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A sulfate group from the sulfate carrier 3ʹ-phosphoadenosine-5ʹphosphosulfate ([PAPS], Fig. 17.16) is added by a sulfotransferase to the 3ʹ-hydroxyl group of the galactose in a galactocerebroside, forming the sulfatide galactocerebroside 3-sulfate (Fig. 17.17). [Note: PAPS is also the sulfur donor in glycosaminoglycan synthesis (see p. 162) and steroid hormone catabolism (see p. 240).] An overview of the synthesis of sphingolipids is shown in Figure 17.18.
C. Glycosphingolipid degradation
Glycosphingolipids are internalized by phagocytosis as described for the glycosaminoglycans (see p. 163). All of the enzymes required for the degradative process are present in lysosomes, which fuse with the phagosomes. The lysosomal enzymes hydrolytically and irreversibly cleave specific bonds in the glycosphingolipid. As seen with the glycosaminoglycans and glycoproteins (see p. 170), degradation is a sequential process following the rule “last on, first off,” in which the last group added during synthesis is the first group removed in degradation. Therefore, defects in the degradation of the polysaccharide chains in these three glycoconjugates result in lysosomal storage diseases.
D. Sphingolipidoses
In a normal individual, synthesis and degradation of glycosphingolipids are balanced, so that the amount of these compounds present in membranes is constant. If a specific lysosomal acid hydrolase required for degradation is partially or totally missing, a sphingolipid accumulates. Lysosomal lipid storage diseases caused by these deficiencies are called sphingolipidoses. The result of a specific acid hydrolase deficiency may be seen dramatically in nerve tissue, where neurologic deterioration can lead to early death. Figure 17.20 provides an outline of the pathway of sphingolipid degradation and descriptions of some sphingolipidoses. [Note: Some sphingolipidoses can also result from defects in lysosomal activator proteins (for example, the saposins) that facilitate access of the hydrolases to short carbohydrate chains as degradation proceeds.] 1.
Common properties: A specific lysosomal hydrolytic enzyme is deficient in the classic form of each disorder. Therefore, usually, only a single sphingolipid (the substrate for the deficient enzyme) accumulates in the involved organs in each disease. [Note: The rate of biosynthesis of the accumulating lipid is normal.] The disorders are progressive and, although many are fatal in childhood, extensive phenotypic variability is seen leading to the designation of different clinical types, such as types A and B in Niemann-Pick disease. Genetic variability is also seen because a given disorder can be caused by any one of a variety of mutations within a single gene. The sphingolipidoses are autosomal-recessive disorders, except for Fabry disease, which is X linked. The incidence of the sphingolipidoses is low in most populations, except for Gaucher and Tay-Sachs diseases, which, like Niemann-Pick disease, show a high frequency in the Ashkenazi Jewish population. [Note: Tay-Sachs also has a high frequency in Irish American, French Canadian, and Louisiana Cajun populations.] 2.
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Diagnosis and treatment: A specific sphingolipidosis can be diagnosed by measuring enzyme activity in cultured fibroblasts or peripheral leukocytes or by analyzing DNA (see Chapter 34). Histologic examination of the affected tissue is also useful. [Note: Shell-like inclusion bodies are seen in Tay-Sachs, and a crumpled tissue paper appearance of the cytosol is seen in Gaucher disease (Fig. 17.19).] Prenatal diagnosis, using cultured amniocytes or chorionic villi, is available. Gaucher disease, in which macrophages become engorged with glucocerebroside, and Fabry disease, in which globosides accumulate in the vascular endothelial lysosomes of the brain, heart, kidneys, and skin, are treated by recombinant human enzyme replacement therapy, but the monetary cost is extremely high. Gaucher has also been treated by bone marrow transplantation (because macrophages are derived from hematopoietic stem cells) and by substrate reduction therapy through pharmacologic reduction of glucosylceramide, the substrate for the deficient enzyme.
VIII. EICOSANOIDS: PROSTAGLANDINS, THROMBOXANES, AND LEUKOTRIENES
Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT) are collectively known as eicosanoids to reflect their origin from ω-3 and ω-6 polyunsaturated FA with 20 carbons (eicosa = 20). They are extremely potent compounds that elicit a wide range of responses, both physiologic (inflammatory response) and pathologic (hypersensitivity). They insure gastric integrity and renal function, regulate smooth muscle contraction (the intestine and uterus are key sites) and blood vessel diameter, and maintain platelet homeostasis. Although they have been compared to hormones in terms of their actions, eicosanoids differ from endocrine hormones in that they are produced in very small amounts in almost all tissues rather than in specialized glands and act locally rather than after transport in the blood to distant sites. Eicosanoids are not stored, and they have an extremely short half-life, being rapidly metabolized to inactive products. Their biologic actions are mediated by plasma membrane GPCR (see p. 94), which are different in different organ systems and typically result in changes in cyclic adenosine monophosphate production. Examples of eicosanoid structures are shown in Figure 17.21.
life. Cer = ceramide; Gal = galactose; Glc = glucose; GalNAc = Nacetylgalactosamine; NANA = N-acetylneuraminic acid; CNS = central nervous system. = sulfate; ERT = enzyme replacement therapy.
known as prostacyclin. Thromboxanes are designated by TX and leukotrienes by LT.]
A. Prostaglandin and thromboxane synthesis
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Arachidonic acid, an ω-6 FA containing 20 carbons and four double bonds (an eicosatetraenoic FA), is the immediate precursor of the predominant type of human PG (series 2 or those with two double bonds, as shown in Fig. 17.22). It is derived by the elongation and desaturation of the essential FA linoleic acid, also an ω-6 FA. Arachidonic acid is incorporated into membrane phospholipids (typically PI) at carbon 2, from which it is released by phospholipase A2 (Fig. 17.23) in response to a variety of signals, such as a rise in calcium. [Note: Series 1 PG contain one double bond and are derived from an ω-6 eicosatrienoic FA, dihomo-γ-linolenic acid, whereas series 3 PG contain three double bonds and are derived from eicosapentaenoic acid (EPA), an ω-3 FA. See p. 363.] activities (cyclooxygenase and peroxidase) of PGH2 synthase (prostaglandin endoperoxide synthase). G-SH = reduced glutathione; G-S-S-G = oxidized glutathione; PG = prostaglandin.
1.
Prostaglandin H2 synthase: The first step in PG and TX synthesis is the oxidative cyclization of free arachidonic acid to yield PGH2 by PGH2 synthase (or, prostaglandin endoperoxide synthase). This enzyme is an ER membrane-bound protein that has two catalytic activities: fatty acid cyclooxygenase (COX), which requires two molecules of O2, and peroxidase, which requires reduced glutathione (see p. 148). PGH2 is converted to a variety of PG and TX, as shown in Figure 17.23, by cell-specific synthases. [Note: PG contain a five-carbon ring, whereas TX contain a heterocyclic six-membered oxane ring (see Fig. 17.21).] Two isozymes of PGH2 synthase, usually denoted as COX-1 and COX-2, are known. COX-1 is made constitutively in most tissues and is required for maintenance of healthy gastric tissue, renal homeostasis, and platelet aggregation. COX-2 is inducible in a limited number of tissues in response to products of activated immune and inflammatory cells. [Note: The increase in PG synthesis subsequent to the induction of COX-2 mediates the pain, heat, redness, and swelling of inflammation and the fever of infection.] 2.
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Synthesis inhibition: The synthesis of PG and TX can be inhibited by unrelated compounds. For example, cortisol (a steroidal anti-inflammatory agent) inhibits phospholipase A2 activity (see Fig. 17.23) and, therefore, arachidonic acid, the substrate for PG and TX synthesis, is not released from membrane phospholipids. Aspirin, indomethacin, and phenylbutazone (all nonsteroidal anti-inflammatory drugs [NSAID]) inhibit both COX-1 and COX-2 and, thus, prevent the synthesis of the parent molecule, PGH2. [Note: Systemic inhibition of COX-1, with subsequent damage to the stomach and the kidneys and impaired clotting of blood, is the basis of aspirin’s toxicity.] Aspirin (but not other NSAID) also induces synthesis of lipoxins (anti-inflammatory mediators made from arachidonic acid) and resolvins and protectins (inflammationresolving mediators made from EPA). Inhibitors specific for COX-2 (the coxibs, for example, celecoxib) were designed to reduce pathologic inflammatory processes mediated by COX-2 while maintaining the physiologic functions of COX-1. However, their use has been associated with increased risk of heart attacks, likely as a result of decreased PGI2 synthesis (see B. below), and some have been withdrawn from the market.
B. Thromboxanes and prostaglandins in platelet homeostasis
Thromboxane A2 (TXA2) is produced by COX-1 in activated platelets. It promotes platelet adhesion and aggregation and contraction of vascular smooth muscle, thereby promoting formation of blood clots (thrombi). (See online Chapter 35.) Prostacyclin (PGI2), produced by COX-2 in vascular endothelial cells, inhibits platelet aggregation and stimulates vasodilation and, so, impedes thrombogenesis. The opposing effects of TXA2 and PGI2 limit thrombi formation to sites of vascular injury. [Note: Aspirin has an antithrombogenic effect. It inhibits TXA2 synthesis by COX-1 in platelets and PGI2 synthesis by COX-2 in endothelial cells through irreversible acetylation of these isozymes (Fig. 17.24). COX-1 inhibition cannot be overcome in platelets, which lack nuclei. However, COX-2 inhibition can be overcome in endothelial cells because they have a nucleus and, therefore, can generate more of the enzyme. This difference is the basis of low-dose aspirin therapy used to lower the risk of stroke and heart attacks by decreasing formation of thrombi.]
C. Leukotriene synthesis
Arachidonic acid is converted to a variety of linear hydroperoxy (–OOH) acids by a separate pathway involving a family of lipoxygenases (LOX). For example, 5-LOX converts arachidonic acid to 5-hydroperoxy-6,8,11,14 eicosatetraenoic acid ([5-HPETE]; see Fig. 17.23). 5-HPETE is converted to a series of LT containing four double bonds, the nature of the final products varying according to the tissue. LT are mediators of allergic response and inflammation. Inhibitors of 5-LOX and LT-receptor antagonists are used in the treatment of asthma. [Note: LT synthesis is inhibited by cortisol and not by NSAID. Aspirin-exacerbated respiratory disease is a response to LT overproduction with NSAID use in ~10% of individuals with asthma.]
IX. CHAPTER SUMMARY
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Phospholipids are polar, ionic compounds composed of an alcohol (for example, choline or ethanolamine) attached by a phosphodiester bond either to diacylglycerol (DAG), producing phosphatidylcholine or phosphatidylethanolamine, or to the amino alcohol sphingosine (Fig. 17.25). Addition of a long-chain fatty acid to sphingosine produces a ceramide. Addition of phosphorylcholine produces the phospholipid sphingomyelin. Phospholipids are the predominant lipids of cell membranes. Nonmembrane phospholipids serve as components of lung surfactant and bile. Dipalmitoylphosphatidylcholine, also called dipalmitoyl lecithin, is the major lipid component of lung surfactant. Insufficient surfactant production causes respiratory distress syndrome. Phosphatidylinositol (PI) serves as a reservoir for arachidonic acid in membranes. The phosphorylation of membrane-bound PI produces phosphatidylinositol 4,5-bisphosphate (PIP2). This compound is degraded by phospholipase C in response to the binding of various neurotransmitters, hormones, and growth factors to membrane G protein– coupled receptors. The products of this degradation, inositol 1,4,5trisphosphate (IP3) and DAG, mediate the mobilization of intracellular calcium and the activation of protein kinase C, which act synergistically to evoke cellular responses. Specific proteins can be covalently attached via a carbohydrate bridge to membrane-bound PI, forming a glycosyl phosphatidylinositol (GPI) anchor. A deficiency in GPI synthesis in hematopoietic cells results in the hemolytic disease paroxysmal nocturnal hemoglobinuria. The degradation of phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice. Sphingomyelin is degraded to a ceramide plus phosphorylcholine by the lysosomal enzyme sphingomyelinase, a deficiency of which causes Niemann-Pick (A and B) disease. Glycosphingolipids are derivatives of ceramides to which carbohydrates have been attached. Adding one sugar molecule to the ceramide produces a cerebroside, adding an oligosaccharide produces a globoside, and adding an acidic N-acetylneuraminic acid molecule produces a ganglioside. Glycosphingolipids are found predominantly in cell membranes of the brain and peripheral nervous tissue, with high concentrations in the myelin sheath. They are antigenic. Glycolipids are degraded in the lysosomes by acid hydrolases. A deficiency of any one of these enzymes causes a sphingolipidosis, in which a characteristic sphingolipid accumulates. Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT), the eicosanoids, are produced in very small amounts in almost all tissues, act locally, and have an extremely short half-life. They serve as mediators of the inflammatory response. Arachidonic acid is the immediate precursor of the predominant class of human PG (those with two double bonds). It is derived by the elongation and desaturation of the essential fatty acid linoleic acid and is stored in the membrane as a component of a phospholipid, generally PI. Arachidonic acid is released from the phospholipid by phospholipase A2 (inhibited by cortisol). Synthesis of the PG and TX begins with the oxidative cyclization of free arachidonic acid to yield PGH2 by PGH2 synthase (or, prostaglandin endoperoxide synthase), an endoplasmic reticular membrane protein that has two catalytic activities: fatty acid cyclooxygenase (COX) and peroxidase. There are two isozymes of PGH2 synthase: COX-1 (constitutive) and COX-2 (inducible). Aspirin irreversibly inhibits both.
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Opposing effects of PGI2 and TXA2 limit clot formation. LT are linear molecules produced from arachidonic acid by the 5-lipoxygenase (5-LOX) pathway. They mediate allergic response. Their synthesis is inhibited by cortisol and not by aspirin.
glycosphingolipids, and eicosanoids. PLA2 = phospholipase A2; = sulfate ion; NSAID = nonsteroidal anti-inflammatory drugs.
Choose the ONE best answer.
7.1. Aspirin-exacerbated respiratory disease (AERD) is a severe reaction to nonsteroidal anti-inflammatory drugs (NSAID) characterized by bronchoconstriction 30 minutes to several hours after ingestion. Which of the following statements best explains the symptoms seen in patients with AERD? NSAID:
A. inhibit the activity of the cystic fibrosis transmembrane conductance regulator protein, resulting in thickened mucus that block airways.
B. inhibit cyclooxygenase but not lipoxygenase, resulting in the flow of arachidonic acid to leukotriene synthesis.
C. activate the cyclooxygenase activity of prostaglandin H2 synthase, resulting in increased synthesis of prostaglandins that promote vasodilation.
D. activate phospholipases, resulting in decreased amounts of dipalmitoylphosphatidylcholine and alveolar collapse (atelectasis).
Correct answer = B. NSAID inhibit cyclooxygenase but not lipoxygenase, so any arachidonic acid available is used for the synthesis of bronchoconstricting leukotrienes. NSAID have no effect on the cystic fibrosis (CF) transmembrane conductance regulator protein, defects in which are the cause of CF. Steroids, not NSAID, inhibit phospholipase A2. Cyclooxygenase is inhibited by NSAID, not activated. NSAID have no effect on phospholipases.
7.2. An infant, born at 28 weeks’ gestation, rapidly gave evidence of respiratory distress. Clinical laboratory and imaging results supported the diagnosis of infant respiratory distress syndrome. Which of the following statements about this syndrome is true?
A. It is unrelated to the baby’s premature birth.
B. It is a consequence of too few type II pneumocytes.
C. The lecithin/sphingomyelin ratio in the amniotic fluid is likely to be high (>2).
D. The concentration of dipalmitoylphosphatidylcholine in the amniotic fluid would be expected to be lower than that of a full-term baby.
E. It is an easily treated disorder with low mortality.
F. It is treated by administering surfactant to the mother just before she gives birth.
Correct answer = D. Dipalmitoylphosphatidylcholine (DPPC or, dipalmitoyl lecithin) is the lung surfactant found in mature, healthy lungs. Respiratory distress syndrome (RDS) can occur in lungs that make too little of this compound. If the lecithin/sphingomyelin (L/S) ratio in amniotic fluid is ≥2, a newborn’s lungs are considered to be sufficiently mature (premature lungs would be expected to have a ratio <2). The RDS would not be due to too few type II pneumocytes, which would simply be secreting sphingomyelin rather than DPPC at 28 weeks’ gestation. The mother is given a glucocorticoid, not surfactant, prior to giving birth (antenatally). Surfactant would be administered to the baby postnatally to reduce surface tension.
7.3. A 10-year-old boy was evaluated for burning sensations in his feet and clusters of small, red-purple spots on his skin. Laboratory studies revealed protein in his urine. Enzymatic analysis revealed a deficiency of αgalactosidase, and enzyme replacement therapy was recommended. The most likely diagnosis is:
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A. Fabry disease.
B. Farber disease.
C. Gaucher disease.
D. Krabbe disease.
E. Niemann-Pick disease.
Correct answer = A. Fabry disease, a deficiency of α-galactosidase, is the only X-linked sphingolipidosis. It is characterized by pain in the extremities, a red-purple skin rash (generalized angiokeratomas), and kidney and cardiac complications. Protein in his urine indicates kidney damage. Enzyme replacement therapy is available.
7.4. Current medical advice for individuals experiencing chest pain is to call emergency medical services and chew a regular strength, noncoated aspirin. What is the basis for recommending aspirin?
Aspirin has an antithrombogenic effect: It prevents formation of blood clots that could occlude heart vessels. Aspirin inhibits thromboxane A2 synthesis by cyclooxygenase-1 in platelets through irreversible acetylation, thereby inhibiting platelet activation and vasoconstriction. Chewing a noncoated aspirin increases the rate of its absorption.
Cholesterol, Lipoprotein, and Steroid Metabolism 18
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I. OVERVIEW
Cholesterol, the characteristic steroid alcohol of animal tissues, performs a number of essential functions in the body. For example, cholesterol is a structural component of all cell membranes, modulating their fluidity, and, in specialized tissues, cholesterol is a precursor of bile acids, steroid hormones, and vitamin D. Therefore, it is critically important that the cells of the body be assured an appropriate supply of cholesterol. To meet this need, a complex series of transport, biosynthetic, and regulatory mechanisms has evolved. The liver plays a central role in the regulation of the body’s cholesterol homeostasis. For example, cholesterol enters the hepatic cholesterol pool from a number of sources including dietary cholesterol as well as that synthesized de novo by extrahepatic tissues and by the liver itself. Cholesterol is eliminated from the liver as unmodified cholesterol in the bile, or it can be converted to bile salts that are secreted into the intestinal lumen. It can also serve as a component of plasma lipoproteins that carry lipids to the peripheral tissues. In humans, the balance between cholesterol influx and efflux is not precise, resulting in a gradual deposition of cholesterol in the tissues, particularly in the endothelial linings of blood vessels. This is a potentially life-threatening occurrence when the lipid deposition leads to plaque formation, causing the narrowing of blood vessels (atherosclerosis) and increased risk of cardio-, cerebro-, and peripheral vascular disease. Figure 18.1 summarizes the major sources of liver cholesterol and the routes by which cholesterol leaves the liver.
II. CHOLESTEROL STRUCTURE
Cholesterol is a very hydrophobic compound. It consists of four fused hydrocarbon rings (A–D) called the steroid nucleus, and it has an eight-carbon, branched hydrocarbon chain attached to carbon 17 of the D ring. Ring A has a hydroxyl group at carbon 3, and ring B has a double bond between carbon 5 and carbon 6 (Fig. 18.2).
A. Sterols
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Steroids with 8 to 10 carbon atoms in the side chain at carbon 17 and a hydroxyl group at carbon 3 are classified as sterols. Cholesterol is the major sterol in animal tissues. It arises from de novo synthesis and absorption of dietary cholesterol. Intestinal uptake of cholesterol is mediated by the Niemann-Pick C1-like 1 protein, the target of the drug ezetimibe that reduces absorption of dietary cholesterol (see p. 176). [Note: Plant sterols (phytosterols), such as β-sitosterol, are poorly absorbed by humans (5% absorbed as compared to 40% for cholesterol). After entering the enterocytes, they are actively transported back into the intestinal lumen. Defects in the efflux transporter (ABCG5/8) result in the rare condition of sitosterolemia. Because some cholesterol is transported back as well, plant sterols reduce the absorption of dietary cholesterol. Daily ingestion of plant sterol esters supplied, for example, in spreads, is one of a number of dietary strategies to reduce plasma cholesterol levels (see p. 363).]
B. Cholesteryl esters
Most plasma cholesterol is in an esterified form (with a fatty acid [FA] attached at carbon 3, as shown in Fig. 18.2), which makes the structure even more hydrophobic than free (nonesterified) cholesterol. Cholesteryl esters are not found in membranes and are normally present only in low levels in most cells. Because of their hydrophobicity, cholesterol and its esters must be transported in association with protein as a component of a lipoprotein particle (see p. 227) or be solubilized by phospholipids and bile salts in the bile (see p. 226).
III. CHOLESTEROL SYNTHESIS
Cholesterol is synthesized by virtually all tissues in humans, although liver, intestine, adrenal cortex, and reproductive tissues, including ovaries, testes, and placenta, make the largest contributions to the cholesterol pool. As with FA, all the carbon atoms in cholesterol are provided by acetyl coenzyme A (CoA), and nicotinamide adenine dinucleotide phosphate (NADPH) provides the reducing equivalents. The pathway is endergonic, being driven by hydrolysis of the high-energy thioester bond of acetyl CoA and the terminal phosphate bond of ATP. Synthesis requires enzymes in the cytosol, the membrane of the smooth endoplasmic reticulum (SER), and the peroxisome. The pathway is responsive to changes in cholesterol concentration, and regulatory mechanisms exist to balance the rate of cholesterol synthesis against the rate of cholesterol excretion. An imbalance in this regulation can lead to an elevation in circulating levels of plasma cholesterol, with the potential for vascular disease.
A. 3-Hydroxy-3-methylglutaryl coenzyme A synthesis
The first two reactions in the cholesterol biosynthetic pathway are similar to those in the pathway that produces ketone bodies (see Fig. 16.22, p. 196). They result in the production of 3-hydroxy-3-methylglutaryl CoA ([HMG CoA], Fig. 18.3). First, two acetyl CoA molecules condense to form acetoacetyl CoA. Next, a third molecule of acetyl CoA is added by HMG CoA synthase, producing HMG CoA, a six-carbon compound. [Note: Liver parenchymal cells contain two isoenzymes of the synthase. The cytosolic enzyme participates in cholesterol synthesis, whereas the mitochondrial enzyme functions in the pathway for ketone body synthesis.]
B. Mevalonate synthesis
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HMG CoA is reduced to mevalonate by HMG CoA reductase. This is the rate-limiting and key regulated step in cholesterol synthesis. It occurs in the cytosol, uses two molecules of NADPH as the reducing agent, and releases CoA, making the reaction irreversible (Fig. 18.4). [Note: HMG CoA reductase is an integral membrane protein of the SER, with its catalytic domain projecting into the cytosol. Regulation of reductase activity is discussed in D. below.]
C. Cholesterol synthesis from mevalonate
The reactions and enzymes involved in the synthesis of cholesterol from mevalonate are illustrated in Figure 18.5. [Note: The numbers shown in brackets below correspond to numbered reactions shown in this figure.] [1] Mevalonate is converted to 5-pyrophosphomevalonate in two steps, each of which transfers a phosphate group from ATP.
[2] A five-carbon isoprene unit, isopentenyl pyrophosphate (IPP), is formed by the decarboxylation of 5-pyrophosphomevalonate. The reaction requires ATP. [Note: IPP is the precursor of a family of molecules with diverse functions, the isoprenoids. Cholesterol is a sterol isoprenoid. Nonsterol isoprenoids include dolichol (see p. 167) and ubiquinone (or, coenzyme Q; see p. 75).] [3] IPP is isomerized to 3,3-dimethylallyl pyrophosphate (DPP).
[4] IPP and DPP condense to form 10-carbon geranyl pyrophosphate (GPP).
[5] A second molecule of IPP then condenses with GPP to form 15-carbon farnesyl pyrophosphate (FPP). [Note: Covalent attachment of farnesyl to proteins, a process known as prenylation, is one mechanism for anchoring proteins (for example, ras) to the inner face of plasma membranes.] [6] Two molecules of FPP combine, releasing pyrophosphate, and are reduced, forming the 30-carbon compound squalene. [Note: Squalene is formed from six isoprenoid units. Because 3 ATP are hydrolyzed per mevalonate residue converted to IPP, a total of 18 ATP are required to make the polyisoprenoid squalene.] [7] Squalene is converted to the sterol lanosterol by a sequence of two reactions catalyzed by SER-associated enzymes that use molecular oxygen (O2) and NADPH. The hydroxylation of linear squalene triggers the cyclization of the structure to lanosterol.
The conversion of lanosterol to cholesterol is a multistep process involving shortening of the side chain, oxidative removal of methyl groups, reduction of double bonds, and migration of a double bond. Smith-Lemli-Opitz syndrome (SLOS), an autosomal-recessive disorder of cholesterol biosynthesis, is caused by a partial deficiency in 7-dehydrocholesterol-7reductase, the enzyme that reduces the double bond in 7dehydrocholesterol (7-DHC), thereby converting it to cholesterol. SLOS is one of several multisystem, embryonic malformation syndromes associated with impaired cholesterol synthesis. [Note: 7-DHC is converted to vitamin D3 in the skin (see p. 390).]
D.
HMG CoA reductase is the major control point for cholesterol biosynthesis and is subject to different kinds of metabolic control.
1.
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Sterol-dependent regulation of gene expression: Expression of the gene for HMG CoA reductase is controlled by the trans-acting factor, sterol regulatory element–binding protein-2 (SREBP-2), which binds DNA at the cis-acting sterol regulatory element (SRE) upstream of the reductase gene. Inactive SREBP-2 is an integral protein of the SER membrane and associates with a second SER membrane protein, SREBP cleavage– activating protein (SCAP). When sterol levels in the SER are low, the SREBP-2–SCAP complex moves from the ER to the Golgi. In the Golgi membrane, SREBP-2 is sequentially acted upon by two proteases, which generate a soluble fragment that enters the nucleus, binds the SRE, and functions as a transcription factor. This results in increased synthesis of HMG CoA reductase and, therefore, increased cholesterol synthesis (Fig. 18.6). However, if sterols are abundant, they bind SCAP at its sterolsensing domain and induce the binding of SCAP to yet other ER membrane proteins, the insulin-induced gene proteins (INSIG). This results in the retention of the SCAP–SREBP complex in the SER, thereby preventing the activation of SREBP-2 and leading to downregulation of cholesterol synthesis. [Note: SREBP-1c upregulates expression of enzymes involved in FA synthesis in response to insulin (see p. 184).] 2.
Sterol-accelerated enzyme degradation: The reductase itself is a sterolsensing integral protein of the SER membrane. When sterol levels in the SER are high, the enzyme binds to INSIG proteins. Binding leads to cytosolic transfer, ubiquitination, and proteasomal degradation of the reductase (see p. 247).
3.
Sterol-independent phosphorylation/dephosphorylation: HMG CoA reductase activity is controlled covalently through the actions of adenosine monophosphate (AMP)–activated protein kinase ([AMPK] see p. 183) and a phosphoprotein phosphatase (see Fig. 18.6). The phosphorylated form of the enzyme is inactive, whereas the dephosphorylated form is active. [Note: Because AMPK is activated by AMP, cholesterol synthesis, like FA synthesis, is decreased when ATP availability is decreased.] 4.
Hormonal regulation: The activity of HMG CoA reductase is controlled hormonally. An increase in insulin favors dephosphorylation (activation) of the reductase, whereas an increase in glucagon and epinephrine has the opposite effect.
5.
Drug inhibition: The statin drugs (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin) are structural analogs of HMG CoA and are (or are metabolized to) reversible, competitive inhibitors of HMG CoA reductase (Fig. 18.7). They are used to decrease plasma cholesterol levels in patients with hypercholesterolemia.
IV. CHOLESTEROL DEGRADATION
Humans cannot metabolize the cholesterol ring structure to carbon dioxide and water. Rather, the intact steroid nucleus is eliminated from the body by conversion to bile acids and bile salts, a small percentage of which is excreted in the feces, and by secretion of cholesterol into the bile, which transports it to the intestine for elimination. [Note: Some of the cholesterol in the intestine is modified by bacteria before excretion. The primary compounds made are the isomers coprostanol and cholestanol, which are reduced derivatives of cholesterol. Together with cholesterol, these compounds make up the bulk of neutral fecal sterols.]
V. BILE ACIDS AND BILE SALTS
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Bile consists of a watery mixture of organic and inorganic compounds. Phosphatidylcholine (PC), or lecithin (see p. 202), and conjugated bile salts are quantitatively the most important organic components of bile. Bile can either pass directly from the liver, where it is synthesized, into the duodenum through the common bile duct, or be stored in the gallbladder when not immediately needed for digestion.
A. Structure
The bile acids contain 24 carbons, with two or three hydroxyl groups and a side chain that terminates in a carboxyl group. The carboxyl group has a pKa (see p. 6) of ~6. In the duodenum (pH ~6), this group will be protonated in half of the molecules (the bile acids) and deprotonated in the rest (the bile salts). The terms bile acid and bile salt are frequently used interchangeably, however. Both forms have hydroxyl groups that are α in orientation (they lie below the plane of the rings) and methyl groups that are β (they lie above the plane of the rings). Therefore, the molecules have both a polar and a nonpolar surface and can act as emulsifying agents in the intestine, helping prepare dietary fat (triacylglycerol [TAG]) and other complex lipids for degradation by pancreatic digestive enzymes.
B. Synthesis
Bile acids are synthesized in the liver by a multistep, multiorganelle pathway in which hydroxyl groups are inserted at specific positions on the steroid structure; the double bond of the cholesterol B ring is reduced; and the hydrocarbon chain is shortened by three carbons, introducing a carboxyl group at the end of the chain. The most common resulting compounds, cholic acid (a triol) and chenodeoxycholic acid (a diol), as shown in Figure 18.8, are called primary bile acids. [Note: The rate-limiting step in bile acid synthesis is the introduction of a hydroxyl group at carbon 7 of the steroid nucleus by 7-α-hydroxylase, a SER-associated cytochrome P450 monooxygenase found only in liver. Expression of the enzyme is downregulated by bile acids (Fig. 18.9)].
C. Conjugation
Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine (an end product of cysteine metabolism) by an amide bond between the carboxyl group of the bile acid and the amino group of the added compound. These new structures include glycocholic and glycochenodeoxycholic acids and taurocholic and taurochenodeoxycholic acids (Fig. 18.10). The ratio of glycine to taurine forms in the bile is ~3/1. Addition of glycine or taurine results in the presence of a carboxyl group with a lower pKa (from glycine) or a sulfonate group (from taurine), both of which are fully ionized (negatively charged) at the alkaline pH of bile. The conjugated, ionized bile salts are more effective detergents than the unconjugated ones because of their enhanced amphipathic nature. Therefore, only the conjugated forms are found in the bile. Individuals with genetic deficiencies in the conversion of cholesterol to bile acids are treated with exogenously supplied chenodeoxycholic acid.
Bile salts provide the only significant mechanism for cholesterol excretion, both as a metabolic product of cholesterol and as a solubilizer of cholesterol in bile.
D. Bacterial action on bile salts
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Bacteria of the intestinal microbiota (see p. 372) can deconjugate (remove glycine and taurine) bile salts. They can also dehydroxylate carbon 7, producing secondary bile salts such as deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid.
E. Enterohepatic circulation
Bile salts secreted into the intestine are efficiently reabsorbed (>95%) and reused. The liver actively secretes bile salts via the bile salt export pump. In the intestine, they are reabsorbed in the terminal ileum via the apical sodium (Na+)–bile salt cotransporter and returned to the blood via a separate transport system. [Note: Lithocholic acid is only poorly absorbed.] They are efficiently taken up from blood by the hepatocytes via an isoform of the cotransporter and reused. [Note: Albumin binds bile salts and transports them through the blood as was seen with FA (see p. 181).] The continuous process of secretion of bile salts into the bile, their passage through the duodenum where some are deconjugated then dehydroxylated to secondary bile salts, their uptake in the ileum, and their subsequent return to the liver as a mixture of primary and secondary forms is termed the enterohepatic circulation (Fig. 18.11). Between 15 and 30 g of bile salts are secreted from the liver into the duodenum each day, yet only ~0.5 g (<3%) is lost daily in the feces. Approximately 0.5 g/day is synthesized from cholesterol in the liver to replace the amount lost. Bile acid sequestrants, such as cholestyramine, bind bile salts in the gut and prevent their reabsorption, thereby promoting their excretion. They are used in the treatment of hypercholesterolemia because the removal of bile salts relieves the inhibition on bile acid synthesis in the liver, thereby diverting additional cholesterol into that pathway. [Note: Dietary fiber also binds bile salts and increases their excretion (see p. 365).]
F. Bile salt deficiency: Cholelithiasis
The movement of cholesterol from the liver into the bile must be accompanied by the simultaneous secretion of phospholipid and bile salts. If this dual process is disrupted and more cholesterol is present than can be solubilized by the bile salts and PC present, the cholesterol may precipitate in the gallbladder, leading to cholesterol gallstone disease or cholelithiasis (Fig. 18.12). This disorder is typically caused by a decrease of bile acids in the bile. Cholelithiasis also may result from increased secretion of cholesterol into bile, as seen with the use of fibrates (for example, gemfibrozil) to reduce cholesterol (and TAG) in the blood. Laparoscopic cholecystectomy (surgical removal of the gallbladder through a small incision) is currently the treatment of choice. However, for patients who are unable to undergo surgery, oral administration of chenodeoxycholic acid to supplement the body’s supply of bile acids results in a gradual (months to years) dissolution of the gallstones. [Note: Cholesterol stones account for >85% of cases of cholelithiasis, with bilirubin and mixed stones accounting for the rest].
