BCM225 Lipid Metabolism (2021/2022) Part II PDF

# COLLEGE OF HEALTH SCIENCES ## FACULTY OF BASIC MEDICAL SCIENCES ### DEPARTMENT OF MEDICAL BIOCHEMISTRY ### BCM 225 LIPID METABOLISM (2021/2022) PART II ## De Novo Synthesis of Fatty acids (Lipogenesis) The majority of fatty acids required by the body is supplied by the diet. Fatty acids are synt...

# COLLEGE OF HEALTH SCIENCES ## FACULTY OF BASIC MEDICAL SCIENCES ### DEPARTMENT OF MEDICAL BIOCHEMISTRY ### BCM 225 LIPID METABOLISM (2021/2022) PART II ## De Novo Synthesis of Fatty acids (Lipogenesis) The majority of fatty acids required by the body is supplied by the diet. Fatty acids are synthesized whenever there is a caloric excess in the diet. Excess amounts of carbohydrate and protein obtained from the diet can be converted to fatty acids which are stored as triacylglycerol. In humans, fatty acids synthesis occurs mainly in the liver and lactating mammary glands and, to a lesser extent, in adipose tissue, kidney and brain. De novo synthesis means new synthesis from amphibolic compounds. Fatty acid synthesis is not simply a reversal of the degradative pathway. Rather it consists of a new set of reactions. Some important features of the pathways for the biosynthesis and degradation of fatty acids are, listed in Table 1. ## Table 1: Important features of the biosynthesis and degradation pathways of fatty acid | Biosynthesis of fatty acids | Degradation of fatty acids | |---|---| | Occurs in cytosol | Occurs in mitochondrial matrix | | Intermediates are covalently linked to the sulmydryl Group of an acyl carrier protein (ACP) | Intermediates are bonded to coenzyme-A (COA-SH) | | Enzymes are joined in a single polypeptide chain to form a multienzyme complex called fatty acid synthase | Enzymes do not seem to be associated | | Reducing equivalent involved is NADPH | Reducing equivalents involved are NAD and FAD | | Fatty acids are synthesized by an elongation process in which two carbon units (acetyl-CoA) are added in the form of malonyl-CoA sequentially to the carboxyl end of the growing fatty acid chain. | Fatty acids are degraded by the sequential removal of two carbon units, acetyl-CoA. | | CO participates in the formation of malonyl-CoA from Acetyl-CoA | No participation of CO | | Stercosomeric forın of hydroxylacyl is D(-) | Stereosomeric form of hydroxyl acyl group is L() | | - In fatty acid biosynthesis, the acetyl-CoA, used as a primer, forms carbon atoms 15 and 16 of palmitate. - The addition of all the subsequent 2-C units is via malonyl-CoA formation. | | ## Phases of De Novo Fatty Acid Synthesis Fatty acid synthesis occurs in three phases. - **Phase 1:** Transport of substrates into the cytosol and carboxylation of acetyl-CoA to malonyl-CoA by an enzyme complex, acetyl-CoA carboxylase. - **Phase II:** Utilization of substrate to form palmitate by the enzyme complex, fatty acid synthase. - **Phase III:** The elongation and desaturation (addition of double bond) of palmitate to generale different fatty acids. ### PHASE 1 - Acetyl-CoA is the intermediate substrate for fatty acid synthesis. - The initial two carbons incorporated into fatty acids are donated by acetyl-CoA and are found at the end of the fatty acid. - All other carbon atoms are donated by malonyl-CoA formed from acetyl-CoA. - The synthesis of palmitate C-16 saturated fatty acid requires the input of 8 molecules of acetyl-CoA, 14 NADPH and 7 ATP. ## Transport of Acetyl-CoA from Mitochondria to Cytosol Fatty acids are synthesized in the cytosol, whereas acetyl-CoA is formed from pyruvate in mitochondria, hence, acetyl-CoA must be transferred froin mitochondria to the cytosol. The barrier to acetyl CoA is bypassed by citrate, which carries acetyl-CoA across the inner mitochondrial membrane. - Citrate is formed in the mitochondrial matrix by the, condensation of acetyl-CoA