Lipid Metabolism

How does β-oxidation metabolize fatty acids to generate ATP?

• Fatty acids destined for mitochondrial degradation are activated by coenzyme A on the cytosolic side of the outer mitochondrial membrane and then transported into the mitochondrial matrix by the carnitine transport cycle.

• Fatty acyl-CoA synthetases activate short-, medium-, and long-chain fatty acids in the cytosol by using an ATP-dependent reaction involving formation of a fatty acyl-adenylate intermediate. The thermodynamic favorability of this reaction is enhanced by pyrophosphate hydrolysis.

• The carnitine transport cycle provides a mechanism to control the flux of fatty acids either into the degradative pathway inside the mitochondrial matrix or toward the synthesis of triacylglycerols and membrane lipids in the cytosol. It also serves to maintain separate pools of coenzyme A in the cytosol and mitochondrial matrix.

• Fatty acid degradation converts redox energy available in electron-rich fatty acids into ATP by producing acetyl-CoA, which is oxidized by the citrate cycle, as well as FADH₂ and NADH, which are oxidized by the electron transport system.

• The β-oxidation pathway of fatty acid degradation involves the sequential thiolytic cleavage of fatty acyl-CoA substrates at the β carbon of the fatty acid, releasing acetyl-CoA and a fatty acyl-CoA product that is two carbons (C2) shorter.

• Each cycle of the β-oxidation pathway involves four reactions: (1) an oxidation catalyzed by acyl-CoA dehydrogenase, (2) a hydration catalyzed by enoyl-CoA hydratase, (3) an oxidation catalyzed by 3-hydroxyacyl-CoA dehydrogenase, and (4) a thiolysis catalyzed by β-ketoacyl-CoA thiolase.

• Hypoglycin A is a toxin present in unripe ackee fruit and is metabolized in liver mitochondria to produce methylenecyclopropylacetyl-CoA (MCPA-CoA), a potent inhibitor of acyl-CoA dehydrogenases and fatty acid oxidation.

• Degradation of the fully saturated C16 fatty acid palmitate by the β-oxidation pathway generates 8 acetyl-CoA, 7 FADH₂, and 7 NADH, which are then oxidized by the citrate cycle and electron transport system in the mitochondrial matrix. The overall yield is 106 net ATP per palmitate after subtracting the 2 ATP equivalents invested in the cytosolic acyl-CoA synthetase reaction.

• Oxidation of unsaturated fatty acids requires that cis C=C bonds be converted to trans C=C bonds by an isomerase reaction in order for enzymes in the β-oxidation pathway to completely degrade the unsaturated fatty acid to acetyl-CoA.

• Oxidation of odd-numbered fatty acids such as tricosanoate (23:0) results in the production of propionyl-CoA, which is converted to succinyl-CoA and used by the citrate cycle to generate malate. If the energy charge in the cell is low, the malate can be converted to pyruvate by cytosolic malic enzyme, and the pyruvate can be oxidized.

• Animal cell peroxisomes carry out β-oxidation of saturated very-long-chain fatty acids such as hexacosanoate (26:0). This process does not result in energy conversion because peroxisomes lack the necessary enzymes, but it does produce acetyl-CoA for cholesterol biosynthesis in the cytosol.

• X-linked adrenoleukodystrophy (X-ALD) is caused by a defect in the peroxisomal adrenoleukodystrophy protein (ALDP), which is required for peroxisomal import of saturated very-long-chain fatty acids. Elevated levels of serum very-long-chain fatty acids in individuals with X-ALD causes destruction of neuronal myelin sheaths.

How does the liver produce ketone bodies during carbohydrate starvation?

• When carbohydrate sources are limited, ongoing β-oxidation in liver cell mitochondria results in the buildup of excess acetyl-CoA, which is converted to acetoacetate and D-β-hydroxybutyrate in the ketogenic pathway.

• Acetoacetate and D-β-hydroxybutyrate are exported from the liver and used by skeletal and heart muscle to generate acetyl-CoA for energy conversion reactions. The brain, which prefers glucose as an energy source, can adapt to use of ketone bodies as chemical energy during times of extreme starvation.

• Ketoacidosis is a condition caused by low blood pH that occurs when ketogenesis produces more acetoacetate and D-β-hydroxybutyrate than can be used by the peripheral tissues. Ketoacidosis is associated with hypoglycemia, delirium, nausea, vomiting, and a fruity odor on the breath from high acetone levels in the blood.

How is palmitate synthesized from acetyl-CoA?

• The liver synthesizes triacylglycerols from fatty acids when glucose levels are high and the amount of acetyl-CoA produced exceeds the energy requirements of the cell. Enzymatic reactions linking glycolysis, the citrate shuttle, and fatty acid synthesis efficiently convert excess carbohydrates into stored fat in adipose tissue.

