Metabolism of lipids -2. Lipogenesis. Metabolism of cholesterol. Regulation and pathology of lipid metabolism: obesity, atherosclerosis.

Activation and transfer of fatty acid Fatty acids must be activated in the cytoplasm before being oxidized in the mitochondria. Activation is catalyzed by fatty acyl-CoA ligase (also called acyl-CoA synthetase or thiokinase). The net result of this activation process is the consumption of 2 molar equivalents of ATP. Fatty acid + ATP + CoA -------> Acyl-CoA + PPi + AMP Oxidation of fatty acids occurs in the mitochondria. The transport of fatty acyl-CoA into the mitochondria is accomplished via an acyl-carnitine intermediate, which itself is generated by the action of carnitine acyltransferase I, an enzyme that resides in the outer mitochondrial membrane, from acyl-CoA and carnitine. The acyl-carnitine molecule then is transported into the mitochondria where carnitine acyltransferase II catalyzes the regeneration of the fatty acyl-CoA molecule.

Reaction sequence in the b-oxidation

Connections to Electron Transport and ATP:One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is Step 1 - FAD into e.t.c. = 2 ATP Step 3 - NAD+ into e.t.c. = 3 ATP Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. Example with Palmitic Acid = 16 carbons = 8 acetyl groupsNumber of turns of fatty acid spiral = 8-1 = 7 turnsATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.NET ATP from Fatty Acid Spiral = 35 - 1 = 34 ATP

Lipogenesis1. Production of fat, either fatty degeneration or fatty infiltration. Also called adipogenesis.2. The normal deposition of fat or the conversion of carbohydrate or protein to fat.

The pathway of fatty acid synthesis In the opposite to fatty acid degradation, which is located within the mitochondria, de novo synthesis of fatty acids takes place within the cytosol.The precursor of fatty acid synthesis is acetyl-CoA, that is transported out of the mitochondria into the cytosol via the citrate shuttle.The NADPH necessary for fatty acid synthesis derives from the conversions of malic enzyme, glucose 6-P dehydrogenase, gluconate 6-P dehydrogenase and NADP-dependent isocitrate dehydrogenase

Lipogenesis is a collective name for the complex process of producing lipids (fatty acids) from smaller precursor molecules.The main ingredient in the production of fatty acids is glucose.Glucose is what most of the carbohydrates are turned into before entering the blood stream, so in a way, lipogenesis is the conversion of carbohydrates into fat.55% of carbs are involved in fat synthesis.Fat metabolism is in a constant state of dynamic equilibrium, where some fats are constantly oxidized to meet energy requirements, and some fats are synthesised and stored.In rats, a single lipid molecule lasts for between 2 and 10 days, this is likely to be the same in humans.

Lipogenesis starts with an acetyl CoA which builds with the addition of two carbon units.It takes place in the cytoplasm, unlike oxidation which happens in the mitochondria.Many enzymes involved are in a multienzyme complex called fatty acid synthetase.Important points to remember are that lipogenesis requires ATP and that the reactions involved are reductions (the addition of H+ and the use of NADPH), which is the opposite to fatty acid spiral oxidations.

All reactions of fatty acid synthesis is catalyzed by a complex multienzyme system in the cytosol the fatty-acid synthetase complex.Acetyl-CoA is the ultimate precursor of all the carbon atoms of the fatty acid chain. However, of the all acetyl units required for biosynthesis of fatty acid, only one is provided by acetyl-CoA; the others arrive in the form of malonyl-CoA. Malonyl-CoA is formed from acetyl-CoA and CO2 in a carboxylation reaction.

A distinctive feature of the mechanism of fatty acid biosynthesis is that the low-molecular-weight conjugated protein called acyl carrier protein (ACP) is required. This protein can form a complex or complexes with the six other enzyme proteins required for the complete synthesis of fatty acid. In most eukariotic cells all seven proteins of the fatty acid synthetase complex are associated in a multienzymes cluster.

In most organisms the end product of the fatty-acid synthetase system is palmitic acid, the precursor of all other higher saturated fatty acids and of all unsaturated fatty acids.Before the acetyl groups of acetyl-CoA can be utilized by the fatty-acid synthetase complex, an important preparatory reaction must take place to convert acetyl-CoA into malonyl-CoA. Malonyl-CoA is formed from acetyl-CoA and bicarbonate in the cytosol by the action of acetyl-CoA carboxylase, a complex enzyme that catalyzes the reaction:

Formation of malonyl-CoA

carboxylase contains biotin as its prosthetic group. Acetyl-CoA Malonyl-CoA

Once malonyl-CoA has been generated from acetyl-CoA the ensuing reactions of fatty acid synthesis occur in a sequence of six successive steps catalyzed by the six enzymes of the fatty-acid synthetase system. The seventh protein of this system, which has no enzymatic activity itself, is acyl carrier protein (ACP), to which the growing fatty acid chain is covalently attached.