with oxaloacetate (first reaction in the citric acid cycle). - When present at high levels, citrate is transported to the cytosol by translocase, where it is cleaved by citrate lyase at expense of ATP to oxaloacetate and acetyl-CoA. - Oxaloacetate forined in this reaction must be returned to the mitochondria. The inner mitochondrial membrane is impermeable to oxaloacetate, therefore, a series of bypass reactions are needed, which generate NADPII, needed for fatty acid synthesis. - First, oxaloacetate is reduced to malate by NADII, catalyzed by cytosolic malate dehyrogenase. - Second, malate is oxidatively decarboxylated to pyruvate by NADP-linked malate enzyme, also called malic enzyme. - The pyruvate formed in this reaction readily diffuses into mitochondria, where it is carboxylated to oxaloacetate by pyruvate carboxylase. Thus, one NADPH is generated for each acetyl-CoA that is transferred froin mitochondria to the cytosol. Consequently, eight NADPHs are formed when eight molecules of acetyl-CoA are transferred to the cytosol for the synthesis of palmitate. The additional six NADPH required for synthesis of palmitate comes from the pentose phosphate pathway. ## Carboxylation of Acetyl-CoA to Malonyl-CoA - Carboxylation of acetyl-CoA to malonyl-CoA is the initial and rate limiting reaction in fatty acid synthesis. - This irreversible reaction is catalyzed by an enzyme complex, acetyl-CoA carboxylase, that contains biotin (prosthetic group) and utilizes bicarbonate (as a source of CO) in presence of ATP (Figure 2). - Acetyl-CoA carboxylase is an allosteric enzyme that is activated by citrate and inhibited by its end product palmitoyl-CoA. In addition to allosteric control, a high carbohydrate and low fat dict stimulates the synthesis of the enzyme. ## PHASE II Malonyl-CoA is the substrate for fatty acid synthase complex. Fatty acid synthase sequentially adds 2-carbon units from malonyl-CoA to the growing fatty acyl chain to form palinitate. ## Fatty Acid Synthase Multienzyme Complex Fatty acid synthase complex is a polypeptide containing seven enzyme activities and an acyl carrier protein (ACP) segment. The seven enzyme activities are: - Acetyl transacylase - Malonyl transacylase - 3-ketoacyl synthase - 3-ketoacyl reductase - 3-hydroxylacyl hydratase - Enoyl reductase - Thioesterase Fatty acid synthase complex is a dimer (Figure 3) composed of two identical monomer units. - Each monomer is identical consisting of one polypeptide chain containing all seven enzyme activities of fatty acid synthase. - The ACP -segment contains a 4-phosphopantetheine group. This provides the sulfhydryl (-SH) group to which the growing fatty acid chain is attached as it is synthesized. - Thus, the function of ACP in fatty acid biosynthesis is analogous to the role of coenzyme-A in fatty acid oxidation. - Both -SH groups participate in fatty acid biosynthesis. - The dimmers (2- monomers) associate in a head-to-tail arrangement so that the phosphopantetheine (-SH) group on one subunit and a cysteinyl –SH group on another subunit are closely aligned (Figure 3). Though each monomer contains all the activities of the reaction sequence, the actual functional unit consist of one half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously. Since both thiols (-SH groups) participate in the synthase activity, only the dimer is active. ## Reactions of Phase II (Figure 4) - In the first reaction, catalyzed by acetyl transacylase, the acctyl group of acetyl-CoA is transferred to the cysteine- SH group of the 3-ketoacyl synthase of fatty acid synthase complex (designated E). - In the second reaction, the malonyl group of malonyl-CoA is transferred to the phosphopantetheine -SH group (Pan -) SH of ACP of other monomer in a reaction catalyzed by malonyl transacylase to form acetyl-malonyl enzyme. - The malonyl-CoA binds