VI. PLASMA LIPOPROTEINS
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The plasma lipoproteins are spherical macromolecular complexes of lipids and proteins (apolipoproteins). The lipoprotein particles include chylomicrons, verylow-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They differ in lipid and protein composition, size, density (Fig. 18.13), and site of origin. [Note: Because lipoprotein particles constantly interchange lipids and apolipoproteins, the actual apolipoprotein and lipid content of each class of particles is somewhat variable.] Lipoproteins function both to keep their component lipids soluble as they transport them in the plasma and to provide an efficient mechanism for transporting their lipid contents to (and from) the tissues. In humans, the transport system is less perfect than in other animals and, as a result, humans experience a gradual deposition of lipid (especially cholesterol) in tissues.
A. Composition
Lipoproteins are composed of a neutral lipid core (containing TAG and cholesteryl esters) surrounded by a shell of amphipathic apolipoproteins, phospholipid, and nonesterified (free) cholesterol (Fig. 18.14). These amphipathic compounds are oriented such that their polar portions are exposed on the surface of the lipoprotein, thereby rendering the particle soluble in aqueous solution. The TAG and cholesterol carried by the lipoproteins are obtained either from the diet (exogenous source) or from de novo synthesis (endogenous source). [Note: The cholesterol (C) content of plasma lipoproteins is now routinely measured in fasting blood. Total C = LDL-C + HDL-C + VLDL-C, where VLDL-C is calculated by dividing TAG by 5 because the TAG/cholesterol ratio is 5/1 in VLDL. The goal value for total cholesterol is <200 mg/dl.] 1.
Size and density: Chylomicrons are the lipoprotein particles lowest in density and largest in size and that contain the highest percentage of lipid (as TAG) and the lowest percentage of protein. VLDL and LDL are successively denser, having higher ratios of protein to lipid. HDL particles are the smallest and densest. Plasma lipoproteins can be separated on the basis of their electrophoretic mobility, as shown in Figure 18.15, or on the basis of their density by ultracentrifugation.
2.
Apolipoproteins: The apolipoproteins associated with lipoprotein particles have a number of diverse functions, such as providing recognition sites for cell-surface receptors and serving as activators or coenzymes for enzymes involved in lipoprotein metabolism. Some of the apolipoproteins are required as essential structural components of the particles and cannot be removed (in fact, the particles cannot be produced without them), whereas others are transferred freely between lipoproteins. Apolipoproteins are divided by structure and function into several major classes, denoted by letters, with each class having subclasses (for example, apolipoprotein [apo] C-I, apo C-II, and apo CIII). [Note: The functions of all the apolipoproteins are not yet known.]
B. Chylomicron metabolism
Chylomicrons are assembled in intestinal mucosal cells and carry dietary (exogenous) TAG, cholesterol, fat-soluble vitamins, and cholesteryl esters to the peripheral tissues (Fig. 18.16). [Note: TAG account for close to 90% of the lipids in a chylomicron.] 1.
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Apolipoprotein synthesis: Apo B-48 is unique to chylomicrons. Its synthesis begins on the rough ER (RER), and it is glycosylated as it moves through the RER and Golgi. [Note: Apo B-48 is so named because it constitutes the N-terminal 48% of the protein encoded by the gene for apo B. Apo B-100, which is synthesized by the liver and found in VLDL and LDL, represents the entire protein encoded by this gene. Posttranscriptional editing (see p. 474) of a cytosine to a uracil in intestinal apo B-100 messenger RNA (mRNA) creates a nonsense (stop) codon (see p. 449), allowing translation of only 48% of the mRNA.] 2.
Chylomicron assembly: Many enzymes involved in TAG, cholesterol, and phospholipid synthesis are located in the SER. Assembly of the apolipoprotein and lipid into chylomicrons requires microsomal triglyceride transfer protein ([MTP] see p. 177), which loads apo B-48 with lipid. This occurs before transition from the ER to the Golgi, where the particles are packaged in secretory vesicles. These fuse with the plasma membrane releasing the lipoproteins, which then enter the lymphatic system and, ultimately, the blood. [Note: Chylomicrons leave the lymphatic system via the thoracic duct that empties into the left subclavian vein.] 3.
Nascent chylomicron modification: The particle released by the intestinal mucosal cell is called a nascent chylomicron because it is functionally incomplete. When it reaches the plasma, the particle is rapidly modified, receiving apo E (which is recognized by hepatic receptors) and apo C. The latter includes apo C-II, which is necessary for the activation of lipoprotein lipase (LPL), the enzyme that degrades the TAG contained in the chylomicron. The source of these apolipoproteins is circulating HDL (see Fig. 18.16). [Note: Apo C-III on TAG-rich lipoproteins inhibits LPL.] 4.
Triacylglycerol degradation by lipoprotein lipase: LPL is an extracellular enzyme that is anchored to the capillary walls of most tissues but predominantly those of adipose tissue and cardiac and skeletal muscle.
The adult liver does not express this enzyme. [Note: A hepatic lipase is found on the surface of endothelial cells of the liver. It plays a role in TAG degradation in chylomicrons and VLDL and is important in HDL metabolism (see p. 234).] LPL, activated by apo C-II on circulating chylomicrons, hydrolyzes the TAG in these particles to FA and glycerol. The FA are stored (in adipose) or used for energy (in muscle). The glycerol is taken up by the liver, converted to dihydroxyacetone phosphate (an intermediate of glycolysis), and used in lipid synthesis or gluconeogenesis. [Note: Patients with a deficiency of LPL or apo C-II (type I hyperlipoproteinemia or familial chylomicronemia) show a dramatic accumulation (≥1,000 mg/dl) of chylomicron-TAG in the plasma (hypertriacylglycerolemia) even in the fasted state. They are at increased risk for acute pancreatitis. Treatment is the reduction of dietary fat.] 5.
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Lipoprotein lipase expression: LPL is synthesized by adipose tissue and by cardiac and skeletal muscle. Expression of the tissue-specific isozymes is regulated by nutritional state and hormonal level. For example, in the fed state (elevated insulin levels), LPL synthesis is increased in adipose but decreased in muscle tissue. Fasting (decreased insulin) favors LPL synthesis in muscle. [Note: The highest concentration of LPL is in cardiac muscle, reflecting the use of FA to provide much of the energy needed for cardiac function.] 6.
Chylomicron remnant formation: As the chylomicron circulates, and >90% of the TAG in its core is degraded by LPL, the particle decreases in size and increases in density. In addition, the C apolipoproteins (but not apo B or E) are returned to HDL. The remaining particle, called a remnant, is rapidly removed from the circulation by the liver, whose cell membranes contain lipoprotein receptors that recognize apo E (see Fig. 18.16). Chylomicron remnants bind to these receptors and are taken into the hepatocytes by endocytosis. The endocytosed vesicle then fuses with a lysosome, and the apolipoproteins, cholesteryl esters, and other components of the remnant are hydrolytically degraded, releasing amino acids, free cholesterol, and FA. The receptor is recycled. [Note: The mechanism of receptor-mediated endocytosis is illustrated for LDL in Fig. 18.20.]
C. Very-low-density lipoprotein metabolism
VLDL are produced in the liver (Fig. 18.17). They are composed predominantly of endogenous TAG (~60%), and their function is to carry this lipid from the liver (site of synthesis) to the peripheral tissues. There, the TAG is degraded by LPL, as discussed for chylomicrons (see p. 228). [Note: Nonalcoholic fatty liver (hepatic steatosis) occurs in conditions in which there is an imbalance between hepatic TAG synthesis and the secretion of VLDL. Such conditions include obesity and type 2 diabetes mellitus (see p. 343).] 1.
Release from the liver: VLDL are secreted directly into the blood by the liver as nascent particles containing apo B-100. They must obtain apo CII and apo E from circulating HDL (see Fig. 18.17). As with chylomicrons, apo C-II is required for activation of LPL. [Note: Abetalipoproteinemia is a rare hypolipoproteinemia caused by a defect in MTP, leading to an inability to load apo B with lipid. Consequently, few VLDL or chylomicrons are formed, and TAG accumulates in the liver and intestine. Absorption of fat-soluble vitamins is decreased. LDL are low.] 2.
Modification in the circulation: As VLDL pass through the circulation, TAG is degraded by LPL, causing the VLDL to decrease in size and become denser. Surface components, including the C and E apolipoproteins, are returned to HDL, but the particles retain apo B-100. Additionally, some TAG are transferred from VLDL to HDL in an exchange reaction that concomitantly transfers cholesteryl esters from HDL to VLDL. This exchange is accomplished by cholesteryl ester transfer protein (CETP), as shown in Figure 18.18.
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3. Conversion to low-density lipoproteins: With these modifications, the VLDL is converted in the plasma to LDL. Intermediate-density lipoproteins (IDL) of varying sizes are formed during this transition. IDL can also be taken up by liver cells through receptor-mediated endocytosis that uses apo E as the ligand. Apo E is normally present in three isoforms, E-2 (the least common), E-3 (the most common), and E-4. Apo E-2 binds poorly to receptors, and patients who are homozygotic for apo E-2 are deficient in the clearance of IDL and chylomicron remnants. These individuals have familial type III hyperlipoproteinemia (familial dysbetalipoproteinemia or broad beta disease), with hypercholesterolemia and premature atherosclerosis. [Note: The apo E-4 isoform confers increased susceptibility to an earlier age of onset of the late-onset form of Alzheimer disease. The effect is dose dependent, with homozygotes being at greatest risk. Estimates of the risk vary.]
D. Low-density lipoprotein metabolism
LDL particles contain much less TAG than their VLDL predecessors and have a high concentration of cholesterol and cholesteryl esters (Fig. 18.19). About 70% of plasma cholesterol is in LDL.
1. Receptor-mediated endocytosis: The primary function of LDL particles is to provide cholesterol to the peripheral tissues (or return it to the liver). They do so by binding to plasma membrane LDL receptors that recognize apo B-100 (but not apo B-48). Because these LDL receptors can also bind apo E, they are known as apo B-100/apo E receptors. A summary of the uptake and degradation of LDL particles is presented in Figure 18.20. [Note: The numbers in brackets below refer to corresponding numbers on that figure.] A similar mechanism of receptor-mediated endocytosis is used for the uptake and degradation of chylomicron remnants and IDL by the liver.
[1] LDL receptors are negatively charged glycoproteins that are clustered in pits on cell membranes. The cytosolic side of the pit is coated with the protein clathrin, which stabilizes the pit.
[2] After binding, the LDL–receptor complex is endocytosed. [Note: Defects in the synthesis of functional LDL receptors causes a significant elevation in plasma LDL-C. Patients with such deficiencies have type IIa hyperlipidemia (familial hypercholesterolemia [FH]) and premature atherosclerosis. Autosomal dominant hypercholesterolemia can also be caused by defects in apo B-100 that reduce its binding to the receptor and by increased activity of a protease, proprotein convertase subtilisin/kexin type 9 (PCSK9), which promotes internalization and lysosomal degradation of the receptor. PCSK9 inhibitors are now available for the treatment of hypercholesterolemia.] [3] The vesicle containing LDL loses its clathrin coat and fuses with other similar vesicles, forming larger vesicles called endosomes.
[4] The pH of the endosome falls (due to the proton-pumping activity of endosomal ATPase), which allows separation of the LDL from its receptor. The receptors then migrate to one side of the endosome, whereas the LDL stay free within the lumen of the vesicle.
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[5] The receptors can be recycled, whereas the lipoprotein remnants in the vesicle are transferred to lysosomes and degraded by lysosomal acid hydrolases, releasing free cholesterol, amino acids, FA, and phospholipids. These compounds can be reutilized by the cell. [Note: Lysosomal storage diseases result from rare autosomal-recessive deficiencies in the ability to hydrolyze lysosomal cholesteryl esters (Wolman disease) or to transport free cholesterol out of the lysosome (Niemann-Pick disease, type C).] 2. Endocytosed cholesterol and cholesterol homeostasis: The chylomicron remnant–, IDL-, and LDL-derived cholesterol affects cellular cholesterol content in several ways (see Fig. 18.20). First, expression of the gene for HMG CoA reductase is inhibited by high cholesterol, and de novo cholesterol synthesis decreases as a result. Additionally, degradation of the reductase is accelerated. Second, synthesis of new LDL receptor protein is reduced by decreasing the expression of the LDL receptor gene, thus limiting further entry of LDL-C into cells. [Note: As was seen with the reductase gene (see p. 222), transcriptional regulation of the LDL receptor gene involves an SRE and SREBP-2. This allows coordinate regulation of the expression of these proteins.] Third, if the cholesterol is not required immediately for some structural or synthetic purpose, it is esterified by acyl CoA:cholesterol acyltransferase (ACAT). ACAT transfers a FA from a fatty acyl CoA to cholesterol, producing a cholesteryl ester that can be stored in the cell (Fig. 18.21). The activity of ACAT is enhanced in the presence of increased intracellular cholesterol.
3. Uptake by macrophage scavenger receptors: In addition to the highly specific and regulated receptor-mediated pathway for LDL uptake described above, macrophages possess high levels of scavenger receptor activity. These receptors, known as scavenger receptor class A (SR-A), can bind a broad range of ligands and mediate the endocytosis of chemically modified LDL in which the lipid or apo B component has been oxidized. Unlike the LDL receptor, the scavenger receptor is not downregulated in response to increased intracellular cholesterol. Cholesteryl esters accumulate in macrophages and cause their transformation into “foam” cells, which participate in the formation of atherosclerotic plaque (Fig. 18.22). LDL-C is the primary cause of atherosclerosis.
E. High-density lipoprotein metabolism
HDL comprise a heterogeneous family of lipoproteins with a complex metabolism that is not yet completely understood. HDL particles are formed in the blood by the addition of lipid to apo A-1, an apolipoprotein made and secreted by the liver and intestine. Apo A-1 accounts for ~70% of the apolipoproteins in HDL. HDL perform a number of important functions, including the following.
1.
Apolipoprotein supply: HDL particles serve as a circulating reservoir of apo C-II (the apolipoprotein that is transferred to VLDL and chylomicrons and is an activator of LPL) and apo E (the apolipoprotein required for the receptor-mediated endocytosis of IDL and chylomicron remnants).
2.
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Nonesterified cholesterol uptake: Nascent HDL are disc-shaped particles containing primarily phospholipid (largely PC) and apo A, C, and E. They take up cholesterol from nonhepatic (peripheral) tissues and return it to the liver as cholesteryl esters (Fig. 18.23). [Note: HDL particles are excellent acceptors of nonesterified cholesterol as a result of their high concentration of phospholipids, which are important solubilizers of cholesterol.] 3.
Cholesterol esterification: The cholesterol taken up by HDL is immediately esterified by the plasma enzyme lecithin:cholesterol acyltransferase (LCAT, also known as PCAT, in which P stands for phosphatidylcholine, the source of the FA). This enzyme is synthesized and secreted by the liver. LCAT binds to nascent HDL and is activated by apo A-I. LCAT transfers the FA from carbon 2 of PC to cholesterol. This produces a hydrophobic cholesteryl ester, which is sequestered in the core of the HDL, and lysophosphatidylcholine, which binds to albumin. [Note: Esterification maintains the cholesterol concentration gradient, allowing continued efflux of cholesterol to HDL.] As the discoidal nascent HDL accumulates cholesteryl esters, it first becomes a spherical, relatively cholesteryl ester–poor HDL3 and, eventually, a cholesteryl ester–rich HDL2 particle that carries these esters to the liver. Hepatic lipase, which degrades TAG and phospholipids, participates in the conversion of HDL2 to HDL3 (see Fig. 18.23). CETP (see p. 231) transfers some of the cholesteryl esters from HDL to VLDL in exchange for TAG, relieving product inhibition of LCAT. Because VLDL are catabolized to LDL, the cholesteryl esters transferred by CETP are ultimately taken up by the liver (see p. 231).
4.
Reverse cholesterol transport: The selective transfer of cholesterol from peripheral cells to HDL and from HDL to the liver for bile acid synthesis or disposal via the bile is a key component of cholesterol homeostasis. This process of reverse cholesterol transport (RCT) is, in part, the basis for the inverse relationship seen between plasma HDL concentration and atherosclerosis and for the designation of HDL as the “good” cholesterol carrier. [Note: Exercise and estrogen raise HDL levels.] RCT involves efflux of cholesterol from peripheral cells to HDL, esterification of the cholesterol by LCAT, binding of the cholesteryl ester–rich HDL (HDL2) to liver (and, perhaps, steroidogenic cells), selective transfer of the cholesteryl esters into these cells, and release of lipid-depleted HDL (HDL3). The efflux of cholesterol from peripheral cells is mediated primarily by the transport protein ABCA1. [Note: Tangier disease is a very rare deficiency of ABCA1 and is characterized by the virtual absence of HDL particles due to degradation of lipid-poor apo A-1.] Cholesteryl ester uptake by the liver is mediated by the cell-surface receptor SR-B1 (scavenger receptor class B type 1) that binds HDL (see p. 232 for SR-A receptors). The HDL particle itself is not taken up. Instead, there is selective uptake of the cholesteryl ester from the HDL particle. [Note: Low HDL-C is a risk factor for atherosclerosis.]
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ABCA1 is an ATP-binding cassette (ABC) protein. ABC proteins use energy from ATP hydrolysis to transport materials, including lipids, in and out of cells and across intracellular compartments. In addition to Tangier disease, defects in specific ABC proteins result in sitosterolemia, cystic fibrosis, X-linked adrenoleukodystrophy, respiratory distress syndrome due to decreased surfactant secretion, and liver disease due to decreased bile salt secretion.
F. Lipoprotein (a) and heart disease
Lipoprotein (a), or Lp(a), is nearly identical in structure to an LDL particle. Its distinguishing feature is the presence of an additional apolipoprotein molecule, apo(a), which is covalently linked at a single site to apo B-100. Circulating levels of Lp(a) are determined primarily by genetics. However, factors such as diet may play some role, as trans FA have been reported to increase it. The physiologic function of Lp(a) is unknown. When present in large quantities in the plasma, Lp(a) is associated with an increased risk of coronary heart disease. [Note: Apo(a) is structurally homologous to plasminogen, the precursor of a blood protease whose target is fibrin, the main protein component of blood clots (see Chapter 35 online). It is hypothesized that elevated Lp(a) slows the breakdown of blood clots that trigger heart attacks because it competes with plasminogen for binding to fibrin.] Niacin reduces Lp(a), as well as LDL-C and TAG, and raises HDLC.
VII. STEROID HORMONES
Cholesterol is the precursor of all classes of steroid hormones: glucocorticoids (for example, cortisol), mineralocorticoids (for example, aldosterone), and the sex hormones (that is, androgens, estrogens, and progestins), as shown in Figure 18.24. [Note: Glucocorticoids and mineralocorticoids are collectively called corticosteroids.] Synthesis and secretion occur in the adrenal cortex (cortisol, aldosterone, and androgens), ovaries and placenta (estrogens and progestins), and testes (testosterone). Steroid hormones are transported by the blood from their sites of synthesis to their target organs. Because of their hydrophobicity, they must be complexed with a plasma protein. Albumin can act as a nonspecific carrier and does carry aldosterone. However, specific steroid-carrier plasma proteins bind the steroid hormones more tightly than does albumin (for example, corticosteroid-binding globulin, or transcortin, is responsible for transporting cortisol). A number of genetic diseases are caused by deficiencies in specific steps in the biosynthesis of steroid hormones. Some representative diseases are described in Figure 18.25.
A. Synthesis
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Synthesis involves shortening the hydrocarbon chain of cholesterol and hydroxylating the steroid nucleus. The initial and rate-limiting reaction converts cholesterol to the 21-carbon pregnenolone. It is catalyzed by the cholesterol side-chain cleavage enzyme, a cytochrome P450 (CYP) mixed function oxidase of the inner mitochondrial membrane (see p. 149) that is also known as P450scc and desmolase. NADPH and O2 are required for the reaction. The cholesterol substrate can be newly synthesized, taken up from lipoproteins, or released by an esterase from cholesteryl esters stored in the cytosol of steroidogenic tissues. The cholesterol moves to the outer mitochondrial membrane. An important control point is the subsequent movement from the outer to the inner mitochondrial membrane. This process is mediated by StAR (steroidogenic acute regulatory) protein. Pregnenolone is the parent compound for all steroid hormones (see Fig. 18.25). It is oxidized and then isomerized to progesterone, which is further modified to the other steroid hormones by CYP protein–catalyzed hydroxylation reactions in the SER and mitochondria. A defect in the activity or amount of an enzyme in this pathway can lead to a deficiency in the synthesis of hormones beyond the affected step and to an excess in the hormones or metabolites before that step. Because all members of the pathway have potent biologic activity, serious metabolic imbalances occur with enzyme deficiencies (see Fig. 18.25). Collectively, these disorders are known as the congenital adrenal hyperplasias (CAH), because they result in enlarged adrenals. [Note: Addison disease, due to autoimmune destruction of the adrenal cortex, is characterized by adrenocortical insufficiency.]
B. Adrenal cortical steroid hormones
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Steroid hormones are synthesized and secreted in response to hormonal signals. The corticosteroids and androgens are made in different regions of the adrenal cortex and are secreted into blood in response to different signals. [Note: The adrenal medulla makes catecholamines (see p. 285).] 1. Cortisol: Its production in the middle layer (zona fasciculata) of the adrenal cortex is controlled by the hypothalamus, to which the pituitary gland is attached (Fig. 18.26). In response to severe stress (for example, infection), corticotropin-releasing hormone (CRH), produced by the hypothalamus, travels through capillaries to the anterior lobe of the pituitary, where it induces the production and secretion of adrenocorticotropic hormone (ACTH), a peptide. ACTH stimulates the adrenal cortex to synthesize and secrete the glucocorticoid cortisol, the stress hormone. [Note: ACTH binds to a membrane G protein–coupled receptor, resulting in cyclic AMP (cAMP) production and activation of protein kinase A ([PKA] see p. 94). PKA phosphorylates and activates both the esterase that converts cholesteryl ester to free cholesterol and StAR protein.] Cortisol allows the body to respond to stress through its effects on intermediary metabolism (for example, increased gluconeogenesis) and the inflammatory and immune responses (which are decreased). As cortisol levels rise, the release of CRH and ACTH is inhibited. [Note: The reduction of cortisol in CAH results in a rise in ACTH that causes adrenal hyperplasia.] 2. Aldosterone: Its production in the outer layer (zona glomerulosa) of the adrenal cortex is induced by a decrease in the plasma Na+/potassium (K+) ratio and by the hormone angiotensin II (Ang-II). Ang-II (an octapeptide) is produced from angiotensin I ([Ang-I] a decapeptide) by angiotensinconverting enzyme (ACE), an enzyme found predominantly in the lungs but also distributed widely in the body. [Note: Ang-I is produced in the blood by cleavage of an inactive precursor, angiotensinogen, secreted by the liver. Cleavage is catalyzed by renin, made and secreted by the kidneys.] Ang-II binds to cell surface receptors. However, in contrast to ACTH, its effects are mediated through the phosphatidylinositol 4,5bisphosphate pathway (see p. 205) and not by cAMP. Aldosterone’s primary effect is on the kidney tubules, where it stimulates Na+ and water uptake and K+ excretion (Fig. 18.27). [Note: An effect of aldosterone is an increase in blood pressure. Competitive inhibitors of ACE are used to treat renin-dependent hypertension.] 3. Androgens: Both the inner (zona reticularis) and middle layers of the adrenal cortex produce androgens, primarily dehydroepiandrosterone and androstenedione. Although adrenal androgens themselves are weak, they are converted by aromatase (CYP19) to testosterone, a stronger androgen, in the testes and to estrogens in the ovaries (primarily) of premenopausal women. [Note: Postmenopausal women produce estrogen at extragonadal sites such as the breast. Aromatase inhibitors are used in the treatment of estrogen-responsive breast cancer in these women.]
C. Gonadal steroid hormones
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The testes and ovaries (gonads) synthesize hormones necessary for sexual differentiation and reproduction. A single hypothalamic-releasing factor, gonadotropin-releasing hormone, stimulates the anterior pituitary to release the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Like ACTH, LH and FSH bind to surface receptors and cause an increase in cAMP. LH stimulates the testes to produce testosterone and the ovaries to produce estrogens and progesterone (see Fig. 18.27). FSH regulates the growth of ovarian follicles and stimulates testicular spermatogenesis.
D. Mechanism
Each steroid hormone diffuses across the plasma membrane of its target cell and binds to a specific cytosolic or nuclear receptor. These receptor–ligand complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone response elements [HRE]) in association with coactivator proteins, thereby causing increased transcription of targeted genes (Fig. 18.28). An HRE is found in the promoter or an enhancer element (see p. 440) for genes that respond to a specific steroid hormone, thus insuring coordinated regulation of these genes. Hormone–receptor complexes can also inhibit transcription in association with corepressors. [Note: The binding of a hormone to its receptor causes a conformational change in the receptor that uncovers its DNA-binding domain, allowing the complex to interact through a zinc finger motif with the appropriate DNA sequence. Receptors for the steroid hormones, plus those for thyroid hormone, retinoic acid (see p. 386), and 1,25-dihydroxycholecalciferol (vitamin D; see p. 390), are members of a superfamily of structurally related gene regulators that function in a similar way.]
E. Further metabolism
Steroid hormones are generally converted into inactive metabolic excretion products in the liver. Reactions include reduction of unsaturated bonds and the introduction of additional hydroxyl groups. The resulting structures are made more soluble by conjugation with glucuronic acid or sulfate (from 3′phosphoadenosyl-5′-phosphosulfate; see p. 162). These conjugated metabolites are fairly water soluble and do not need protein carriers. They are eliminated in feces and urine.
VIII. CHAPTER SUMMARY
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Cholesterol is a hydrophobic compound, with a single hydroxyl group located at carbon 3 of the A ring, to which a fatty acid (FA) can be attached, producing an even more hydrophobic cholesteryl ester. Cholesterol is synthesized by virtually all human tissues, although primarily by the liver, intestine, adrenal cortex, and reproductive tissues (Fig. 18.29). All the carbon atoms are provided by acetyl coenzyme A (CoA), and nicotinamide adenine dinucleotide phosphate provides the reducing equivalents. The pathway is driven by hydrolysis of the high-energy thioester bond of acetyl CoA and the terminal phosphate bond of ATP. Synthesis requires enzymes of the cytosol, smooth endoplasmic reticulum (SER), and peroxisomes. The rate-limiting and regulated step in cholesterol synthesis is catalyzed by the SER-membrane protein hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, which produces mevalonate from HMG CoA. The enzyme is regulated by a number of mechanisms: 1) increased expression of the reductase gene when cholesterol levels are low, via the transcription factor, sterol regulatory element–binding protein-2 (SREBP-2), bound to a sterol regulatory element (SRE), resulting in increased enzyme and, therefore, cholesterol, synthesis; 2) accelerated degradation of the reductase protein when cholesterol levels are high; 3) phosphorylation (causing inactivation of reductase activity) by adenosine monophosphate–activated protein kinase [AMPK] and dephosphorylation (activation) by a phosphoprotein phosphatase; and 4) hormonal regulation by insulin and glucagon. Statins are competitive inhibitors of HMG CoA reductase. These drugs are used to decrease plasma cholesterol in patients with hypercholesterolemia. The ring structure of cholesterol cannot be degraded in humans.
Cholesterol is eliminated from the body either by conversion to bile salts or by secretion into the bile. Bile salts and phosphatidylcholine (PC) are quantitatively the most important organic components of bile. The rate-limiting step in bile acid synthesis is catalyzed by cholesterol-7-αhydroxylase, which is inhibited by bile acids. Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine, producing the conjugated bile salts glycocholic or taurocholic acid and glycochenodeoxycholic or taurochenodeoxycholic acid. Bile salts (deprotonated) are more amphipathic than bile acids (protonated) and, therefore, are more effective emulsifiers of dietary fat. Intestinal bacteria can remove the glycine and taurine as well as a hydroxyl group from the steroid nucleus, producing the secondary bile salts, deoxycholic and lithocholic acids. Bile salts are efficiently reabsorbed (>95%) in the intestinal ileum by a sodium–bile salt cotransporter, returned to the blood, and carried by albumin back to the liver where they are taken up by the hepatic isoform of the cotransporter and reused (enterohepatic circulation, which bile acid sequestrants reduce). If more cholesterol enters the bile than can be solubilized by the available bile salts and PC, cholesterol gallstone disease (cholelithiasis) can occur.
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The plasma lipoproteins (see Fig. 18.29) include chylomicrons, verylow-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They function to keep lipids (primarily triacylglycerol [TAG] and cholesteryl esters) soluble as they transport them between tissues. Lipoproteins are composed of a neutral lipid (TAG, cholesteryl esters, or both) core surrounded by a shell of amphipathic apolipoproteins, phospholipid, and nonesterified cholesterol. Chylomicrons are assembled in intestinal mucosal cells from dietary lipids (primarily TAG). Each nascent chylomicron particle has one molecule of apolipoprotein (apo) B-48. They are released from the cells into the lymphatic system and travel to the blood, where they receive apo C-II and apo E from HDL. Apo C-II activates endothelial lipoprotein lipase (LPL), which degrades the TAG in chylomicrons to FA and glycerol. The FA that are released are stored (in adipose tissue) or used for energy (in muscle). The glycerol is metabolized by the liver. Patients with a deficiency of LPL or apo C-II show a dramatic accumulation of chylomicrons in the plasma (type I hyperlipoproteinemia or familial chylomicronemia) even if fasted. After most of the TAG is removed, apo C-II is returned to HDL, and the chylomicron remnant, carrying most of the dietary cholesterol, binds to a liver receptor that recognizes apo E. The particle is endocytosed, and its contents degraded by lysosomal enzymes. Defective uptake of these remnants (and IDL) causes type III hyperlipoproteinemia or dysbetalipoproteinemia. Nascent VLDL are produced in the liver and are composed predominantly of TAG. They contain a single molecule of apo B-100. Like chylomicrons, VLDL receive apo C-II and apo E from HDL in the plasma. VLDL carry hepatic TAG to the peripheral tissues where LPL degrades the lipid. Additionally, the VLDL particle receives cholesteryl esters from HDL in exchange for TAG. This process is accomplished by cholesteryl ester transfer protein (CETP). VLDL in the plasma is first converted to IDL and then to LDL, a much smaller, denser particle. Apo C-II and apo E are returned to HDL, but the LDL retains apo B-100, which is recognized by receptors on peripheral tissues and the liver. LDL undergo receptor-mediated endocytosis, and their contents are degraded in the lysosomes. The protease proprotein convertase subtilisin/kexin type 9 (PCSK9) prevents receptor recycling. Defects in the synthesis of functional LDL receptors causes type IIa hyperlipoproteinemia (familial hypercholesterolemia [FH]). The endocytosed cholesterol decreases expression of HMG CoA reductase (and LDL receptors) through prevention of SREBP-2 binding to the SRE. Some of it can be esterified by acyl CoA:cholesterol acyltransferase (ACAT) and stored. HDL are created by lipidation of apo A-1 synthesized in the liver and intestine. They have a number of functions, including 1) serving as a circulating reservoir of apo C-II and apo E for chylomicrons and VLDL; 2) removing cholesterol from peripheral tissues via ABCA1 and esterifying it using lecithin:cholesterol acyl transferase (LCAT), a liver-synthesized plasma enzyme that is activated by apo A-1; and 3) delivering these cholesteryl esters to the liver (reverse cholesterol transport) for uptake via scavenger receptor-B1 (SR-B1).
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Cholesterol is the precursor of all classes of steroid hormones, which include glucocorticoids, mineralocorticoids, and the sex hormones (androgens, estrogens, and progestins). Synthesis, using primarily cytochrome P450 mixed function oxidases, occurs in the adrenal cortex (cortisol in the zona fasciculata, aldosterone in the zona glomerulosa, and androgens in the zona reticularis), ovaries and placenta (estrogens and progestins), and testes (testosterone). The initial and rate-limiting step is the conversion of cholesterol to pregnenolone by the side-chain cleavage enzyme P450scc. Deficiencies in synthesis lead to congenital adrenal hyperplasia (CAH). Each steroid hormone diffuses across the plasma membrane of its target cell and binds to a specific intracellular receptor. These receptor–hormone complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone response elements) in association with coactivator proteins, thereby causing increased transcription of targeted genes. In association with corepressors, transcription is decreased.
lipoproteins; TAG = triacylglycerol; NADPH = nicotinamide adenine dinucleotide phosphate; C = carbon.
Choose the ONE best answer.
8.1. Mice were genetically engineered to contain hydroxymethylglutaryl coenzyme A reductase in which serine 871, a phosphorylation site, was replaced by alanine. Which of the following statements concerning the modified form of the enzyme is most likely to be correct?
A. The enzyme is nonresponsive to ATP depletion.
B. The enzyme is nonresponsive to statin drugs.
C. The enzyme is nonresponsive to the sterol response element–sterol response element–binding protein system.
D. The enzyme is unable to be degraded by the ubiquitin–proteasome system.
Correct answer = A. The reductase is regulated by covalent phosphorylation and dephosphorylation. Depletion of ATP results in a rise in adenosine monophosphate (AMP), which activates AMP kinase (AMPK), thereby phosphorylating and inactivating the reductase. In the absence of the serine, a common phosphorylation site, the enzyme cannot be phosphorylated by AMPK. The enzyme is also regulated physiologically through changes in transcription and degradation and pharmacologically by statin drugs (competitive inhibitors), but none of these depends on serine phosphorylation.
8.2. Calculate the amount of cholesterol in the low-density lipoproteins in an individual whose fasting blood gave the following lipid-panel test results: total cholesterol = 300 mg/dl, high-density lipoprotein cholesterol = 25 mg/dl, triglycerides = 150 mg/dl.
A. 55 mg/dl B. 95 mg/dl C. 125 mg/dl D. 245 mg/dl
Correct answer = D. The total cholesterol in the blood of a fasted individual is equal to the sum of the cholesterol in low-density lipoproteins plus the cholesterol in high-density lipoproteins plus the cholesterol in very-lowdensity lipoproteins (VLDL). This last term is calculated by dividing the triacylglycerol value by 5 because cholesterol accounts for about 1/5 of the volume of VLDL in fasted blood.
For Questions 18.3 and 18.4, use the following scenario.
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A young girl with a history of severe abdominal pain was taken to her local hospital at 5 a.m. in severe distress. Blood was drawn, and the plasma appeared milky, with the triacylglycerol level >2,000 mg/dl (normal = 4–150 mg/dl). The patient was placed on a diet extremely limited in fat but supplemented with medium-chain triglycerides.