• The citrate shuttle provides a mechanism to stimulate fatty acid synthesis in the cytosol when acetyl-CoA accumulates in the mitochondrial matrix. Citrate export to the cytosol is balanced by malate and pyruvate import, thereby maintaining a steady supply of carbohydrate-derived C2 acetate units for fatty acid synthesis.

• Fatty acid degradation and fatty acid synthesis both require a four-step reaction cycle, and each pathway involves the removal or addition of C2 units attached to coenzyme A.

• Malonyl-CoA is the activated substrate in fatty acid synthesis and is produced by the rate-limiting enzyme acetyl-CoA carboxylase. This is a biotin-dependent enzyme that has three functional activities: a biotin carboxylase, a biotin carboxyl carrier, and a carboxyltransferase.

• The acyl carrier protein (ACP) subunit in fatty acid synthase requires an enzyme-linked phosphopantetheine coenzyme group derived from vitamin B5 (pantothenate), which provides a sulfhydryl attachment site for the growing hydrocarbon chain.

• The first step in the fatty acid synthesis pathway requires malonyl/acetyl-CoA ACP transacylase (MAT) to catalyze the priming reaction, which transfers the acetyl group from acetyl-CoA to the sulfhydryl group in ACP. This acetyl group primer is then linked to a cysteine residue in the KS domain to free up the phosphopantetheine group on ACP for the first malonyl group.

• Each cycle of the fatty acid synthesis pathway involves four reactions: (1) condensation catalyzed by β-ketoacyl-ACP synthase (KS), (2) reduction catalyzed by β-ketoacyl-ACP reductase (KR), (3) dehydration catalyzed by β-hydroxyacyl-ACP dehydratase (DH), and (4) reduction catalyzed by enoyl-ACP reductase (ER).

• The mammalian fatty acid synthase enzyme is a homodimer encoding all seven protein functions in each monomer subunit on a single polypeptide chain. The upper lobe of the fatty acid synthase complex contains the KR, DH, and ER catalytic sites responsible for the modifying functions, whereas the lower lobe contains the condensing functions encoded by the KS and MAT catalytic sites.

• The primary product of fatty acid synthesis is palmitate, which is used as a substrate for elongation and desaturation reactions localized to the endoplasmic reticulum (ER) in animal cells. The elongation reactions use coenzyme A as the carrier molecule rather than acyl carrier protein. The desaturating enzymes use molecular oxygen (O₂) as the oxidant and are called mixed-function oxidases.

How are triacylglycerols and membrane lipids synthesized?

• Triacylglycerols and glycerophospholipids are derived from diacylglycerol-3-phosphate, also called phosphatidic acid, whereas sphingolipids are derived from ceramide.

• Membrane lipids are synthesized by enzymes associated with the smooth endoplasmic reticulum and must be transported to various membrane targets in the cell.

• The most abundant membrane lipids in cells are glycerophospholipids, which include phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. Phosphatidic acid is the building block for all three of these glycerophospholipids.

• Attachment of a phosphocholine head group to ceramide leads to the production of sphingomyelin. Ceramide can also be modified by the addition of monosaccharides, such as glucose, via the enzyme ceramide glucosyltransferase to generate the membrane lipid glucocerebroside.

How does regulation of acetyl-CoA carboxylase control fatty acid synthesis ?

• The primary control point for regulating flux through the fatty acid biosynthetic pathway is modulation of acetyl-CoA carboxylase activity. The activity of acetyl-CoA carboxylase is controlled by both allosteric mechanisms (metabolic control) and covalent modification (hormonal control).

• Metabolic regulation of acetyl-CoA carboxylase activity is mediated by citrate and palmitoyl-CoA, which are allosteric regulators that bind to the enzyme and alter the equilibrium between polymerization (active) and depolymerization (inactive).

• Insulin stimulates dephosphorylation and polymerization of acetyl-CoA carboxylase through activation of protein phosphatase 2A, whereas glucagon stimulates phosphorylation and depolymerization of acetyl-CoA carboxylase through activation of AMP-activated protein kinase (AMPK).

• Citrate binding to phosphorylated acetyl-CoA carboxylase partially activates the enzyme by stimulating polymerization in the absence of insulin signaling.

• Low energy charge activates AMPK through AMP binding, leading to AMPK-mediated phosphorylation and inactivation of acetyl-CoA carboxylase, thus decreasing flux through the fatty acid synthesis pathway.

• High glucose levels stimulate insulin signaling, which leads to dephosphorylation and inactivation of AMPK, resulting in increased flux through the fatty acid synthesis pathway.

• Three metabolic control mechanisms regulate flux through the fatty acid synthesis pathway: (1) citrate export from the mitochondrial matrix activates acetyl-CoA carboxylase activity, (2) malonyl-CoA inhibits carnitine acyltransferase I activity to prevent mitochondrial import of fatty acyl-CoA molecules, and (3) palmitoyl-CoA inhibits acetyl-CoA carboxylase activity to decrease fatty acid synthesis.