only to the pantetheine SH group. The fatty acid synthase now has two covalently bound acyl groups, an acetyl group at the cysteine –SH group and a malonyl group at the Pan -SH group, which is now ready for the actual chain lengthening process. Chain lengthening or elongation of the fatty acid chain requires four steps: - Condensation - Reduction - Dehydration and - Saturation ### Condensation The acetyl and malonyl groups, covalently bonded to –SH groups of the synthase, undergo a condensation reaction to form an acetoacetyl group bound to the phosphopantethine -SH group, simultaneously a molecule of CO set free, forming 3-ketoacyl enzyme (acetoacetyl enzyme). This reaction is catalyzed by 3-ketoacyl synthase. - Here acetyl group is transferred from the Cysteine- SH group to the malonyl group on the -SH of pantetheine. - The CO formed in this reaction is the same CO that was originally introduced into malonyl-CoA by the acetyl-CoA carboxylase reaction; described above. - Thus, CO is not permanently fixed in covalent linkage during fatty acid biosynthesis. The loss of CO from the malonyl group momentarily makes the remaining 2 carbon portion reactive, enabling it to react readily with the acetyl group. ### Reduction The 3-ketoacyl enzyme (acetoacetyl enzyme) undergoes reduction at the 3-keto group, at the expense of NADPH as electron donor to form D(-) 3-Hydroxyacyl enzyme, catalyzed by 3-ketoacyl reductase. - The D(-)3-hydroxyacyl group is not the same stereoisomeric form as the ( )3-hydroxyacyl intermediate in fatty acid oxidation. - The main source of NADPH for fatty acid synthesis is the pentose phosphate pathway and from malic enzyme (Figure 1). ### Dehydration D(-3)- hydroxyacyl enzyme is dehydrated by 3-hydroxy-acyl hydratase to yield 2,3 unsaturated acyl enzyme. ### Saturation 2,3 unsaturated acyl enzyme is reduced or saturated to form acyl enzyme containing 4-carbon, by the action of enoyl reductase and NADPH is the electron donor. - This acyl group (4 carbon) is now transferred from pantetheine -SH group to the cysteine –SH group. Thus newly lengthened fatty acyl group now occupies the –SH group originally occupied by the acetyl group. - To lengthen the chain by another 2-carbon unit, the sequence of reactions is repeated, a new malonyl residue being incorporated during each sequence, until a saturated 16-carbon acyl radical (palmityl) has been assembled. - Thus, after a total of seven such cycles, palmitoyl enzyme is formed, which is liberated from the enzyme complex by the activity of a seventh enzyme in the complex thioesterase (deacylase). ## Fate of Palmitate The free palmitate, the norinal product of fatty acid synthase, must be activated to acyl-CoA, before it can proceed via any other metabolic pathway. Its usual fate is: - Elther esterification into acylglycerols or - It acts as a precursor of other long chain fatty acids. ## PHASE III Chain Elongation and Desaturation Elongation and desaturation of fatty acids occurs in the: - Microsomes of endoplasmic reticulum. - Mitochondria. The major product of fatty acid synthesis is palmitate. Longer fatty acids are formed by elongation reactions either in endoplasmic reticulum (microsomes) or in mitochondria. ### Microsomal elongation of fatty acids The microsomal enzyme fatty acid elongase elongates palmitate by the addition of 2-carbon fragments derived from malonyl-CoA and NADPH provides the reducing equivalents. The elongation reaction resembles those of fatty acid synthesis, except that the fatty acyl chain is attached to coenzyme-A rather than to phosphopantetheine group of ACP. ### Mitochondrial elongation of fatty acids Fatty acids can be elongated in mitochondria, but mitochondrial elongation of fatty acids is less active. In this case, the source of the 2-carbon units is acetyl-CoA and the substrates are usually fatty