To prime the fatty-acid synthetase system, acetyl-CoA first reacts with the sulfhydryl group of ACP by the action of one of the six enzymes of the synthetase system, ACP-acyltransferase, which catalyzes the reaction:The malonyl transfer step

In the next reaction, catalyzed by ACP malonyltransferase, malonyl-S-CoA formed in the acetyl-CoA carboxylase reaction reacts with the SH group of the 4'-phosphopantetheine arm of ACP, with loss of free CoA, to form malonyl-S-ACP:MalonylSCoA + ACPSH malonylSACP + CoASHAs a result of this step and of the preceding priming reaction, a malonyl group is now esterified to ACP and an acetyl group is esterified to an SH group on the ACP molecule.

The condensation reactionIn the next reaction of the sequence, catalyzed by -ketoacyl-ACP synthase, the acetyl group esterified to the cysteine residue is transferred to carbon atom 2 of the malonyl group on ACP, with release of the free carboxyl group of the malonyl residue as CO2:

The condensation reaction

The first reduction reactionThe acetoacetyl-S-ACP now undergoes reduction by NADPH to form -hydroxybutyryl-S-ACP. This reaction is catalyzed by -ketoacyl-ACP reductase:NADPH2NADP

The dehydration step-Hydroxybutyryl-S-ACP is next dehydrated to the corresponding unsaturated acyl-S-ACP, namely, crotonyl-S-ACP, by -hydroxyacylACP-dehydratase:

The second reduction stepCrotonyl-S-ACP is now reduced to butyryl-S-ACP by enoil-ACP reductase (NADPH); the electron donor is NADPH in animal tissues:NADPH2NADP

The formation of butyryl-ACP completes the first of seven cycles en route to palmitoyl-S-ACP.To start the next cycle the butyryl group is transferred from SH group of phosphopantetheine to the SH group of cysteine, thus allowing SH group of ACP phosphopantetheine to accept a malonyl group from another molecule of malonyl-CoA.

Then the cycle repeats, the next step being the condensation of malonyl-S-ACP with butyryl-S-ACP. After seven complete cycles, palmitoyl-ACP is the end product. The palmitoyl group may be removed to yield free palmitic acid by the action of a thioesterase, or it may be transferred from ACP to CoA, or it may be incorporated directly into phosphatidic acid in the pathway to phospholipids and triacylglycerols

The overall equation for palmitic acid biosynthesis starting from acetyl-S-CoA:8 AcetylSCoA + 14NADPH + 14H+ + 7ATP + H2O palmitic acid + 8CoA + 14NADP+ + 7ADP + 7P.

Palmitic acid, the normal end product of the fatty-acid synthetase system, is the precursor of the other long-chain saturated and unsaturated fatty acids in most organisms. Elongation of palmitic acid to longer-chain saturated fatty acids, of which stearic acid is most abundant, occurs by the action of two different types of enzyme systems, one in the mitochondria and the other in the endoplasmic reticulum.

The ways of formation of active form of glycerol.There are two ways of formation of active form of glycerol.1. Phosphorilation of glycerol through the action of glycerol kinase:ATP + glycerol glycerol 3-phosphate + ADP2. Reduction of dihydroxyacetone phosphate which is the product of the aldolase reaction of glycolysis. Dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the NAD-linked glycerol-3-phosphate dehydrogenase of the cytosol:Dihydroxyacetone phosphate + NADH + H+ glycerol 3-phosphate + NAD

Biosynthesis of triacylglycerols

The first stage in triacyglycerol formation is the acylation of the free hydroxyl groups of glycerol phosphate by two molecules of fatty acyl-CoA to yield first a lysophosphotidic acid and then a phosphatidic acid:

Lysophosphotidic acid Phosphatidic acid

Free glycerol is not acylated. Phosphatidic acids occur only in trace amounts in cells, but they are important intermediates in the biosynthesis of triacylglycerols and phosphoglycerides.In the pathway to triacylglycerols, phosphatidic acid undergoes hydrolysis by phosphatidate phosphatase to form a diacylglycerol:

Phosphatidic acid Diacylglycerol

The diacylglycerol then reacts with a third molecule of a fatty acyl-CoA to yield a triacylglycerol by the action of diacylglycerol acyltransferase:

Regulation of lipogenesis by AMP The key regulating enzyme of lipogenesis is acetyl-CoA carboxylase, which catalyzes the synthesis of malonyl-CoA from acetyl-CoA and CO2.

