIDS BY ADIPOCYTES TO FORM TGs (slide 85) Under well-fed situations, fatty acids for TG synthesis originate from the circulating lipoproteins, mainly from chylomicron, VLDL, and IDL. Insulin signaling activates the formation of lipoprotein lipase (LpL) on the surface of the endothelial cells in the adipose tissue. When heparan sulfate-anchored LpL is present, Apolipoprotein C of the lipoprotein particles activates LpL, which hydrolyzes TGs allowing free fatty acids to be absorbed. Glycerol released from TGs is transported to the liver for utilization. 12 White adipocytes passively transport the free fatty acids (FFAs) using a specific transporter (CD36). The Fatty Acid Binding Protein (FABP) binds to FFAs in the cytosol and transports them. STEPS OF THE SYNTHESIS: I. ACTIVATION OF FATTY ACIDS: SYNTHESIS OF ACYL-CoAs (slide 87) An FFA must be converted to its activated form (bound to CoA through a thioester link) before it can participate in metabolic processes such as TAG synthesis. This reaction is catalyzed by a family of fatty acyl CoA synthetases using 2 high-energy bonds of ATP. II. GLYCEROL-3-PHOSPHATE IS NEEDED FOR TG SYNTHESIS (slide 88) Glycerol 3-phosphate is the initial acceptor of fatty acids during TAG synthesis. There are two major pathways for its production. (Note: A third process, glyceroneogenesis, is described later). In both liver and adipose tissue, glycerol 3-phosphate can be produced from glucose, first using the reactions of the glycolytic pathway to produce dihydroxyacetone phosphate (DHAP). DHAP is reduced by glycerol 3-phosphate dehydrogenase to glycerol 3-phosphate. A second pathway found in the liver, but not in adipose tissue, uses glycerol kinase to convert free glycerol to glycerol 3-phosphate. (Note: The glucose transporter in adipocytes (GLUT-4) is insulin dependent. Thus, when plasma glucose levels are low, adipocytes have only a limited ability to synthesize glycerol phosphate and cannot produce TAG de novo.) III. FORMATION OF TRIACYLGLYCEROLS (TGs) IS CARRIED OUT BY ACYL-TRANSFERASES (slides 89-91) The synthesis of TG is carried out mainly by acyl-transferases transferring an acyl group from acyl-CoAs to hydroxyl groups of glycerol molecules. The synthesis of triacylglycerols involves the formation of phosphatidic acid, which is produced by two sequential acylations of glycerol 3-phosphate first to form monoacylglycerol-3- phosphate (lysophosphatidic acid) by glycerol-3-phosphate acyl-transferase (GPAT) and then phosphatidic acid by acylglycerol-3-phosphate acyltransferase. Phosphatidic acid is used for triacylglycerol synthesis by hydrolysis of the phosphate group by phosphatidic acid phosphatase to yield diacylglycerol, which is then acylated to triacylglycerol by diacylglycerol-acyltransferase. Phosphatidic acid and diacylglycerol are also key intermediates in the synthesis of other glycerolipids necessary for the formation of membrane structures. TG synthesis follows a different pathway in enterocytes of the small intestine. These cells take up 2-monoacylglycerols and free fatty acids from the gut which are the major digestion products of dietary triacylglycerols by pancreatic lipase. Acylglycerol acyltransferase (AGAT) in the mucosal cells acylates these monoacylglycerols using acyl-CoAs as substrates. The resulting diacylglycerols can then be acylated to form triacylglycerols that are packaged into chylomicrons. 13 GLYCERONEOGENESIS IS A SHORTENED GLUCONEOGENIC PATHWAY (slide 92) It frequently occurs in the case of starvation, that too high amounts of lipids are mobilized from adipose tissue compared to the actual energy requirements of the body. Excess of free fatty acids must be taken back into the adipocytes or hepatocytes. TG synthesis needs glycerol-3-phosphate, but because of the effects of glucagon, glycolysis is inactive in these cells, and cannot produce the precursor dihydroxyacetone phosphate. In this case, dihydroxyacetone phosphate is formed by the shortened gluconeogenic pathway both in adipocytes and hepatocytes. (Note: In adipocytes, the process is stopped at the formation of dihydroxyacetone phosphate, therefore adipocytes cannot synthesize glucose.) Consequently, during starvation synthesis of TGs may occur from fatty acids and glycerol in case of need; however, the synthesis of fatty acids is completely inhibited because of phosphorylated (and inactive) acetyl-CoA carboxylase. LIPOPROTEINS ARE FORMED IN THE ROUGH ENDOPLASMIC RETICULUM (slide 93) Enzymes of triacylglycerol (TG) synthesis are localized on the rough endoplasmic reticulum (RER). TGs being synthesized and introduced into the hydrophobic parts of the membrane first form a lens-like structure between the bilayer halves of the RER membrane, then bud out into the cytoplasm and are released from the membrane. Apolipoproteins are also synthesized on the RER and packed into the membrane. Transport of lipoproteins (chylomicron in enterocytes and VLDL in hepatocytes) to the cell surface and their release need functional microfilament structures. TRIACYLGLYCEROL FATE DIFFERS IN LIVER AND ADIPOSE TISSUE (slide 94) TGs are not stored in a healthy liver. Instead, most are exported, and packaged with other lipids and apolipoproteins to form lipoprotein particles called very–low-density lipoproteins (VLDL). Nascent VLDLs are secreted directly into the blood where they mature and function to deliver the lipids to the peripheral tissues. When the liver cannot form or release VLDL under pathological conditions, lipid droplets can be observed in the tissue, this condition is known as fatty liver or steatosis caused by chronic alcoholism and some genetic factors. LIPID DROPLETS ARE DYNAMIC ORGANELLES (slide 95) In white adipose tissue, TAG is stored in a nearly anhydrous form as fat droplets in the cytosol of the cells. The fat droplets are coated with a family of proteins known as perilipins that sequester and protect TAG from lipolysis until the body requires fatty acids for fuel. The biogenesis of small lipid droplets in the endoplasmic reticulum is followed by their subsequent fusion in the cytoplasm of white adipocytes. Lipid droplet proteins (dark blue) such as perilipin, are involved in packaging the lipids in multilocular lipid droplets; however, other Fat Specific Proteins (FSPs, like FSP27) play a key role in the formation of unilocular lipid droplets in adipocytes of White Adipose Tissue (WAT). The exact ratio of these proteins will determine the average size of droplets in adipocytes. 14 References: Denise R. Ferrier: Lippincotts’ Illustrated Review s of Biochemistry, Wolters Kluwer/Lippincott Williams and Wilkins, 8th edition, 2021. Rodwell, V.W., Bender, D.A., Botham, K.M., Kennelly, P.J., Weil, P.A.: Harper’s Illustarted Biochemistry, Lange, 31th edition, 2018. Devlin, T.M.: Textbook of Biochemistry with Clinical Correlations, John Wiley and Sons, 2011. Baynes, J.W., Baynes, Dominiczak, M.H.: Medical Biochemistry, Elsevier, 5th edition, 2019. 15