acids containing less than 16-carbons, mainly short and medium chain fatty acids. ## Regulation of Fatty Acid Synthesis De novo fatty acid synthesis is regulated by Acetyl-CoA Carboxylase enzyme. - Acetyl-CoA carboxylase which catalyzes the formation of malonyl-CoA is the rate limiting enzyme in the fatty acid synthesis. It is regulated by following ways: - **Allosteric regulation:** Acetyl-CoA carboxylase is allosterically activated by citrate and inhibited by its own product palmitoyl-CoA. - **Hormonal regulation:** The activity of acetyl-CoA carboxylase is also controlled by hormones. Glucagon and epinephrine inactivate the enzyme. In contrast, insulin activates the enzyme. Thus, fatty acid synthesis is stimulated by insulin and inhibited by glucagon and epinephrine. - **Nutritional regulation:** Synthesis of acetyl-CoA carboxylase is increased with high carbohydrate and low fat diet, which promote fatty acid synthesis. By contrast, sythesis of acetyl-CoA carboxylase decreases during starvation, diabetes mellitus or high fat diet. ## SYNTHESIS OF ESSENTIAL FATTY ACIDS - Plants are able to introduce double bonds into fatty acids in the region between Cand end, and therefore, can synthesize 6-3 and 6-6 polyunsaturated fatty acids. - Arachidonic acid listed as an essential fatty acid although is an 4-6 fatty acid is not essential in the diet if linoleic acid is present because arachidonic acid can be synthesized from dietary linoleic acid. ## Importance of Essential Fatty acids The essential fatty acid is required in the diet because: - It serves as a precursor of arachidonic acid from which eicosanoids are produced. - It covalently binds another fatty acid attached to cerebrosides in the skin forming an unusual lipid (acylglucosylceremide) that helps to make the skin impermeable to water. This formation of linoleic acid may help explain the red scaly dermatitis and other skin problems associated with a dietary deficiency of essential free fatty acids. ## Triglycerol Metabolism Triglycerols are esters of the alcohol glycerol and fatty acids. Fatty acids derived from endogenous synthesis or from the diet are stored in the adipose tissue in the form of triglycerol, called as “neutral fat". Fatty acids are esterified through their carboxyl group with alcohol glycerol, resulting in a loss of negative charge and formation of neutral fat. Triacylgycerol serves as the body's major fuel storage reserve. ### Biosynthesis of Triacylglycerols In both liver and adipose tissue, triacylglycerols are produced by a pathway, involving glycerol -3-phosphate (Figure 9). - First fatty acids are activated to acyl-CoA (Figure 8). - Then two molecules of acyl-CoA combine with glycerol-3-phosphate to form phosphatidic acid (1,2- diacylglycerol phosphate) via formation of lysophosphatidic acid. - Phosphatidic acid is the common precursor in the biosynthesis of the triacylglycerols and many phosphoglycerides and cardiolipin. - Dephosphorylation of phosphatidic acid produces diacylglycerol. - A further molecule of acyl-CoA is esterified with diacylglycerol to form triacylglycerol. The sources of glycerol-3-phosphate, which provides the glycerol moiety for triacylgycerol synthesis, differ in liver and adipose tissue. - In liver glycerol-3-phosphate is produced from the phosphorylation of glycerol by glycerol kinase or from the reduction of dihydroxyacetone phosphate (DHAP), derived from glycolysis. - Adipose tissue lacks glycerol kinase and can produce glycerol-3-phosphate only from glucose via DHAP. Thus, adipose tissue can store fatty acids only when glycolysis is activated, i.e. in the fed state. ## FATE OF TRIACYLGLYCEROL FORMED IN LIVER AND ADIPOSE TISSUE The fates of triacylglycerol in liver and adipose tissue are different. - In the liver, little triacylglycerol is stored; instead most is exported in the