The activity of acetyl-CoA carboxylase depends on its phosphorylation status .In its inactive form acetyl-CoA carboxylase is phosphorylated in serine, whereas the active form is not phosphorylated.

The phosporylation of acetyl CoA carboxylase is catalyzed by an AMP-dependent protein kinase (AMPK).High AMP levels induce the phosphorylation and inactivation of acetyl-CoA carboxylase.

Therefore, low AMP levels activate acetyl-CoA carboxylase by inhibition of AMP-dependent protein kinase. The activation of acetyl-CoA carboxylase leads to an increase of malonyl-CoA.Tumor cells are characterizedby high malonyl-CoA concentrations.Malonyl-CoA itself inhibits carnitine acyl-CoA transferase-1, the enzyme that is responsible for the transport of fatty acids into the mitochondria, which leads to an inhibition of mitochondrial fatty acid degradation.

Cholesterol and Bile Metabolism

Cholesterol is an extremely important biological molecule that has roles in membrane structure as well as being a precursor for the synthesis of the steroid hormones and bile acids. Both dietary cholesterol and that synthesized de novo are transported through the circulation in lipoprotein particles. The same is true of cholesteryl esters, the form in which cholesterol is stored in cells.

Synthesis of Cholesterol and other Steroids:Without going into detail, acetyl CoA forms the basis from which the fairly complicated steroids are synthesized. Some steroids of importance include cholesterol, bile salts, sex hormones, aldosterone, and cortisol.

The major concern about cholesterol in the diet is muted somewhat by the knowledge that the liver can and does synthesize all of the cholesterol that the body needs. Excess cholesterol, whether from food or synthesized by the liver, ends up in the blood stream where it builds up on the artery walls. It has been determined that cholesterol levels can be controlled by lowering the amount of saturated fat and increasing the unsaturated fats. Unsaturated fats seem to speed the rate at which cholesterol breaks down in the blood. Controlling fats and cholesterol in the diet can significantly affect the levels of these substances in the blood.

The synthesis and utilization of cholesterol must be tightly regulated in order to prevent over-accumulation and abnormal deposition within the body. Of particular importance clinically is the abnormal deposition of cholesterol and cholesterol-rich lipoproteins in the coronary arteries. Such deposition, eventually leading to atherosclerosis, is the leading contributory factor in diseases of the coronary arteries.

Biosynthesis of Cholesterol Slightly less than half of the cholesterol in the body derives from biosynthesis de novo. Biosynthesis in the liver accounts for approximately 10%, and in the intestines approximately 15%, of the amount produced each day. Cholesterol synthesis occurs in the cytoplasm and microsomes from the two-carbon acetate group of acetyl-CoA.

The process has major steps:

1. Acetyl-CoAs are converted to 3-hydroxy- 3-methyl glutaryl-CoA (HMG-CoA) 2. HMG-CoA is converted to mevalonate 3. Mevalonate is converted to the isoprene based molecule, isopentenyl pyrophosphate (IPP), with the concomitant loss of CO2 4. IPP is converted to geranylpyrophosphate 5. geranylpyrophosphate to farnesylpyrophosphate 6. and farnesylpyrophosphate to squalene 7. Squalene is converted to lanosterol and lanosterol to cholesterol

Pathway of cholesterol biosynthesis.

Pathway of cholesterol biosynthesis. . Synthesis begins with the transport of acetyl-CoA from the mitochondrion to the cytosol. The rate limiting step occurs at the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase catalyzed step. The phosphorylation reactions are required to solubilize the isoprenoid intermediates in the pathway. Intermediates in the pathway are used for the synthesis of prenylated proteins, dolichol, coenzyme Q and the side chain of heme a.

The acetyl-CoA utilized for cholesterol biosynthesis is derived from an oxidation reaction ( fatty acids or pyruvate) in the mitochondria and is transported to the cytoplasm . Acetyl-CoA can also be derived from cytoplasmic oxidation of ethanol by acetyl-CoA synthetase. All the reduction reactions of cholesterol biosynthesis use NADPH as a cofactor. The isoprenoid intermediates of cholesterol biosynthesis can be diverted to other synthesis reactions, such as those for dolichol (used in the synthesis of N-linked glycoproteins, coenzyme Q (of the oxidative phosphorylation) pathway or the side chain of heme a. Additionally, these intermediates are used in the lipid modification of some proteins.

