Lipid Metabolism Lecture 15 PDF

1 Lecture 15 Lipid Metabolism  Utilization and Transport of Fat and Cholesterol 2 Overview of intermediary metabolism with fatty acid and triacylglycerol pathways highlighted: 3 Utilization and Transport of Fat and Cholesterol...

1 Lecture 15 Lipid Metabolism  Utilization and Transport of Fat and Cholesterol 2 Overview of intermediary metabolism with fatty acid and triacylglycerol pathways highlighted: 3 Utilization and Transport of Fat and Cholesterol Fat has six times more caloric content by weight than carbohydrate because fat is more highly reduced and is anhydrous.  Utilization and Transport of Fat and Cholesterol 4 Overview of fat digestion, absorption, storage, and mobilization in the human:  Utilization and Transport of Fat and Cholesterol 5 Action of bile salts in emulsifying fats in the intestine: Cholic acid, a typical bile acid, ionizes to give its cognate bile salt. The hydrophobic surface of the bile salt molecule associates with triacylglycerol, and several such complexes aggregate to form a micelle. The polar surface of the bile salts faces outward, allowing the micelle to associate with pancreatic lipase/colipase. Hydrolytic action of this enzyme frees the fatty acids to associate in a much smaller micelle that can be absorbed through the intestinal mucosa. 6 Utilization and Transport of Fat and Cholesterol Lipoproteins are lipid–protein complexes that allow movement of apolar lipids through aqueous environments. Generalized structure of a plasma lipoprotein: The spherical particle, part of which is shown, contains neutral lipids in the interior and phospholipids, cholesterol, and protein at the surface. 7 Utilization and Transport of Fat and Cholesterol 8 Utilization and Transport of Fat and Cholesterol  Utilization and Transport of Fat and Cholesterol 9 Binding of a chylomicron to lipoprotein lipase on the inner surface of a capillary: The chylomicron is anchored by lipoprotein lipase, which is linked by a polysaccharide chain to the lumenal surface of the endothelial cell. When activated by apoprotein C-II, the lipase hydrolyzes the triacylglycerols in the chylomicron, allowing uptake into the cell of the glycerol and the free fatty acids.  Utilization and Transport of Fat and Cholesterol 10 Overview of lipoprotein transport pathways and fates: 11 Utilization and Transport of Fat and Cholesterol A major consequence of liver dysfunction is an inability to synthesize apolipoproteins and, hence, to transport fat out of the liver. Cholesterol accumulation in the blood is correlated with development of atherosclerotic plaque. 12 Utilization and Transport of Fat and Cholesterol HMG-CoA reductase is the rate- controlling enzyme of the mevalonate pathway, the metabolic pathway that produces cholesterol and other isoprenoids. HMGCR catalyzes the conversion of HMG-CoA to mevalonic acid, a necessary step in the biosynthesis of cholesterol. Normally in mammalian cells this enzyme is competitively suppressed so that its effect is controlled. This enzyme is the target of the widely available cholesterol-lowering drugs known collectively as the statins 13 Utilization and Transport of Fat and Cholesterol Cholesterol esters are synthesized in plasma from cholesterol and an acyl chain on phosphatidylcholine (lecithin), through the action of lecithin:cholesterol acyltransferase (LCAT), an enzyme that is secreted from liver into the bloodstream, bound to HDL and LDL. Cholesterol esters are considerably more hydrophobic than cholesterol itself.  Utilization and Transport of Fat and Cholesterol 14 Feedback regulation of HMG-CoA reductase activity: Fibroblasts obtained from a normal subject or from a patient homozygous for familial hypercholesterolemia (FH Homozygote) were grown in monolayer cultures. a)At time zero, the medium was replaced with fresh medium depleted of lipoproteins, and HMG-CoA reductase activity was measured in extracts prepared at the indicated times. a)Twenty-four hours after addition of the lipoprotein- deficient medium, human LDL was added to the cells at the indicated levels, and HMG-CoA reductase activity was measured at the indicated time.  Utilization and Transport of Fat and Cholesterol 15 Uptake of cholesterol from the blood occurs at the LDL receptor via receptor-mediated endocytosis. Intracellular cholesterol regulates its own level by controlling: 1. de novo cholesterol biosynthesis, 2. formation and storage of cholesterol esters 3. LDL receptor density Uptake of oxidized LDL by a scavenger receptor is a key event in atherogenesis. Fat mobilization in adipose cells is hormonally controlled, via the cAMP–dependent phosphorylation of lipolytic enzymes and lipid droplet-associated proteins. 16 Utilization and Transport of Fat and Cholesterol Receptor-mediated endocytosis of LDL: LDL was conjugated with ferritin to permit electron microscopic visualization. a)The LDL–ferritin (dark dots) binds to a coated pit on the surface of a cultured human fibroblast. a)The plasma membrane closes over the coated pit, forming an endocytotic vesicle. 17 Utilization and Transport of Fat and Cholesterol Structure of a clathrin-coated pit: a)Clathrin, the major protein in coated pits, forms triskelions (named after the symbol of three legs radiating from the center), which assemble into polyhedral lattices composed of hexagons and pentagons, such as the barrel shown in the next panel. a)Image reconstruction from electron cryomicroscopy of a clathrin barrel formed from 36 triskelions. A single clathrin triskelion is highlighted in light blue. a)A coated pit on the inner surface of the plasma membrane of a cultured mammalian cell is visualized by freeze-fracture electron microscopy. The cage-like structure of the pit is due to the clathrin lattice.  Utilization and Transport of Fat and Cholesterol 18 Involvement of LDL receptors in cholesterol uptake and metabolism: 19 Utilization and Transport of Fat and Cholesterol Polyunsaturated fat (PUFA) ingestion is correlated with low plasma cholesterol levels. The mechanisms involved are not completely understood.  Utilization and Transport of Fat and Cholesterol 20 Mobilization of adipose cell triacylglycerols by lipolysis: Three lipases act sequentially to hydrolyze TG to glycerol and FFA. These enzymes act at the oil–water interface of the lipid droplet. FFA are exported to the blood plasma, where they are bound to albumin for transport to liver and other tissues for subsequent oxidation. Glycerol is released to the blood to be taken up by liver cells, where it serves as a gluconeogenic substrate.  Utilization and Transport of Fat and Cholesterol 21 Control of lipolysis in adipose cells by a cAMP- mediated cascade system: Hormonal activation of a b–adrenergic G-protein coupled receptor on the plasma membrane leads to elevation of cAMP levels, which in turn, activates protein kinase A (PKA). PKA phosphorylates perilipin (PL) and HSL. 1.CGI-58 dissociates from phosphorylated-PL, and binds ATGL. 1.Phosphorylated HSL is recruited to the lipid droplet and activated by phosphorylated-PL. 1.Phosphorylated-PL also recruits the ATGL/CGI-58 complex to the lipid droplet, activating this lipase. 1.Activated ATGL hydrolyzes TG to activated HSL hydrolyzes DG to cytoplasmic MGL hydrolyzes MG to free glycerol + FFA.  Fatty Acid Oxidation 22 Knoop’s experiments to determine the b- oxidation of fatty acids: When dogs fed fatty acids that had an even- numbered carbon chain, the final breakdown product, recovered from urine, was phenylacetic acid. When the fed fatty acid had an odd-numbered chain, the product was benzoic acid. These results led Knoop to propose that fatty acids are oxidized in a stepwise fashion, with initial attack on carbon 3 (the b-carbon with respect to the carboxyl group). This attack would release the terminal two carbons, and the remainder of the fatty acid molecule could undergo another oxidation.  Fatty Acid Oxidation 23 Fatty acids are activated for oxidation by ATP-dependent acylation of coenzyme A. The loss of pyrophosphate is equivalent to 2 ATP’s used for activation. The further hydrolysis of pyrophosphate makes the activation step irreversible.  Fatty Acid Oxidation 24 Overview of the fatty acid oxidation pathway: 25 Fatty Acid Oxidation Mechanism of acyl-CoA synthetase reactions: The figure shows reversible formation of the activated fatty acyl adenylate, nucleophilic attack by the thiol sulfur of CoA-SH on the activated carboxyl group, and the quasi-irreversible pyrophosphatase reaction, which draws the overall reaction toward fatty acyl-CoA. 26 Fatty Acid Oxidation Carnitine transports acyl-CoAs into mitochondria for oxidation.  Fatty Acid Oxidation 27 The carnitine acyltransferase system, for transport of fatty acyl-CoAs into mitochondria: 28 Fatty Acid Oxidation Outline of the b-oxidation of fatty acids: In the diagram a 16-carbon saturated fatty acyl-CoA (palmitoyl-CoA) undergoes seven cycles of oxidation to yield eight molecules of acetyl-CoA.  Fatty Acid Oxidation 29 30 Fatty Acid Oxidation Fatty acids are oxidized by repeated cycles of dehydrogenation, hydration, dehydrogenation, and thiolytic cleavage, with each cycle yielding acetyl-CoA and a fatty acyl-CoA shorter by two carbons than the input acyl-CoA. 31 Fatty Acid Oxidation Energy Yield from Fatty Acid Oxidation: 32 Fatty Acid Oxidation Two enzymes, enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase, play essential roles in the oxidation of unsaturated fatty acids. 33 Fatty Acid Oxidation b-Oxidation pathway for polyunsaturated fatty acids: This example, using linoleyl-CoA, shows sites of action of enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase, enzymes specific to unsaturated fatty acid oxidation (identified with red type). 34 Fatty Acid Oxidation Odd-numbered fatty acid chains yield upon b-oxidation 1 mole of propionyl-CoA, whose conversion to succinyl- CoA involves a biotin-dependent carboxylation and a coenzyme B12–dependent rearrangement. 35 Fatty Acid Oxidation Pathway for catabolism of propionyl-CoA:  Fatty Acid Oxidation 36  Fatty Acid Oxidation During fasting or starvation, when carbohydrate intake is too low, oxaloacetate levels fall 37 so that flux through citrate synthase is impaired, causing acetyl-CoA levels to rise. Under these conditions, 2 moles of acetyl-CoA undergo a reversal of the thiolase reaction to give acetoacetyl-CoA. Acetoacetyl-CoA can react in turn with a 3rd mole of acetyl-CoA to give 3-hydroxy-3- methylglutaryl-CoA (HMG-CoA), catalyzed by HMG-CoA synthase. In the cytosol, HMG-CoA is an early intermediate in cholesterol biosynthesis. In the mitochondria, HMG-CoA is acted on by HMG-CoA lyase to yield acetoacetate plus acetyl-CoA. Acetoacetate undergoes NADH-dependent reduction to give D-b-hydroxybutyrate or spontaneous decarboxylation to acetone. Collectively, acetoacetate, acetone, and b-hydroxybutyrate are called ketone bodies. 38 Fatty Acid Oxidation Biosynthesis of ketone bodies in the liver: 39 Fatty Acid Biosynthesis When carbohydrate catabolism is limited, acetyl-CoA is converted to ketone bodies, mainly acetoacetate and b- hydroxybutyrate—important metabolic fuels in some circumstances. Animals readily convert carbohydrate to fat, but cannot carry out net conversion of fat to carbohydrate. Fatty acid synthesis occurs through intermediates similar to those of fatty acid oxidation, but with differences in electron carriers, carboxyl group activation, stereochemistry, and cellular location.  Fatty Acid Biosynthesis 40 Acetyl-CoA as a key intermediate between fat and carbohydrate metabolism: Citrate serves as a carrier to transport acetyl units from the mitochondrion to the cytosol for fatty acid synthesis.  Fatty Acid Biosynthesis 41 Biosynthesis of Palmitate from Acetyl-CoA: Synthesis of malonyl-CoA is the first committed step in fatty acid biosynthesis from acetyl-CoA and bicarbonate, catalyzed by acetyl- CoA carboxylase (ACC).  Fatty Acid Biosynthesis 42 Chemical similarities between oxidation and synthesis of a fatty acid:  Fatty Acid Biosynthesis 43 Structure and swinging arm mechanism in the mammalian fatty acid synthase complex: 44 Fatty Acid Biosynthesis Malonyl-CoA represents an activated source of two-carbon fragments for fatty acid biosynthesis, with the loss of CO2 driving C-C bond formation. In eukaryotes, fatty acid synthesis is carried out by a megasynthase, an organized multienzyme complex that contains multifunctional proteins.  Fatty Acid Biosynthesis 45 Transport of acetyl units and reducing equivalents used in fatty acid synthesis: Citrate serves as a carrier of two-carbon fragments from mitochondria to cytosol for fatty acid biosynthesis.  Fatty Acid Biosynthesis 46 Fatty acid desaturation system: The black arrows indicate the path of electron flow as the two substrates are oxidized. D5 and D6 desaturases use the same mechanism.  Fatty Acid Biosynthesis 47 Pathway for synthesis of polyunsaturated fatty acids (PUFAs) in plants and animals:  Fatty Acid Biosynthesis 48 Regulation of fatty acid synthesis in animal cells: The rate-limiting enzyme, acetyl-CoA carboxylase (ACC), is controlled by both allosteric (citrate and long- chain fatty acids) and covalent modification mechanisms. Phosphorylation by AMP-activated protein kinase (AMPK) or cyclic AMP–dependent protein kinase (PKA) inactivates ACC. Insulin stimulates fatty acid synthesis by increasing glucose uptake and increasing flux through pyruvate dehydrogenase to produce acetyl-CoA. The dephosphorylated form of PDH is the enzymatically active form.  Fatty Acid Biosynthesis 49 A related series of pathways in bacteria and fungi is involved in the biosynthesis of a class of antibiotics called polyketides. Examples include Erythromycin, from Saccharopolyspora erythraea, and oxytetracycline, from Streptomyces rimosus. These polyketide antibiotics are potent inhibitors of bacterial protein synthesis. Other polyketides, such as lovastatin and simvastatin have found clinical use as cholesterol- lowering drugs by inhibiting HMG-CoA reductase. Polyketides are synthesized in assembly-line fashion by giant enzyme megasynthases that consist of individual modules for rounds of carbon addition, with each module closely resembling the process whereby two carbons are added in a cycle of the fatty acid synthesis pathway.  Interdependence of the Major Organs 50 in Vertebrate Fuel Metabolism Metabolite concentrations represent a significant intracellular control mechanism. The major fuel depots are triacylglycerols (adipose tissue), protein (muscle), and glycogen (muscle and liver).  Interdependence of the Major Organs 51 in Vertebrate Fuel Metabolism Metabolic interactions among the major fuel-metabolizing organs:  Interdependence of the Major Organs 52 in Vertebrate Fuel Metabolism 53 Hormonal Regulation of Fuel Metabolism  Hormonal Regulation of Fuel Metabolism 54 One of the most important roles of liver is to serve as a “glucostat” monitoring and stabilizing blood glucose levels. Maintenance of blood glucose within narrow limits is critical to brain function. The key hormones regulating fuel metabolism are insulin, which promotes glucose use, and glucagon and epinephrine, which increase blood glucose. 55 Hormonal Regulation of Fuel Metabolism Actions of glucagon in liver that lead to a rise in blood glucose:  Responses to Metabolic Stress: 56 Starvation, Diabetes Major events in the storage, retrieval, and use of fuels in the fed and unfed states and in early starvation:  Responses to Metabolic Stress: 57 Starvation, Diabetes Metabolic adaptations promote alternative fuel use during starvation so that glucose homeostasis is maintained for several weeks.  Responses to Metabolic Stress: 58 Starvation, Diabetes Diabetes results either from insulin deficiency or from defects in the insulin response mechanism. Two related mechanisms have been proposed as causes of type 2 diabetes: the lipid overload hypothesis and the inflammation hypothesis. Diabetes can be thought of as "starvation in the midst of plenty" because cells are unable to utilize the glucose that accumulates in the blood.  Responses to Metabolic Stress: 59 Starvation, Diabetes The metabolic abnormalities in diabetes: The insulin deficiency blocks the uptake of glucose into muscle and adipose tissue and reduces glucose catabolism in all tissues. Proteolysis in muscle and lipolysis in adipose tissue are enhanced. In the liver, gluconeogenesis from amino acids and citric acid cycle intermediates is stimulated as the cells attempt to remedy the perceived lack of usable glucose, and fatty acid oxidation and ketogenesis are also increased.  Steroid Metabolism 60 We turn now to an extraordinarily large and diverse group of lipids, the isoprenoids, or terpenes. These compounds are built up from one or more five- carbon activated derivatives of isoprene. The family includes: o Steroids o Bile acids o Lipid-soluble vitamins o Dolichol and undecaprenol phosphates o Phytol o Gibberellins o Insect juvenile hormones o Components of rubber o Coenzyme Q o And many more compounds  Steroid Metabolism 61 Ring identification system (a) Carbon numbering system (b) used for steroids. Structural conventions (c), with cholestanol as the example. a-Substituents project below the plane of the steroid ring system (blue dashed wedge), and b-substituents project above that plane (red solid wedge). The hydrogens at positions 5, 9, and 14 have the a-configuration, whereas the hydroxyl, the two methyl groups, the hydrogen at C-8, and the aliphatic side chain at C-17 are all b-substituents. 62 Steroid Metabolism Cholesterol, the precursor to all steroids, derives all of its carbon atoms from acetate. 63 Steroid Metabolism The five carbons of isoprene could be derived metabolically from three molecules of acetate, and the prediction that cholesterol was a product of the cyclization of the linear hydrocarbon squalene. Squalene contains six isoprene units (delineated by red marks on the structures below), and its configuration makes it a plausible steroid precursor. 64 Steroid Metabolism Cholesterol biosynthesis can be considered as three distinct processes: 1.Conversion of C2 fragments (acetate) to a C6 isoprenoid precursor (mevalonate). 2. Conversion of six C6 mevalonates, via activated intermediates, to the C30 squalene. 3. Cyclization of squalene and its transformation to the C27 cholesterol. 65 Biosynthesis of mevalonate and conversion to isopentenyl pyrophosphate and dimethylallyl pyrophosphate: The two carbons of the third acetyl group are shown in red.  Steroid Metabolism 66 Stage 2: Synthesis of Squalene from Mevalonate The next several reactions occur in the cytosol. First, mevalonate is activated by three successive phosphorylations. The first two are simple nucleophilic substitutions on the g-phosphorous of ATP. The third phosphorylation, at position 3, sets the stage for a decarboxylation to give the five-carbon isopentenyl pyrophosphate  Steroid Metabolism 67 Conversion of farnesyl pyrophosphate to squalene: Catalyzed by squalene synthase. Dissociation of the PPi group on one of the molecules of farnesyl-PPi yields an allylic carbocation. The double bond nucleophilically attacks the carbocation, forming a tertiary cation at C3 of the first farnesyl-PPi. Loss of a proton from C1 gives the activated cyclopropane intermediate, presqualene-PPi. Dissociation of the second pyrophosphate leads to rearrangement and formation of another tertiary carbocation intermediate. Hydride transfer from NADPH completes the rearrangement, giving squalene.  Steroid Metabolism 68  Steroid Metabolism 69 Regulation of HMG-CoA reductase by ubiquitinmediated proteolysis: Sterol binding leads to rapid degradation of the enzyme.  Steroid Metabolism 70 Once the rate-limiting role of HMG-CoA reductase in cholesterol biosynthesis was understood, specific inhibitors were sought to lower blood cholesterol levels. The compounds discovered are collectively called statins. They act by competitively inhibiting HMG-CoA reductase. Lovastatin and simvastatin are fungal polyketides and atorvastatin (Lipitor®) is synthetic. Each statin carries a mevalonate-like moiety (blue), explaining the competitive nature of their activity. Inhibition of HMG-CoA reductase depresses de novo cholesterol biosynthesis and, hence, intracellular cholesterol levels. This in turn leads to increased production of LDL receptors, allowing more rapid clearance of extracellular cholesterol from the blood, thus lowering blood cholesterol levels. 71 Steroid Metabolism Diethylstilbestrol, a synthetic estrogen, was widely used to promote growth of beef cattle, until it was found to be potentially carcinogenic at the levels found in meat from treated cattle.  Other Isoprenoid Compounds 72 There are three active forms of vitamin A: all-trans- retinol, -retinal, and –retinoic acid. Collectively, these are referred to as retinoids. The vitamin can be either consumed in the diet as esterified retinol, or biosynthesized from b-carotene, a plant isoprenoid especially abundant in carrots. b-carotene is cleaved in the intestine by a monooxygenase to form two molecules of all-trans- retinal (retinaldehyde), which are then reduced to retinol. All-trans-retinol is the form which circulates in the blood and which has the highest biological activity.  Other Isoprenoid Compounds 73 Structures of other important isoprenoids:  Other Isoprenoid Compounds 74 Some terpene compounds: These examples are representative of an enormous class of natural products.  Eicosanoids: Prostaglandins, 75 Thromboxanes, and Leukotrienes Structures of the major prostaglandins and thromboxane A2: The most abundant prostaglandins are those of the 2-series are shown. They are derived from arachidonic acid, as is thromboxane A2. Numbering of carbons begins with the carboxyl group as shown for the structure of PGG2.  Eicosanoids: Prostaglandins, 76 Thromboxanes, and Leukotrienes COX-1 is constitutively expressed in most tissues, and is responsible for the physiological production of prostaglandins. COX-2 is induced by cytokines, mitogens, and endotoxins in inflammatory cells and is responsible for the elevated production of prostaglandins during inflammation. Both isoforms are covalently modified, and hence inactivated, by reaction with aspirin (acetylsalicylic acid). As shown, aspirin acetylates a specific serine residue, which in turn blocks access of the fatty acid substrate to the cyclooxygenase active site.  Eicosanoids: Prostaglandins, 77 Thromboxanes, and Leukotrienes Nonsteroidal anti-inflammatory drugs (NSAIDs): Ibuprofen and naproxen are examples of nonselective COX Inhibitors. Rofecoxib (Vioxx®) and celecoxib (Celebrex®) are selective COX-2 inhibitors. o The phenylsulphonamide moieties, which contribute to their selectivity, are highlighted in red.  Eicosanoids: Prostaglandins, 78 Thromboxanes, and Leukotrienes Biosynthesis of leukotrienes: Leukotriene C was originally discovered in the class of white blood cells called polymorphonuclear leukocytes (PMNs) and was named after the source (leukocytes) and the triene structure. It is a potent muscle contractant that is involved in the pathogenesis of asthma, through constriction of the small airways in the lung. LTs are formed from the initial attack on arachidonate of a lipoxygenase, which adds O2 to C-5, giving 5-HPETE. A dehydration to give the epoxide coupled with isomerization of double bonds gives leukotriene A4. Hydrolysis of the epoxide ring yields leukotriene B4. Transfer of the thiol group of glutathione yields leukotriene C4. Subsequent modifications of the peptide chain yield related compounds, leukotrienes D and E.