form of very low density lipoprotein (VLDL). - In adipose tissue triacylglycerol is stored in the cells. - It serves as "depot fat" ready for mobilization when the body requires it for fuel. ## FATE OF TRIACYLGLYCEROL FORMED IN LIVER - The triacylglycerol produced in the liver is packaged with cholesterol, phospholipids and proteins (apolipoprotein, apoB-100) to form VLDL and released into blood stream and delivered to the peripheral tissue. - Once released into the blood stream, triacylglycerol of VLDL is hydrolyzed by lipoprotein lipase enzyme, which is located on the walls of blood capillaries. - It clears the triacylglycerol in VLDL, forming free fatty acids and glycerol. - Lipoprotein lipase is found in heart, adipose tissue, spleen, lungs, renal medulla, aorta and lactating mammary gland. It is not active in adult liver. ## FATE OF TRIACYLGLYCEROL FORMED IN ADIPOSE TISSUE - This triacylglycerol stored in adipose tissue are continually undergoing lipolysis (hydrolysis) and reesterificaiton. - Triacylglycerol undergoes hydrolysis by a hormone sensitive lipase to form free fatty acids and glycerol. - This lipase is distinct from lipoprotein lipase that catalyzes lipoprotein triacylglycerol hydrolysis before its uptake into extrahepatic tissues. - The glycerol, released in adipose tissue, cannot be metabolized by adipocytes because they lack glycerol kinase. - Rather, glycerol is transported through the blood to the liver, which can phosphorylate it. - The resulting glycerol phosphate can be used to form triacylglycerol in the liver to be converted to DHAP. Hormone sensitive lipase is activated directly following its phosphorylation through c-AMP. In the presence of insulin and glucose, hormone sensitive lipase is dephosphorylated and become inactive. Epinephrine, norephinephrine, glucagon, and ACTH stimulates hormone sensitive lipase by increasing c-AMP. ## Phospholipid Metabolism Phospholipids are the major class of membrane lipids. There are two classes of phospholipids: - Those that have glycerol, 3-carbon alcohol, as a backbone, called glycerolphospholipids or phosphoglycerides, e.g. - Phosphatidylserine - Phosphatidylinositol - Phosphatidylcholine (Lecithins) - Phosphatidylethanolamine (Cephalins) - Cardiolipin (Diphosphatidyl glycerol) - Plasmalogens (Glycerol ether phospholipid) - Those that contain sphingosine, a more complex amino alcohol called “sphingophospholipids, c.g. Sphingomyelin. ## BIOSYNTHESIS OF GLYCEROPHOSPHOLIPIDS ### Biosynthesis of Glycerophospholipids The initial steps in the synthesis of glycerophospholipids are similar to those of triacylglycerol synthesis (Figure 9). Phosphatidate (diacylglycerol-3-phosphate) is a common intermediate in the synthesis of glycerophospholipids and triacylglycerols. - The starting point is glycerol-3-phosphate, which is formed mainly by reduction of dihydroxyacetone phosphate (DHAP) and to a lesser extent by phosphorylation of glycerol by glycerol kinase. - Glycerol-3-phosphate reacts with fatty acyl-CoA to form lysophosphatidate, which again reacts with second molecule of fatty acyl-CoA to form phosphatidate. These reactions are catalyzed by glycerophosphate acyl transferase. ### Synthesis of phosphatidylserine and phosphatidylinositol - Synthesis of phosphatidylserine and phosphatidylinositol starts with the formation of cytidine diacylglycerol (CDP – diacylglycerol) an activated phosphatidyl unit, from phosphatidate and cytidine triphosphate (CTP). - The activated phosphatidyl unit then reacts with the hydroxyl group of alcohol. - If alcohol is serine, it forins phosphatidylserine - Likewise if the alcohol is inositol, the product is phosphatidylinositol. - Phosphatidylserine can also be formed from phosphatidylethanolamine directly by reactions with serine (Figure 9). By subsequent phosphorylations catalysed by specific