Acetyl-CoA units are converted to mevalonate by a series of reactions that begins with the formation of HMG-CoA. Unlike the HMG-CoA formed during ketone body synthesis in the mitochondria, this form is synthesized in the cytoplasm. However, the pathway and the necessary enzymes are the same as those in the mitochondria. Two moles of acetyl-CoA are condensed in a reversal of the thiolase reaction, forming acetoacetyl-CoA. Acetoacetyl-CoA and a third mole of acetyl-CoA are converted to HMG-CoA by the action of HMG-CoA synthase. HMG-CoA is converted to mevalonate by HMG-CoA reductase (this enzyme is bound to the endoplasmic reticulum).

HMG-CoA reductase absolutely requires NADPH as a cofactor and two moles of NADPH are consumed during the conversion of HMG-CoA to mevalonate. The reaction catalyzed by HMG-CoA reductase is the rate limiting step of cholesterol biosynthesis, and this enzyme is subject to complex regulatory controls. Mevalonate is then activated by three successive phosphorylations, yielding 5-pyrophosphomevalonate. In addition to activating mevalonate, the phosphorylations maintain its solubility, since otherwise it is insoluble in water.

. After phosphorylation, an ATP-dependent decarboxylation yields isopentenyl pyrophosphate, IPP, an activated isoprenoid molecule. Isopentenyl pyrophosphate is in equilibrium with its isomer, dimethylallyl pyrophosphate, DMPP. One molecule of IPP condenses with one molecule of DMPP to generate geranyl pyrophosphate, GPP. GPP further condenses with another IPP molecule to yield farnesyl pyrophosphate, FPP. Finally, the NADPH-requiring enzyme, squalene synthase catalyzes the head-to-tail condensation of two molecules of FPP, yielding squalene (squalene synthase also is tightly associated with the endoplasmic reticulum). Squalene undergoes a two step cyclization to yield lanosterol.

The first reaction is catalyzed by squalene monooxygenase. This enzyme uses NADPH as a cofactor to introduce molecular oxygen as an epoxide at the 2,3 position of squalene. Through a series of 19 additional reactions, lanosterol is converted to cholesterol.

Regulating Cholesterol Synthesis The cellular supply of cholesterol is maintained at a steady level by three distinct mechanisms: 1. Regulation of HMG-CoA reductase activity and levels 2. Regulation of excess intracellular free cholesterol through the activity of acyl-CoA:cholesterol acyltransferase, ACAT 3. Regulation of plasma cholesterol levels via LDL receptor-mediated uptake and HDL-mediated reverse transport.

Regulation of HMG-CoA reductase activity is the primary means for controlling the level of cholesterol biosynthesis Regulation of HMG-CoA reductase by covalent modification. HMG-CoA reductase is most active in the dephosphorylated state. Phosphorylation is catalyzed by reductase kinase (RK), an enzymes whose activity is also regulated by phosphorylation. Phosphorylation of RK is catalyzed by reductase kinase kinase (RKK). Hormones such as glucagon and epinephrine negatively affect cholesterol biosynthesis by increasing the activity of the inhibitor of phosphoprotein phosphatase-1, PPI-1. Conversely, insulin stimulates the removal of phosphates and, thereby, activates HMG-CoA reductase activity. Additional regulation of HMG-CoA reductase occurs through an inhibition of synthesis of the enzyme by elevation in intracellular cholesterol levels.

The Utilization of Cholesterol Cholesterol is transported in the plasma predominantly as cholesteryl esters associated with lipoproteins. Dietary cholesterol is transported from the small intestine to the liver within chylomicrons. Cholesterol synthesized by the liver, as well as any dietary cholesterol in the liver that exceeds hepatic needs, is transported in the serum within LDLs. The liver synthesizes VLDLs and these are converted to LDLs through the action of endothelial cell-associated lipoprotein lipase.

. Cholesterol found in plasma membranes can be extracted by HDLs and esterified by the HDL-associated enzyme LCAT. The cholesterol acquired from peripheral tissues by HDLs can then be transferred to VLDLs and LDLs via the action of cholesteryl ester transfer protein (apo-D) which is associated with HDLs. Reverse cholesterol transport allows peripheral cholesterol to be returned to the liver in LDLs. Ultimately, cholesterol is excreted in the bile as free cholesterol or as bile salts following conversion to bile acids in the liver.