8.3. Which of the following lipoprotein particles are most likely responsible for the appearance of the patient’s plasma?
A. Chylomicrons
B. High-density lipoproteins
C. Intermediate-density lipoproteins
D. Low-density lipoproteins
E. Very-low-density lipoproteins
Correct answer = A. The milky appearance of her plasma was a result of triacylglycerol-rich chylomicrons. Because 5 a.m. is presumably several hours after her evening meal, the patient must have difficulty degrading these lipoprotein particles. Intermediate-, low-, and high-density lipoproteins contain primarily cholesteryl esters, and, if one or more of these particles was elevated, it would cause hypercholesterolemia. Very-low-density lipoproteins do not cause the described milky appearance of plasma.
8.4. Which one of the following proteins is most likely to be deficient in this patient?
A. Apolipoprotein A-I B. Apolipoprotein B-48
C. Apolipoprotein C-II
D. Cholesteryl ester transfer protein
E. Microsomal triglyceride transfer protein
Correct answer = C. The triacylglycerol (TAG) in chylomicrons is degraded by endothelial lipoprotein lipase (LPL), which requires apolipoprotein (apo) C-II as a coenzyme. Deficiency of LPL or apo C-II results in decreased ability to degrade chylomicrons to their remnants, which get cleared (via apo E) by liver receptors. Apo A-I is the coenzyme for lecithin:cholesterol acyltransferase; apo B-48 is the characteristic structural protein of chylomicrons; cholesteryl ester transfer protein catalyzes the cholesteryl ester–TAG exchange between high-density and very-low-density lipoproteins (VLDL); and microsomal triglyceride transfer protein is involved in the formation, not degradation, of chylomicrons (and VLDL).
8.5. Complete the table below for an individual with classic 21-α-hydroxylase deficiency relative to a normal individual.
How might the results be changed if this individual were deficient in 17-αhydroxylase, rather than 21-α-hydroxylase?
Classic 21-α-hydroxylase deficiency causes mineralocorticoids (aldosterone) and glucocorticoids (cortisol) to be virtually absent. Because aldosterone increases blood pressure, and cortisol increases blood glucose, their deficiencies result in a decrease in blood pressure and blood glucose, respectively. Cortisol normally feeds back to inhibit adrenocorticotropic hormone (ACTH) release by the pituitary, and, so, its absence results in an elevation in ACTH. The loss of 21-α-hydroxylase pushes progesterone and pregnenolonetoandrogensynthesisand,therefore,causesandrostenedionelevelstorise.With17-α-hydroxylasedeficiency,sexhormonesynthesiswouldbedecreased.Mineralocorticoidproductionwouldbeincreased,leadingtohypertension.
UNIT IV Nitrogen Metabolism Amino Acids: Nitrogen Disposal 19
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I. OVERVIEW
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Unlike fats and carbohydrates, amino acids are not stored by the body. That is, no protein exists whose sole function is to maintain a supply of amino acids for future use. Therefore, amino acids must be obtained from the diet, synthesized de novo, or produced from the degradation of body protein. Any amino acids in excess of the biosynthetic needs of the cell are rapidly degraded. The first phase of catabolism involves the removal of the α-amino groups (usually by transamination and subsequent oxidative deamination), forming ammonia and the corresponding α-keto acids, the carbon skeletons of amino acids. A portion of the free ammonia is excreted in the urine, but most is used in the synthesis of urea (Fig. 19.1), which is quantitatively the most important route for disposing of nitrogen from the body. In the second phase of amino acid catabolism, described in Chapter 20, the carbon skeletons of the α-keto acids are converted to common intermediates of energy-producing metabolic pathways. These compounds can be metabolized to carbon dioxide (CO2) and water (H2O), glucose, fatty acids, or ketone bodies by the central pathways of metabolism described in Chapters 8–13 and 16.
II. OVERALL NITROGEN METABOLISM
Amino acid catabolism is part of the larger process of the metabolism of nitrogen-containing molecules. Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism (such as creatinine, see p. 287). The role of body proteins in these transformations involves two important concepts: the amino acid pool and protein turnover.
A. Amino acid pool
Free amino acids are present throughout the body, such as in cells, blood, and the extracellular fluids. For the purpose of this discussion, envision all of these amino acids as if they belonged to a single entity, called the amino acid pool. This pool is supplied by three sources: 1) amino acids provided by the degradation of endogenous (body) proteins, most of which are reutilized; 2) amino acids derived from exogenous (dietary) protein; and 3) nonessential amino acids synthesized from simple intermediates of metabolism (Fig. 19.2). Conversely, the amino acid pool is depleted by three routes: 1) synthesis of body protein, 2) consumption of amino acids as precursors of essential nitrogen-containing small molecules, and 3) conversion of amino acids to glucose, glycogen, fatty acids, and ketone bodies or oxidation to CO2 + H2O (see Fig. 19.2). Although the amino acid pool is small (comprising ~90–100 g of amino acids) in comparison with the amount of protein in the body (~12 kg in a 70-kg man), it is conceptually at the center of whole-body nitrogen metabolism.
In healthy, well-fed individuals, the input to the amino acid pool is balanced by the output. That is, the amount of amino acids contained in the pool is constant. The amino acid pool is said to be in a steady state, and the individual is said to be in nitrogen balance (see p. 367).
B. Protein turnover
Most proteins in the body are constantly being synthesized and then degraded (turned over), permitting the removal of abnormal or unneeded proteins. For many proteins, regulation of synthesis determines the concentration of protein in the cell, with protein degradation assuming a minor role. For other proteins, the rate of synthesis is constitutive (that is, essentially constant), and cellular levels of the protein are controlled by selective degradation.
1.
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Rate: In healthy adults, the total amount of protein in the body remains constant because the rate of protein synthesis is just sufficient to replace the protein that is degraded. This process, called protein turnover, leads to the hydrolysis and resynthesis of 300–400 g of body protein each day. The rate of protein turnover varies widely for individual proteins. Short-lived proteins (for example, many regulatory proteins and misfolded proteins) are rapidly degraded, having half-lives measured in minutes or hours. Long-lived proteins, with half-lives of days to weeks, constitute the majority of proteins in the cell. Structural proteins, such as collagen, are metabolically stable and have half-lives measured in months or years.
2.
Protein degradation: There are two major enzyme systems responsible for degrading proteins: the ATP-dependent ubiquitin (Ub)–proteasome system of the cytosol and the ATP-independent degradative enzyme system of the lysosomes. Proteasomes selectively degrade damaged or short-lived proteins. Lysosomes use acid hydrolases (see p. 162) to nonselectively degrade intracellular proteins (autophagy) and extracellular proteins (heterophagy), such as plasma proteins, that are taken into the cell by endocytosis.
a. Ubiquitin–proteasome system: Proteins selected for degradation by the cytosolic ubiquitin–proteasome system are first modified by the covalent attachment of Ub, a small, globular, nonenzymic protein that is highly conserved across eukaryotic species. Ubiquitination of the target substrate occurs through isopeptide linkage of the α-carboxyl group of the C-terminal glycine of Ub to the ε-amino group of a lysine in the protein substrate by a three-step, enzyme-catalyzed, ATP-dependent process. [Note: Enzyme 1 (E1, an activating enzyme) activates Ub, which is then transferred to E2 (a conjugating enzyme). E3 (a ligase) identifies the protein to be degraded and interacts with E2-Ub. There are many more E3 proteins than there are E1 or E2.] The consecutive addition of four or more Ub molecules to the target protein generates a polyubiquitin chain. Proteins tagged with Ub chains are recognized by a large, barrel-shaped, macromolecular, proteolytic complex called a proteasome (Fig. 19.3). The proteasome unfolds, deubiquitinates, and cuts the target protein into fragments that are then further degraded by cytosolic proteases to amino acids, which enter the amino acid pool. The Ub is recycled. It is noteworthy that the selective degradation of proteins by the ubiquitin–proteosome complex (unlike simple hydrolysis by proteolytic enzymes) requires ATP hydrolysis.
b. Degradation signals: Because proteins have different half-lives, it is clear that protein degradation cannot be random but, rather, is influenced by some structural aspect of the protein that serves as a degradation signal, which is recognized and bound by an E3. The half-life of a protein is also influenced by the amino (N)-terminal residue, the so-called N-end rule, and ranges from minutes to hours. Destabilizing N-terminal amino acids include arginine and posttranslationally modified amino acids such as acetylated alanine. In contrast, serine is a stabilizing amino acid. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences after the one-letter designations for these amino acids) are rapidly ubiquitinated and degraded and, therefore, have short half-lives.
III. DIETARY PROTEIN DIGESTION
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Most of the nitrogen in the diet is consumed in the form of protein, typically amounting to 70–100 g/day in the American diet (see Fig. 19.2). Proteins are generally too large to be absorbed by the intestine. [Note: An example of an exception to this rule is that newborns can take up maternal antibodies in breast milk.] Therefore, proteins must be hydrolyzed to yield di-and tripeptides as well as individual amino acids, which can be absorbed. Proteolytic enzymes responsible for degrading proteins are produced by three different organs: the stomach, the pancreas, and the small intestine (Fig. 19.4).
A. Digestion by gastric secretion
The digestion of proteins begins in the stomach, which secretes gastric juice, a unique solution containing hydrochloric acid (HCl) and the proenzyme pepsinogen.
1.
Hydrochloric acid: Stomach HCl is too dilute (pH 2–3) to hydrolyze proteins. The acid, secreted by the parietal cells of the stomach, functions instead to kill some bacteria and to denature proteins, thereby making them more susceptible to subsequent hydrolysis by proteases.
2.
Pepsin: This acid-stable endopeptidase is secreted by the chief cells of the stomach as an inactive zymogen (or proenzyme), pepsinogen. [Note: In general, zymogens contain extra amino acids in their sequences that prevent them from being catalytically active. Removal of these amino acids permits the proper folding required for an active enzyme.] In the presence of HCl, pepsinogen undergoes a conformational change that allows it to cleave itself (autocatalysis) to the active form, pepsin, which releases polypeptides and a few free amino acids from dietary proteins.
B. Digestion by pancreatic enzymes
On entering the small intestine, the polypeptides produced in the stomach by the action of pepsin are further cleaved to oligopeptides and amino acids by a group of pancreatic proteases that include both endopeptidases (that cleave within) and exopeptidases (that cut at an end). [Note: Bicarbonate (HCO3−), secreted by the pancreas in response to the intestinal hormone secretin, raises the intestinal pH.] 1.
Specificity: Each of these enzymes has a different specificity for the amino acid R-groups adjacent to the susceptible peptide bond (Fig. 19.5). For example, trypsin cleaves only when the carbonyl group of the peptide bond is contributed by arginine or lysine. These enzymes, like pepsin described above, are synthesized and secreted as inactive zymogens.
2.
Zymogen release: The release and activation of the pancreatic zymogens are mediated by the secretion of cholecystokinin, a polypeptide hormone of the small intestine (see p. 176).
3.
Zymogen activation: Enteropeptidase (also called enterokinase), a serine protease synthesized by and present on the luminal (apical) surface of intestinal mucosal cells (enterocytes) of the brush border, converts the pancreatic zymogen trypsinogen to trypsin by removal of a hexapeptide from the N-terminus of trypsinogen. Trypsin subsequently converts other trypsinogen molecules to trypsin by cleaving a limited number of specific peptide bonds in the zymogen. Thus, enteropeptidase unleashes a cascade of proteolytic activity because trypsin is the common activator of all the pancreatic zymogens (see Fig. 19.5).
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Digestion abnormalities: In individuals with a deficiency in pancreatic secretion (for example, because of chronic pancreatitis, cystic fibrosis, or surgical removal of the pancreas), the digestion and absorption of fat and protein are incomplete. This results in the abnormal appearance of lipids in the feces (a condition called steatorrhea; see p. 177) as well as undigested protein.
Celiac disease (celiac sprue) is a disease of malabsorption resulting from immune-mediated damage to the small intestine in response to ingestion of gluten (or gliadin produced from gluten), a protein found in wheat, barley, and rye.
C. Digestion of oligopeptides by small intestine enzymes
The luminal surface of the enterocytes contains aminopeptidase, an exopeptidase that repeatedly cleaves the N-terminal residue from oligopeptides to produce even smaller peptides and free amino acids.
D. Amino acid and small peptide intestinal absorption
Most free amino acids are taken into enterocytes via sodium-dependent secondary active transport by solute carrier (SLC) proteins of the apical membrane. At least seven different transport systems with overlapping amino acid specificities are known. Di-and tripeptides, however, are taken up by a proton-linked peptide transporter (PepT1). The peptides are then hydrolyzed to free amino acids. Regardless of their source, free amino acids are released from enterocytes into the portal system by sodium-independent transporters of the basolateral membrane. Therefore, only free amino acids are found in the portal vein after a meal containing protein. These amino acids are either metabolized by the liver or released into the general circulation. [Note: Branched-chain amino acids (BCAA) are not metabolized by the liver but, instead, are sent from the liver to muscle via the blood.]
E. Absorption abnormalities
The small intestine and the proximal tubules of the kidneys have common transport systems for amino acid uptake. Consequently, a defect in any one of these systems results in an inability to absorb particular amino acids into the intestine and into the kidney tubules. For example, one system is responsible for the uptake of cystine and the dibasic amino acids ornithine, arginine, and lysine (represented as COAL). In the inherited disorder cystinuria, this carrier system is defective, and all four amino acids appear in the urine (Fig. 19.6). Cystinuria occurs at a frequency of 1 in 7,000 individuals, making it one of the most common inherited diseases and the most common genetic error of amino acid transport. The disease expresses itself clinically by the precipitation of cystine to form kidney stones (calculi), which can block the urinary tract. Oral hydration is an important part of treatment for this disorder. [Note: Defects in the uptake of tryptophan by a neutral amino acid transporter can result in Hartnup disorder and pellagra-like (see p. 384) dermatologic and neurologic symptoms.]
IV. NITROGEN REMOVAL FROM AMINO ACIDS
The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown. Removing the α-amino group is essential for producing energy from any amino acid and is an obligatory step in the catabolism of all amino acids. Once removed, this nitrogen can be incorporated into other compounds or excreted as urea, with the carbon skeletons being metabolized. This section describes transamination and oxidative deamination, reactions that ultimately provide ammonia and aspartate, the two sources of urea nitrogen (see p. 253).
A. Transamination: Funneling amino groups to glutamate
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The first step in the catabolism of most amino acids is the transfer of their α-amino group to α-ketoglutarate (Fig. 19.7), producing an α-keto acid (derived from the original amino acid) and glutamate. α-Ketoglutarate plays a pivotal role in amino acid metabolism by accepting the amino groups from most amino acids, thereby becoming glutamate. Glutamate produced by transamination can be oxidatively deaminated (see B. below) or used as an amino group donor in the synthesis of nonessential amino acids. This transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases (also called transaminases). These enzymes are found in the cytosol and mitochondria of cells throughout the body. All amino acids, with the exception of lysine and threonine, participate in transamination at some point in their catabolism. [Note: These two amino acids lose their α-amino groups by deamination (see pp. 265–266).] 1. Substrate specificity: Each aminotransferase is specific for one or, at most, a few amino group donors. Aminotransferases are named after the specific amino group donor, because the acceptor of the amino group is almost always α-ketoglutarate. Two important aminotransferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as shown in Figure 19.8.
a.
Alanine aminotransferase: ALT is present in many tissues. The enzyme catalyzes the transfer of the amino group of alanine to α-ketoglutarate, resulting in the formation of pyruvate and glutamate. The reaction is readily reversible. However, during amino acid catabolism, this enzyme (like most aminotransferases) functions in the direction of glutamate synthesis. [Note: In effect, glutamate acts as a collector of nitrogen from most amino acids.] b.
Aspartate aminotransferase: AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate. During amino acid catabolism, AST primarily transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle (see p. 255). Like other transaminations, the AST reaction is reversible.
2. Mechanism: All aminotransferases require the coenzyme pyridoxal phosphate (a derivative of vitamin B6; see p. 382), which is covalently linked to the ε-amino group of a specific lysine residue at the active site of the enzyme. Aminotransferases act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. The pyridoxamine form of the coenzyme then reacts with an α-keto acid to form an amino acid, at the same time regenerating the original aldehyde form of the coenzyme. Figure 19.9 shows these two component reactions for the transamination catalyzed by AST.
3.
Equilibrium: For most transamination reactions, the equilibrium constant is near 1. This allows the reaction to function in both amino acid degradation through removal of α-amino groups (for example, after consumption of a protein-rich meal) and biosynthesis of nonessential amino acids through addition of amino groups to the carbon skeletons of α-keto acids (for example, when the supply of amino acids from the diet is not adequate to meet the synthetic needs of cells).
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Diagnostic value: Aminotransferases are normally intracellular enzymes, with the low levels found in the plasma representing the release of cellular contents during normal cell turnover. Elevated plasma levels of aminotransferases indicate damage to cells rich in these enzymes. For example, physical trauma or a disease process can cause cell lysis, resulting in release of intracellular enzymes into the blood. Two aminotransferases, AST and ALT, are of particular diagnostic value when they are found in the plasma.
a.
Hepatic disease: Plasma AST and ALT are elevated in nearly all hepatic diseases but are particularly high in conditions that cause extensive cell necrosis, such as severe viral hepatitis, toxic injury, and prolonged circulatory collapse. ALT is more specific than AST for liver disease, but the latter is more sensitive because the liver contains larger amounts of AST. Serial measurements of AST and ALT (liver function tests) are often useful in determining the course of liver damage. Figure 19.10 shows the early release of ALT into the blood, following ingestion of a liver toxin. [Note: The elevation in bilirubin results from hepatocellular damage that decreases the hepatic conjugation and excretion of bilirubin (see p. 282).] b.
Nonhepatic disease: Aminotransferases may be elevated in nonhepatic diseases such as those that cause damage to cardiac or skeletal muscle. However, these disorders can usually be distinguished clinically from liver disease.
B. Oxidative deamination: Amino group removal
In contrast to transamination reactions that transfer amino groups, oxidative deamination reactions result in the liberation of the amino group as free ammonia (Fig. 19.11). These reactions occur primarily in the liver and kidney. They provide α-keto acids that can enter the central pathways of energy metabolism and ammonia, which is a source of nitrogen in hepatic urea synthesis. [Note: Ammonia exists primarily as ammonium (NH4+) in aqueous solution, but it is the unionized form (NH3) that crosses membranes.] 1. Glutamate dehydrogenase: As described above, the amino groups of most amino acids are ultimately funneled to glutamate by means of transamination with α-ketoglutarate. Glutamate is unique in that it is the only amino acid that undergoes rapid oxidative deamination, a reaction catalyzed by glutamate dehydrogenase ([GDH], see Fig. 19.11). Therefore, the sequential action of transamination (resulting in the transfer of amino groups from most amino acids to α-ketoglutarate to produce glutamate) and the oxidative deamination of that glutamate (regenerating α-ketoglutarate) provide a pathway whereby the amino groups of most amino acids can be released as ammonia.
a. Coenzymes: GDH, a mitochondrial enzyme, is unusual in that it can use either nicotinamide adenine dinucleotide (NAD+) or its phosphorylated reduced form (NADPH) as a coenzyme (see Fig. 19.11). NAD+ is used primarily in oxidative deamination (the simultaneous loss of ammonia coupled with the oxidation of the carbon skeleton, as shown in Fig. 19.12A), whereas NADPH is used in reductive amination (the simultaneous gain of ammonia coupled with the reduction of the carbon skeleton, as shown in Fig. 19.12B).
NADP(H) = nicotinamide adenine dinucleotide phosphate.
b.
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Reaction direction: The direction of the reaction depends on the relative concentrations of glutamate, α-ketoglutarate, and ammonia and the ratio of oxidized to reduced coenzymes. For example, after ingestion of a meal containing protein, glutamate levels in the liver are elevated, and the reaction proceeds in the direction of amino acid degradation and the formation of ammonia (see Fig. 19.12A). High ammonia levels are required to drive the reaction to glutamate synthesis.
c.
Allosteric regulators: Guanosine triphosphate is an allosteric inhibitor of GDH, whereas adenosine diphosphate is an activator. Therefore, when energy levels are low in the cell, amino acid degradation by GDH is high, facilitating energy production from the carbon skeletons derived from amino acids.
2. d-Amino acid oxidase: D-Amino acids (see p. 5) are supplied by the diet but are not used in the synthesis of mammalian proteins. They are, however, efficiently metabolized to α-keto acids, ammonia, and hydrogen peroxide in the peroxisomes of liver and kidney cells by flavin adenine dinucleotide–dependent D-amino acid oxidase (DAO). The αketo acids can enter the general pathways of amino acid metabolism and be reaminated to L-isomers or catabolized for energy. [Note: DAO degrades D-serine, the isomeric form of serine that modulates N-methylD-aspartate (NMDA)-type glutamate receptors. Increased DAO activity has been linked to increased susceptibility to schizophrenia. DAO also converts glycine to glyoxylate (see p. 263).] L-Amino acid oxidases are found in snake venom.
C. Ammonia transport to the liver
Two mechanisms are available in humans for the transport of ammonia from peripheral tissues to the liver for conversion to urea. Both are important in, but not exclusive to, skeletal muscle. The first uses glutamine synthetase to combine ammonia with glutamate to form glutamine, a nontoxic transport form of ammonia (Fig. 19.13). The glutamine is transported in the blood to the liver where it is cleaved by glutaminase to glutamate and ammonia (see p. 256). The glutamate is oxidatively deaminated to ammonia and α-ketoglutarate by GDH. The ammonia is converted to urea. The second transport mechanism involves the formation of alanine by the transamination of pyruvate produced from both aerobic glycolysis and metabolism of the succinyl coenzyme A (CoA) generated by the catabolism of the BCAA isoleucine and valine. Alanine is transported in the blood to the liver, where it is transaminated by ALT to pyruvate. The pyruvate is used to synthesize glucose, which can enter the blood and be used by muscle, a pathway called the glucose–alanine cycle. The glutamate product of ALT can be deaminated by GDH, generating ammonia. Thus, both alanine and glutamine carry ammonia to the liver.
V. UREA CYCLE is the major disposal form of amino groups derived from amino acids and accounts for ~90% of the nitrogen-containing components of urine. One nitrogen of the urea molecule is supplied by free ammonia and the other nitrogen by aspartate. [Note: Glutamate is the immediate precursor of both ammonia (through oxidative deamination by GDH) and aspartate nitrogen (through transamination of oxaloacetate by AST).] The carbon and oxygen of urea are derived from CO2 (as HCO3−). Urea is produced by the liver and then is transported in the blood to the kidneys for excretion in the urine.
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A. Reactions
The first two reactions leading to the synthesis of urea occur in the mitochondrial matrix, whereas the remaining cycle enzymes are located in the cytosol (Fig. 19.14). [Note: Gluconeogenesis (see p. 117) and heme synthesis (see p. 278) also involve both the mitochondrial matrix and the cytosol.]
Pi = inorganic phosphate; NAD(H) = nicotinamide adenine dinucleotide; MD = malate dehydrogenase.
1.
Carbamoyl phosphate formation: Formation of carbamoyl phosphate by carbamoyl phosphate synthetase I (CPS I) is driven by cleavage of two molecules of ATP. Ammonia incorporated into carbamoyl phosphate is provided primarily by the oxidative deamination of glutamate by mitochondrial GDH (see Fig. 19.11). Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of urea. CPS I requires N-acetylglutamate (NAG) as a positive allosteric activator (see Fig. 19.14). [Note: Carbamoyl phosphate synthetase II participates in the biosynthesis of pyrimidines (see p. 302). It does not require NAG, uses glutamine as the nitrogen source, and occurs in the cytosol.] 2.
Citrulline formation: The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcarbamylase (OTC) as the phosphate is released as inorganic phosphate. The reaction product, citrulline, is transported to the cytosol. [Note: Ornithine and citrulline move across the inner mitochondrial membrane via an antiporter. These basic amino acids are not incorporated into cellular proteins because there are no codons for them (see p. 447).] Ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the tricarboxylic acid (TCA) cycle (see p. 109).
3.
Argininosuccinate formation: Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate. The α-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine monophosphate and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea.
4.
Argininosuccinate cleavage: Argininosuccinate is cleaved by argininosuccinate lyase to yield arginine and fumarate. The arginine serves as the immediate precursor of urea. The fumarate is hydrated to malate, providing a link with several metabolic pathways. Malate can be oxidized by malate dehydrogenase to oxaloacetate, which can be transaminated to aspartate (see Fig. 19.8) and enter the urea cycle (see Fig. 19.14). Alternatively, malate can be transported into mitochondria via the malate–aspartate shuttle (see p. 80), reenter the TCA cycle, and get oxidized to oxaloacetate, which can be used for gluconeogenesis (see p. 120). [Note: Malate oxidation generates NADH for oxidative phosphorylation (see p. 77), thereby reducing the energy cost of the urea cycle.] 5.
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Arginine cleavage to ornithine and urea: Arginase-I hydrolyzes arginine to ornithine and urea and is virtually exclusive to the liver. Therefore, only the liver can cleave arginine, thereby synthesizing urea, whereas other tissues, such as the kidney, can synthesize arginine from citrulline. [Note: Arginase-II in kidneys controls arginine availability for nitric oxide synthesis (see p. 150).] 6.
Fate of urea: Urea diffuses from the liver and is transported in the blood to the kidneys, where it is filtered and excreted in the urine (see Fig. 19.19). A portion of the urea diffuses from the blood into the intestine and is cleaved to CO2 and ammonia by bacterial urease. The ammonia is partly lost in the feces and is partly reabsorbed into the blood. In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of urea from blood into the gut. The intestinal action of urease on this urea becomes a clinically important source of ammonia, contributing to the hyperammonemia often seen in these patients. Oral administration of antibiotics reduces the number of intestinal bacteria responsible for this ammonia production.
B. Overall stoichiometry
Because four high-energy phosphate bonds are consumed in the synthesis of each molecule of urea, the synthesis of urea is irreversible, with a large, negative ∆G (see p. 70). One nitrogen of the urea molecule is supplied by free ammonia and the other nitrogen by aspartate. Glutamate is the immediate precursor of both ammonia (through oxidative deamination by GDH) and aspartate nitrogen (through transamination of oxaloacetate by AST). In effect, both nitrogen atoms of urea arise from glutamate, which, in turn, gathers nitrogen from other amino acids (Fig. 19.15).
C. Regulation
NAG is an essential activator for CPS I, the rate-limiting step in the urea cycle. It increases the affinity of CPS I for ATP. NAG is synthesized from acetyl CoA and glutamate by N-acetylglutamate synthase (NAGS), as shown in Figure 19.16, in a reaction for which arginine is an activator. The cycle is also regulated by substrate availability (short-term regulation) and enzyme induction (long term).
activator of carbamoyl phosphate synthetase I. CoA = coenzyme A.
VI. AMMONIA METABOLISM
Ammonia is produced by all tissues during the metabolism of a variety of compounds, and it is disposed of primarily by formation of urea in the liver. However, the blood ammonia level must be kept very low, because even slightly elevated concentrations (hyperammonemia) are toxic to the central nervous system (CNS). Therefore, a mechanism is required for the transport of nitrogen from the peripheral tissues to the liver for ultimate disposal as urea while keeping circulating levels of free ammonia low.
A. Sources
Amino acids are quantitatively the most important source of ammonia because most Western diets are high in protein and provide excess amino acids, which travel to the liver and undergo transdeamination (that is, the linking of the aminotransferase and GDH reactions), producing ammonia. [Note: The liver catabolizes straight-chain amino acids, primarily.] However, substantial amounts of ammonia can be obtained from other sources.
1. Glutamine: An important source of plasma glutamine is from the catabolism of BCAA in skeletal muscle. This glutamine is taken up by cells of the intestine, the liver, and the kidneys. The liver and kidneys generate ammonia from glutamine by the actions of glutaminase (Fig.
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19.17) and GDH. In the kidneys, most of this ammonia is excreted into + the urine as NH4 , which provides an important mechanism for maintaining the body’s acid–base balance through the excretion of protons. In the liver, the ammonia is detoxified to urea and excreted. [Note: α-Ketoglutarate, the second product of GDH, is a glucogenic precursor in the liver and kidneys.] Ammonia is also generated by intestinal glutaminase. Enterocytes obtain glutamine either from the blood or from digestion of dietary protein. [Note: Intestinal glutamine metabolism also produces alanine, which is used by the liver for gluconeogenesis, and citrulline, which is used by the kidneys to synthesize arginine.] 2.
Intestinal bacteria: Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. This ammonia is absorbed from the intestine by way of the portal vein, and virtually all is removed by the liver via conversion to urea.
3.
Amines: Amines obtained from the diet and monoamines that serve as hormones or neurotransmitters give rise to ammonia by the action of monoamine oxidase (see p. 286).
4.
Purines and pyrimidines: In the catabolism of purines and pyrimidines, amino groups attached to the ring atoms are released as ammonia (see Fig. 22.15 on p. 300).
B. Transport in the circulation
Although ammonia is constantly produced in the tissues, it is present at very low levels in blood. This is due both to the rapid removal of blood ammonia by the liver and to the fact that several tissues, particularly muscle, release amino acid nitrogen in the form of glutamine and alanine, rather than as free ammonia (see Fig. 19.13).
1.
Urea: Formation of urea in the liver is quantitatively the most important disposal route for ammonia. Urea travels in the blood from the liver to the kidneys, where it passes into the glomerular filtrate.
2.
Glutamine: This amide of glutamate provides a nontoxic storage and transport form of ammonia (Fig. 19.18). The ATP-requiring formation of glutamine from glutamate and ammonia by glutamine synthetase occurs primarily in skeletal muscle and the liver but is also important in the CNS, where it is the major mechanism for the removal of ammonia in the brain. Glutamine is found in plasma at concentrations higher than other amino acids, a finding consistent with its transport function. [Note: The liver keeps blood ammonia levels low through glutaminase, GDH, and the urea cycle in periportal (close to inflow of blood) hepatocytes and through glutamine synthetase as an ammonia scavenger in the perivenous hepatocytes.] Ammonia metabolism is summarized in Figure 19.19.
C. Hyperammonemia
The capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation, and the levels of blood ammonia are normally low (5–35 µmol/l). However, when liver function is compromised, due either to genetic defects of the urea cycle or liver disease, blood levels can be >1,000 µmol/l. Such hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS. For example, elevated concentrations of ammonia in the blood cause the symptoms of ammonia intoxication, which include tremors, slurring of speech, somnolence (drowsiness), vomiting, cerebral edema, and blurring of vision. At high concentrations, ammonia can cause coma and death. There are two major types of hyperammonemia.
1.
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Acquired: Liver disease is a common cause of acquired hyperammonemia in adults and may be due, for example, to viral hepatitis or to hepatotoxins such as alcohol. Cirrhosis of the liver may result in formation of collateral circulation around the liver. As a result, portal blood is shunted directly into the systemic circulation and does not have access to the liver. Therefore, the conversion of ammonia to urea is severely impaired, leading to elevated levels of ammonia.
2.
Congenital: Genetic deficiencies of each of the five enzymes of the urea cycle (and of NAGS) have been described, with an overall incidence of ~1:25,000 live births. X-linked OTC deficiency is the most common of these disorders, predominantly affecting males, although female carriers may become symptomatic. All of the other urea cycle disorders follow an autosomal-recessive inheritance pattern. In each case, the failure to synthesize urea leads to hyperammonemia during the first weeks following birth. [Note: The hyperammonemia seen with arginase deficiency is less severe because arginine contains two waste nitrogens and can be excreted in the urine.] Historically, urea cycle defects had high morbidity (neurologic manifestations) and mortality. Treatment included restriction of dietary protein in the presence of sufficient calories to prevent protein catabolism. Administration of compounds that bind covalently to nonessential amino acids, producing nitrogen-containing molecules that are excreted in the urine, has improved survival. For example, phenylbutyrate given orally is converted to phenylacetate. This condenses with glutamine to form phenylacetylglutamine, which is excreted (Fig. 19.20).
VII. CHAPTER SUMMARY
Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism (Fig. 19.21). Free amino acids in the body are produced by hydrolysis of dietary protein by proteases activated from their zymogen form in the stomach and intestine, degradation of tissue proteins, and de novo synthesis. This amino acid pool is consumed in the synthesis of body protein, metabolized for energy, or its members used as precursors for other nitrogen-containing compounds. Free amino acids from digestion are taken up by intestinal enterocytes via sodium-dependent secondary active transport. Small peptides are taken up via proton-linked transport. Note that body protein is simultaneously degraded and resynthesized, a process known as protein turnover. The concentration of a cellular protein may be determined by regulation of its synthesis or degradation. The ATP-dependent, cytosolic, selective ubiquitin–proteasome and ATP-independent, relatively nonselective lysosomal acid hydrolases are the two major enzyme systems that are responsible for degrading proteins. Nitrogen cannot be stored, and amino acids in excess of the biosynthetic needs of the cell are quickly degraded. The first phase of catabolism involves the transfer of the α-amino groups through transamination by pyridoxal phosphate–dependent aminotransferases (transaminases), followed by oxidative deamination of glutamate by glutamate dehydrogenase, forming ammonia and the corresponding α-keto acids. A portion of the free ammonia is excreted in the urine. Some ammonia is used in converting glutamate to glutamine for safe transport, but most is used in the hepatic synthesis of urea, which is quantitatively the most important route for disposing of nitrogen from the body. Alanine also carries nitrogen to the liver for disposal as urea. The two major causes of hyperammonemia (with its neurologic effects) are acquired liver disease and congenital deficiencies of urea cycle enzymes such as X-linked ornithine transcarbamylase.
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Choose the ONE best answer.
9.1. In this transamination reaction (right), which of the following are the products X and Y?
A. Alanine, α-ketoglutarate
B. Aspartate, α-ketoglutarate
C. Glutamate, alanine
D. Pyruvate, aspartate
Correct answer = B. Transamination reactions always have an amino acid and an α-keto acid as substrates. The products of the reaction are also an amino acid (corresponding to the α-keto substrate) and an α-keto acid (corresponding to the amino acid substrate). Three amino acid α-keto acid pairs commonly encountered in metabolism are alanine/pyruvate, aspartate/oxaloacetate, and glutamate/α-ketoglutarate. In this question, glutamate is deaminated to form αketoglutarate, and oxaloacetate is aminated to form aspartate.
9.2. Which one of the following statements about amino acids and their metabolism is correct?
A. Free amino acids are taken into the enterocytes by a single proton-linked transport system.
B. In healthy, well-fed individuals, the input to the amino acid pool exceeds the output.
C. The liver uses ammonia to buffer protons.
D. Muscle-derived glutamine is metabolized in liver and kidney tissue to ammonia + a gluconeogenic precursor.
E. The first step in the catabolism of most amino acids is their oxidative deamination.
F. The toxic ammonia generated from the amide nitrogen of amino acids is transported through blood as arginine.
Correct answer = D. Glutamine, produced by the catabolism of branched-chain amino acids in muscle, is deaminated by glutaminase to ammonia + glutamate. The glutamate is deaminated by glutamate dehydrogenase to ammonia + αketoglutarate, which can be used for gluconeogenesis. Free amino acids are taken into enterocytes by several different sodium-linked transport systems. Healthy, well-fed individuals are in nitrogen balance, in which nitrogen input equals output. The liver converts ammonia to urea, and the kidneys use ammonia to buffer protons. Amino acid catabolism begins with transamination that generates glutamate. The glutamate undergoes oxidative deamination. Toxic ammonia is transported as glutamine and alanine. Arginine is synthesized and hydrolyzed in the hepatic urea cycle.
For Questions 19.3–19.5, use the following scenario.
A female neonate appeared healthy until age ~24 hours, when she became lethargic. A sepsis workup proved negative. At 56 hours, she started showing focal seizure activity. The plasma ammonia level was found to be 887 µmol/l (normal 5–35 µmol/l). Quantitative plasma amino acid levels revealed a marked elevation of citrulline but not argininosuccinate.
9.3. Which one of the following enzymic activities is most likely to be deficient in this patient?
A. Arginase
B. Argininosuccinate lyase
C. Argininosuccinate synthetase
D. Carbamoyl phosphate synthetase I
E. Ornithine transcarbamylase Correct answer = C. Genetic deficiencies of each of the five enzymes of the urea cycle, as well as deficiencies in N-acetylglutamate synthase, have been described. The accumulation of citrulline (but not argininosuccinate) in the plasma of this patient means that the enzyme required for the conversion of citrulline to argininosuccinate (argininosuccinate synthetase) is defective, whereas the enzyme that cleaves argininosuccinate (argininosuccinate lyase) is functional.
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9.4. Which one of the following would also be elevated in the blood of this patient?
A. Asparagine
B. Glutamine
C. Lysine
D. Urea
Correct answer = B. Deficiencies of the enzymes of the urea cycle result in the failure to synthesize urea and lead to hyperammonemia in the first few weeks after birth. Glutamine will also be elevated because it acts as a nontoxic storage and transport form of ammonia. Therefore, elevated glutamine accompanies hyperammonemia. Asparagine and lysine do not serve this sequestering role. Urea would be decreased because of impaired activity of the urea cycle. [Note: Alanine would also be elevated in this patient.] 9.5. Why might supplementation with arginine be of benefit to this patient?
The arginine will be cleaved by arginase to urea and ornithine. Ornithine will be combined with carbamoyl phosphate by ornithine transcarbamylase to form citrulline. Citrulline, containing one waste nitrogen, will be excreted.
Amino Acids: Degradation and Synthesis 20
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I. OVERVIEW
Amino acid degradation involves removal of the α-amino group, followed by the catabolism of the resulting α-keto acids (carbon skeletons). These pathways converge to form seven intermediate products: oxaloacetate, pyruvate, αketoglutarate, fumarate, succinyl coenzyme A (CoA), acetyl CoA, and acetoacetate. The products directly enter the pathways of intermediary metabolism, resulting either in the synthesis of glucose, ketone bodies, or lipids or in the production of energy through their oxidation to carbon dioxide (CO2) by the tricarboxylic acid (TCA) cycle. Figure 20.1 provides an overview of these pathways, with a more detailed summary presented in Figure 20.15 (see p. 269). Nonessential amino acids (Fig. 20.2) can be synthesized in sufficient amounts from the intermediates of metabolism or, as in the case of cysteine and tyrosine, from essential amino acids. In contrast, because the essential amino acids cannot be synthesized (or synthesized in sufficient amounts) by humans, they must be obtained from the diet in order for normal protein synthesis to occur. Genetic defects in the pathways of amino acid metabolism can cause serious disease.
postoperative infections, and immunosuppression.]
II. GLUCOGENIC AND KETOGENIC AMINO ACIDS
Amino acids can be classified as glucogenic, ketogenic, or both, based on which of the seven intermediates are produced during their catabolism (see Fig. 20.2).
A. Glucogenic amino acids
Amino acids whose catabolism yields pyruvate or one of the intermediates of the TCA cycle are termed glucogenic. Because these intermediates are substrates for gluconeogenesis (see p. 118), they can give rise to the net synthesis of glucose in the liver and kidney.
B. Ketogenic amino acids
Amino acids whose catabolism yields either acetoacetate or one of its precursors (acetyl CoA or acetoacetyl CoA) are termed ketogenic (see Fig. 20.2). Acetoacetate is one of the ketone bodies, which also include 3hydroxybutyrate and acetone (see p. 195). Leucine and lysine are the only exclusively ketogenic amino acids found in proteins. Their carbon skeletons are not substrates for gluconeogenesis and, therefore, cannot give rise to the net synthesis of glucose.
III. AMINO ACID CARBON SKELETON
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The pathways by which amino acids are catabolized are conveniently organized according to which one (or more) of the seven intermediates listed above is produced from a particular amino acid.
A. Amino acids that form oxaloacetate
Asparagine is hydrolyzed by asparaginase, liberating ammonia and aspartate (Fig. 20.3). Aspartate loses its amino group by transamination to form oxaloacetate (see Fig. 20.3). [Note: Some rapidly dividing leukemic cells are unable to synthesize sufficient asparagine to support their growth. This makes asparagine an essential amino acid for these cells, which, therefore, require asparagine from the blood. Asparaginase, which hydrolyzes asparagine to aspartate, can be administered systemically to treat leukemia. Asparaginase lowers the level of asparagine in the plasma, thereby depriving cancer cells of a required nutrient.]
B. Amino acids that form α-ketoglutarate via glutamate 1.
Glutamine: This amino acid is hydrolyzed to glutamate and ammonia by the enzyme glutaminase (see p. 256). Glutamate is converted to αketoglutarate by transamination or through oxidative deamination by glutamate dehydrogenase (see p. 252).
2.
Proline: This amino acid is oxidized to glutamate. Glutamate is transaminated or oxidatively deaminated to form α-ketoglutarate.
3.
Arginine: This amino acid is hydrolyzed by arginase to produce ornithine (and urea). [Note: The reaction occurs primarily in the liver as part of the urea cycle (see p. 255).] Ornithine is subsequently converted to α-ketoglutarate, with glutamate semialdehyde as an intermediate.
4.
Histidine: This amino acid is oxidatively deaminated by histidase to urocanic acid, which subsequently forms N-formiminoglutamate ([FIGlu], Fig. 20.4). FIGlu donates its formimino group to tetrahydrofolate (THF), leaving glutamate, which is degraded as described above. [Note: Individuals deficient in folic acid excrete increased amounts of FIGlu in the urine, particularly after ingestion of a large dose of histidine. The FIGlu excretion test has been used in diagnosing a deficiency of folic acid. See p. 267 for a discussion of folic acid, THF, and one-carbon metabolism.]
C. Amino acids that form pyruvate 1.
Alanine: This amino acid loses its amino group by transamination to 2.
Serine: This amino acid can be converted to glycine as THF becomes N5,N10-methylenetetrahydrofolate (N5,N10-MTHF), as shown in Figure 20.6A. Serine can also be converted to pyruvate (see Fig. 20.6B).
3.
Glycine: This amino acid can be converted to serine by the reversible addition of a methylene group from N5,N10-MTHF (see Fig. 20.6A) or oxidized to CO2 and ammonia by the glycine cleavage system. [Note: Glycine can be deaminated to glyoxylate (by a d-amino acid oxidase; see form pyruvate (Fig. 20.5). [Note: Tryptophan catabolism produces alanine and, therefore, pyruvate (see Fig. 20.10 on p. 265).] p. 253), which can be oxidized to oxalate or transaminated to glycine. Deficiency of the transaminase in liver peroxisomes causes overproduction of oxalate, the formation of oxalate stones, and kidney damage (primary oxaluria type 1).] 4.
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Cysteine: This sulfur-containing amino acid undergoes desulfurization to yield pyruvate. [Note: The sulfate released can be used to synthesize 3′phosphoadenosine-5′-phosphosulfate (PAPS), an activated sulfate donor to a variety of acceptors.] Cysteine can also be oxidized to its disulfide derivative, cystine.
5.
Threonine: This amino acid is converted to pyruvate in most organisms but is a minor pathway (at best) in humans.
D. Amino acids that form fumarate 1.
Phenylalanine and tyrosine: Hydroxylation of phenylalanine produces tyrosine (Fig. 20.7). This irreversible reaction, catalyzed by tetrahydrobiopterin-requiring phenylalanine hydroxylase (PAH), initiates the catabolism of phenylalanine. Thus, phenylalanine metabolism and tyrosine metabolism merge, leading ultimately to fumarate and acetoacetate formation. Therefore, phenylalanine and tyrosine are both glucogenic and ketogenic.
2.
Inherited deficiencies: Inherited deficiencies in the enzymes of phenylalanine and tyrosine metabolism lead to the diseases phenylketonuria (PKU) (see p. 270), tyrosinemia (see p. 274), and alkaptonuria (see p. 274) as well as the condition of albinism (see p. 273).
E. Amino acids that form succinyl CoA: Methionine
Methionine is one of four amino acids that form succinyl CoA. This sulfur-containing amino acid deserves special attention because it is converted to S-adenosylmethionine (SAM), the major methyl group donor in one-carbon metabolism (Fig. 20.8). Methionine is also the source of homocysteine (Hcy), a metabolite associated with atherosclerotic vascular disease and thrombosis (see p. 265).
the methyl group carrier and donor.] PPi = pyrophosphate; Pi = inorganic phosphate; NH3 = ammonia.
1.
S-Adenosylmethionine synthesis: Methionine condenses with ATP, forming SAM, a high-energy compound that is unusual in that it contains no phosphate. The formation of SAM is driven by hydrolysis of all three phosphate bonds in ATP (see Fig. 20.8).
2.
Activated methyl group: The methyl group attached to the sulfur in SAM is activated and can be transferred by methyltransferases to a variety of acceptors such as norepinephrine in the synthesis of epinephrine. The methyl group is usually transferred to nitrogen or oxygen atoms (as with epinephrine synthesis and degradation, respectively; see p. 286) and sometimes to carbon atoms (as with cytosine). The reaction product, Sadenosylhomocysteine (SAH), is a simple thioether, analogous to methionine. The resulting loss of free energy makes methyl transfer essentially irreversible.
3.
S-Adenosylhomocysteine hydrolysis: After donation of the methyl group, SAH is hydrolyzed to Hcy and adenosine. Hcy has two fates. If there is a deficiency of methionine, Hcy may be remethylated to methionine (see Fig. 20.8). If methionine stores are adequate, Hcy may enter the transsulfuration pathway, where it is converted to cysteine.
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a. Methionine resynthesis: Hcy accepts a methyl group from N5methyltetrahydrofolate (N5-methyl-THF) in a reaction requiring methylcobalamin, a coenzyme derived from vitamin B12 (see p. 379). [Note: The methyl group is transferred by methionine synthase from the B12 derivative to Hcy, regenerating methionine. Cobalamin is remethylated from N5-methyl-THF.] b. Cysteine synthesis: Hcy condenses with serine, forming cystathionine, which is hydrolyzed to α-ketobutyrate and cysteine (see Fig. 20.8). This vitamin B6–requiring sequence has the net effect of converting serine to cysteine and Hcy to α-ketobutyrate, which is oxidatively decarboxylated to form propionyl CoA. Propionyl CoA is converted to succinyl CoA (see Fig. 16.20 on p. 195). Because Hcy is synthesized from the essential amino acid methionine, cysteine is not an essential amino acid as long as sufficient methionine is available.
4. Relationship of homocysteine to vascular disease: Elevations in plasma Hcy levels promote oxidative damage, inflammation, and endothelial dysfunction and are an independent risk factor for occlusive vascular diseases such as cardiovascular disease (CVD) and stroke (Fig. 20.9). Mild elevations (hyperhomocysteinemia) are seen in ~7% of the population. Epidemiologic studies have shown that plasma Hcy levels are inversely related to plasma levels of folate, B12, and B6, the three vitamins involved in the conversion of Hcy to methionine and cysteine. Supplementation with these vitamins has been shown to reduce circulating levels of Hcy. However, in patients with established CVD, vitamin therapy does not decrease cardiovascular events or death. This raises the question as to whether Hcy is a cause of the vascular damage or merely a marker of such damage. [Note: Large elevations in plasma Hcy as a result of rare deficiencies in cystathionine β-synthase of the transsulfuration pathway are seen in patients with classic homocystinuria (resulting from severe hyperhomocysteinemia [>100 µmol/l], see p. 273).] Deficiencies in the remethylation reaction also result in a rise in Hcy.
Elevated homocysteine and decreased folic acid levels in pregnant women are associated with increased incidence of neural tube defects (improper closure, as in spina bifida) in the fetus. Periconceptual supplementation with folate reduces the risk of such defects.
F. Other amino acids that form succinyl CoA
Degradation of valine, isoleucine, and threonine also results in the production of succinyl CoA, a TCA cycle intermediate and gluconeogenic compound. [Note: It is metabolized to pyruvate.] 1.
Valine and isoleucine: These amino acids are branched-chain amino acids (BCAA) that generate propionyl CoA, which is converted to methylmalonyl CoA and then succinyl CoA by biotin-and vitamin B12 – requiring reactions.
2.
Threonine: This amino acid is dehydrated to α-ketobutyrate, which is converted to propionyl CoA and then to succinyl CoA. Propionyl CoA, then, is generated by the catabolism of the amino acids methionine, valine, isoleucine, and threonine. [Note: Propionyl CoA also is generated by the oxidation of odd-numbered fatty acids (see p. 193).]
G. Amino acids that form acetyl CoA or acetoacetyl CoA
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Tryptophan, leucine, isoleucine, and lysine form acetyl CoA or acetoacetyl CoA directly, without pyruvate serving as an intermediate. As noted earlier, phenylalanine and tyrosine also give rise to acetoacetate during their catabolism (see Fig. 20.7). Therefore, there are a total of six partly or wholly ketogenic amino acids.
1.
Tryptophan: This amino acid is both glucogenic and ketogenic, because its catabolism yields alanine and acetoacetyl CoA (Fig. 20.10). [Note: Quinolinate from tryptophan catabolism is used in the synthesis of nicotinamide adenine dinucleotide ([NAD], see p. 383).] 2.
Leucine: This amino acid is exclusively ketogenic, because its catabolism yields acetyl CoA and acetoacetate (Fig. 20.11). The first two reactions in the catabolism of leucine and the other BCAA, isoleucine and valine, are catalyzed by enzymes that use all three BCAA (or their derivatives) as substrates (see H. below).
3.
Isoleucine: This amino acid is both ketogenic and glucogenic, because its metabolism yields acetyl CoA and propionyl CoA.
4.
Lysine: This amino acid is exclusively ketogenic and is unusual in that neither of its amino groups undergoes transamination as the first step in catabolism. Lysine is ultimately converted to acetoacetyl CoA.
H. Branched-chain amino acid degradation
The BCAA isoleucine, leucine, and valine are essential amino acids. In contrast to other amino acids, they are catabolized primarily by the peripheral tissues (particularly muscle), rather than by the liver. Because these three amino acids have a similar route of degradation, it is convenient to describe them as a group (see Fig. 20.11).
1.
Transamination: Transfer of the amino groups of all three BCAA to α ketoglutarate is catalyzed by a single, vitamin B6–requiring enzyme, branched-chain amino acid aminotransferase, that is expressed primarily in skeletal muscle.
2.
Oxidative decarboxylation: Removal of the carboxyl group of the α-keto acids derived from leucine, valine, and isoleucine is catalyzed by a single multienzyme complex, branched-chain α-keto acid dehydrogenase (BCKD) complex. This complex uses thiamine pyrophosphate, lipoic acid, oxidized flavin adenine dinucleotide (FAD), NAD+, and CoA as its coenzymes and produces NADH. [Note: This reaction is similar to the conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase (PDH) complex (see p. 109) and α-ketoglutarate to succinyl CoA by the α-ketoglutarate dehydrogenase complex (see p. 112). The dihydrolipoyl dehydrogenase (Enzyme 3, or E3) component is identical in all three complexes.] 3.
Dehydrogenations: Oxidation of the products formed in the BCKD reaction produces α-β-unsaturated acyl CoA derivatives and FADH2. These reactions are analogous to the FAD-linked dehydrogenation in the β-oxidation of fatty acids (see p. 192). [Note: Deficiency in the dehydrogenase specific for isovaleryl CoA causes neurologic problems and is associated with a “sweaty feet” odor in body fluids.] 4.
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End products: The catabolism of isoleucine ultimately yields acetyl CoA and succinyl CoA, rendering it both ketogenic and glucogenic. Valine yields succinyl CoA and is glucogenic. Leucine is ketogenic, being metabolized to acetoacetate and acetyl CoA. In addition, NADH and FADH2 are produced in the decarboxylation and dehydrogenation reactions, respectively. [Note: BCAA catabolism also results in glutamine and alanine being synthesized and sent out into the blood from muscle (see p. 253).]
IV. FOLIC ACID AND AMINO ACID METABOLISM
Some synthetic pathways require the addition of single-carbon groups that exist in a variety of oxidation states, including formyl, methenyl, methylene, and methyl. These single-carbon groups can be transferred from carrier compounds such as THF and SAM to specific structures that are being synthesized or modified. The “one-carbon pool” refers to the single-carbon units attached to this group of carriers. [Note: CO2, coming from bicarbonate (HCO3–), is carried by the vitamin biotin (see p. 385), which is a prosthetic group for most carboxylation reactions but is not considered a member of the one-carbon pool. Defects in the ability to add or remove biotin from carboxylases result in multiple carboxylase deficiency. Treatment is supplementation with biotin.]
A. Folic acid and one-carbon metabolism
The active form of folic acid, THF, is produced from folate by dihydrofolate reductase in a two-step reaction requiring two nicotinamide adenine dinucleotide phosphate (NADPH). The one-carbon unit carried by THF is bound to N5 or N10 or to both N5 and N10 . Figure 20.12 shows the structures of the various members of the THF family and their interconversions and indicates the sources of the one-carbon units and the synthetic reactions in which the specific members participate. [Note: Folate deficiency presents as a megaloblastic anemia because of decreased availability of the purines and of the thymidine monophosphate needed for DNA synthesis (see p. 303).]
V. BIOSYNTHESIS OF NONESSENTIAL AMINO ACIDS
Nonessential amino acids are synthesized from intermediates of metabolism or, as in the case of tyrosine and cysteine, from the essential amino acids phenylalanine and methionine, respectively. The synthetic reactions for the nonessential amino acids are described below and are summarized in Figure 20.15. [Note: Some amino acids found in proteins, such as hydroxyproline and hydroxylysine (see p. 45), are produced by posttranslational modification (after incorporation into a protein) of their precursor (parent) amino acids.]
A. Synthesis from α-keto acids
Alanine, aspartate, and glutamate are synthesized by transfer of an amino group to the α-keto acids pyruvate, oxaloacetate, and α-ketoglutarate, respectively. These transamination reactions (Fig. 20.13; also see p. 250) are the most direct of the biosynthetic pathways. Glutamate is unusual in that it can also be synthesized by reversal of oxidative deamination, catalyzed by glutamate dehydrogenase, when ammonia levels are high (see p. 252).
B. Synthesis by amidation 1.
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Glutamine: This amino acid, which contains an amide linkage with ammonia at the γ-carboxyl, is formed from glutamate by glutamine synthetase (see Fig. 19.18, p. 256). The reaction is driven by the hydrolysis of ATP. In addition to producing glutamine for protein synthesis, the reaction also serves as a major mechanism for the transport of ammonia in a nontoxic form. (See p. 256 for a discussion of ammonia metabolism.) 2.
Asparagine: This amino acid, which contains an amide linkage with ammonia at the β-carboxyl, is formed from aspartate by asparagine synthetase, using glutamine as the amide donor. Like the synthesis of glutamine, the reaction requires ATP and has an equilibrium far in the direction of amide synthesis.
C. Proline
Glutamate via glutamate semialdehyde is converted to proline by cyclization and reduction reactions. [Note: The semialdehyde can also be transaminated to ornithine.]
D. Serine, glycine, and cysteineThe pathways of synthesis for these amino acids are interconnected.
1.
Serine: This amino acid arises from 3-phosphoglycerate, a glycolytic intermediate (see Fig. 8.18, p. 101), which is first oxidized to 3phosphopyruvate and then transaminated to 3-phosphoserine. Serine is formed by hydrolysis of the phosphate ester. Serine can also be formed from glycine through transfer of a hydroxymethyl group by serine hydroxymethyltransferase using N5,N10-MTHF as the one-carbon donor (see Fig. 20.6A). [Note: Selenocysteine (Sec), the 21st genetically encoded amino acid, is synthesized from serine and selenium (see p. 407), while serine is attached to transfer RNA. Sec is found in ~25 human proteins including glutathione peroxidase (see p. 148) and thioredoxin reductase (see p. 297).] 2.
Glycine: This amino acid is synthesized from serine by removal of a hydroxymethyl group, also by serine hydroxymethyltransferase (see Fig. 20.6A). THF is the one-carbon acceptor.
3.
Cysteine: This amino acid is synthesized by two consecutive reactions in which Hcy combines with serine, forming cystathionine, which, in turn, is hydrolyzed to α-ketobutyrate and cysteine (see Fig. 20.8). [Note: Hcy is derived from methionine, as described on p. 264. Because methionine is an essential amino acid, cysteine synthesis requires adequate dietary intake of methionine.]
E. Tyrosine
Tyrosine is formed from phenylalanine by PAH (see p. 263). The reaction requires molecular oxygen and the coenzyme tetrahydrobiopterin (BH4), which is synthesized from guanosine triphosphate. One atom of molecular oxygen becomes the hydroxyl group of tyrosine, and the other atom is reduced to water. During the reaction, BH4 is oxidized to dihydrobiopterin (BH2). BH4 is regenerated from BH2 by NADH-requiring dihydropteridine reductase. Tyrosine, like cysteine, is formed from an essential amino acid and is, therefore, nonessential only in the presence of adequate dietary phenylalanine.
VI. AMINO ACID METABOLISM DISORDERS
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These single gene disorders, a subset of the inborn errors of metabolism, are caused by mutations that generally result in abnormal proteins, most often enzymes. The inherited defects may be expressed as a total loss of enzyme activity or, more frequently, as a partial deficiency in catalytic activity. Without treatment, the amino acid disorders almost invariably result in intellectual disability or other developmental abnormalities as a consequence of harmful accumulation of metabolites. Although >50 of these disorders have been described, many are rare, occurring in <1 per 250,000 in most populations (Fig. 20.14). Collectively, however, they constitute a very significant portion of pediatric genetic diseases (Fig. 20.15).
boxes. Classification of amino acids is color coded: Red = glucogenic; brown = glucogenic and ketogenic; green = ketogenic. Compounds in BLUE ALL CAPS are the seven metabolites to which all amino acid metabolism converges. CoA = coenzyme A; NAD(H) = nicotinamide adenine dinucleotide.
A. Phenylketonuria
PKU is the most common clinically encountered inborn error of amino acid metabolism (incidence 1:15,000). It is caused by a deficiency of PAH (Fig. 20.16). Biochemically, PKU is characterized by hyperphenylalaninemia. Phenylalanine is present in high concentrations (ten times normal) not only in plasma but also in urine and body tissues. Tyrosine, which normally is formed from phenylalanine by PAH, is deficient. Treatment includes dietary restriction of phenylalanine and supplementation with tyrosine. [Note: Hyperphenylalaninemia may also be caused by rare deficiencies in any of the several enzymes required to synthesize BH4 or in dihydropteridine reductase, which regenerates BH4 from BH2 (Fig. 20.17). Such deficiencies indirectly raise phenylalanine concentrations, because PAH requires BH4 as a coenzyme. BH4 is also required for tyrosine hydroxylase and tryptophan hydroxylase, which catalyze reactions leading to the synthesis of neurotransmitters, such as serotonin and the catecholamines. Simply restricting dietary phenylalanine does not reverse the central nervous system effects due to deficiencies in neurotransmitters. Supplementation with BH4 and replacement therapy with L-3,4 dihydroxyphenylalanine and 5-hydroxytryptophan (products of the affected tyrosine hydroxylase– and tryptophan hydroxylase–catalyzed reactions) improves the clinical outcome in these variant forms of hyperphenylalaninemia, although the response is unpredictable.]
Screening of newborns for a number of treatable disorders, including inborn errors of amino acid metabolism, is done by tandem mass spectrometry of blood obtained from a heel prick. By law, all states must screen for >20 disorders, with some screening for >50. All states screen for PKU.
1. Additional characteristics: As the name suggests, PKU is also characterized by elevated levels of a phenylketone in the urine.
a. Elevated phenylalanine metabolites: Phenylpyruvate (a phenylketone), phenylacetate, and phenyllactate, which are not normally produced in significant amounts in the presence of functional PAH, are elevated in PKU (Fig. 20.18). These metabolites give urine a characteristic musty (“mousy”) odor.
b.
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Central nervous system effects: Severe intellectual disability, developmental delay, microcephaly, and seizures are characteristic findings in untreated PKU. The affected individual typically shows symptoms of intellectual disability by age 1 year and rarely achieves an intelligence quotient (IQ) >50 (Fig. 20.19). [Note: These clinical manifestations are now rarely seen as a result of newborn screening programs, which allow early diagnosis and treatment.] c.
Hypopigmentation: Patients with untreated PKU may show a deficiency of pigmentation (fair hair, light skin color, and blue eyes). The hydroxylation of tyrosine by copper-requiring tyrosinase, which is the first step in the formation of the pigment melanin, is decreased in PKU because tyrosine is decreased.
2.
Newborn screening and diagnosis: Early diagnosis of PKU is important because the disease is treatable by dietary means. Because of the lack of neonatal symptoms, laboratory testing for elevated blood levels of phenylalanine is mandatory for detection. However, the infant with PKU frequently has normal blood levels of phenylalanine at birth because the mother clears increased blood phenylalanine in her affected fetus through the placenta. Normal levels of phenylalanine may persist until the newborn is exposed to 24–48 hours of protein feeding. Thus, screening tests are typically done after this time to avoid false negatives. For newborns with a positive screening test, diagnosis is confirmed through quantitative determination of phenylalanine levels.
3.
Prenatal diagnosis: Classic PKU is caused by any of 100 or more different mutations in the gene that encodes PAH. The frequency of any given mutation varies among populations, and the disease is often doubly heterozygous (that is, the PAH gene has a different mutation in each allele). Despite this complexity, prenatal diagnosis is possible (see p. 493).
4.
Treatment: Because most natural protein contains phenylalanine, an essential amino acid, it is impossible to satisfy the body’s protein requirement without exceeding the phenylalanine limit when ingesting a normal diet. Therefore, in PKU, blood phenylalanine level is maintained close to the normal range by feeding synthetic amino acid preparations free of phenylalanine, supplemented with some natural foods (such as fruits, vegetables, and certain cereals) selected for their low phenylalanine content. The amount is adjusted according to the tolerance of the individual as measured by blood phenylalanine levels. The earlier treatment is started, the more completely neurologic damage can be prevented. Individuals who are appropriately treated can have normal intelligence. [Note: Treatment must begin during the first 7–10 days of life to prevent cognitive impairment.] Because phenylalanine is an essential amino acid, overzealous treatment that results in blood phenylalanine levels below normal is avoided. In patients with PKU, tyrosine cannot be synthesized from phenylalanine, and, therefore, it becomes an essential amino acid and must be supplied in the diet. Discontinuance of the phenylalanine-restricted diet in early childhood is associated with poor performance on IQ tests. Adult PKU patients show deterioration of IQ scores after discontinuation of the diet (Fig. 20.20). Therefore, lifelong restriction of dietary phenylalanine is recommended. [Note: Individuals with PKU are advised to avoid aspartame, an artificial sweetener that contains phenylalanine.] 5. Maternal phenylketonuria: If women with PKU who are not on a low-phenylalanine diet become pregnant, the offspring can be affected with maternal PKU syndrome. High blood phenylalanine in the mother has a teratogenic effect, causing microcephaly and congenital heart abnormalities in the fetus. Because these developmental responses to high phenylalanine occur during the first months of pregnancy, dietary control of blood phenylalanine must begin prior to conception and be maintained throughout the pregnancy.
B. Maple syrup urine disease
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Maple syrup urine disease (MSUD) is a rare (1:185,000), autosomalrecessive disorder in which there is a partial or complete deficiency in BCKD, the mitochondrial enzyme complex that oxidatively decarboxylates leucine, isoleucine, and valine (see Fig. 20.11). These BCAA and their corresponding α-keto acids accumulate in the blood, causing a toxic effect that interferes with brain functions. The disease is characterized by feeding problems, vomiting, ketoacidosis, changes in muscle tone, neurologic problems that can result in coma (primarily because of the rise in leucine), and a characteristic maple syrup–like odor of the urine because of the rise in isoleucine. If untreated, the disease is fatal. If treatment is delayed, intellectual disability results.
1.
Classification: MSUD includes a classic type and several variant forms. The classic, neonatal-onset form is the most common type of MSUD. Leukocytes or cultured skin fibroblasts from these patients show little or no BCKD activity. Infants with classic MSUD show symptoms within the first several days of life. If not diagnosed and treated, classic MSUD is lethal in the first weeks of life. Patients with intermediate forms have a higher level of enzyme activity (up to 30% of normal). The symptoms are milder and show an onset from infancy to adolescence. Patients with the rare thiamine-dependent variant of MSUD respond to large doses of this vitamin.
2.
Screening and diagnosis: As with PKU, prenatal diagnosis and newborn screening are available, and most affected individuals are compound heterozygotes.
3.
Treatment: MSUD is treated with a synthetic formula that is free of BCAA, supplemented with limited amounts of leucine, isoleucine, and valine to allow for normal growth and development without producing toxic levels. [Note: Elevated leucine is the cause of the neurologic damage in MSUD, and its level is carefully monitored.] Early diagnosis and lifelong dietary treatment are essential if the child with MSUD is to develop normally. [Note: BCAA are an important energy source in times of metabolic need, and individuals with MSUD are at risk of decompensation during periods of increased protein catabolism.]
C. Albinism
Albinism refers to a group of conditions in which a defect in tyrosine metabolism results in a deficiency in the production of melanin. These defects result in the partial or full absence of pigment from the skin, hair, and eyes. Albinism appears in different forms, and it may be inherited by one of several modes: autosomal recessive (primary mode), autosomal dominant, or X linked. Total absence of pigment from the hair, eyes, and skin (Fig. 20.21), tyrosinase-negative oculocutaneous albinism (type 1 albinism), results from an absent or defective copper-requiring tyrosinase. It is the most severe form of the condition. In addition to hypopigmentation, affected individuals have vision defects and photophobia (sunlight hurts their eyes). They are at increased risk for skin cancer.
D. Homocystinuria
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The homocystinurias are a group of disorders involving defects in the metabolism of Hcy. These autosomal-recessive diseases are characterized by high urinary levels of Hcy, high plasma levels of Hcy and methionine, and low plasma levels of cysteine. The most common cause of homocystinuria is a defect in the enzyme cystathionine β-synthase, which converts Hcy to cystathionine (Fig. 20.22). Individuals homozygous for cystathionine β-synthase deficiency exhibit dislocation of the lens (ectopia lentis), skeletal anomalies (long limbs and fingers), intellectual disability, and an increased risk for developing thrombi (blood clots). Thrombosis is the major cause of early death in these individuals. Treatment includes restriction of methionine and supplementation with vitamin B12 and folate. Additionally, some patients are responsive to oral administration of pyridoxine (vitamin B6), which is converted to pyridoxal phosphate, the coenzyme of cystathionine β-synthase. These patients usually have a milder and later onset of clinical symptoms compared with B6nonresponsive patients. [Note: Deficiencies in methylcobalamin (see Fig.
20.8) or N5,N10-MTHF reductase ([MTHFR]; see Fig. 20.12) also result in elevated Hcy.]
E. Alkaptonuria
Alkaptonuria is a rare organic aciduria involving a deficiency in homogentisic acid oxidase, resulting in the accumulation of homogentisic acid (HA), an intermediate in the degradative pathway of tyrosine (see Fig.
20.15 on p. 269). The condition has three characteristic symptoms: homogentisic aciduria (the urine contains elevated levels of HA, which is oxidized to a dark pigment on standing, as shown in Fig. 20.23A), early onset of arthritis in the large joints, and deposition of black pigment (ochronosis) in cartilage and collagenous tissue (see Fig. 20.23B). Dark staining of diapers can indicate the disease in infants, but usually no symptoms are present until about age 40 years. Treatment includes dietary restriction of phenylalanine and tyrosine to reduce HA levels. Although alkaptonuria is not life threatening, the associated arthritis may be severely crippling. [Note: Deficiencies in fumarylacetoacetate hydrolase, the terminal enzyme of tyrosine metabolism, result in tyrosinemia type I (see Fig. 20.15) and a characteristic cabbage-like odor to urine.]
VII. CHAPTER SUMMARY
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Amino acids whose catabolism yields pyruvate or an intermediate of the tricarboxylic acid cycle are termed glucogenic (Fig. 20.24). They can give rise to the net formation of glucose in the liver and kidneys. The solely glucogenic amino acids are glutamine, glutamate, proline, arginine, histidine, alanine, serine, glycine, cysteine, methionine, valine, threonine, aspartate, and asparagine. Amino acids whose catabolism yields either acetoacetate or one of its precursors, acetyl coenzyme A (CoA) or acetoacetyl CoA, are termed ketogenic. Leucine and lysine are solely ketogenic. Tyrosine, phenylalanine, tryptophan, and isoleucine are both ketogenic and glucogenic. Nonessential amino acids can be synthesized from metabolic intermediates or from the carbon skeletons of essential amino acids. Essential amino acids need to be obtained from the diet. They include histidine, methionine, threonine, valine, isoleucine, phenylalanine, tryptophan, leucine, and lysine. Phenylketonuria (PKU) is caused by a deficiency of phenylalanine hydroxylase (PAH), which converts phenylalanine to tyrosine. Hyperphenylalaninemia may also be caused by deficiencies in the enzymes that synthesize or regenerate the coenzyme for PAH, tetrahydrobiopterin. Untreated individuals with PKU suffer from severe intellectual disability, developmental delay, microcephaly, seizures, and a characteristic musty (mousy) smell of the urine. Treatment involves controlling dietary phenylalanine. Tyrosine becomes an essential dietary component for people with PKU. Maple syrup urine disease (MSUD) is caused by a partial or complete deficiency in branched-chain a-keto acid dehydrogenase, the enzyme that decarboxylates the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. Symptoms include feeding problems, vomiting, ketoacidosis, changes in muscle tone, and a characteristic sweet smell of the urine. If untreated, the disease leads to neurologic problems that result in death. Treatment involves controlling BCAA intake. Other important genetic diseases associated with amino acid metabolism include albinism, homocystinuria, methylmalonic acidemia, alkaptonuria, histidinemia, tyrosinemia, and cystathioninuria.
Choose the ONE best answer.
For Questions 20.1–20.3, match the deficient enzyme with the associated clinical sign or laboratory finding in urine.
0.1. Cystathionine β-synthase 0.2. Homogentisic acid oxidase 0.3. Tyrosinase
Correct answers = F, A, D. A deficiency in cystathionine β-synthase of methionine degradation results in a rise in homocysteine. A deficiency in homogentisic acid oxidase of tyrosine degradation results in a rise in homogentisic acid, which forms a black pigment that is deposited in connective tissue (ochronosis). A deficiency in tyrosinase results in decreased formation of melanin from tyrosine in the skin, hair, and eyes. A sweaty feet– like odor is characteristic of isovaleryl coenzyme A dehydrogenase deficiency. Cystine crystals in urine are seen with cystinuria, a defect in intestinal and renal cystine absorption. Increased branched-chain amino acids are seen in maple syrup urine disease, increased methionine is seen in defects in homocysteine metabolism, and increased phenylalanine is seen in phenylketonuria.
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0.4. A 1-week-old infant, who was born at home in a rural, medicallyunderserved area, has undetected classic phenylketonuria. Which statement about this baby and/or her treatment is correct?
A. A diet devoid of phenylalanine should be initiated immediately.
B. Dietary treatment will be discontinued in adulthood.
C. Supplementation with vitamin B6 is required.
D. Tyrosine is an essential amino acid.
Correct answer = D. In patients with phenylketonuria, tyrosine cannot be synthesized from phenylalanine and, hence, becomes essential and must be supplied in the diet. Phenylalanine in the diet must be controlled but cannot be eliminated entirely because it is an essential amino acid. Dietary treatment must begin during the first 7–10 days of life to prevent intellectual disability, and lifelong restriction of phenylalanine is recommended to prevent cognitive decline. Additionally, elevated levels of phenylalanine are teratogenic to a developing fetus.
0.5. Which one of the following statements concerning amino acids is correct?
A. Alanine is ketogenic.
B. Amino acids that are catabolized to acetyl coenzyme A are glucogenic.
C. Branched-chain amino acids are catabolized primarily in the liver.
D. Cysteine is essential for individuals consuming a diet severely limited in methionine.
Correct answer = D. Methionine is the precursor of cysteine, which becomes essential if methionine is severely restricted. Alanine is a key glucogenic amino acid. Acetyl coenzyme A (CoA) cannot be used for the net synthesis of glucose. Amino acids catabolized to acetyl CoA are ketogenic. Branched-chain amino acids are catabolized primarily in skeletal muscle.
0.6. In an individual with the dihydrolipoyl dehydrogenase (E3)-deficient form of maple syrup urine disease, why would lactic acidosis be an expected finding?
The three α-keto acid dehydrogenase complexes (pyruvate dehydrogenase [PDH], α-ketoglutarate dehydrogenase, and branched-chain α-keto acid dehydrogenase [BCKD]) have dihydrolipoyl dehydrogenase (Enzyme 3, or E3) in common. In E3-deficient maple syrup urine disease, in addition to the branched-chain amino acids and their α-keto acid derivatives accumulating as a result of decreased activity of BCKD, lactate will also be increased because of decreased activity of PDH.
0.7. In contrast to the vitamin B6–derived pyridoxal phosphate required in most enzymic reactions involving amino acids, what coenzyme is required by the aromatic amino acid hydroxylases?
Tetrahydrobiopterin, made from guanosine triphosphate, is the required coenzyme.
Amino Acids: Conversion to Specialized Products 21
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I. OVERVIEW
In addition to serving as building blocks for proteins, amino acids are precursors of many nitrogen-containing compounds that have important physiologic functions (Fig. 21.1). These molecules include porphyrins, neurotransmitters, hormones, purines, and pyrimidines. [Note: See p. 151 for the synthesis of nitric oxide from arginine.]
II. PORPHYRIN METABOLISM
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Porphyrins are cyclic compounds that readily bind metal ions, usually ferrous (Fe2+) or ferric (Fe3+) iron. The most prevalent metalloporphyrin in humans is heme, which consists of one Fe2+ coordinated in the center of the tetrapyrrole ring of protoporphyrin IX (see p. 279). Heme is the prosthetic group for hemoglobin (Hb), myoglobin, the cytochromes, the cytochrome P450 (CYP) monooxygenase system, catalase, nitric oxide synthase, and peroxidase. These hemeproteins are rapidly synthesized and degraded. For example, 6–7 g of Hb is synthesized each day to replace heme lost through the normal turnover of erythrocytes. The synthesis and degradation of the associated porphyrins and recycling of the iron are coordinated with the turnover of hemeproteins.
A. Structure
Porphyrins are cyclic planar molecules formed by the linkage of four pyrrole rings through methenyl bridges (Fig. 21.2). Three structural features of these molecules are relevant to understanding their medical significance.
1. Side chains: Different porphyrins vary in the nature of the side chains attached to each of the four pyrrole rings. Uroporphyrin contains acetate (−CH2–COO–) and propionate (−CH2–CH2–COO–) side chains; coproporphyrin contains methyl (−CH3) and propionate groups; and protoporphyrin IX (and heme b, the most common heme) contains vinyl (−CH=CH2), methyl, and propionate groups. [Note: The methyl and vinyl groups are produced by decarboxylation of acetate and propionate side chains, respectively.] 2.
Side chain distribution: The side chains of porphyrins can be ordered around the tetrapyrrole nucleus in four different ways, designated by Roman numerals I to IV. Only type III porphyrins, which contain an asymmetric substitution on ring D (see Fig. 21.2), are physiologically important in humans. [Note: Protoporphyrin IX is a member of the type III series.] 3.
Porphyrinogens: These porphyrin precursors (for example, uroporphyrinogen) exist in a chemically reduced, colorless form and serve as intermediates between porphobilinogen (PBG) and the oxidized, colored protoporphyrins in heme biosynthesis.
B. Heme biosynthesis
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The major sites of heme biosynthesis are the liver, which synthesizes a number of heme proteins (particularly the CYP proteins), and the erythrocyte-producing cells of the bone marrow, which are active in Hb synthesis. In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for hemeproteins. In contrast, heme synthesis in erythroid cells is relatively constant and is matched to the rate of globin synthesis. [Note: Over 85% of all heme synthesis occurs in erythroid tissue. Mature red blood cells (RBC) lack mitochondria and are unable to synthesize heme.] The initial reaction and the last three steps in the formation of porphyrins occur in mitochondria, whereas the intermediate steps of the biosynthetic pathway occur in the cytosol. [Note: Fig. 21.8 summarizes heme synthesis.] 1. δ-Aminolevulinic acid formation: All the carbon and nitrogen atoms of the porphyrin molecule are provided by glycine (a nonessential amino acid) and succinyl coenzyme A (a tricarboxylic acid cycle intermediate) that condense to form δ-aminolevulinic acid (ALA) in a reaction catalyzed by ALA synthase ([ALAS], Fig. 21.3). This reaction requires pyridoxal phosphate ([PLP] see p. 382) as a coenzyme and is the committed and rate-limiting step in porphyrin biosynthesis. [Note: There are two ALAS isoforms, each produced by different genes and controlled by different mechanisms. ALAS1 is found in all tissues, whereas ALAS2 is erythroid specific. Loss-of-function mutations in ALAS2 result in X-linked sideroblastic anemia and iron overload.] (Continued in Figs. 21.4 and 21.5.) a.
Heme (hemin) effects: When porphyrin production exceeds the availability of the apoproteins that require it, heme accumulates and is converted to hemin by the oxidation of Fe2+ to Fe3+ . Hemin decreases the amount (and, thus, the activity) of ALAS1 by repressing transcription of its gene, increasing degradation of its messenger RNA, and decreasing import of the enzyme into mitochondria. [Note: In erythroid cells, ALAS2 is controlled by the availability of intracellular iron (see p. 475).] b.
Drug effects: Administration of any of a large number of drugs results in a significant increase in hepatic ALAS1 activity. These drugs are metabolized by the microsomal CYP monooxygenase system, a hemeprotein oxidase system found in the liver (see p. 149). In response to these drugs, the synthesis of CYP proteins increases, leading to an enhanced consumption of heme, a component of these proteins. This, in turn, causes a decrease in the concentration of heme in liver cells. The lower intracellular heme concentration leads to an increase in the synthesis of ALAS1 and prompts a corresponding increase in the synthesis of ALA.
2.
Porphobilinogen formation: The cytosolic condensation of two ALA to form PBG by zinc-containing ALA dehydratase (PBG synthase) is extremely sensitive to inhibition by heavy metal ions (for example, lead) that replace the zinc (see Fig. 21.3). This inhibition is, in part, responsible for the elevation in ALA and the anemia seen in lead poisoning.
3.
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Uroporphyrinogen formation: The condensation of four PBG produces the linear tetrapyrrole hydroxymethylbilane, which is cyclized and isomerized by uroporphyrinogen III synthase to produce the asymmetric uroporphyrinogen III. This cyclic tetrapyrrole undergoes decarboxylation of its acetate groups by uroporphyrinogen III decarboxylase (UROD), generating coproporphyrinogen III (Fig. 21.4). The reactions occur in the cytosol.
4. Heme formation: Coproporphyrinogen III enters the mitochondrion, and two propionate side chains are decarboxylated by coproporphyrinogen III oxidase to vinyl groups generating protoporphyrinogen IX, which is oxidized to protoporphyrin IX. The introduction of iron (as Fe2+) into protoporphyrin IX produces heme. This step can occur spontaneously, but the rate is enhanced by ferrochelatase, an enzyme that, like ALA dehydratase, is inhibited by lead (Fig. 21.5).
C. Porphyrias
Porphyrias are rare, inherited (or sometimes acquired) defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors (see Fig. 21.8). [Note: Inherited porphyrias are autosominal-dominant (AD) or autosomal-recessive (AR) disorders.] Each porphyria results in the accumulation of a unique pattern of intermediates caused by the deficiency of an enzyme in the heme synthetic pathway. [Note: Porphyria, derived from the Greek for purple, refers to the red-blue color caused by pigment-like porphyrins in the urine of some patients with defects in heme synthesis.] 1. Clinical manifestations: The porphyrias are classified as erythropoietic or hepatic, depending on whether the enzyme deficiency occurs in the erythropoietic cells of the bone marrow or in the liver. Hepatic porphyrias can be further classified as chronic or acute. In general, individuals with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and neuropsychiatric signs, whereas those with enzyme defects leading to the accumulation of tetrapyrrole intermediates show photosensitivity (that is, their skin itches and burns [pruritus] when exposed to sunlight). [Note: Photosensitivity is a result of the oxidation of colorless porphyrinogens to colored porphyrins, which are photosensitizing molecules thought to participate in the formation of superoxide radicals from oxygen. These radicals can oxidatively damage membranes and cause the release of destructive enzymes from lysosomes.] a. Chronic hepatic porphyria: Porphyria cutanea tarda, the most common porphyria, is a chronic disease of the liver. The disease is associated with severe deficiency of UROD, but clinical expression of the deficiency is influenced by various factors, such as hepatic iron overload, exposure to sunlight, alcohol ingestion, estrogen therapy, and the presence of hepatitis B or C or HIV infections. [Note: Mutations to UROD are found in only 20% of affected individuals. Inheritance is AD.] Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms (Fig. 21.6) as well as urine that is red to brown in natural light (Fig.
21.7) and pink to red in fluorescent light.
b.
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Acute hepatic porphyrias: Acute hepatic porphyrias (ALA dehydratase–deficiency porphyria, acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria) are characterized by acute attacks of gastrointestinal (GI), neuropsychiatric, and motor symptoms that may be accompanied by photosensitivity (Fig. 21.8). Porphyrias leading to accumulation of ALA and PBG, such as acute intermittent porphyria, cause abdominal pain and neuropsychiatric disturbances, ranging from anxiety to delirium. Symptoms of the acute hepatic porphyrias are often precipitated by use of drugs, such as barbiturates and ethanol, which induce the synthesis of the heme-containing CYP microsomal drug-oxidation system. This further decreases the amount of available heme, which, in turn, promotes increased synthesis of ALAS1.
c.
Erythropoietic porphyrias: The chronic erythropoietic porphyrias (congenital erythropoietic porphyria and erythropoietic protoporphyria) cause photosensitivity characterized by skin rashes and blisters that appear in early childhood (see Fig. 21.8).
2.
Increased δ-aminolevulinic acid synthase activity: One common feature of the hepatic porphyrias is decreased synthesis of heme. In the liver, heme normally functions as a repressor of the ALAS1 gene. Therefore, the absence of this end product results in an increase in the synthesis of ALAS1 (derepression). This causes an increased synthesis of intermediates that occur prior to the genetic block. The accumulation of these toxic intermediates is the major pathophysiology of the porphyrias.
3.
Treatment: During acute porphyria attacks, patients require medical support, particularly treatment for pain and vomiting. The severity of acute symptoms of the porphyrias can be diminished by intravenous injection of hemin and glucose, which decreases the synthesis of ALAS1. Protection from sunlight, ingestion of β-carotene (provitamin A; see p.
386) that scavenges free radicals, and phlebotomy (removes porphyrins) are helpful in porphyrias with photosensitivity.
D. Heme degradation
After ~120 days in the circulation, RBC are taken up and degraded by the mononuclear phagocyte system (MPS), particularly in the liver and spleen (Fig. 21.9). Approximately 85% of heme destined for degradation comes from senescent RBC. The remainder is from the degradation of hemeproteins other than Hb.
1. Bilirubin formation: The first step in the degradation of heme is catalyzed by microsomal heme oxygenase in macrophages of the MPS. In the presence of nicotinamide adenine dinucleotide phosphate and oxygen, the enzyme catalyzes three successive oxygenations that result in opening of the porphyrin ring (converting cyclic heme to linear biliverdin), production of carbon monoxide (CO), and release of Fe2+ (see Fig. 21.9). [Note: The CO has biologic function, acting as a signaling molecule and anti-inflammatory. Iron is discussed in Chapter 29.] Biliverdin, a green pigment, is reduced, forming the red-orange bilirubin. Bilirubin and its derivatives are collectively termed bile pigments. [Note: The changing colors of a bruise reflect the varying pattern of intermediates that occurs during heme degradation.]
Bilirubin, unique to mammals, appears to function at low levels as an antioxidant. In this role, it is oxidized to biliverdin, which is then reduced by biliverdin reductase, regenerating bilirubin.
2.
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Bilirubin uptake by the liver: Because bilirubin is only slightly soluble in plasma, it is transported through blood to the liver by binding noncovalently to albumin. [Note: Certain anionic drugs, such as salicylates and sulfonamides, can displace bilirubin from albumin, permitting bilirubin to enter the central nervous system (CNS). This causes the potential for neural damage in infants (see p. 285).] Bilirubin dissociates from the carrier albumin molecule, enters a hepatocyte via facilitated diffusion, and binds to intracellular proteins, particularly the protein ligandin.
3.
Bilirubin diglucuronide formation: In the hepatocyte, bilirubin solubility is increased by the sequential addition of two molecules of glucuronic acid in a process called conjugation. The reactions are catalyzed by microsomal bilirubin UDP-glucuronosyltransferase (bilirubin UGT) using uridine diphosphate (UDP)-glucuronic acid as the glucuronate donor. The bilirubin diglucuronide product is referred to as conjugated bilirubin (CB). [Note: Varying degrees of deficiency of bilirubin UGT result in Crigler-Najjar I and II and Gilbert syndrome, with Crigler-Najjar I being the most severe.] 4.
Bilirubin secretion into bile: CB is actively transported against a concentration gradient into the bile canaliculi and then into the bile. This energy-dependent, rate-limiting step is susceptible to impairment in liver disease. [Note: A rare deficiency in the protein required for transport of CB out of the liver results in Dubin-Johnson syndrome.] Unconjugated bilirubin (UCB) is normally not secreted into bile.
5.
Urobilin formation in the intestine: CB is hydrolyzed and reduced by gut bacteria to yield urobilinogen, a colorless compound. Most of the urobilinogen is further oxidized by bacteria to stercobilin, which gives feces the characteristic brown color. However, some is reabsorbed from the gut and enters the portal blood. A portion of this urobilinogen participates in the enterohepatic urobilinogen cycle in which it is taken up by the liver and then resecreted into the bile. The remainder of the urobilinogen is transported by the blood to the kidney, where it is converted to yellow urobilin and excreted, giving urine its characteristic color. The metabolism of bilirubin is summarized in Figure 21.10.
E. Jaundice
Jaundice (or, icterus) refers to the yellow color of skin, nail beds, and sclerae (whites of the eyes) caused by bilirubin deposition, secondary to increased bilirubin levels in the blood (hyperbilirubinemia) as shown in Figure 21.11. Although not a disease, jaundice is usually a symptom of an underlying disorder. [Note: Blood bilirubin levels are normally ≤1 mg/dl. Jaundice is seen at 2–3 mg/dl.] 1. Types: Jaundice can be classified into three major types described below. However, in clinical practice, jaundice is often more complex than indicated in this simple classification. For example, the accumulation of bilirubin may be a result of defects at more than one step in its metabolism.
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a. Hemolytic (prehepatic): The liver has the capacity to conjugate and excrete >3,000 mg of bilirubin/day, whereas the normal production of bilirubin is only 300 mg/day. This excess capacity allows the liver to respond to increased heme degradation with a corresponding increase in conjugation and secretion of CB. However, extensive hemolysis (for example, in patients with sickle cell anemia or deficiency of pyruvate kinase or glucose 6-phosphate dehydrogenase) may produce bilirubin faster than it can be conjugated. UCB levels in the blood become elevated (unconjugated hyperbilirubinemia), causing jaundice (Fig. 21.12A). [Note: With hemolysis, more CB is made and excreted into the bile, the amount of urobilinogen entering the enterohepatic circulation is increased, and urinary urobilinogen is increased.] b.
Hepatocellular (hepatic): Damage to liver cells (for example, in patients with cirrhosis or hepatitis) can cause unconjugated hyperbilirubinemia as a result of decreased conjugation. Urobilinogen is increased in the urine because hepatic damage decreases the enterohepatic circulation of this compound, allowing more to enter the blood, from which it is filtered into the urine. The urine consequently darkens, whereas stools may be a pale, clay color. Plasma levels of alanine and aspartate transaminases (ALT and AST, respectively; see p. 251) are elevated. If CB is made but is not efficiently secreted from the liver into bile (intrahepatic cholestasis), it can leak into the blood (regurgitation), causing a conjugated hyperbilirubinemia.
c.
Obstructive (posthepatic): In this instance, jaundice is not caused by overproduction of bilirubin or decreased conjugation but, instead, results from obstruction of the common bile duct (extrahepatic cholestasis). For example, the presence of a tumor or bile stones may block the duct, preventing passage of CB into the intestine. Patients with obstructive jaundice experience GI pain and nausea and produce stools that are a pale, clay color. The CB regurgitates into the blood (conjugated hyperbilirubinemia). The CB is eventually excreted in the urine (which darkens over time) and is referred to as urinary bilirubin. Urinary urobilinogen is absent.
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2. Jaundice in newborns: Most newborn infants (60% of full term and 80% of preterm) show a rise in UCB in the first postnatal week (and a transient, physiologic jaundice) because the activity of hepatic bilirubin UGT is low at birth (it reaches adult levels in about 4 weeks), as shown in Figures 21.12B and 21.13. Elevated UCB, in excess of the binding capacity of albumin (20–25 mg/dl), can diffuse into the basal ganglia, causing toxic encephalopathy (kernicterus) and a pathologic jaundice. Therefore, newborns with significantly elevated bilirubin levels are treated with blue fluorescent light (phototherapy), as shown in Figure 21.14, which converts bilirubin to more polar and, therefore, water-soluble isomers. These photoisomers can be excreted into the bile without conjugation to glucuronic acid. [Note: Because of solubility differences, only UCB crosses the blood–brain barrier, and only CB appears in urine.] 3. Bilirubin measurement: Bilirubin is commonly measured by the van den Bergh reaction, in which diazotized sulfanilic acid reacts with bilirubin to form red azodipyrroles that are measured colorimetrically. In aqueous solution, the water-soluble CB reacts rapidly with the reagent (within 1 minute) and is said to be direct reacting. The UCB, which is much less soluble in aqueous solution, reacts more slowly. However, when the reaction is carried out in methanol, both CB and UCB are soluble and react with the reagent, providing the total bilirubin value. The indirect-reacting bilirubin, which corresponds to the UCB, is obtained by subtracting the direct-reacting bilirubin from the total bilirubin. [Note: In normal plasma, only ~4% of the total bilirubin is conjugated, or direct reacting, because most is secreted into bile.]
III.
A.
Dopamine, norepinephrine (NE), and epinephrine (or, adrenaline) are biologically active (biogenic) amines that are collectively termed catecholamines. Dopamine and NE are synthesized in the brain and function as neurotransmitters. Epinephrine is synthesized from NE in the adrenal medulla.
1.
Function: Outside the CNS, NE and its methylated derivative, epinephrine, are hormone regulators of carbohydrate and lipid metabolism. NE and epinephrine are released from storage vesicles in the adrenal medulla in response to fright, exercise, cold, and low levels of blood glucose. They increase the degradation of glycogen and triacylglycerol as well as increase blood pressure and the output of the heart. These effects are part of a coordinated response to prepare the individual for stress and are often called the “fight-or-flight” reactions.
2.
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Synthesis: The catecholamines are synthesized from tyrosine, as shown in Figure 21.15. Tyrosine is first hydroxylated by tyrosine hydroxylase to form L-3,4-dihydroxyphenylalanine (DOPA) in a reaction analogous to that described for the hydroxylation of phenylalanine (see p. 263). The tetrahydrobiopterin (BH4)-requiring enzyme is abundant in the CNS, the sympathetic ganglia, and the adrenal medulla, and it catalyzes the rate-limiting step of the pathway. DOPA is decarboxylated in a reaction requiring PLP to form dopamine, which is hydroxylated by dopamine βhydroxylase to yield NE in a reaction that requires ascorbic acid (vitamin C) and copper. Epinephrine is formed from NE by an N-methylation reaction using S-adenosylmethionine (SAM) as the methyl donor (see p. 264).
Parkinson disease, a neurodegenerative movement disorder, is due to insufficient dopamine production as a result of the idiopathic loss of dopamine-producing cells in the brain. Administration of L-DOPA (levodopa) is the most common treatment, because dopamine cannot cross the blood–brain barrier.
3. Degradation: The catecholamines are inactivated by oxidative deamination catalyzed by monoamine oxidase (MAO) and by Omethylation catalyzed by catechol-O-methyltransferase (COMT) using SAM as the methyl donor (Fig. 21.16). The reactions can occur in either order. The aldehyde products of the MAO reaction are oxidized to the corresponding acids. The products of these reactions are excreted in the urine as vanillylmandelic acid (VMA) from epinephrine and NE and homovanillic acid (HVA) from dopamine. [Note: VMA and the metanephrines are increased with pheochromocytomas, rare tumors of the adrenal gland characterized by excessive production of catecholamines.] 4. Monoamine oxidase inhibitors: MAO is found in neural and other tissues, such as the intestine and liver. In the neuron, this enzyme oxidatively deaminates and inactivates any excess neurotransmitter molecules (NE, dopamine, or serotonin) that may leak out of synaptic vesicles when the neuron is at rest. MAO inhibitors (MAOI) may irreversibly or reversibly inactivate the enzyme, permitting neurotransmitter molecules to escape degradation and, therefore, both to accumulate within the presynaptic neuron and to leak into the synaptic space. This causes activation of NE and serotonin receptors and may be responsible for the antidepressant action of MAOI. [Note: The interaction of MAOI with tyraminecontaining foods is discussed on p. 373.]
B. Histamine
Histamine is a chemical messenger that mediates a wide range of cellular responses, including allergic and inflammatory reactions and gastric acid secretion. A powerful vasodilator, histamine is formed by decarboxylation of histidine in a reaction requiring PLP (Fig. 21.17). It is secreted by mast cells as a result of allergic reactions or trauma. Histamine has no clinical applications, but agents that interfere with the action of histamine have important therapeutic applications.
C. Serotonin
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Serotonin, also called 5-hydroxytryptamine (5-HT), is synthesized and/or stored at several sites in the body (Fig. 21.18). The largest amount by far is found in the intestinal mucosa. Smaller amounts occur in the CNS, where it functions as a neurotransmitter, and in platelets (see online Chapter 35). Serotonin is synthesized from tryptophan, which is hydroxylated in a BH4 requiring reaction analogous to that catalyzed by phenylalanine hydroxylase. The product, 5-hydroxytryptophan, is decarboxylated to 5HT. Serotonin has multiple physiologic roles including pain perception and regulation of sleep, appetite, temperature, blood pressure, cognitive functions, and mood (causes a feeling of well-being). [Note: Selective serotonin reuptake inhibitors (SSRI) maintain serotonin levels, thereby functioning as antidepressants.] Serotonin is degraded by MAO to 5hydroxy-3-indoleacetic acid (5-HIAA).
D. Creatine
Creatine phosphate (also called phosphocreatine), the phosphorylated derivative of creatine found in muscle, is a high-energy compound that provides a small but rapidly mobilized reserve of high-energy phosphates that can be reversibly transferred to adenosine diphosphate (Fig. 21.19) to maintain the intracellular level of ATP during the first few minutes of intense muscular contraction. [Note: The amount of creatine phosphate in the body is proportional to the muscle mass.] 1.
Synthesis: Creatine is synthesized in the liver and kidneys from glycine and the guanidino group of arginine, plus a methyl group from SAM (see Fig. 21.19). Animal products are dietary sources. Creatine is reversibly phosphorylated to creatine phosphate by creatine kinase, using ATP as the phosphate donor. [Note: The presence of creatine kinase (MB isozyme) in the plasma is indicative of heart damage and is used in the diagnosis of myocardial infarction (see p. 65).] 2.
Degradation: Creatine and creatine phosphate spontaneously cyclize at a slow but constant rate to form creatinine, which is excreted in the urine. The amount excreted is proportional to the total creatine phosphate content of the body and, therefore, can be used to estimate muscle mass. When muscle mass decreases for any reason (for example, from paralysis or muscular dystrophy), the creatinine content of the urine falls. In addition, a rise in blood creatinine is a sensitive indicator of kidney malfunction, because creatinine normally is rapidly cleared from the blood and excreted. A typical adult male excretes ~1–2 g of creatinine/day.
E. Melanin
Melanin is a pigment that occurs in several tissues, particularly the eye, hair, and skin. It is synthesized from tyrosine in melanocytes (pigmentforming cells) of the epidermis. It functions to protect underlying cells from the harmful effects of sunlight. [Note: A defect in melanin production results in oculocutaneous albinism, the most common type being due to defects in copper-containing tyrosinase (see p. 273).]
IV. CHAPTER SUMMARY
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Amino acids are precursors of many nitrogen (N)-containing compounds including porphyrins, which, in combination with ferrous (Fe2+) iron, form heme (Fig. 21.20). The major sites of heme biosynthesis are the liver, which synthesizes a number of hemeproteins (particularly cytochrome P450 enzymes), and the erythrocyte-producing cells of the bone marrow, which are active in hemoglobin synthesis. In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for hemeproteins. In contrast, heme synthesis in erythroid cells is relatively constant and is matched to the rate of globin synthesis. Heme synthesis starts with glycine and succinyl coenzyme A. The committed step is the formation of δ-aminolevulinic acid (ALA). This mitochondrial reaction is catalyzed by ALA synthase-1 (ALAS1) in the liver (inhibited by hemin, the oxidized form of heme that accumulates when heme is being underutilized) and ALAS2 in erythroid tissues (regulated by iron). Porphyrias are caused by inherited or acquired (lead poisoning) defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors. Enzymic defects early in the pathway cause abdominal pain and neuropsychiatric symptoms, whereas later defects cause photosensitivity. Degradation of heme occurs in the mononuclear phagocyte system, particularly in the liver and spleen. The first step is the production by heme oxygenase of biliverdin, which is subsequently reduced to bilirubin. Bilirubin is transported by albumin to the liver, where its solubility is increased by the addition of two molecules of glucuronic acid by bilirubin uridine diphosphate-glucuronosyltransferase (bilirubin UGT). Bilirubin diglucuronide (conjugated bilirubin) is transported into the bile canaliculi, where it is first hydrolyzed and reduced by gut bacteria to yield urobilinogen, which is further oxidized by bacteria to stercobilin. Jaundice (icterus) refers to the yellow color of the skin and sclerae that is caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood. Three commonly encountered types of jaundice are hemolytic (prehepatic), obstructive (posthepatic), and hepatocellular (hepatic) (see Fig. 21.20). Other important N-containing compounds derived from amino acids include the catecholamines (dopamine, norepinephrine, and epinephrine), creatine, histamine, serotonin, melanin, and nitric oxide.
bilirubin or decreased secretion of conjugated bilirubin from the liver into bile.] CoA = coenzyme A; CO = carbon monoxide; Fe = iron.
Choose the ONE best answer.
1.1. δ-Aminolevulinic acid synthase activity:
A. catalyzes the committed step in porphyrin biosynthesis.
B. is decreased by iron in erythrocytes.
C. is decreased in the liver in individuals treated with certain drugs such as the barbiturate phenobarbital.
D. occurs in the cytosol.
E. requires tetrahydrobiopterin as a coenzyme.
Correct answer = A. δ-Aminolevulinic acid synthase is mitochondrial and catalyzes the rate-limiting and regulated step of porphyrin synthesis. It requires pyridoxal phosphate as a coenzyme. Iron increases production of the erythroid isozyme. The hepatic isozyme is increased in patients treated with certain drugs.
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1.2. A 50-year-old man presented with painful blisters on the backs of his hands. He was a golf instructor and indicated that the blisters had erupted shortly after the golfing season began. He did not have recent exposure to common skin irritants. He had partial complex seizure disorder that had begun ~3 years earlier after a head injury. The patient had been taking phenytoin (his only medication) since the onset of the seizure disorder. He admitted to an average weekly ethanol intake of ~18 12-oz cans of beer. The patient’s urine was reddish orange. Cultures obtained from skin lesions failed to grow organisms. A 24-hour urine collection showed elevated uroporphyrin (1,000 mg; normal, <27 mg). The most likely diagnosis is:
A. acute intermittent porphyria.
B. congenital erythropoietic porphyria.
C. erythropoietic protoporphyria.
D. hereditary coproporphyria.
E. porphyria cutanea tarda.
Correct answer = E. The disease is associated with a deficiency in uroporphyrinogen III decarboxylase (UROD), but clinical expression of the enzyme deficiency is influenced by hepatic injury caused by environmental (for example, ethanol) and infectious (for example, hepatitis B virus) agents. Exposure to sunlight can also be a precipitating factor. Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms and urine that is red to brown. Treatment of the patient’s seizure disorder with phenytoin caused increased synthesis of δaminolevulinic acid synthase and, therefore, of uroporphyrinogen, the substrate of the deficient UROD. The laboratory and clinical findings are inconsistent with other porphyrias.
21.3. A patient presents with jaundice, abdominal pain, and nausea. Clinical laboratory results are shown below.
What is the most likely cause of the jaundice?
A. Decreased hepatic conjugation of bilirubin
B. Decreased hepatic uptake of bilirubin
C. Decreased secretion of bile into the intestine
D. Increased hemolysis
Correct answer = C. The data are consistent with an obstructive jaundice in which a block in the common bile duct decreases the secretion of bile containing conjugated bilirubin (CB) into the intestine (stool will be pale in color). The CB regurgitates into the blood (conjugated hyperbilirubinemia). The CB is excreted in the urine (which darkens) and is referred to as urinary bilirubin. Urinary urobilinogen is not present because its source is intestinal urobilinogen, which is low. The other choices do not match the data.
1.4. A 2-year-old child was brought to his pediatrician for evaluation of gastrointestinal problems. The parents report that the boy has been listless for the last few weeks. Lab tests reveal a microcytic, hypochromic anemia. Blood lead levels are elevated. Which of the enzymes listed below is most likely to have higher-than-normal activity in the liver of this child?
A. δ-Aminolevulinic acid synthase
B. Bilirubin UDP glucuronosyltransferase
C. Ferrochelatase
D. Heme oxygenase
E. Porphobilinogen synthase
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Correct answer = A. This child has the acquired porphyria of lead poisoning. Lead inhibits both δ-aminolevulinic acid dehydratase and ferrochelatase and, consequently, heme synthesis. The decrease in heme derepresses δaminolevulinic acid synthase-1 (the hepatic isozyme), resulting in an increase in its activity. The decrease in heme also results in decreased hemoglobin synthesis, and anemia is seen. Ferrochelatase is directly inhibited by lead. The other choices are enzymes of heme degradation.
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I. OVERVIEW
Ribonucleoside and deoxyribonucleoside phosphates (nucleotides) are essential for all cells. Without them, neither ribonucleic acid (RNA) nor deoxyribonucleic acid (DNA) can be produced, and, therefore, proteins cannot be synthesized or cells proliferate. Nucleotides also serve as carriers of activated intermediates in the synthesis of some carbohydrates, lipids, and conjugated proteins (for example, uridine diphosphate [UDP]-glucose and cytidine diphosphate [CDP]choline) and are structural components of several essential coenzymes, such as coenzyme A, flavin adenine dinucleotide (FAD[H2]), nicotinamide adenine dinucleotide (NAD[H]), and nicotinamide adenine dinucleotide phosphate (NADP[H]). Nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), serve as second messengers in signal transduction pathways. In addition, nucleotides play an important role as energy sources in the cell. Finally, nucleotides are important regulatory compounds for many of the pathways of intermediary metabolism, inhibiting or activating key enzymes. The purine and pyrimidine bases found in nucleotides can be synthesized de novo or can be obtained through salvage pathways that allow the reuse of the preformed bases resulting from normal cell turnover. [Note: Little of the purines and pyrimidines supplied by diet is utilized and is degraded instead.]
II. STRUCTURE
Nucleotides are composed of a nitrogenous base; a pentose monosaccharide; and one, two, or three phosphate groups. The nitrogen-containing bases belong to two families of compounds: the purines and the pyrimidines.
A. Purine and pyrimidine bases
Both DNA and RNA contain the same purine bases: adenine (A) and guanine (G). Both DNA and RNA contain the pyrimidine cytosine (C), but they differ in their second pyrimidine base: DNA contains thymine (T), whereas RNA contains uracil (U). T and U differ in that only T has a methyl group (Fig. 22.1). Unusual (modified) bases are occasionally found in some species of DNA (for example, in some viral DNA) and RNA (for example, in transfer RNA [tRNA]). Base modifications include methylation, glycosylation, acetylation, and reduction. Some examples of unusual bases are shown in Figure 22.2. [Note: The presence of an unusual base in a nucleotide sequence may aid in its recognition by specific enzymes or protect it from being degraded by nucleases.]
B. Nucleosides
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The addition of a pentose sugar to a base through an N-glycosidic bond (see p. 86) produces a nucleoside. If the sugar is ribose, a ribonucleoside is produced, and if the sugar is 2-deoxyribose, a deoxyribonucleoside is produced (Fig. 22.3A). The ribonucleosides of A, G, C, and U are named adenosine, guanosine, cytidine, and uridine, respectively. The deoxyribonucleosides of A, G, C, and T have the added prefix deoxy-(for example, deoxyadenosine). [Note: The compound deoxythymidine is often simply called thymidine, with the deoxy-prefix being understood, because it is incorporated into DNA only.] The carbon and nitrogen atoms in the rings of the base and the sugar are numbered separately (see Fig. 22.3B). [Note: Carbons in the pentose are numbered 1′ to 5′. Thus, when the 5′carbon of a nucleoside (or nucleotide) is referred to, a carbon atom in the pentose, rather than an atom in the base, is being specified.]
C. Nucleotides
The addition of one or more phosphate groups to a nucleoside produces a nucleotide. The first phosphate group is attached by an ester linkage to the 5′-OH of the pentose, forming a nucleoside 5′-phosphate or a 5′-nucleotide. The type of pentose is denoted by the prefix in the names 5′-ribonucleotide and 5′-deoxyribonucleotide. If one phosphate group is attached to the 5′carbon of the pentose, the structure is a nucleoside monophosphate, like adenosine monophosphate (AMP, or adenylate). If a second or third phosphate is added to the nucleoside, a nucleoside diphosphate (for example, adenosine diphosphate [ADP] or triphosphate, for example, ATP) results (Fig. 22.4). The second and third phosphates are each connected to the nucleotide by a “high-energy bond” (a bond with a large, negative change in free energy [–∆G, see p. 70] of hydrolysis). [Note: The phosphate groups are responsible for the negative charges associated with nucleotides and cause DNA and RNA to be referred to as nucleic acids.]
III. PURINE NUCLEOTIDE SYNTHESIS
The atoms of the purine ring are contributed by a number of compounds, including amino acids (aspartate, glycine, and glutamine), carbon dioxide (CO2), and N10-formyltetrahydrofolate (N10-formyl-THF), as shown in Figure 22.5. The purine ring is constructed primarily in the liver by a series of reactions that add the donated carbons and nitrogens to a preformed ribose 5-phosphate. [Note: Synthesis of ribose 5-phosphate from glucose 6-phosphate by the pentose phosphate pathway is discussed on p. 147.]
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A. 5-Phosphoribosyl-1-pyrophosphate synthesis 5-Phosphoribosyl-1-pyrophosphate (PRPP) is an activated pentose that participates in the synthesis and salvage of purines and pyrimidines. Synthesis of PRPP from ATP and ribose 5-phosphate is catalyzed by PRPP synthetase (Fig. 22.6). This X-linked enzyme is activated by inorganic phosphate and inhibited by purine nucleotides (end-product inhibition). [Note: Because the sugar moiety of PRPP is ribose, ribonucleotides are the end products of de novo purine synthesis. When deoxyribonucleotides are required for DNA synthesis, the ribose sugar moiety is reduced (see p. 297).] reaction. [Note: This is not the committed step of purine synthesis because PRPP is used in other pathways such as salvage (see p. 296).] = phosphate; Pi = inorganic phosphate; AMP = adenosine monophosphate; Mg = magnesium.
B. 5-Phosphoribosylamine synthesis
Synthesis of 5-phosphoribosylamine from PRPP and glutamine is shown in Figure 22.7. The amide group of glutamine replaces the pyrophosphate group attached to carbon 1 of PRPP. This is the committed step in purine nucleotide biosynthesis. The enzyme that catalyzes the reaction, glutamine:phosphoribosylpyrophosphate amidotransferase (GPAT), is inhibited by the purine 5′-nucleotides AMP and guanosine monophosphate (GMP, or guanylate), the end products of the pathway. The rate of the reaction is also controlled by the intracellular concentration of PRPP. [Note: The concentration of PRPP is normally far below the Michaelis constant (Km) for the GPAT. Therefore, any small change in the PRPP concentration causes a proportional change in rate of the reaction (see p. 59).] dioxide.
C. Inosine monophosphate synthesis
The next nine steps in purine nucleotide biosynthesis leading to the synthesis of inosine monophosphate ([IMP] whose base is hypoxanthine) are illustrated in Figure 22.7. IMP is the parent purine nucleotide for AMP and GMP. Four steps in this pathway require ATP as an energy source, and two steps in the pathway require N10-formyl-THF as a one-carbon donor (see p. 267). [Note: Hypoxanthine is found in tRNA (see Fig. 32.9 on p. 453).]
D. Synthetic inhibitors
Some synthetic inhibitors of purine synthesis (for example, the sulfonamides) are designed to inhibit the growth of rapidly dividing microorganisms without interfering with human cell functions (see Fig. 22.7). Other purine synthesis inhibitors, such as structural analogs of folic acid (for example, methotrexate), are used pharmacologically to control the spread of cancer by interfering with the synthesis of nucleotides and, therefore, of DNA and RNA (see Fig. 22.7).
Inhibitors of human purine synthesis are extremely toxic to tissues, especially to developing structures such as in a fetus, or to cell types that normally replicate rapidly, including those of bone marrow, skin, gastrointestinal (GI) tract, immune system, or hair follicles. As a result, individuals taking such anticancer drugs can experience adverse effects, including anemia, scaly skin, GI tract disturbance, immunodeficiency, and hair loss.
E. Adenosine and guanosine monophosphate synthesis
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The conversion of IMP to either AMP or GMP uses a two-step, energy-and nitrogen-requiring pathway (Fig. 22.8). [Note: AMP synthesis requires guanosine triphosphate (GTP) as an energy source and aspartate as a nitrogen source, whereas GMP synthesis requires ATP and glutamine.] Also, the first reaction in each pathway is inhibited by the end product of that pathway. This provides a mechanism for diverting IMP to the synthesis of the purine present in lesser amounts. If both AMP and GMP are present in adequate amounts, the de novo pathway of purine nucleotide synthesis is inhibited at the GPAT step.
showing feedback inhibition. NAD(H) = nicotinamide adenine dinucleotide; = pyrophosphate.
F. Nucleoside di-and triphosphate synthesis
Nucleoside diphosphates are synthesized from the corresponding nucleoside monophosphates by base-specific nucleoside monophosphate kinases (Fig. 22.9). [Note: These kinases do not discriminate between ribose or deoxyribose in the substrate.] ATP is generally the source of the transferred phosphate because it is present in higher concentrations than the other nucleoside triphosphates. Adenylate kinase is particularly active in the liver and in muscle, where the turnover of energy from ATP is high. Its function is to maintain equilibrium among the adenine nucleotides (AMP, ADP, and ATP). Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase, an enzyme that, unlike the monophosphate kinases, has broad substrate specificity.
G. Purine salvage pathway
Purines that result from the normal turnover of cellular nucleic acids, or the small amount that is obtained from the diet and not degraded, can be converted to nucleoside triphosphates and used by the body. This is referred to as the salvage pathway for purines. [Note: Salvage is particularly important in the brain.] 1. purine base salvage to nucleotides: Two enzymes are involved: adenine phosphoribosyltransferase (APRT) and X-linked hypoxanthine– guanine phosphoribosyltransferase (HGPRT). Both use PRPP as the source of the ribose 5-phosphate group (Fig. 22.10). The release of pyrophosphate and its subsequent hydrolysis by pyrophosphatase makes these reactions irreversible. [Note: Adenosine is the only purine nucleoside to be salvaged. It is phosphorylated to AMP by adenosine kinase.] deficiencies of HGPRT are known. As the amount of functional enzyme increases, the severity of the symptoms decreases.] IMP, GMP, and AMP = inosine, guanosine, and adenosine monophosphates; PRPP = 5-phosphoribosyl1-pyrophosphate; PPi = pyrophosphate.
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2. Lesch-Nyhan syndrome: This is a rare, X-linked recessive disorder associated with a virtually complete deficiency of HGPRT. The deficiency results in an inability to salvage hypoxanthine or guanine, from which excessive amounts of uric acid, the end product of purine degradation, are then produced (see p. 298). In addition, the lack of this salvage pathway causes increased PRPP levels and decreased IMP and GMP levels. As a result, GPAT (the regulated step in purine synthesis) has excess substrate and decreased inhibitors available, and de novo purine synthesis is increased. The combination of decreased purine reutilization and increased purine synthesis results in increased degradation of purines and the production of large amounts of uric acid, making HGPRT deficiency an inherited cause of hyperuricemia. In patients with Lesch-Nyhan syndrome, the hyperuricemia frequently results in the formation of uric acid stones in the kidneys (urolithiasis) and the deposition of urate crystals in the joints (gouty arthritis) and soft tissues. In addition, the syndrome is characterized by motor dysfunction, cognitive deficits, and behavioral disturbances that include self-mutilation (for example, biting of lips and fingers), as shown in Figure 22.11.
IV. DEOXYRIBONUCLEOTIDE SYNTHESIS
The nucleotides described thus far all contain ribose (ribonucleotides). DNA synthesis, however, requires 2′-deoxyribonucleotides, which are produced from ribonucleoside diphosphates by the enzyme ribonucleotide reductase during the S-phase of the cell cycle (see p. 423). [Note: The same enzyme acts on pyrimidine ribonucleotides.]
A. Ribonucleotide reductase
Ribonucleotide reductase (ribonucleoside diphosphate reductase) is a dimer composed of two nonidentical subunits, R1 (or, α) and the smaller R2 (or, β), and is specific for the reduction of purine nucleoside diphosphates (ADP and GDP) and pyrimidine nucleoside diphosphates (CDP and UDP) to their deoxy forms (dADP, dGDP, dCDP, and dUDP). The immediate donors of the hydrogen atoms needed for the reduction of the 2′-hydroxyl group are two sulfhydryl (–SH) groups on the enzyme itself (R1 subunit), which form a disulfide bond during the reaction (see p. 19). [Note: R2 contains the stable tyrosyl radical required for catalysis at R1.] 1.
Reduced enzyme regeneration: In order for ribonucleotide reductase to continue to produce deoxyribonucleotides at R1, the disulfide bond created during the production of the 2′-deoxy carbon must be reduced. The source of the reducing equivalents is thioredoxin, a protein coenzyme of ribonucleotide reductase. Thioredoxin contains two cysteine residues separated by two amino acids in the peptide chain. The two –SH groups of thioredoxin donate their hydrogen atoms to ribonucleotide reductase, forming a disulfide bond in the process (Fig. 22.12).
2.
Reduced thioredoxin regeneration: Thioredoxin must be converted back to its reduced form in order to continue performing its function. The reducing equivalents are provided by NADPH + H+, and the reaction is catalyzed by thioredoxin reductase, a selenoprotein (see p. 268).
B. Deoxyribonucleotide synthesis regulation
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Ribonucleotide reductase is responsible for maintaining a balanced supply of the deoxyribonucleotides required for DNA synthesis. Consequently, the regulation of the enzyme is complex. In addition to the catalytic site, R1 contains two distinct allosteric sites involved in regulating enzymic activity (Fig. 22.13).
1.
Activity sites: The binding of dATP to allosteric sites (known as activity sites) on R1 inhibits the overall catalytic activity of the enzyme and, therefore, prevents the reduction of any of the four nucleoside diphosphates. This effectively prevents DNA synthesis and explains the toxicity of increased levels of dATP seen in conditions such as adenosine deaminase (ADA) deficiency (see p. 301). In contrast, ATP bound to these sites activates the enzyme.
2.
Substrate specificity sites: The binding of nucleoside triphosphates to additional allosteric sites (known as substrate specificity sites) on R1 regulates substrate specificity, causing an increase in the conversion of different species of ribonucleotides to deoxyribonucleotides as they are required for DNA synthesis. For example, deoxythymidine triphosphate binding at the specificity site causes a conformational change that allows reduction of GDP to dGDP at the catalytic site when ATP is at the activity site.
The drug hydroxyurea (hydroxycarbamide) inhibits ribonucleotide reductase, thereby inhibiting the generation of substrates for DNA synthesis. The drug is an antineoplastic agent and is used in the treatment of cancers such as melanoma. Hydroxyurea is also used in the treatment of sickle cell anemia (see p. 36). However, the increase in fetal hemoglobin seen with hydroxyurea is because of changes in gene expression and not to ribonucleotide reductase inhibition.
V. PURINE NUCLEOTIDE DEGRADATION
Degradation of dietary nucleic acids occurs in the small intestine, where pancreatic nucleases hydrolyze them to nucleotides. The nucleotides are sequentially degraded by intestinal enzymes to nucleosides, phosphorylated sugars, and free bases. Uric acid is the end product of intestinal purine degradation. [Note: Purine nucleotides from de novo synthesis are degraded in the liver primarily. The free bases are sent out from the liver and salvaged by peripheral tissues.]
A. Degradation in the small intestine
Ribonucleases and deoxyribonucleases, secreted by the pancreas, hydrolyze dietary RNA and DNA to oligonucleotides that are further hydrolyzed by pancreatic phosphodiesterases, producing a mixture of 3′and 5′-mononucleotides. At the intestinal mucosal surface, nucleotidases remove the phosphate groups hydrolytically, releasing nucleosides that are taken into enterocytes by sodium-dependent transporters and degraded by nucleosidases (nucleoside phosphorylases) to free bases plus (deoxy) ribose 1-phosphate. Dietary purine bases are not used to any appreciable extent for the synthesis of tissue nucleic acids. Instead, they are degraded to uric acid in the enterocytes. Most of the uric acid enters the blood and is eventually excreted in the urine. A summary of this pathway is shown in Figure 22.14. [Note: Mammals other than primates express urate oxidase (uricase), which cleaves the purine ring, generating allantoin. Modified recombinant urate oxidase is now used clinically to lower urate levels.]
B. Uric acid formation
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A summary of the steps in the production of uric acid and the genetic diseases associated with deficiencies of specific degradative enzymes are shown in Figure 22.15. [Note: The bracketed numbers refer to specific reactions in the figure.] some of the genetic diseases associated with this pathway. [Note: The numbers in brackets refer to the corresponding numbered citations in the text.] BMT = bone marrow transplantation; ERT = enzyme replacement therapy; Pi = inorganic phosphate; H2O2 = hydrogen peroxide; NH3 = ammonia.
[1] An amino group is removed from AMP to produce IMP by AMP (adenylate) deaminase or from adenosine to produce inosine (hypoxanthine-ribose) by adenosine deaminase.
[2] IMP and GMP are converted into their respective nucleoside forms, inosine and guanosine, by the action of 5′-nucleotidase.
[3] Purine nucleoside phosphorylase converts inosine and guanosine into their respective purine bases, hypoxanthine and guanine. [Note: A mutase interconverts ribose 1-and ribose 5-phosphate.] [4] Guanine is deaminated to form xanthine.
[5] Hypoxanthine is oxidized by molybdenum-containing xanthine oxidase (XO) to xanthine, which is further oxidized by XO to uric acid, the final product of human purine degradation. Uric acid is excreted primarily in the urine.
C. Diseases associated with purine degradation 1. Gout: Gout is a disorder initiated by high levels of uric acid (the end product of purine catabolism) in blood (hyperuricemia), as a result of either the overproduction or underexcretion of uric acid. The hyperuricemia can lead to the deposition of monosodium urate (MSU) crystals in the joints and an inflammatory response to the crystals, causing first acute and then progressing to chronic gouty arthritis. Nodular masses of MSU crystals (tophi) may be deposited in the soft tissues, resulting in chronic tophaceous gout (Fig. 22.16). Formation of uric acid stones in the kidney (urolithiasis) may also be seen. [Note: Hyperuricemia is not sufficient to cause gout, but gout is always preceded by hyperuricemia. Hyperuricemia is typically asymptomatic but may be indicative of comorbid conditions such as hypertension.] The definitive diagnosis of gout requires aspiration and examination of synovial fluid (Fig. 22.17) from an affected joint (or material from a tophus) using polarized light microscopy to confirm the presence of needle-shaped MSU crystals (Fig. 22.18).
a.
Uric acid underexcretion: In >90% of individuals with hyperuricemia, the cause is underexcretion of uric acid. Underexcretion can be primary, because of as-yet-unidentified inherent excretory defects, or secondary to known disease processes that affect how the kidney handles urate (for example, in lactic acidosis, lactate increases renal urate reabsorption, thereby decreasing its excretion) and to environmental factors such as the use of drugs (for example, thiazide diuretics) or exposure to lead (saturnine gout).
b.
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Uric acid overproduction: A less common cause of hyperuricemia is from the overproduction of uric acid. Primary hyperuricemia is, for the most part, idiopathic (having no known cause). However, several identified mutations in the gene for X-linked PRPP synthetase result in the enzyme having an increased maximal velocity ([Vmax] see p. 57) for the production of PRPP, a lower Km (see p. 59) for ribose 5phosphate, or a decreased sensitivity to purine nucleotides, its allosteric inhibitors (see p. 62). In each case, increased availability of PRPP increases purine production, resulting in elevated levels of plasma uric acid. Lesch-Nyhan syndrome (see p. 296) also causes hyperuricemia as a result of the decreased salvage of hypoxanthine and guanine and the subsequent increased availability of PRPP. Secondary hyperuricemia is typically the consequence of increased availability of purines (for example, in patients with myeloproliferative disorders or who are undergoing chemotherapy and so have a high rate of cell turnover). Hyperuricemia can also be the result of seemingly unrelated metabolic diseases, such as von Gierke disease (see Fig. 11.8 on p.
130) or hereditary fructose intolerance (see p. 138).
A diet rich in meat, seafood (particularly shellfish), and ethanol is associated with increased risk of gout, whereas a diet rich in low-fat dairy products is associated with a decreased risk.
c. Treatment: Acute attacks of gout are treated with anti-inflammatory agents. Colchicine, steroidal drugs such as prednisone, and nonsteroidal drugs such as indomethacin are used. [Note: Colchicine prevents formation of microtubules, thereby decreasing the movement of neutrophils into the affected area. Like the other anti-inflammatory drugs, it has no effect on uric acid levels.] Long-term therapeutic strategies for gout involve lowering the uric acid level below its saturation point (6.5 mg/dl), thereby preventing the deposition of MSU crystals. Uricosuric agents, such as probenecid or sulfinpyrazone, that increase renal excretion of uric acid, are used in patients who are underexcretors of uric acid. Allopurinol, a structural analog of hypoxanthine, inhibits uric acid synthesis and is used in patients who are overproducers of uric acid. Allopurinol is oxidized to oxypurinol, a long-lived inhibitor of XO. This results in an accumulation of hypoxanthine and xanthine (see Fig. 22.15), compounds more soluble than uric acid and, therefore, less likely to initiate an inflammatory response. In patients with normal levels of HGPRT, the hypoxanthine can be salvaged, reducing the levels of PRPP and, therefore, de novo purine synthesis. Febuxostat, a nonpurine inhibitor of XO, is also available. [Note: Uric acid levels in the blood normally are close to the saturation point. One reason for this may be the strong antioxidant effects of uric acid.] 2. Adenosine deaminase deficiency
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ADA is expressed in a variety of tissues, but, in humans, lymphocytes have the highest activity of this cytoplasmic enzyme. A deficiency of ADA results in an accumulation of adenosine, which is converted to its ribonucleotide or deoxyribonucleotide forms by cellular kinases. As dATP levels rise, ribonucleotide reductase is inhibited, thereby preventing the production of all deoxyribose-containing nucleotides (see p. 297). Consequently, cells cannot make DNA and divide. [Note: The dATP and adenosine that accumulate in ADA deficiency lead to developmental arrest and apoptosis of lymphocytes.] In its most severe form, this autosomal-recessive disorder causes a type of severe combined immunodeficiency disease (SCID), involving a decrease in T cells, B cells, and natural killer cells. ADA deficiency accounts for ~14% of cases of SCID in the United States. Treatments include bone marrow transplantation, enzyme replacement therapy, and gene therapy (see p. 501). Without appropriate treatment, children with this disorder usually die from infection by age 2 years. [Note: Purine nucleoside phosphorylase deficiency results in a less severe immunodeficiency primarily involving T cells.]
VI. PYRIMIDINE SYNTHESIS AND DEGRADATION
Unlike the synthesis of the purine ring, which is constructed on a pre-existing ribose 5-phosphate, the pyrimidine ring is synthesized before being attached to ribose 5-phosphate, which is donated by PRPP. The sources of the atoms in the pyrimidine ring are glutamine, CO2, and aspartate (Fig. 22.19).
A. Carbamoyl phosphate synthesis
The regulated step of this pathway in mammalian cells is the synthesis of carbamoyl phosphate from glutamine and CO2, catalyzed by carbamoyl phosphate synthetase (CPS) II. CPS II is inhibited by uridine triphosphate (the end product of this pathway, which can be converted into the other pyrimidine nucleotides) and is activated by PRPP. [Note: Carbamoyl phosphate, synthesized by CPS I, is also a precursor of urea (see p. 253). Defects in ornithine transcarbamylase of the urea cycle promote pyrimidine synthesis because of increased availability of carbamoyl phosphate. A comparison of the two enzymes is presented in Figure 22.20.]
B. Orotic acid synthesis
The second step in pyrimidine synthesis is the formation of carbamoylaspartate, catalyzed by aspartate transcarbamoylase. The pyrimidine ring is then closed by dihydroorotase. The resulting dihydroorotate is oxidized to produce orotic acid (orotate), as shown in Figure 22.21. The human enzyme that produces orotate, dihydroorotate dehydrogenase, is a flavin mononucleotide-containing protein of the inner mitochondrial membrane. All other enzymes in pyrimidine biosynthesis are cytosolic. [Note: The first three enzymic activities in this pathway (CPS II, aspartate transcarbamoylase, and dihydroorotase) are actually three different catalytic domains of a single polypeptide known as CAD from the first letter in the name of each domain. (See p. 18 for a discussion of domains.) This is an example of a multifunctional or multicatalytic polypeptide that facilitates the ordered synthesis of an important compound. Synthesis of the purine nucleotide IMP also involves multifunctional proteins.]
C. Pyrimidine nucleotide synthesis
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The completed pyrimidine ring is converted to the nucleotide orotidine monophosphate (OMP) in the second stage of pyrimidine nucleotide synthesis (see Fig. 22.21). As seen with the purines, PRPP is the ribose 5phosphate donor. The enzyme orotate phosphoribosyltransferase produces OMP and releases pyrophosphate, thereby making the reaction biologically irreversible. [Note: Both purine and pyrimidine synthesis require glutamine, aspartic acid, and PRPP as essential precursors.] OMP (orotidylate) is decarboxylated to uridine monophosphate (UMP) by orotidylate decarboxylase. The phosphoribosyltransferase and decarboxylase activities are separate catalytic domains of a single polypeptide called UMP synthase. Hereditary orotic aciduria (a very rare disorder) may be caused by a deficiency of one or both activities of this bifunctional enzyme, resulting in orotic acid in the urine (see Fig. 22.21). UMP is sequentially phosphorylated to UDP and UTP. [Note: The UDP is a substrate for ribonucleotide reductase, which generates dUDP. The dUDP is phosphorylated to dUTP, which is rapidly hydrolyzed to dUMP by UTP diphosphatase (dUTPase). Thus, dUTPase plays an important role in reducing availability of dUTP as a substrate for DNA synthesis, thereby preventing erroneous incorporation of uracil into DNA.]
D. Cytidine triphosphate synthesis
Cytidine triphosphate (CTP) is produced by amination of UTP by CTP synthetase (Fig. 22.22), with glutamine providing the nitrogen. Some of this CTP is dephosphorylated to CDP, which is a substrate for ribonucleotide reductase. The dCDP product can be phosphorylated to dCTP for DNA synthesis or dephosphorylated to dCMP that is deaminated to dUMP.
E. Deoxythymidine monophosphate synthesis dUMP is converted to deoxythymidine monophosphate (dTMP) by thymidylate synthase, which uses N5,N10-methylene-THF as the source of the methyl group (see p. 267). This is an unusual reaction in that THF contributes not only a one-carbon unit but also two hydrogen atoms from the pteridine ring, resulting in the oxidation of THF to dihydrofolate ([DHF], Fig. 22.23). Inhibitors of thymidylate synthase include thymine analogs such as 5-fluorouracil, which serve as antitumor agents. 5Fluorouracil is metabolically converted to 5-fluorodeoxyuridine monophosphate (5-FdUMP), which becomes permanently bound to the inactivated thymidylate synthase, making the drug a suicide inhibitor (see p. 60). DHF can be reduced to THF by dihydrofolate reductase (see Fig.
28.2, p. 378), an enzyme that is inhibited by folate analogs such as methotrexate. By decreasing the supply of THF, these drugs not only inhibit purine synthesis (see Fig. 22.7), but, by preventing methylation of dUMP to dTMP, they also decrease the availability of this essential component of DNA. DNA synthesis is inhibited and cell growth slowed. Thus, these drugs are used to treat cancer. [Note: Acyclovir (a purine analog) and AZT (3′-azido-3′-deoxythymidine, a pyrimidine analog) are used to treat infections of herpes simplex virus and human immunodeficiency virus, respectively. Each inhibits the viral DNA polymerase.]
F. Pyrimidine salvage and degradation
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Unlike the purine ring, which is not cleaved in humans and is excreted as poorly soluble uric acid, the pyrimidine ring is opened and degraded to highly soluble products, β-alanine (from the degradation of CMP and UMP) and β-aminoisobutyrate (from TMP degradation), with the production of ammonia and CO2. Pyrimidine bases can be salvaged to nucleosides, which are phosphorylated to nucleotides. However, their high solubility makes pyrimidine salvage less significant clinically than purine salvage. [Note: The salvage of pyrimidine nucleosides is the basis for using uridine in the treatment of hereditary orotic aciduria (see p. 302).]
VII. CHAPTER SUMMARY
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Nucleotides are composed of a nitrogenous base (adenine = A, guanine = G, cytosine = C, uracil = U, and thymine = T); a pentose sugar; and one, two, or three phosphate groups (Fig. 22.24). A and G are purines, and C, U, and T are pyrimidines. If the sugar is ribose, the nucleotide is a ribonucleoside phosphate (for example, adenosine monophosphate [AMP]), and it can have several functions in the cell, including being a component of RNA. If the sugar is deoxyribose, the nucleotide is a deoxyribonucleoside phosphate (for example, deoxyAMP) and will be found almost exclusively as a component of DNA. The committed step in purine synthesis uses 5-phosphoribosyl-1-pyrophosphate ([PRPP], an activated pentose that provides the ribose 5-phosphate for de novo purine and pyrimidine synthesis and salvage) and nitrogen from glutamine to produce phosphoribosylamine. The enzyme is glutamine:phosphoribosylpyrophosphate amidotransferase and is inhibited by AMP and guanosine monophosphate (the end products of the pathway) and activated by PRPP. Purine nucleotides can also be produced from preformed purine bases by using salvage reactions catalyzed by adenine phosphoribosyltransferase (APRT) and hypoxanthine–guanine phosphoribosyltransferase (HGPRT). A near-total deficiency of HGPRT causes Lesch-Nyhan syndrome, a severe, inherited form of hyperuricemia accompanied by compulsive self-mutilation. All deoxyribonucleotides are synthesized from ribonucleotides by the enzyme ribonucleotide reductase. This enzyme is highly regulated (for example, it is strongly inhibited by deoxyadenosine triphosphate [dATP], a compound that is overproduced in bone marrow cells in individuals with adenosine deaminase [ADA] deficiency). ADA deficiency causes severe combined immunodeficiency disease. The end product of purine degradation is uric acid, a compound of low solubility whose overproduction or undersecretion causes hyperuricemia that, if accompanied by the deposition of monosodium urate crystals in joints and soft tissues and an inflammatory response to those crystals, results in gout. The first step in pyrimidine synthesis, the production of carbamoyl phosphate by carbamoyl phosphate synthetase II, is the regulated step in this pathway (it is inhibited by uridine triphosphate [UTP] and activated by PRPP). The UTP produced by this pathway can be converted to cytidine triphosphate. Deoxyuridine monophosphate can be converted to deoxythymidine monophosphate by thymidylate synthase, an enzyme targeted by anticancer drugs such as 5-fluorouracil. The regeneration of tetrahydrofolate from dihydrofolate produced in the thymidylate synthase reaction requires dihydrofolate reductase, an enzyme targeted by the drug methotrexate. Pyrimidine degradation results in soluble products.
guanosine, cytidine, thymidine, and inosine monophosphates; d = deoxy; PPi = pyrophosphate; PRPP = 5-phosphoribosyl-1-pyrophosphate.
Choose the ONE best answer.
2.1. Azaserine, a drug with research applications, inhibits glutamine-dependent enzymes. Incorporation of which of the ring nitrogens (N) in the generic purine structure shown would most likely be affected by azaserine?
A. 1 B. 3 C. 7 D. 9
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Correct answer = D. The N at position 9 is supplied by glutamine in the first step of purine de novo synthesis, and its incorporation would be affected by azaserine. The N at position 1 is supplied by aspartate and at position 7 by glycine. The N at position 3 is also supplied by glutamine, but azaserine would have inhibited purine synthesis prior to this step.
2.2. A 42-year-old male patient undergoing radiation therapy for prostate cancer develops severe pain in the metatarsal phalangeal joint of his right big toe. Monosodium urate crystals are detected by polarized light microscopy in fluid obtained from this joint by arthrocentesis. This patient’s pain is directly caused by the overproduction of the end product of which of the following metabolic pathways?
A. De novo pyrimidine biosynthesis
B. Pyrimidine degradation
C. De novo purine biosynthesis
D. Purine salvage
E. Purine degradation
Correct answer = E. The patient’s pain is caused by gout, resulting from an inflammatory response to the crystallization of excess urate (as monosodium urate) in his joints. Radiation therapy caused cell death, with degradation of nucleic acids and their constituent purines. Uric acid, the end product of purine degradation, is a relatively insoluble compound that can cause gout (and kidney stones). Pyrimidine metabolism is not associated with uric acid production. Overproduction of purines can indirectly result in hyperuricemia. Purine salvage decreases uric acid production.
2.3. Which one of the following enzymes of nucleotide metabolism is correctly paired with its pharmacologic inhibitor?
A. Dihydrofolate reductase—methotrexate
B. Inosine monophosphate dehydrogenase—hydroxyurea C. Ribonucleotide reductase—5-fluorouracil
D. Thymidylate synthase—allopurinol
E. Xanthine oxidase—probenecid
Correct answer = A. Methotrexate interferes with folate metabolism by acting as a competitive inhibitor of the enzyme dihydrofolate reductase. This starves cells for tetrahydrofolate and makes them unable to synthesize purines and thymidine monophosphate. Inosine monophosphate dehydrogenase is inhibited by mycophenolic acid. Ribonucleotide reductase is inhibited by hydroxyurea. Thymidylate synthase is inhibited by 5-fluorouracil. Xanthine oxidase is inhibited by allopurinol. Probenecid increases renal excretion of urate but does not inhibit its production.
2.4. A 1-year-old female patient is lethargic, weak, and anemic. Her height and weight are low for her age. Her urine contains an elevated level of orotic acid. Activity of uridine monophosphate synthase is low. Administration of which of the following is most likely to alleviate her symptoms?
A. Adenine
B. Guanine
C. Hypoxanthine
D. Thymidine
E. Uridine
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Correct answer = E. The elevated excretion of orotic acid and low activity of uridine monophosphate (UMP) synthase indicate that the patient has orotic aciduria, a very rare genetic disorder affecting de novo pyrimidine synthesis. Deficiencies in one or both catalytic domains of UMP synthase leave the patient unable to synthesize pyrimidines. Uridine, a pyrimidine nucleoside, is a useful treatment because it bypasses the missing activities and can be salvaged to UMP, which can be converted to all the other pyrimidines. Although thymidine is a pyrimidine nucleoside, it cannot be converted to other pyrimidines. Hypoxanthine, guanine, and adenine are all purine bases and cannot be converted to pyrimidines.
2.5. What laboratory test would help in distinguishing an orotic aciduria caused by ornithine transcarbamylase deficiency from that caused by uridine monophosphate synthase deficiency?
Blood ammonia level would be expected to be elevated in ornithine transcarbamylase deficiency that affects the urea cycle but not in uridine monophosphate synthase deficiency.
UNIT V Integration of Metabolism Metabolic Effects of Insulin and Glucagon 23
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I. OVERVIEW
Four major tissues play a dominant role in fuel metabolism: liver, adipose, muscle, and brain. These tissues contain unique sets of enzymes, such that each tissue is specialized for the storage, use, or generation of specific fuels. These tissues do not function in isolation but rather form part of a network in which one tissue may provide substrates to another or process compounds produced by other tissues. Communication between tissues is mediated by the nervous system, by the availability of circulating substrates, and by variation in the levels of plasma hormones (Fig. 23.1). The integration of energy metabolism is controlled primarily by the actions of two peptide hormones, insulin and glucagon (secreted in response to changing substrate levels in the blood), with the catecholamines epinephrine and norepinephrine (secreted in response to neural signals) playing a supporting role. Changes in the circulating levels of these hormones allow the body to store energy when food is abundant or to make stored energy available such as during survival crises (for example, famine, severe injury, and “fight-or-flight” situations). This chapter describes the structure, secretion, and metabolic effects of the two hormones that most profoundly affect energy metabolism.
II. INSULIN
Insulin is a peptide hormone produced by the β cells of the islets of Langerhans, which are clusters of cells embedded in the endocrine portion of the pancreas (Fig. 23.2). [Note: “Insulin” is from the Latin for island.] The islets make up only about 1%–2% of the total cells of the pancreas. Insulin is the most important hormone coordinating the use of fuels by tissues. Its metabolic effects are anabolic, favoring, for example, synthesis of glycogen, triacylglycerol (TAG), and protein.
A. Structure
Insulin is composed of 51 amino acids arranged in two polypeptide chains, designated A (21 amino acids) and B, which are linked together by two disulfide bonds (Fig. 23.3A). The insulin molecule also contains an intramolecular disulfide bond between amino acid residues of the A chain. [Note: Insulin was the first peptide for which the primary structure was determined and the first therapeutic molecule made by recombinant DNA technology (see p. 486).]
B. Synthesis
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The processing and transport of intermediates that occur during the synthesis of insulin are shown in Figures 23.3B and 23.4. Biosynthesis involves production of two inactive precursors, preproinsulin and proinsulin, which are sequentially cleaved to form the active hormone plus the connecting or C-peptide in a 1:1 ratio (see Fig. 23.4). [Note: The C-peptide is essential for proper insulin folding. Also, because its half-life in plasma is longer than that of insulin, the C-peptide level is a good indicator of insulin production and secretion.] Insulin is stored in cytosolic granules that, given the proper stimulus (see C.1. below), are released by exocytosis. (See p. 459 for a discussion of the synthesis of secreted proteins.) Insulin is degraded by insulin-degrading enzyme, which is present in the liver and, to a lesser extent, in the kidneys. Insulin has a plasma half-life of ~6 minutes. This short duration of action permits rapid changes in circulating levels of the hormone.
C. Secretion regulation
Secretion of insulin is regulated by bloodborne fuels and hormones.
1. Increased secretion: Insulin secretion by the pancreatic β cells is closely coordinated with the secretion of glucagon by pancreatic α cells (Fig. 23.5). The relative amounts of glucagon and insulin released are normally regulated such that the rate of hepatic glucose production is kept equal to the use of glucose by peripheral tissues. This maintains blood glucose between 70 and 140 mg/dl. In view of its coordinating role, it is not surprising that the β cell responds to a variety of stimuli. In particular, insulin secretion is increased by glucose, amino acids, and gastrointestinal peptide hormones.
a.
Glucose: Ingestion of a carbohydrate-rich meal leads to a rise in blood glucose, the primary stimulus for insulin secretion (see Fig. 23.5). The β cells are the most important glucose-sensing cells in the body. Like the liver, β cells contain GLUT-2 transporters and express glucokinase (hexokinase IV; see p. 98). At blood glucose levels >45 mg/dl, glucokinase phosphorylates glucose in amounts proportional to the glucose concentration. Proportionality results from the lack of direct inhibition of glucokinase by glucose 6-phosphate, its product. Additionally, the sigmoidal relationship between the velocity of the reaction and substrate concentration (see p. 98) maximizes the enzyme’s responsiveness to changes in blood glucose level. Metabolism of glucose 6-phosphate generates ATP, leading to insulin secretion (see blue box below).
b.
Amino acids: Ingestion of protein causes a transient rise in plasma amino acid levels (for example, arginine) that enhances the glucose-stimulated secretion of insulin. [Note: Fatty acids have a similar effect.] c.
Gastrointestinal peptide hormones: The intestinal peptides glucagonlike peptide-1 (GLP-1) and gastric inhibitory polypeptide ([GIP] also called glucose-dependent insulinotropic peptide) increase the sensitivity of β cells to glucose. They are released from the small intestine after the ingestion of food, causing an anticipatory rise in insulin levels and, thus, are referred to as incretins. Their action may account for the fact that the same amount of glucose given orally induces a much greater secretion of insulin than if given intravenously (IV).
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Glucose-dependent release of insulin into blood is mediated through a rise in calcium (Ca2+) concentration in the β cell. Glucose taken into β cells by GLUT-2 is phosphorylated and metabolized, with subsequent production of ATP. ATP-sensitive potassium (K+) channels close, causing depolarization of the plasma membrane, opening of voltage-gated Ca2+ channels, and influx of Ca2+ into the cell. Ca2+ causes vesicles containing insulin to be exocytosed from the β cell. Sulfonylureas, oral agents used to treat type 2 diabetes, increase insulin secretion by closing ATP-sensitive K+ channels.
2. Decreased secretion: The synthesis and release of insulin are decreased when there is a scarcity of dietary fuels and also during periods of physiologic stress (for example, infection, hypoxia, and vigorous exercise), thereby preventing hypoglycemia. These effects are mediated primarily by the catecholamines norepinephrine and epinephrine, which are made from tyrosine in the sympathetic nervous system (SNS) and the adrenal medulla and then secreted. Secretion is largely controlled by neural signals. The catecholamines (primarily epinephrine) have a direct effect on energy metabolism, causing a rapid mobilization of energy-yielding fuels, including glucose from the liver (produced by glycogenolysis or gluconeogenesis; see p. 121) and fatty acids (FA) from adipose tissue (produced by lipolysis; see p. 189). In addition, these biogenic amines can override the normal glucose-stimulated release of insulin. Thus, in emergency situations, the SNS largely replaces the plasma glucose concentration as the controlling influence over β-cell secretion. The regulation of insulin secretion is summarized in Figure 23.6.
D. Metabolic effects
Insulin promotes the storage of nutrients as glycogen, TAG, and protein and inhibits their mobilization.
1. Effects on carbohydrate metabolism: The effects of insulin on glucose metabolism promote its storage and are most prominent in three tissues: liver, muscle, and adipose. In liver and muscle, insulin increases glycogen synthesis. In muscle and adipose, insulin increases glucose uptake by increasing the number of glucose transporters (GLUT-4; see p.
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97) in the cell membrane. Thus, the IV administration of insulin causes an immediate decrease in blood glucose level. In the liver, insulin decreases the production of glucose through the inhibition of glycogenolysis and gluconeogenesis. [Note: The effects of insulin are due not just to changes in enzyme activity but also in enzyme amount insofar as insulin alters gene transcription.] 2. Effects on lipid metabolism: A rise in insulin rapidly causes a significant reduction in the release of FA from adipose tissue by inhibiting the activity of hormone-sensitive lipase, a key enzyme of TAG degradation in adipocytes. Insulin acts by promoting the dephosphorylation and, hence, inactivation of the enzyme (see p. 190). Insulin also increases the transport and metabolism of glucose into adipocytes, providing the glycerol 3-phosphate substrate for TAG synthesis (see p. 188). Expression of the gene for lipoprotein lipase, which degrades TAG in circulating chylomicrons and very-low-density lipoproteins ([VLDL] see p. 229), is increased by insulin in adipose, thereby providing FA for esterification to the glycerol. [Note: Insulin also promotes the conversion of glucose to TAG in the liver. The TAG are secreted in VLDL.] 3. Effects on protein synthesis: In most tissues, insulin stimulates both the entry of amino acids into cells and protein synthesis (translation). [Note: Insulin stimulates protein synthesis through covalent activation of factors required for translation initiation.]
E. Mechanism
Insulin binds to specific, high-affinity receptors in the cell membrane of most tissues, including liver, muscle, and adipose. This is the first step in a cascade of reactions ultimately leading to a diverse array of biologic actions (Fig. 23.7).
1.
Insulin receptor: The insulin receptor is synthesized as a single polypeptide that is glycosylated and cleaved into α and β subunits, which are then assembled into a tetramer linked by disulfide bonds (see Fig. 23.7). The extracellular α subunits contain the insulin-binding site. A hydrophobic domain in each β subunit spans the plasma membrane. The cytosolic domain of the β subunit is a tyrosine kinase, which is activated by insulin. As a result, the insulin receptor is classified as a tyrosine kinase receptor.
2.
Signal transduction: The binding of insulin to the α subunits of the insulin receptor induces conformational changes that are transmitted to the β subunits. This promotes a rapid autophosphorylation of specific tyrosine residues on each β subunit (see Fig. 23.7). Autophosphorylation initiates a cascade of cell-signaling responses, including phosphorylation of a family of proteins called insulin receptor substrates (IRS). At least four IRS have been identified that show similar structures but different tissue distributions. Phosphorylated IRS proteins interact with other signaling molecules through specific domains (known as SH2), activating a number of pathways that affect gene expression, cell metabolism, and growth. The actions of insulin are terminated by dephosphorylation of the receptor.
3.
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Membrane effects: Glucose transport in some tissues, such as muscle and adipose, increases in the presence of insulin (Fig. 23.8). Insulin promotes movement of insulin-sensitive glucose transporters (GLUT-4) from a pool located in intracellular vesicles to the cell membrane. [Note: Movement is the result of a signaling cascade in which an IRS binds to and activates a kinase (phosphoinositide 3-kinase), leading to phosphorylation of the membrane phospholipid phosphatidylinositol 4,5bisphosphate (PIP2) to the 3,4,5-trisphosphate form (PIP3) that binds to and activates phosphoinositide-dependent kinase 1. This kinase, in turn, activates Akt (or protein kinase B), resulting in GLUT-4 movement.] In contrast, other tissues have insulin-insensitive systems for glucose transport (Fig. 23.9). For example, hepatocytes, erythrocytes, and cells of the nervous system, intestinal mucosa, renal tubules, and cornea do not require insulin for glucose uptake.
4.
Receptor regulation: Binding of insulin is followed by internalization of the hormone–receptor complex. Once inside the cell, insulin is degraded in the lysosomes. The receptors may be degraded, but most are recycled to the cell surface. [Note: Elevated levels of insulin promote the degradation of receptors, thereby decreasing the number of surface receptors. This is one type of downregulation.] 5.
Time course: The binding of insulin provokes a wide range of actions. The most immediate response is an increase in glucose transport into adipocytes and skeletal and cardiac muscle cells that occurs within seconds of insulin binding to its membrane receptor. Insulin-induced changes in enzymic activity in many cell types occur over minutes to hours and reflect changes in the phosphorylation states of existing proteins. Insulin-induced increase in the amount of many enzymes, such as glucokinase, liver pyruvate kinase, acetyl coenzyme A (CoA) carboxylase (ACC), and fatty acid synthase, requires hours to days. These changes reflect an increase in gene expression through increased transcription (mediated by sterol regulatory element–binding protein-1c; see p. 184) and translation.
III. GLUCAGON
Glucagon is a peptide hormone secreted by the α cells of the pancreatic islets of Langerhans. Glucagon, along with epinephrine, norepinephrine, cortisol, and growth hormone (the counterregulatory hormones), opposes many of the actions of insulin (Fig. 23.10). Most importantly, glucagon acts to maintain blood glucose levels by activation of hepatic glycogenolysis and gluconeogenesis. Glucagon is composed of 29 amino acids arranged in a single polypeptide chain. [Note: Unlike insulin, the amino acid sequence of glucagon is the same in all mammalian species examined to date.] Glucagon is synthesized as a large precursor molecule (preproglucagon) that is converted to glucagon through a series of selective proteolytic cleavages, similar to those described for insulin biosynthesis (see Fig. 23.3). In contrast to insulin, preproglucagon is processed to different products in different tissues, for example, GLP-1 in intestinal L cells. Like insulin, glucagon has a short half-life.
A. Increased secretion
The α cell is responsive to a variety of stimuli that signal actual or potential hypoglycemia (Fig. 23.11). Specifically, glucagon secretion is increased by low blood glucose, amino acids, and catecholamines.
1.
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Low blood glucose: A decrease in plasma glucose concentration is the primary stimulus for glucagon release. During an overnight or prolonged fast, elevated glucagon levels prevent hypoglycemia (see Section IV below for a discussion of hypoglycemia).
2.
Amino acids: Amino acids (for example, arginine) derived from a meal containing protein stimulate the release of glucagon. The glucagon effectively prevents the hypoglycemia that would otherwise occur as a result of the increased insulin secretion that also occurs after a protein meal.
3.
Catecholamines: Elevated levels of circulating epinephrine (from the adrenal medulla), norepinephrine (from sympathetic innervation of the pancreas), or both stimulate the release of glucagon. Thus, during periods of physiologic stress, the elevated catecholamine levels can override the effect on the α cell of circulating substrates. In these situations, regardless of the concentration of blood glucose, glucagon levels are elevated in anticipation of increased glucose use. In contrast, insulin levels are depressed.
B. Decreased secretion
Glucagon secretion is significantly decreased by elevated blood glucose and by insulin. Both substances are increased following ingestion of glucose or a carbohydrate-rich meal (see Fig. 23.5). The regulation of glucagon secretion is summarized in Figure 23.11.
C. Metabolic effects
Glucagon is a catabolic hormone that promotes the maintenance of blood glucose levels. Its primary target is the liver.
1.
Effects on carbohydrate metabolism: The IV administration of glucagon leads to an immediate rise in blood glucose. This results from an increase in the degradation of liver glycogen and an increase in hepatic gluconeogenesis.
2.
Effects on lipid metabolism: The primary effect of glucagon on lipid metabolism is inhibition of FA synthesis through phosphorylation and subsequent inactivation of ACC by adenosine monophosphate (AMP)– activated protein kinase (see p. 184). The resulting decrease in malonyl CoA production removes the inhibition on long-chain FA β-oxidation (see p. 191). Glucagon also plays a role in lipolysis in adipocytes, but the major activators of hormone-sensitive lipase (via phosphorylation by protein kinase A) are the catecholamines. The free FA released are taken up by liver and oxidized to acetyl CoA, which is used in ketone body synthesis.
3.
Effects on protein metabolism: Glucagon increases uptake by the liver of amino acids supplied by muscle, resulting in increased availability of carbon skeletons for gluconeogenesis. As a consequence, plasma levels of amino acids are decreased.
D. Mechanism
Glucagon binds to high-affinity G protein–coupled receptors (GPCR) on the cell membrane of hepatocytes. The GPCR for glucagon is distinct from the GPCR that bind epinephrine. [Note: Glucagon receptors are not found on skeletal muscle.] Glucagon binding results in activation of adenylyl cyclase in the plasma membrane (Fig. 23.12; also see p. 94). This causes a rise in cyclic AMP (cAMP), which, in turn, activates cAMP-dependent protein kinase A and increases the phosphorylation of specific enzymes or other proteins. This cascade of increasing enzymic activities results in the phosphorylation-mediated activation or inhibition of key regulatory enzymes involved in carbohydrate and lipid metabolism. An example of such a cascade in glycogen degradation is shown in Figure 11.9 on p. 131. [Note: Glucagon, like insulin, affects gene transcription. For example, glucagon induces expression of phosphoenolpyruvate carboxykinase (see p. 122).]
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IV. HYPOGLYCEMIA
Hypoglycemia is characterized by 1) central nervous system (CNS) symptoms, including confusion, aberrant behavior, or coma; 2) a simultaneous blood glucose level ≤50 mg/dl; and 3) symptoms being resolved within minutes following glucose administration (Fig. 23.13). Hypoglycemia is a medical emergency because the CNS has an absolute requirement for a continuous supply of bloodborne glucose to serve as a metabolic fuel. Transient hypoglycemia can cause cerebral dysfunction, whereas severe, prolonged hypoglycemia causes brain damage. Therefore, it is not surprising that the body has multiple overlapping mechanisms to prevent or correct hypoglycemia. The most important hormone changes in combating hypoglycemia are increased secretion of glucagon and the catecholamines, combined with decreased insulin secretion.
A. Symptoms
The symptoms of hypoglycemia can be divided into two categories. Adrenergic (neurogenic, autonomic) symptoms, such as anxiety, palpitation, tremor, and sweating, are mediated by catecholamine release (primarily epinephrine) regulated by the hypothalamus in response to hypoglycemia. Adrenergic symptoms typically occur when blood glucose levels fall abruptly. The second category of hypoglycemic symptoms is neuroglycopenic. The impaired delivery of glucose to the brain (neuroglycopenia) results in impairment of brain function, causing headache, confusion, slurred speech, seizures, coma, and death. Neuroglycopenic symptoms often result from a gradual decline in blood glucose, often to levels <50 mg/dl. The slow decline in glucose deprives the CNS of fuel but fails to trigger an adequate adrenergic response.
B. Glucoregulatory systems
Humans have two overlapping glucose-regulating systems that are activated by hypoglycemia: 1) the pancreatic α cells, which release glucagon, and 2) receptors in the hypothalamus, which respond to abnormally low concentrations of blood glucose. The hypothalamic glucoreceptors can trigger both the secretion of catecholamines (mediated by the sympathetic division of the autonomic nervous system) and release of adrenocorticotropic hormone (ACTH) and growth hormone by the anterior pituitary (see Fig. 23.13). [Note: ACTH increases cortisol synthesis and secretion in the adrenal cortex (see p. 239).] Glucagon, the catecholamines, cortisol, and growth hormone are sometimes called the counterregulatory hormones because each opposes the action of insulin on glucose use.
1.
Glucagon and epinephrine: Secretion of these counterregulatory hormones is most important in the acute, short-term regulation of blood glucose levels. Glucagon stimulates hepatic glycogenolysis and gluconeogenesis. Epinephrine promotes glycogenolysis and lipolysis. It inhibits insulin secretion, thereby preventing GLUT-4–mediated uptake of glucose by muscle and adipose tissues. Epinephrine assumes a critical role in hypoglycemia when glucagon secretion is deficient, for example, in the late stages of type 1 diabetes mellitus (see p. 340). The prevention or correction of hypoglycemia fails when the secretion of both glucagon and epinephrine is deficient.
2.
Cortisol and growth hormone: These counterregulatory hormones are less important in the short-term maintenance of blood glucose concentrations. They do, however, play a role in the long-term (transcriptional) management of glucose metabolism.
C. Types
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Hypoglycemia may be divided into four types: 1) insulin induced, 2) postprandial (sometimes called reactive hypoglycemia), 3) fasting hypoglycemia, and 4) alcohol related.
1. Insulin-induced hypoglycemia: Hypoglycemia occurs frequently in patients with diabetes who are receiving insulin treatment, particularly those striving to achieve tight control of blood glucose levels. Mild hypoglycemia in fully conscious patients is treated by oral administration of carbohydrate. Unconscious patients are typically given glucagon subcutaneously or intramuscularly (Fig. 23.14).
2.
Postprandial hypoglycemia: This is the second most common form of hypoglycemia. It is caused by an exaggerated insulin release following a meal, prompting transient hypoglycemia with mild adrenergic symptoms. The plasma glucose level returns to normal even if the patient is not fed. The only treatment usually required is that the patient eats frequent small meals rather than the usual three large meals.
3.
Fasting hypoglycemia: Low blood glucose during fasting is rare but is more likely to present as a serious medical problem. Fasting hypoglycemia, which tends to produce neuroglycopenic symptoms, may result from a reduction in the rate of glucose production by hepatic glycogenolysis or gluconeogenesis. Thus, low blood glucose levels are often seen in patients with hepatocellular damage or adrenal insufficiency or in fasting individuals who have consumed large quantities of ethanol (see 4. below). Alternately, fasting hypoglycemia may be the result of an increased rate of glucose use by the peripheral tissues because of overproduction of insulin by rare pancreatic tumors. If left untreated, a patient with fasting hypoglycemia may lose consciousness and experience convulsions and coma. [Note: Certain inborn errors of metabolism, for example, defects in FA oxidation, result in fasting hypoglycemia.] 4.
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Alcohol-related hypoglycemia: Alcohol (ethanol) is metabolized in the liver by two oxidation reactions (Fig. 23.15). Ethanol is first converted to acetaldehyde by zinc-containing alcohol dehydrogenase. Acetaldehyde is subsequently oxidized to acetate by aldehyde dehydrogenase (ALDH). [Note: ALDH is inhibited by disulfiram, a drug that is used in the treatment of chronic alcoholism. The resulting rise in acetaldehyde results in flushing, tachycardia, hyperventilation, and nausea.] In each reaction, electrons are transferred to oxidized nicotinamide adenine dinucleotide (NAD+), resulting in an increase in the ratio of the reduced form (NADH) to NAD+. The abundance of NADH favors the reduction of pyruvate to lactate and of oxaloacetate (OAA) to malate. Recall from p. 118 that pyruvate and OAA are substrates in the synthesis of glucose. Thus, the ethanol-mediated increase in NADH causes these gluconeogenic precursors to be diverted into alternate pathways, resulting in the decreased synthesis of glucose. This can precipitate hypoglycemia, particularly in individuals who have depleted their stores of liver glycogen. [Note: Decreased availability of OAA allows acetyl CoA to be diverted to ketone body synthesis in the liver (see p. 195) and can result in alcoholic ketosis that may result in ketoacidosis.] Hypoglycemia can produce many of the behaviors associated with alcohol intoxication, such as agitation, impaired judgment, and combativeness. Therefore, alcohol consumption in vulnerable individuals (such as those who are fasted or have engaged in prolonged, strenuous exercise) can produce hypoglycemia that may contribute to the behavioral effects of alcohol. Because alcohol consumption can also increase the risk for hypoglycemia in patients using insulin, those in an intensive insulin treatment protocol (see p. 340) are counseled about the increased risk of hypoglycemia that generally occurs many hours after alcohol ingestion.
B. Inhibition of gluconeogenesis resulting from hepatic metabolism of ethanol. NAD(H) = nicotinamide adenine dinucleotide.
Chronic alcohol consumption can also result in alcoholic fatty liver because of increased hepatic synthesis of TAG coupled with impaired formation or release of VLDL. This occurs as a result of decreased FA oxidation because of a fall in the NAD+/NADH ratio and increased lipogenesis because of the increased availability of FA (decreased catabolism) and of glyceraldehyde 3-phosphate (the dehydrogenase is inhibited by the low NAD+/NADH ratio; see p. 101). With continued alcohol consumption, alcoholic fatty liver can progress first to alcoholic hepatitis and then to alcoholic cirrhosis.
V. CHAPTER SUMMARY
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The integration of energy metabolism is controlled primarily by insulin and the opposing actions of glucagon and the catecholamines, particularly epinephrine (Fig. 23.16). Changes in the circulating levels of these hormones allow the body to store energy when food is abundant or to make stored energy available in times of physiologic stress (for example, during survival crises, such as famine). Insulin is a peptide hormone produced by the β cells of the islets of Langerhans of the pancreas. It consists of disulfide-linked A and B chains. A rise in blood glucose is the most important signal for insulin secretion. The catecholamines, secreted in response to stress, trauma, or extreme exercise, inhibit insulin secretion. Insulin increases glucose uptake (by glucose transporters (GLUT-4) in muscle and adipose tissue) and the synthesis of glycogen, protein, and triacylglycerol: It is an anabolic hormone. These actions are mediated by binding to its membrane tyrosine kinase receptor. Binding initiates a cascade of cell-signaling responses, including phosphorylation of a family of proteins called insulin receptor substrate proteins. Glucagon is a monomeric peptide hormone produced by the α cells of the pancreatic islets (both insulin and glucagon synthesis involve formation of inactive precursors that are cleaved to form the active hormones). Glucagon, along with epinephrine, norepinephrine, cortisol, and growth hormone (the counterregulatory hormones), opposes many of the actions of insulin. Glucagon acts to maintain blood glucose during periods of potential hypoglycemia. Glucagon increases glycogenolysis, gluconeogenesis, fatty acid oxidation, ketogenesis, and amino acid uptake: It is a catabolic hormone. Glucagon secretion is stimulated by low blood glucose, amino acids, and the catecholamines. Its secretion is inhibited by elevated blood glucose and by insulin. Glucagon binds to high-affinity G protein–coupled receptors on the cell membrane of hepatocytes. Binding results in the activation of adenylyl cyclase, which produces the second messenger cyclic adenosine monophosphate (cAMP). Subsequent activation of cAMPdependent protein kinase A results in the phosphorylation-mediated activation or inhibition of key regulatory enzymes involved in carbohydrate and lipid metabolism. Both insulin and glucagon affect gene transcription.
Hypoglycemia is characterized by low blood glucose accompanied by adrenergic and neuroglycopenic symptoms that are rapidly resolved by the administration of glucose. Insulin-induced, postprandial, and fasting hypoglycemia result in release of glucagon and epinephrine. The rise in nicotinamide adenine dinucleotide (NADH) that accompanies ethanol metabolism inhibits gluconeogenesis, leading to hypoglycemia in individuals with depleted stores. Alcohol consumption also increases the risk for hypoglycemia in patients using insulin. Chronic alcohol consumption can cause fatty liver disease.
Choose the ONE best answer.
3.1. Which of the following statements is true for insulin but not for glucagon?
A. It is a peptide hormone secreted by pancreatic cells.
B. Its actions are mediated by binding to a receptor found on the cell membrane of liver cells.
C. Its effects include alterations in gene expression.
D. Its secretion is decreased by the catecholamines.
E. Its secretion is increased by amino acids.
F. Its synthesis involves a nonfunctional precursor that gets cleaved to yield a functional molecule.
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Correct answer = D. Secretion of insulin by pancreatic β cells is inhibited by the catecholamines, whereas glucagon secretion by the α cells is stimulated by them. All of the other statements are true for both insulin and glucagon.
3.2. In which one of the following tissues is glucose transport into the cell insulin dependent?
A. Adipose
B. Brain
C. Liver
D. Red blood cells
Correct answer = A. The glucose transporter (GLUT-4) in adipose (and muscle) tissue is dependent on insulin. Insulin results in movement of GLUT-4 from intracellular vesicles to the cell membrane. The other tissues in the list contain GLUT that are independent of insulin because they are always located on the cell membrane.
3.3. A 39-year-old woman is brought to the emergency room complaining of weakness and dizziness. She recalls getting up early that morning to do her weekly errands and had skipped breakfast. She drank a cup of coffee for lunch and had nothing to eat during the day. She met with friends at 8 p.m.
and had a few drinks. As the evening progressed, she soon became weak and dizzy and was taken to the hospital. Laboratory tests revealed her blood glucose to be 45 mg/dl (normal = 70–99). She was given orange juice and immediately felt better. The biochemical basis of her alcohol-induced hypoglycemia is an increase in:
A. fatty acid oxidation.
B. the ratio of the reduced oxidized forms of nicotinamide adenine dinucleotide.
C. oxaloacetate and pyruvate.
D. use of acetyl coenzyme A in fatty acid synthesis.
Correct answer = B. The oxidation of ethanol to acetate by dehydrogenases is accompanied by the reduction of nicotinamide adenine dinucleotide (NAD+) to NADH. The rise in the NADH/NAD+ ratio shifts pyruvate to lactate and oxaloacetate (OAA) to malate, decreasing the availability of substrates for gluconeogenesis and resulting in hypoglycemia. The rise in NADH also reduces the NAD+ needed for fatty acid (FA) oxidation. The decrease in OAA shunts any acetyl coenzyme A produced to ketogenesis. Note that the inhibition of FA degradation results in their reesterification into triacylglycerol that can result in fatty liver.
3.4. A patient is diagnosed with an insulinoma, a rare neuroendocrine tumor, the cells of which are derived primarily from pancreatic β cells. Which of the following would logically be characteristic of an insulinoma?
A. Decreased body weight
B. Decreased connecting peptide in the blood
C. Decreased glucose in the blood
D. Decreased insulin in the blood
Correct answer = C. Insulinomas are characterized by constant production of insulin (and, therefore, of C-peptide) by the tumor cells. The increase in insulin drives glucose uptake by tissues such as muscle and adipose that have insulin-dependent glucose transporters, resulting in hypoglycemia. However, the hypoglycemia is insufficient to suppress insulin production and secretion. Insulinomas, then, are characterized by increased blood insulin and decreased blood glucose. Insulin, as an anabolic hormone, results in weight gain.
3.5. In a patient with an even rarer glucagon-secreting tumor derived from the α cells of the pancreas, how would the presentation be expected to differ relative to the patient in Question 23.4?
A glucagon-secreting tumor of the pancreas (glucagonoma) would result in hyperglycemia, not hypoglycemia. The constant production of glucagon would result in constant gluconeogenesis, using amino acids from proteolysis as substrates. This results in loss of body weight.
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The Feed–Fast Cycle 24
The absorptive (well-fed) state is the 2-to 4-hour period after ingestion of a normal meal. During this interval, transient increases in plasma glucose, amino acids, and triacylglycerols (TAG) occur, the latter primarily as components of chylomicrons synthesized and secreted by the intestinal mucosal cells (see p. 228). Islet tissue of the pancreas responds to the elevated level of glucose with increased secretion of insulin and decreased secretion of glucagon. The elevated insulin/glucagon ratio and the ready availability of circulating substrates make the absorptive state an anabolic period characterized by increased synthesis of TAG and glycogen to replenish fuel stores as well as increased synthesis of protein. During this absorptive period, virtually all tissues use glucose as a fuel, and the metabolic response of the body is dominated by alterations in the metabolism of liver, adipose tissue, skeletal muscle, and brain. In this chapter, an “organ map” is introduced that traces the movement of metabolites between tissues. The goal is to create an expanded and clinically useful vision of whole-body metabolism.
II. REGULATORY MECHANISMS
The flow of intermediates through metabolic pathways is controlled by four mechanisms: 1) the availability of substrates, 2) allosteric regulation of enzymes, 3) covalent modification of enzymes, and 4) induction-repression of enzyme synthesis, primarily through regulation of transcription. Although this scheme may at first seem redundant, each mechanism operates on a different timescale (Fig. 24.1) and allows the body to adapt to a wide variety of physiologic situations. In the absorptive state, these regulatory mechanisms insure that available nutrients are captured as glycogen, TAG, and protein.
A. Allosteric effectors
Allosteric changes usually involve rate-determining reactions. For example, glycolysis in the liver is stimulated following a meal by an increase in fructose 2,6-bisphosphate, an allosteric activator of phosphofructokinase-1 ([PFK-1] see p. 99). In contrast, gluconeogenesis is decreased by fructose 2,6-bisphosphate, an allosteric inhibitor of fructose 1,6-bisphosphatase (see p. 122).
B. Covalent modification
The activity of many enzymes is regulated by the addition (via kinases, such as cyclic adenosine monophosphate [cAMP ]–dependent protein kinase A [PKA] and adenosine monophosphate–activated protein kinase [AMPK]) or removal (via phosphatases) of phosphate groups from specific serine, threonine, or tyrosine residues of the protein. In the absorptive state, most of the covalently regulated enzymes are in the dephosphorylated form and are active (Fig. 24.2). Three exceptions are glycogen phosphorylase kinase (see p. 132), glycogen phosphorylase (see p. 132), and hormone-sensitive lipase (HSL) (see p. 189), which are inactive in their dephosphorylated form. [Note: In the liver, the phosphatase domain of bifunctional phosphofructokinase-2 (PFK-2) is inactive when the protein is dephosphorylated (see p. 100).]
C. Induction and repression of enzyme synthesis
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Increased (induction of) or decreased (repression of) enzyme synthesis leads to changes in the number of enzyme molecules, rather than changing the activity of existing enzyme molecules. Enzymes subject to synthesis regulation are often those that are needed under specific physiologic conditions. For example, in the well-fed state, elevated insulin levels result in an increase in the synthesis of key enzymes, such as acetyl coenzyme A (CoA) carboxylase (ACC) and fatty acid synthase (see p. 313), involved in anabolic metabolism. In the fasted state, glucagon induces expression of phosphoenolpyruvate carboxykinase (PEPCK) of gluconeogenesis (see p. 314). [Note: Both hormones affect gene transcription.]
III. LIVER: NUTRIENT DISTRIBUTION CENTER
The liver is uniquely situated to process and distribute dietary nutrients because the venous drainage of the gut and pancreas passes through the hepatic portal vein before entry into the general circulation. Thus, after a meal, the liver is bathed in blood containing absorbed nutrients and elevated levels of insulin secreted by the pancreas. During the absorptive period, the liver takes up carbohydrates, lipids, and most amino acids. These nutrients are then metabolized, stored, or routed to other tissues. In this way, the liver smooths out potentially broad fluctuations in the availability of nutrients for the peripheral tissues.
A. Carbohydrate metabolism
The liver is normally a glucose-producing rather than a glucose-using organ. However, after a meal containing carbohydrate, the liver becomes a net consumer, retaining roughly 60 g of every 100 g of glucose presented by the portal system. This increased use reflects increased glucose uptake by the hepatocytes. Their insulin-independent glucose transporter (GLUT 2) has a low affinity (high Km [Michaelis constant]) for glucose and, therefore, takes up glucose only when blood glucose is high (see p. 98).
Processes that are upregulated when hepatic glucose is increased include the following.
1. Increased glucose phosphorylation: The elevated levels of glucose within the hepatocyte (as a result of elevated extracellular levels) allow glucokinase to phosphorylate glucose to glucose 6-phosphate (Fig. 24.3, ). [Note: Glucokinase has a high Km for glucose, is not subject to direct product inhibition, and has a sigmoidal reaction curve (see p. 98).] 2.
Increased glycogenesis: The conversion of glucose 6-phosphate to glycogen is favored by the activation of glycogen synthase, both by dephosphorylation and by increased availability of glucose 6-phosphate, its positive allosteric effector (see Fig. 24.3, ).
3.
Increased pentose phosphate pathway activity: The increased availability of glucose 6-phosphate, combined with the active use of nicotinamide adenine dinucleotide phosphate (NADPH) in hepatic lipogenesis, stimulates the pentose phosphate pathway (see p. 145). This pathway typically accounts for 5%–10% of the glucose metabolized by the liver (see Fig. 24.3, ).
4.
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Increased glycolysis: In the liver, glycolysis is significant only during the absorptive period following a carbohydrate-rich meal. The conversion of glucose to pyruvate is stimulated by the elevated insulin/glucagon ratio that results in increased amounts of the regulated enzymes of glycolysis: glucokinase, PFK-1, and pyruvate kinase ([PK] see p. 105). Additionally, PFK-1 is allosterically activated by fructose 2,6bisphosphate generated by the active (dephosphorylated) kinase domain of bifunctional PFK-2. PK is dephosphorylated and active. Pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl CoA, is active (dephosphorylated) because pyruvate inhibits
PDH kinase (see Fig. 24.3, ). The acetyl CoA either is used as a substrate for fatty acid (FA) synthesis or is oxidized for energy in the tricarboxylic acid (TCA) cycle. (See Fig. 24.4 for the central role of glucose 6-phosphate.) 5. Decreased glucose production: While glycolysis and glycogenesis (pathways that promote glucose storage) are being stimulated in the liver in the absorptive state, gluconeogenesis and glycogenolysis (pathways that generate glucose) are being inhibited. Pyruvate carboxylase (PC), which catalyzes the first step in gluconeogenesis, is largely inactive because of low levels of acetyl CoA, its allosteric activator (see p. 119). [Note: The acetyl CoA is being used for FA synthesis.] The high insulin/glucagon ratio also favors inactivation of other gluconeogenic enzymes such as fructose 1,6-bisphosphatase (see Fig. 8.17, p. 100). Glycogenolysis is inhibited by dephosphorylation of glycogen phosphorylase and phosphorylase kinase. [Note: The increased uptake and decreased production of blood glucose in the absorptive period prevents hyperglycemia.]
B. Fat metabolism 1. Increased fatty acid synthesis: Liver is the primary site of de novo synthesis of FA (see Fig. 24.3, ). FA synthesis, a cytosolic process, is favored in the absorptive period by availability of the substrates acetyl CoA (from glucose and amino acid metabolism) and NADPH (from glucose metabolism in the pentose phosphate pathway) and by the activation of ACC, both by dephosphorylation and by the presence of its allosteric activator, citrate. [Note: Inactivity of AMPK favors dephosphorylation.] ACC catalyzes the formation of malonyl CoA from acetyl CoA, the rate-limiting reaction for FA synthesis (see p. 183). [Note: Malonyl CoA inhibits carnitine palmitoyltransferase-I (CPT-I) of FA oxidation (see p. 191). Thus, citrate directly activates FA synthesis and indirectly inhibits FA degradation.] a.
Source of cytosolic acetyl coenzyme A: Pyruvate from aerobic glycolysis enters mitochondria and is decarboxylated by PDH. The acetyl CoA product is combined with oxaloacetate (OAA) to form citrate via citrate synthase of the TCA cycle. Citrate leaves the mitochondria (as a result of the inhibition of isocitrate dehydrogenase by ATP) and enters the cytosol. Citrate is cleaved by ATP citrate lyase (induced by insulin), producing the acetyl CoA substrate of ACC plus OAA.
b.
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Additional source of NADPH: The OAA is reduced to malate, which is oxidatively decarboxylated to pyruvate by malic enzyme as NADPH is formed (see Fig. 16.11 on p. 187).
2. Increased triacylglycerol synthesis: TAG synthesis is favored because fatty acyl CoA are available both from de novo synthesis and from hydrolysis of the TAG component of chylomicron remnants removed from the blood by hepatocytes (see p. 178). Glycerol 3-phosphate, the backbone for TAG synthesis, is provided by glycolysis (see p. 189). The liver packages these endogenous TAG into very-low-density lipoprotein (VLDL) particles that are secreted into the blood for use by extrahepatic tissues, particularly adipose and muscle tissues (see Fig. 24.3, ).
C. Amino acid metabolism 1.
Increased amino acid degradation: In the absorptive period, more amino acids are present than the liver can use in the synthesis of proteins and other nitrogen-containing molecules. The surplus amino acids are not stored but are either released into the blood for other tissues to use in protein synthesis or deaminated, with the resulting carbon skeletons being degraded by the liver to pyruvate, acetyl CoA, or TCA cycle intermediates. These metabolites can be oxidized for energy or used in FA synthesis (see Fig. 24.3, ). The liver has limited capacity to initiate degradation of the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. They pass through the liver essentially unchanged and are metabolized in muscle (see p. 266).
2.
Increased protein synthesis: The body does not store protein for energy in the same way that it maintains glycogen or TAG reserves. However, a transient increase in the synthesis of hepatic proteins does occur in the absorptive state, resulting in replacement of any proteins that may have been degraded during the previous period of fasting (see Fig. 24.3, ).
IV. ADIPOSE TISSUE: ENERGY STORAGE DEPOT
Adipose tissue is second only to the liver in its ability to distribute fuel molecules. In a 70-kg man, white adipose tissue (WAT) weighs ~14 kg, or about half as much as the total muscle mass. Nearly the entire volume of each adipocyte in WAT can be occupied by a droplet of anhydrous, calorically dense TAG (Fig. 24.5).
A. Carbohydrate metabolism 1. Increased glucose transport: Circulating insulin levels are elevated in the absorptive state, resulting in an influx of glucose into adipocytes via insulin-sensitive GLUT-4 recruited to the cell surface from intracellular vesicles (Fig. 24.6, ). The glucose is phosphorylated by hexokinase.
2.
Increased glycolysis: The increased intracellular availability of glucose results in an enhanced rate of glycolysis (see Fig. 24.6, ). In adipose tissue, glycolysis serves a synthetic function by supplying glycerol 3phosphate for TAG synthesis (see p. 188). [Note: Adipose tissue lacks glycerol kinase.] 3.
Increased pentose phosphate pathway activity: Adipose tissue can metabolize glucose by means of the pentose phosphate pathway, thereby producing NADPH, which is essential for FA synthesis (see p. 186 and Fig. 24.6, ). However, in humans, de novo synthesis is not a major source of FA in adipose tissue, except when refeeding a previously fasted individual (see Fig. 24.6, ).
B. Fat metabolism
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Most of the FA added to the TAG stores of adipocytes after consumption of a lipid-containing meal are provided by the degradation of exogenous (dietary) TAG in chylomicrons sent out by the intestine and endogenous TAG in VLDL sent out by the liver (see Fig. 24.6, ). The FA are released from the lipoproteins by lipoprotein lipase (LPL), an extracellular enzyme attached to the endothelial cells of capillary walls in many tissues, particularly adipose and muscle (see p. 228). In adipose tissue, LPL is upregulated by insulin. Thus, in the fed state, elevated levels of glucose and insulin favor storage of TAG (see Fig. 24.6, ), all the carbons of which are supplied by glucose. [Note: Elevated insulin favors the dephosphorylated (inactive) form of HSL (see p. 189), thereby inhibiting lipolysis in the well-fed state.]
V. RESTING SKELETAL MUSCLE
Skeletal muscle accounts for ~40% of the body mass in individuals of healthy weight, and it can use glucose, amino acids, FA, and ketone bodies as fuel. In the well-fed state, muscle takes up glucose via GLUT-4 (for energy and glycogen synthesis) and amino acids (for energy and protein synthesis). In contrast to liver, there is no covalent regulation of PFK-2 in skeletal muscle. However, in the cardiac isozyme, the kinase domain is activated by epinephrine-mediated phosphorylation (see p. 100).
Skeletal muscle is unique in being able to respond to substantial changes in the demand for ATP that accompanies contraction. At rest, muscle accounts for ~25% of the oxygen (O2) consumption of the body, whereas during vigorous exercise, it is responsible for up to 90%. This underscores the fact that skeletal muscle, despite its potential for transient periods of anaerobic glycolysis, is an oxidative tissue.
A. Carbohydrate metabolism 1. Increased glucose transport: The transient increase in plasma glucose and insulin after a carbohydrate-rich meal leads to an increase in glucose transport into muscle cells (myocytes) by GLUT-4 (see p. 97 and Fig. 24.7, ), thereby reducing blood glucose. Glucose is phosphorylated to glucose 6-phosphate by hexokinase and metabolized to meet the energy needs of myocytes.
2. Increased glycogenesis: The increased insulin/glucagon ratio and the availability of glucose 6-phosphate favor glycogen synthesis, particularly if glycogen stores have been depleted as a result of exercise (see p. 126 and Fig. 24.7, ).
B. Fat metabolism
FA are released from chylomicrons and VLDL by the action of LPL (see pp. 228 and 231). However, FA are of secondary importance as a fuel for resting muscle during the well-fed state, in which glucose is the primary source of energy.
C. Amino acid metabolism 1.
Increased protein synthesis: An increase in amino acid uptake and protein synthesis occurs in the absorptive period after ingestion of a meal containing protein (see Fig. 24.7, and ). This synthesis replaces protein degraded since the previous meal.
2.
Increased branched-chain amino acid uptake: Muscle is the principal site for degradation of the BCAA because it contains the required transaminase (see p. 266). The dietary BCAA escape metabolism by the liver and are taken up by muscle, where they are used for protein synthesis (see Fig. 24.7, ) and as energy sources.
VI. BRAIN
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Although contributing only 2% of the adult weight, the brain accounts for a consistent 20% of the basal O2 consumption of the body at rest. Because the brain is vital to the proper functioning of all organs of the body, special priority is given to its fuel needs. To provide energy, substrates must be able to cross the endothelial cells that line the blood vessels in the brain (the blood–brain barrier [BBB]). In the fed state, the brain exclusively uses glucose as a fuel (GLUT-1 of the BBB is insulin independent), completely oxidizing ~140 g/day to carbon dioxide and water. Because the brain contains no significant stores of glycogen, it is completely dependent on the availability of blood glucose (Fig. 24.8, ). [Note: If blood glucose levels fall to <50 mg/dl (normal fasted blood glucose is 70–99 mg/dl), cerebral function is impaired (see p. 315).] The brain also lacks significant stores of TAG, and the FA circulating in the blood make little contribution to energy production for reasons that are unclear. The intertissue exchanges characteristic of the absorptive period are summarized in Figure 24.9.
VII. OVERVIEW OF THE FASTED STATE
Fasting begins if no food is ingested after the absorptive period. It may result from an inability to obtain food, the desire to lose weight rapidly, or clinical situations in which an individual cannot eat (for example, because of trauma, surgery, cancer, or burns). In the absence of food, plasma levels of glucose, amino acids, and TAG fall, triggering a decline in insulin secretion and an increase in glucagon, epinephrine, and cortisol secretion. The decreased insulin/counterregulatory hormone ratio and the decreased availability of circulating substrates make the postabsorptive period of nutrient deprivation a catabolic period characterized by degradation of TAG, glycogen, and protein. This sets into motion an exchange of substrates among the liver, adipose tissue, skeletal muscle, and brain that is guided by two priorities: 1) the need to maintain adequate plasma levels of glucose to sustain energy metabolism in the brain, red blood cells, and other glucose-requiring tissues and 2) the need to mobilize FA from TAG in WAT for the synthesis and release of ketone bodies by the liver to supply energy to other tissues and spare body protein. As a result, blood glucose levels are maintained within a narrow range in fasting, while FA and ketone body levels increase. [Note: Maintaining glucose requires that the substrates for gluconeogenesis (such as pyruvate, alanine, and glycerol) be available.]
A. Fuel stores
The metabolic fuels available in a normal 70-kg man at the beginning of a fast are shown in Figure 24.10. Observe the enormous caloric stores available in the form of TAG compared with those contained in glycogen. [Note: Although protein is listed as an energy source, each protein also has a function unrelated to energy metabolism (for example, as a structural component of the body or as an enzyme). Therefore, only about one third of the body’s protein can be used for energy production without fatally compromising vital functions.]
B. Enzymic changes
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In fasting (as in the well-fed state), the flow of intermediates through the pathways of energy metabolism is controlled by four mechanisms: 1) the availability of substrates, 2) allosteric regulation of enzymes, 3) covalent modification of enzymes, and 4) induction-repression of enzyme synthesis. The metabolic changes observed in fasting are generally opposite those described for the absorptive state (see Fig. 24.9). For example, although most of the enzymes regulated by covalent modification are dephosphorylated and active in the well-fed state, they are phosphorylated and inactive in the fasted state. Three exceptions are glycogen phosphorylase (see p. 132), glycogen phosphorylase kinase (see p. 132), and HSL (see p. 189), which are active in their phosphorylated states. In fasting, substrates are not provided by the diet but are available from the breakdown of stores and/or tissues, such as glycogenolysis with release of glucose from the liver, lipolysis with release of FA and glycerol from TAG in adipose tissue, and proteolysis with release of amino acids from muscle. Recognition that the changes in fasting are the reciprocal of those in the fed state is helpful in understanding the ebb and flow of metabolism.
VIII. LIVER IN FASTING
The primary role of the liver in fasting is maintenance of blood glucose through the production of glucose (from glycogenolysis and gluconeogenesis) for glucose-requiring tissues and the synthesis and distribution of ketone bodies for use by other tissues. Therefore, hepatic metabolism is distinguished from peripheral (or extrahepatic) metabolism.
A. Carbohydrate metabolism
The liver first uses glycogen degradation and then gluconeogenesis to maintain blood glucose levels to sustain energy metabolism of the brain and other glucose-requiring tissues in the fasted state. [Note: Recall that the presence of glucose-6-phosphatase in the liver allows the production of free glucose both from glycogenolysis and from gluconeogenesis (see Fig. 24.4).] 1. Increased glycogenolysis: Figure 24.11 shows the sources of blood glucose after ingestion of 100 g of glucose. During the brief absorptive period, ingested glucose is the major source of blood glucose. Several hours later, blood glucose levels have declined sufficiently to cause increased secretion of glucagon and decreased secretion of insulin. The increased glucagon/insulin ratio causes a rapid mobilization of liver glycogen stores (which contain ~80 g of glycogen in the fed state) because of PKA-mediated phosphorylation (and activation) of glycogen phosphorylase kinase that phosphorylates (and activates) glycogen phosphorylase (see p. 132). Figure 24.11 shows that because liver glycogen is exhausted by 24 hours of fasting, hepatic glycogenolysis is a transient response to early fasting. Figure 24.12, , shows glycogen degradation as part of the overall metabolic response of the liver during fasting. [Note: Phosphorylation of glycogen synthase simultaneously inhibits glycogenesis.]
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See Section B.2 for an explanation of the decline in gluconeogenesis.] 2. Increased gluconeogenesis: The synthesis of glucose and its release into the circulation are vital hepatic functions during short-and long-term fasting (see Fig. 24.12, ). The carbon skeletons for gluconeogenesis are derived primarily from glucogenic amino acids and lactate from muscle and glycerol from adipose tissue. Gluconeogenesis, favored by activation of fructose 1,6-bisphosphatase (because of decreased availability of its inhibitor fructose 2,6-bisphosphate; see p. 121) and by induction of PEPCK by glucagon (see p. 122), begins 4–6 hours after the last meal and becomes fully active as stores of liver glycogen are depleted (see Fig. 24.11). [Note: The decrease in fructose 2,6-bisphosphate simultaneously inhibits glycolysis at PFK-1 (see p. 99).]
B. Fat metabolism 1. Increased fatty acid oxidation: The oxidation of FA obtained from TAG hydrolysis in adipose tissue is the major source of energy in hepatic tissue in the fasted state (see Fig. 24.12, ). The fall in malonyl CoA because of phosphorylation (inactivation) of ACC by AMPK removes the brake on CPT-I, allowing β-oxidation to occur (see p. 191). FA oxidation generates NADH, flavin adenine dinucleotide (FADH2), and acetyl CoA.
The NADH inhibits the TCA cycle and shifts OAA to malate. This results in acetyl CoA being available for ketogenesis. The acetyl CoA is also an allosteric activator of PC and an allosteric inhibitor of PDH, thereby favoring use of pyruvate in gluconeogenesis (see Fig. 10.9, p. 122). [Note: Acetyl CoA cannot be used as a substrate for gluconeogenesis, in part because the PDH reaction is irreversible.] Oxidation of NADH and FADH2 coupled with oxidative phosphorylation supplies the energy required by the PC and PEPCK reactions of gluconeogenesis.
2. Increased ketogenesis: The liver is unique in being able to synthesize and release ketone bodies, primarily 3-hydroxybutyrate but also acetoacetate, for use as fuel by peripheral tissues but not by the liver itself because liver lacks thiophorase (see p. 197). Ketogenesis, which starts during the first days of fasting (Fig. 24.13), is favored when the concentration of acetyl CoA from FA oxidation exceeds the oxidative capacity of the TCA cycle. [Note: Ketogenesis releases CoA, insuring its availability for continued FA oxidation.] The availability of circulating water-soluble ketone bodies is important in fasting because they can be used for fuel by most tissues, including the brain, once their blood level is high enough. Ketone body concentration in blood increases from ~50 µM to ~6 mM in fasting. This reduces the need for gluconeogenesis from amino acid carbon skeletons, thus preserving essential protein (see Fig. 24.11). Ketogenesis as part of the overall hepatic response to fasting is shown in Figure 24.12, . [Note: Ketone bodies are organic acids and, when present at high concentrations, can cause ketoacidosis.]
IX.
A.
Glucose transport by insulin-sensitive GLUT-4 into the adipocyte and its subsequent metabolism are decreased because of low levels of circulating insulin. This results in decreased TAG synthesis.
B. Fat metabolism 1.
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Increased fat degradation: The PKA-mediated phosphorylation and activation of HSL (see p. 189) and subsequent hydrolysis of stored fat (TAG) are enhanced by the elevated catecholamines norepinephrine and epinephrine. These hormones, which are secreted from the sympathetic nerve endings in adipose tissue and/or from the adrenal medulla, are physiologically important activators of HSL (Fig. 24.14, ).
2.
Increased fatty acid release: FA obtained from hydrolysis of TAG stored in adipocytes are primarily released into the blood (see Fig. 24.14, ). Bound to albumin, they are transported to a variety of tissues for use as fuel. The glycerol produced from TAG degradation is used as a gluconeogenic precursor by the liver, which contains glycerol kinase. [Note: FA can also be oxidized to acetyl CoA, which can enter the TCA cycle, thereby producing energy for the adipocyte. They also can be reesterified to glycerol 3-phosphate (from glyceroneogenesis, see p. 190), generating TAG and reducing plasma FA concentration.] 3.
Decreased fatty acid uptake: In fasting, LPL activity of adipose tissue is low. Consequently, FA in circulating TAG of lipoproteins are less available to adipose tissue than to muscle.
X. RESTING SKELETAL MUSCLE IN FASTING
Resting muscle switches from glucose to FA as its major fuel source in fasting. [Note: By contrast, exercising muscle initially uses creatine phosphate and its glycogen stores. During intense exercise, glucose 6-phosphate from glycogenolysis is converted to lactate by anaerobic glycolysis (see p. 118). The lactate is used by the liver for gluconeogenesis (Cori cycle; see p. 118). As these glycogen reserves are depleted, free FA provided by the degradation of TAG in adipose tissue become the dominant energy source. The contraction-based rise in AMP activates AMPK that phosphorylates and inactivates the muscle isozyme of ACC, decreasing malonyl CoA and allowing FA oxidation (see p. 183).]
A. Carbohydrate metabolism p. 97) and subsequent glucose metabolism are decreased because circulating insulin levels are low. Therefore, the glucose from hepatic gluconeogenesis is unavailable to muscle and adipose.
B. Lipid metabolism
Early in fasting, muscle uses FA from adipose tissue and ketone bodies from the liver as fuels (Fig. 24.15, and ). In prolonged fasting, muscle decreases its use of ketone bodies (thus sparing them for the brain) and oxidizes FA almost exclusively. [Note: The acetyl CoA from FA oxidation indirectly inhibits PDH (by activation of PDH kinase) and spares pyruvate, which is transaminated to alanine and used by the liver for gluconeogenesis (glucose–alanine cycle; see p. 253).]
C. Protein metabolism
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During the first few days of fasting, there is a rapid breakdown of muscle protein (for example, glycolytic enzymes), providing amino acids that are used by the liver for gluconeogenesis (see Fig. 24.15, ). Because muscle does not have glucagon receptors, muscle proteolysis is initiated by a fall in insulin and sustained by a rise in glucocorticoids. [Note: Alanine and glutamine are quantitatively the most important glucogenic amino acids released from muscle. They are produced by the catabolism of BCAA (see p. 267). The glutamine is used as a fuel by enterocytes, for example, which send out alanine that is used in hepatic gluconeogenesis (glucose–alanine cycle)]. In the second week of fasting, the rate of muscle proteolysis decreases, paralleling a decline in the need for glucose as a fuel for the brain, which has begun using ketone bodies as a source of energy.
XI. BRAIN IN FASTING
During the early days of fasting, the brain continues to use only glucose as a fuel (Fig. 24.16, ). Blood glucose is maintained by hepatic gluconeogenesis from glucogenic precursors, such as amino acids from proteolysis and glycerol from lipolysis. In prolonged fasting (beyond 2–3 weeks), plasma ketone bodies (see Fig. 24.12) reach significantly elevated levels and replace glucose as the primary fuel for the brain (see Figs. 24.16, and 24.17). This reduces the need for protein catabolism for gluconeogenesis: Ketone bodies spare glucose and, thus, muscle protein. [Note: As the duration of a fast extends from overnight to days to weeks, blood glucose levels initially drop and then are maintained at the lower level (65–70 mg/dl).] The metabolic changes that occur during fasting insure that all tissues have an adequate supply of fuel molecules. The response of the major tissues involved in energy metabolism during fasting is summarized in Figure 24.18.
XII. KIDNEY IN LONG-TERM FASTING
As fasting continues into early starvation and beyond, the kidney plays important roles. The renal cortex expresses the enzymes of gluconeogenesis, including glucose 6-phosphatase, and, in late fasting, ~50% of gluconeogenesis occurs here. [Note: A portion of this glucose is used by the kidney itself.] The kidney also provides compensation for the acidosis that accompanies the increased production of ketone bodies (organic acids). The glutamine released from the muscle’s metabolism of BCAA is taken up by the kidney and acted upon by renal glutaminase and glutamate dehydrogenase (see p. 256), producing αketoglutarate, which can be used as a substrate for gluconeogenesis, plus ammonia (NH3). The NH3 picks up protons from ketone body dissociation and is excreted in the urine as ammonium (NH4+), thereby decreasing the acid load in the body (Fig. 24.19). Therefore, in long-term fasting, there is a switch from nitrogen disposal in the form of urea to disposal in the form of NH4 . [Note: As ketone body concentration rises, enterocytes, typically consumers of glutamine, become consumers of ketone bodies. This allows more glutamine to be available to the kidney.]
XIII. CHAPTER SUMMARY
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The flow of intermediates through metabolic pathways is controlled by four regulatory mechanisms: 1) the availability of substrates, 2) allosteric activation and inhibition of enzymes, 3) covalent modification of enzymes, and 4) induction-repression of enzyme synthesis. In the absorptive state, the 2-to 4-hour period after ingestion of a meal, these mechanisms insure that available nutrients are captured as glycogen, triacylglycerol (TAG), and protein (Fig. 24.20). During this interval, transient increases in plasma glucose, amino acids, and TAG occur, the last primarily as components of chylomicrons synthesized by the intestinal mucosal cells. The pancreas responds to the elevated levels of glucose with an increased secretion of insulin and a decreased secretion of glucagon. The elevated insulin/glucagon ratio and the ready availability of circulating substrates make the absorptive state an anabolic period during which virtually all tissues use glucose as a fuel. In addition, the liver replenishes its glycogen stores, replaces any needed hepatic proteins, and increases TAG synthesis. The latter are packaged in very-low-density lipoproteins, which are exported to the peripheral tissues. Adipose tissue increases TAG synthesis and storage, whereas muscle increases protein synthesis to replace protein degraded since the previous meal. In the fed state, the brain uses glucose exclusively as a fuel. In fasting, plasma levels of glucose, amino acids, and TAG fall, triggering a decline in insulin secretion and an increase in glucagon and epinephrine secretion. The decreased insulin/counterregulatory hormone ratio and the decreased availability of circulating substrates make the fasting state a catabolic period. This sets into motion an exchange of substrates among the liver, adipose tissue, skeletal muscle, and brain that is guided by two priorities: 1) the need to maintain adequate plasma levels of glucose to sustain energy metabolism of the brain and other glucose-requiring tissues and 2) the need to mobilize fatty acids (FA) from adipose tissue and release ketone bodies from liver to supply energy to other tissues. To accomplish these goals, the liver degrades glycogen and initiates gluconeogenesis, using increased FA oxidation to supply the energy and reducing equivalents needed for gluconeogenesis and the acetyl coenzyme A building blocks for ketogenesis. The adipose tissue degrades stored TAG, thus providing FA and glycerol to the liver. The muscle can also use FA as fuel as well as ketone bodies supplied by the liver. The liver uses the glycerol for gluconeogenesis. Muscle protein is degraded to supply amino acids for the liver to use in gluconeogenesis but decreases as ketone bodies increase. The brain can use both glucose and ketone bodies as fuels. From late fasting into starvation, the kidneys play important roles by synthesizing glucose and excreting the protons from ketone body dissociation as ammonium (NH4+).
Choose the ONE best answer.
4.1. Which one of the following is elevated in plasma during the absorptive (well-fed) state as compared with the postabsorptive (fasted) state?
A. Acetoacetate
B. Chylomicrons
C. Free fatty acids
D. Glucagon
Correct answer = B. Triacylglycerol-rich chylomicrons are synthesized in (and released from) the intestine following ingestion of a meal. Acetoacetate, free fatty acids, and glucagon are elevated in the fasted state, not the absorptive state.
4.2. Which one of the following statements concerning liver in the absorptive state is correct?
A. Fructose 2,6-bisphosphate is elevated.
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B. Insulin stimulates the uptake of glucose.
C. Most enzymes that are regulated by covalent modification are in the phosphorylated state.
D. The oxidation of acetyl coenzyme A is increased.
E. The synthesis of glucokinase is repressed.
Correct answer = A. The increased insulin and decreased glucagon levels characteristic of the absorptive state promote the synthesis of fructose 2,6bisphosphate, which allosterically activates phosphofructokinase-1 of glycolysis. Most covalently modified enzymes are in the dephosphorylated state and are active. Acetyl coenzyme A is not oxidized in the well-fed state because it is being used in fatty acid synthesis. Uptake of glucose (by glucose transporter-2) into the liver is insulin independent. Synthesis of glucokinase is induced by insulin in the well-fed state.
4.3. Which one of the following enzymes is phosphorylated and active in an individual who has been fasting for 12 hours?
A. Arginase
B. Carnitine palmitoyltransferase-I
C. Fatty acid synthase
D. Glycogen synthase
E. Hormone-sensitive lipase F. Phosphofructokinase-1
G. Pyruvate dehydrogenase
Correct answer = E. Hormone-sensitive lipase of adipocytes is phosphorylated and activated by protein kinase A in response to epinephrine. Choices A, B, C, and F are not regulated covalently. Choices D and G are regulated covalently but are inactive if phosphorylated.
4.4. For a 70-kg man, in which one of the periods listed below do ketone bodies supply the major portion of the caloric needs of brain?
A. Absorptive period
B. Overnight fast
C. Three-day fast
D. Four-week fast
E. Five-month fast
Correct answer = D. Ketone bodies, made from the acetyl coenzyme A product of fatty acid oxidation, increase in the blood in fasting but must reach a critical level to cross the blood–brain barrier. Typically, this occurs in the second to third week of a fast. Fat stores in a 70-kg (~154-lb) man would not be able to supply his energy needs for 5 months.
24.5. The diagram below shows inputs to and outputs from pyruvate, a central molecule in energy metabolism.
Which letter on the diagram represents a reaction that requires biotin and is activated by acetyl coenzyme A?
Correct answer = C. Pyruvate carboxylase, a mitochondrial enzyme of gluconeogenesis, requires biotin (and ATP) and is allosterically activated by acetyl coenzyme A from fatty acid oxidation. None of the other choices meets these criteria. A = pyruvate kinase; B = pyruvate dehydrogenase complex; D = aspartate aminotransferase; E = alanine aminotransferase; F = lactate dehydrogenase.
For additional ancillary materials related to this chapter, please visit thePoint.
I. OVERVIEW
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Diabetes mellitus (diabetes) is not one disease but rather is a heterogeneous group of multifactorial, primarily polygenic syndromes characterized by an elevated fasting blood glucose (FBG) caused by a relative or absolute deficiency in insulin. Over 29 million people in the United States (~9% of the population) have diabetes. Of this number, ~8 million are as yet undiagnosed. Diabetes is the leading cause of adult blindness and amputation and a major cause of renal failure, nerve damage, heart attacks, and strokes. Most cases of diabetes mellitus can be separated into two groups (Fig. 25.1), type 1 ([T1D] formerly called insulin-dependent diabetes mellitus) and type 2 ([T2D] formerly called non– insulin-dependent diabetes mellitus). The incidence and prevalence of T2D is increasing because of the aging of the U.S. population and the increasing prevalence of obesity and sedentary lifestyles (see p. 349). The increase in children with T2D is particularly disturbing.
II. TYPE 1
T1D constitutes <10% of the ~21 million known cases of diabetes in the United States. The disease is characterized by an absolute deficiency of insulin caused by an autoimmune attack on the islet β cells of the pancreas. In T1D, the islets of Langerhans become infiltrated with activated T lymphocytes, leading to a condition called insulitis. Over a period of years, this autoimmune attack on the β cells leads to gradual depletion of the β-cell population (Fig. 25.2). However, symptoms appear abruptly when 80%–90% of the β cells have been destroyed. At this point, the pancreas fails to respond adequately to ingestion of glucose, and insulin therapy is required to restore metabolic control and prevent life-threatening ketoacidosis. β-Cell destruction requires both a stimulus from the environment (such as a viral infection) and a genetic determinant that causes the β cells to be mistakenly identified as “nonself.” [Note: Among monozygotic (identical) twins, if one sibling develops T1D, the other twin has only a 30%– 50% chance of developing the disease. This contrasts with T2D (see p. 341), in which the genetic influence is stronger and, in virtually all monozygotic twinships, the disease eventually develops in both individuals.]
A. Diagnosis
The onset of T1D is typically during childhood or puberty, and symptoms develop suddenly. Individuals with T1D can usually be recognized by the abrupt appearance of polyuria (frequent urination), polydipsia (excessive thirst), and polyphagia (excessive hunger), often triggered by physiologic stress such as an infection. These symptoms are usually accompanied by fatigue and weight loss. The diagnosis is confirmed by a FBG ≥126 mg/dl (normal is 70–99). [Note: Fasting is defined as no caloric intake for at least 8 hours.] A FBG of 100–125 mg/dl is categorized as an impaired FBG. Individuals with impaired FBG are considered prediabetic and are at increased risk for developing T2D. Diagnosis can also be made on the basis of a nonfasting (random) blood glucose level >200 mg/dl or a glycated hemoglobin (see p. 340) concentration ≥6.5 mg/dl (normal is <5.7) in an individual with symptoms of hyperglycemia. [Note: The oral glucose tolerance test, in which blood glucose is measured 2 hours after ingestion of a solution containing 75 g of glucose, also is used but is less convenient. It is most typically used to screen pregnant women for gestational diabetes (see p. 342).]
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When blood glucose is >180 mg/dl, the ability of renal sodium-dependent glucose transporters (SGLT) to reclaim glucose is impaired, and glucose “spills” into urine. The loss of glucose is accompanied by the loss of water, resulting in the characteristic polyuria (with dehydration) and polydipsia of diabetes.
B. Metabolic changes
The metabolic abnormalities of T1D result from a deficiency of insulin that profoundly affects metabolism in three tissues: liver, skeletal muscle, and white adipose (Fig. 25.3).
1.
Hyperglycemia and ketonemia: Elevated levels of blood glucose and ketone bodies are the hallmarks of untreated T1D (see Fig. 25.3). Hyperglycemia is caused by increased hepatic production of glucose via gluconeogenesis, combined with diminished peripheral utilization (muscle and adipose tissue have the insulin-regulated glucose transporter GLUT-4; see p. 97). Ketonemia results from increased mobilization of fatty acids (FA) from triacylglycerol (TAG) in adipose tissue, combined with accelerated hepatic FA β-oxidation and synthesis of 3hydroxybutyrate and acetoacetate (ketone bodies; see p. 196). [Note: Acetyl coenzyme A from β-oxidation is the substrate for ketogenesis and the allosteric activator of pyruvate carboxylase, a gluconeogenic enzyme.] Diabetic ketoacidosis (DKA), a type of metabolic acidosis caused by an imbalance between ketone body production and use, occurs in 25%–40% of those newly diagnosed with T1D and may recur if the patient becomes ill (most commonly with an infection) or does not comply with therapy. DKA is treated by replacing fluid and electrolytes and administering short-acting insulin to gradually correct hyperglycemia without precipitating hypoglycemia.
2.
Hypertriacylglycerolemia: Not all of the FA flooding the liver can be disposed of through oxidation and ketone body synthesis. These excess FA are converted to TAG, which are packaged and secreted in very-lowdensity lipoproteins ([VLDL] see p. 230). Chylomicrons rich in dietary TAG are secreted by the intestinal mucosal cells following a meal (see p. 227). Because lipoprotein TAG degradation catalyzed by lipoprotein lipase in the capillary beds of adipose tissue (see p. 228) is low in diabetes (synthesis of the enzyme is decreased when insulin levels are low), the plasma chylomicron and VLDL levels are elevated, resulting in hypertriacylglycerolemia (see Fig. 25.3).
C. Treatment
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