kinases, phosphatidylinositol is transformed to phosphatidylinositol 4,5 -bisphosphate, which is broken down into diacylglycerol and inositoltriphosphate by hormones, e.g., vasopressin. These two products act as second messengers in the action of the hormone. ### Synthesis of phosphatidylcholine (lecithins) and phosphatidylethanolamine (cephalins) Phosphatidylcholine is synthesized by a pathway that utilises choline obtained from the diet - Choline must first be converted to active choline. This is a stage process, first is the phosphorylation with ATP to form phosphocholine, which then reacts with CTP to form CDP-choline (active form). - The phosphorylcholine unit of CDP- choline is then transferred to a diacylglycerol to form, phosphatidylcholine (Figure 9) the activated species in this pathway is the cytidine derivative of phosphorylcholine rather than phosphatidate.. - Likewise phosphatidylethanolamine can be synthesized from ethanolamine by forming a CDP-ethanolamine intermediate by analogous reactions. - Phosphatidylserine may form phosphatidylethanolamine by decarboxylation. - An alternative pathway in liver enables -phosphatidylethanolamine to give rise directly to phosphatidylcholine by methylation of the ethanolamine residue utilising S-adenosyl-methionine (SAM) as the methyl donor. **Assignment**: Write on the synthesis of Cardiolipin (Diphosphatidylglycerol) and plasmalogens (glycerol ether phospholipids). ## Glycolipid (Glycosphingolipid Metabolism) Glycolipid or glycosphingolipid is a sphingolipid which is formed from ceramide. Glycolipids, as their name implies, are sugar containing lipids. Glycolipids like sphingomyelin are derived from sphingosine. The amino group of the sphingosine backbone is acylated by a fatty acid as in sphingomyelin. Characteristically, C24 fatty acids (lignoceric, cerebronic and nervonic acid) occur in many glycosphingolipids. Cerebroside, sulfatides, globosides and ganglosides are the different types of glycolipids. ### Biosynthesis of glycolipids Synthesis of cerebrosides, sulfatides and globosides (Figure 11). - Cerebroside is the simplest glycosphingolipid. In a cerebroside, glucose or galactose is linked to the terminal hydroxyl group of ceremide to form glucocerebroside or galactocerebroside. - Galactocerebroside is a major lipid of myelin, whereas glucocerebroside is the major glycolipid of extraneural tissue and a precursor of most of the more complex glycosphingolipids. - Ceremide reacts with UDP -glucose or UDP galactose to form glucocerebroside or galactocerebroside respectively. - Galactocerebroside may react with active sulphate 3' – phosphoadenosine 5'-phosphosulfate (PAPS) to form sulfatides, the major sulfolipids of the brain. - Additional sugars may be added to ceremide to form globosides. ### Biosynthesis of sphingomyelin Sphingomyelin is a sphingophospholipid. The backbone of sphingolipid is sphingosine rather than glycerol: - The synthesis of all sphingolipids begins with the formation of ceramide which is synthesised in endoplasmic reticulum (Figure 10). - Palmitoyl-CoA and serine condense to form 3-keto-sphinganine. The enzyme catalysing this reaction requires pyridoxal phosphate. - 3-ketosphinganine, is then converted to sphingosine. - In all sphingolipids, the amino group of sphingosine is acylated, a long chain acyl-CoA reacts with sphingosine to form ceramide (N- acylsphingosine). - Ceramide reacts with phosphatidylcholine to form sphingomyclin. This occurs mainly in the Golgi apparatus. ### Synthesis of gangliosides - Gangliosides are the more complex glycolipids, contain a branched chain, an oligosaccharide of as many as seven sugar residues. - Gangliosides are produced from ceramide by the stepwise addition of activated sugar, e.g. UDP- glucose, UDP-galactose and sialic acid usually N-acetylneuraminic acid (NANA) (Figure 11).