Regulation of Cellular Sterol Content The continual alteration of the intracellular sterol content occurs through the regulation of key sterol synthetic enzymes as well as by altering the levels of cell-surface LDL receptors. As cells need more sterol they will induce their synthesis and uptake, conversely when the need declines synthesis and uptake are decreased. Regulation of these events is brought about primarily by sterol-regulated transcription of key rate limiting enzymes and by the regulated degradation of HMGR. Activation of transcriptional control occurs through the regulated cleavage of the membrane-bound transcription factor sterol regulated element binding protein, SREBP.

Sterol control of transcription affects more than 30 genes involved in the biosynthesis of cholesterol, triacylglycerols, phospholipids and fatty acids. Transcriptional control requires the presence of an octamer sequence in the gene termed the sterol regulatory element, SRE-1.

Bile Acids Synthesis and Utilization The end products of cholesterol utilization are the bile acids, synthesized in the liver. Synthesis of bile acids is the predominant mechanisms for the excretion of excess cholesterol. However, the excretion of cholesterol in the form of bile acids is insufficient to compensate for an excess dietary intake of cholesterol.

The most abundant bile acids in human bile are chenodeoxycholic acid (45%) and cholic acid (31%). The carboxyl group of bile acids is conjugated via an amide bond to either glycine or taurine before their secretion into the bile canaliculi.

Clinical Significance of Bile Acid SynthesisBile acids perform four physiologically significant functions: 1. their synthesis and subsequent excretion in the feces represent the only significant mechanism for the elimination of excess cholesterol.2. bile acids and phospholipids solubilize cholesterol in the bile, thereby preventing the precipitation of cholesterol in the gallbladder.3. they facilitate the digestion of dietary triacylglycerols by acting as emulsifying agents that render fats accessible to pancreatic lipases.4. they facilitate the intestinal absorption of fat-soluble vitamins.

These conjugation reactions yield glycocholic acid and taurocholic acid, respectively. The bile canaliculi join with the bile ductules, which then form the bile ducts. Bile acids are carried from the liver through these ducts to the gallbladder, where they are stored for future use. The ultimate fate of bile acids is secretion into the intestine, where they aid in the emulsification of dietary lipids. In the gut the glycine and taurine residues are removed and the bile acids are either excreted (only a small percentage) or reabsorbed by the gut and returned to the liver. This process is termed the enterohepatic circulation.

Obesity and Overweight

Excessive deposits of lipids lead to an obese condition. Extensive blood capillary networks in these deposits mean that they are quite active metabolically. Obesity puts a strain on the heart by causing it to pump blood through extra capillaries. Generally, obesity results from overeating, but a few people have malfunctioning endocrine glands.

Obesity is now recognized as a major risk factor for coronary heart disease, which can lead to heart attack. Some reasons for this higher risk are known, but others are not. For example, obesityraises blood cholesterol and triglyceride levels. lowers HDL "good" cholesterol. HDL cholesterol is linked with lower heart disease and stroke risk, so reducing it tends to raise the risk. raises blood pressure levels. can induce diabetes. In some people, diabetes makes these other risk factors much worse. The danger of heart attack is especially high for these people. . It's a major cause of gallstones and can worsen degenerative joint disease.

AtherosclerosisAtherosclerosis comes from the Greek words athero (meaning gruel or paste) and sclerosis (hardness). It's the name of the process in whichdeposits of fatty substances, cholesterol, cellular waste products, calcium and other substances build up in the inner lining of an artery. This buildup is called plaque. It usually affects large and medium-sized arteries. Some hardening of arteries often occurs when people grow older.

Plaques can grow large enough to significantly reduce the blood's flow through an artery. But most of the damage occurs when theybecome fragile and rupture. Plaques that rupture causeblood clotsto form that can block blood flow or break off and travel to another part of the body. If either happens and blocks a blood vessel that feeds the heart, it causes a heart attack. If it blocks a blood vessel that feeds the brain, it causes a stroke. And if blood supply to the arms or legs is reduced, it can cause difficulty walking and eventually lead to gangrene.

How does atherosclerosis start?Atherosclerosis is a slow, complex disease that typically starts in childhood and often progresses when people grow older. In some people it progresses rapidly, even in their third decade. Many scientists think it begins with damage to the innermost layer of the artery. This layer is called the endothelium .Causes of damage to the arterial wall includeelevated levels of cholesterol and triglyceride in the blood high blood pressure. tobacco smoke diabetes

Because of the damage to the endothelium, fats, cholesterol, platelets, cellular waste products, calcium and other substances are deposited in the artery wall. These may stimulate artery wall cells to produce other substances that result in further buildup of cells.These cells and surrounding material thicken the endothelium significantly. The artery's diameter shrinks and blood flow decreases, reducing the oxygen supply. Often a blood clot forms near this plaque and blocks the artery, stopping the blood flow.