How is cholesterol synthesized and transported through the body?

• Cholesterol has a critical role in the function of animal cell membranes and as a precursor of cell signaling molecules, but it is also a contributing factor to cardiovascular disease.

• Cholesterol synthesis occurs in all cells, with the largest amounts made in the liver. Because of this de novo cholesterol biosynthetic pathway, cholesterol is not an essential lipid in the diet.

• De novo cholesterol biosynthesis consists of four stages: (1) synthesis of mevalonate, a C6 compound, which is generated by the rate-limiting enzyme HMG-CoA reductase; (2) conversion of mevalonate to isopentenyl pyrophosphate (C5) and dimethylallyl pyrophosphate; (3) formation of squalene (C30) from isopentenyl pyrophosphate and dimethylallyl pyrophosphate; and (4) squalene cyclization to form cholesterol (C27).

• Liver cholesterol has three metabolic fates: (1) it is stored as cholesteryl esters in lipid droplets; (2) it is packaged into lipoproteins and exported to the circulatory system; and (3) it is converted into bile acids, which aid in digestion of fatty foods.

• Most of the bile acid secreted into the small intestine is returned to the liver and reused; however, some of it is excreted as waste, which provides the only mechanism to rid the body of excess cholesterol.

• Ingestion of insoluble bile acid resins can be used to decrease serum cholesterol by depleting the body of bile acids and thereby shunting more cholesterol toward bile acid synthesis.

• Steady-state levels of circulating cholesterol are determined by the balance of cholesterol input (diet and de novo biosynthesis), cholesterol recycling (returning tissue cholesterol to the liver), and cholesterol output (excretion of bile acids).

• Triacylglycerols and cholesterol are transported through the circulatory system as components of plasma lipoprotein particles, which are membrane-bound vesicles containing a hydrophobic core and apolipoproteins. The sizes of lipoproteins decrease as their lipid cargo is transferred to endothelial cells during circulation.

• There are five major classes of lipoproteins, which differ in density depending on their size and the ratio of protein to lipid: (1) chylomicrons contain large amounts of dietary triacylglycerols obtained in the small intestine, (2) very-low-density lipoproteins (VLDLs) are synthesized in the liver and transport fatty acids in the form of triacylglycerols, (3) intermediate-density lipoproteins (IDLs) are VLDL remnants, (4) low-density lipoproteins (LDLs) transport large amounts of cholesteryl esters and are known as “bad cholesterol,” and (5) high-density lipoproteins (HDLs) are cholesterol scavengers and are known as “good cholesterol.”

• HDL particles function in reverse cholesterol transport by removing cholesterol from peripheral tissues through activation of serum lecithin–cholesterol acyltransferase, which generates cholesteryl esters that are taken back to the liver by HDL.

• Cardiovascular disease is characterized by the buildup of fibrous tissue and cholesterol deposits in arterial walls, a condition called atherosclerosis. Atherosclerotic plaques can rupture, resulting in myocardial infarction (heart attack) or stroke (blockage of the carotid artery to the brain).

How do statin drugs and PCSK9 inhibitors lower serum LDL cholesterol levels?

• In addition to de novo cholesterol biosynthesis, liver cholesterol is also obtained by endocytosis of serum LDL, which is initiated by LDL binding to LDL receptors on the cell surface. Low levels of intracellular liver cholesterol stimulate LDL receptor expression, leading to increased LDL endocytosis and decreased serum LDL levels.

• Inhibition of HMG-CoA reductase activity by statin drugs such as simvastatin decreases liver cholesterol and induces LDL receptor expression through proteolytic activation of sterol regulatory element binding proteins (SREBPs). Statin drugs therefore decrease the risk of cardiovascular disease by lowering serum LDL levels.

• Ezetimibe blocks cholesterol transport in the small intestine, which reduces serum LDL levels by decreasing uptake of dietary cholesterol.

• The proprotein convertase subtilisin/kexin type 9 (PCSK9) protein increases the rate at which LDL receptors are degraded in liver cells, which lowers the amount of LDL receptor on the cell surface and thereby leads to increased serum LDL levels. Antibody therapy to block PCSK9 function is an effective treatment to lower serum LDL in individuals who cannot tolerate statin drugs owing to their side effects.

• SREBPs are embedded in the ER membrane as inactive precursors containing three functional domains: (1) an N-terminal DNA binding domain; (2) a membrane anchoring domain; and (3) a C-terminal regulatory domain, which interacts with a cholesterol-sensing protein embedded in the ER membrane.

• Low cholesterol levels in liver cells disrupt the association of SREBPs with two SREBP regulatory proteins in the ER membrane called SCAP and INSIG.

• Cleavage of precursor SREBPs by proteolytic enzymes in the Golgi apparatus releases the N-terminal transcriptional regulatory domain, which then enters the nucleus. It induces expression of genes that modulate enzymes of the cholesterol biosynthetic pathway.

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