27.6 What Regulates Our Eating Behavior? 853 brain, and other tissues. If energy demands are low, fatty acids are incorporated into triacylglycerols that are carried to adipose tissue for deposition as fat. Cholesterol is also synthesized in the liver from two-carbon units derived from acetyl-CoA. In addition to these central functions in carbohydrate and fat-based energy me- tabolism, the liver serves other purposes. For example, the liver can use amino acids as metabolic fuels. Amino acids are first converted to their corresponding ␣-keto acids by aminotransferases. The amino group is excreted after incorporation into urea in the urea cycle. The carbon skeletons of glucogenic amino acids can be used for glu- cose synthesis, whereas those of ketogenic amino acids appear in ketone bodies (see Figure 25.41). The liver is also the principal detoxification organ in the body. The endoplasmic reticulum of liver cells is rich in enzymes that convert biologically active substances such as hormones, poisons, and drugs into less harmful by-products. Liver disease leads to serious metabolic derangements, particularly in amino acid metabolism. In cirrhosis, the liver becomes defective in converting NH 4 ϩ to urea for excretion, and blood levels of NH 4 ϩ rise. Ammonia is toxic to the central nervous system, and coma ensues. 27.6 What Regulates Our Eating Behavior? Approximately 65% of Americans are overweight, and one in three Americans is clin- ically obese (overweight by 20% or more). Obesity is the single most important cause of type 2 (adult-onset insulin-independent) diabetes. Research into the regulatory controls that govern our feeding behavior has become a medical urgency with great financial incentives, given the epidemic proportions of obesity and widespread pre- occupation with dieting and weight loss. The Hormones That Control Eating Behavior Come From Many Different Tissues Appetite and weight regulation are determined by a complex neuroendocrine sys- tem that involves hormones produced in the stomach, small intestines, pancreas, adi- pose tissue, and central nervous system. These hormones act in the brain, principally Glycogen Glucose-6-phosphate Pyruvate Glycolysis Acetyl-CoA Oxidative phosphorylation Citric acid cycle CO 2 + H 2 O Cholesterol Fatty acids Fatty acid synthesis Triacylglycerols, phospholipids Lipid synthesis Blood glucose and pentose phosphates Pentose phosphate pathway ATP ATP NADPH FIGURE 27.11 Metabolic conversions of glucose-6- phosphate in the liver. 854 Chapter 27 Metabolic Integration and Organ Specialization on neurons within the arcuate nucleus region of the hypothalamus. The arcuate nu- cleus is an anatomically distinct brain area that functions in homeostasis of body weight, body temperature, blood pressure, and other vital functions (Figure 27.12). The neurons respond to these signals by activating, or not, pathways involved in eat- ing (food intake) and energy expenditure. Hormones that regulate eating behavior can be divided into short-term regulators that determine individual meals and long- term regulators that act to stabilize the levels of body fat deposits. Two subsets of neurons are involved: (1) the NPY/AgRP-producing neurons that release NPY (neuropeptide Y), the protein that stimulates the neurons that trigger eating behav- ior, and (2) the melanocortin-producing neurons, whose products inhibit the neu- rons initiating eating behavior. AgRP is agouti-related peptide, a protein that blocks the activity of melanocortin-producing neurons. Melanocortins are a group of peptide hormones that includes ␣- and -melanocyte–stimulating hormones (␣-MSH and -MSH). Melanocortins act on melanocortin receptors (MCRs), which are members of the 7-TMS G-protein–coupled receptor (GPCR) family of membrane receptors; MCRs trigger cellular responses through adenylyl cyclase activation (see Chapters 15 and 32). Ghrelin and Cholecystokinin Are Short-Term Regulators of Eating Behavior Short-term regulators of eating include ghrelin and cholecystokinin. Ghrelin is an appetite-stimulating peptide hormone produced in the stomach. Production of ghrelin is maximal when the stomach is empty, but ghrelin levels fall quickly once food is consumed. Cholecystokinin is a peptide hormone released from the gas- Insulin Pancreas Fat tissue Melanocortin- producing neuron Hypothalamus Food intake Energy expenditure Neuron NPY/ AgRP Arcuate nucleus Melanocortin receptor (MC4R) (blocked by AgRP) Ghrelin receptor NPY/PYY 3-36 receptor Y2R Melanocortin receptor (MC3R) Leptin receptor or insulin receptor NPY receptor Y1R Stomach Leptin Ghrelin PYY 3-36 ؉ ؉ ؊ ؊ ؊ Intestines FIGURE 27.12 The regulatory pathways that control eating. (Adapted from Figure 1 in Schwartz, M.W., and Morton, G. J., 2002. Obesity: Keeping hunger at bay. Nature 418:595–597.) 27.6 What Regulates Our Eating Behavior? 855 trointestinal tract during eating. In contrast to ghrelin, cholecystokinin signals sati- ety (the sense of fullness) and tends to curtail further eating. Together, ghrelin and cholecystokinin constitute a meal-to-meal control system that regulates the onset and end of eating behavior. The activity of this control system is also modulated by the long-term regulators. Insulin and Leptin Are Long-Term Regulators of Eating Behavior Long-term regulators include insulin and leptin, both of which inhibit eating and promote energy expenditure. Insulin is produced in the -cells of the pancreas when blood glucose levels rise. A major role of insulin is to stimulate glucose uptake from the blood into muscle, fat, and other tissues. Blood insulin levels correlate with body fat amounts. Insulin also stimulates fat cells to make leptin. Leptin (from the Greek word lepto, meaning “thin”) is a 16-kD, 146–amino acid residue protein pro- duced principally in adipocytes (fat cells). Leptin has a four-helix bundle tertiary structure similar to that of cytokines (protein hormones involved in cell–cell com- munication). