Biochemistry_Lippinco

Biochemistry_Lippinco. 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.]


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Nucleosides 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.]


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.]


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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).


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Adenosine and guanosine monophosphate synthesis 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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 =


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Deoxyribonucleotide synthesis regulation 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. [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.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Uric acid formation 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. [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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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).


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.]


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.]


Biochemistry_Lippinco

C. Pyrimidine nucleotide synthesis

Biochemistry_Lippinco. C. Pyrimidine nucleotide synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.]


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. F. Pyrimidine salvage and degradation 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. [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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Adenine B. Guanine C. Hypoxanthine D. Thymidine E. Uridine 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?


Biochemistry_Lippinco

Biochemistry_Lippinco. 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 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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).


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.]


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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).] IV. HYPOGLYCEMIA


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Types 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


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Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


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Biochemistry_Lippinco. 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. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


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Biochemistry_Lippinco. 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.


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Biochemistry_Lippinco. 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. The Feed–Fast Cycle 24


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


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Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Induction and repression of enzyme synthesis 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


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Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


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Biochemistry_Lippinco. 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.]


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Biochemistry_Lippinco. 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.


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Biochemistry_Lippinco. 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. 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).


Biochemistry_Lippinco

Biochemistry_Lippinco. b. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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).


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Fat metabolism 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. 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.


Biochemistry_Lippinco

VII. OVERVIEW OF THE FASTED STATE

Biochemistry_Lippinco. VII. OVERVIEW OF THE FASTED STATE


Biochemistry_Lippinco

Biochemistry_Lippinco. 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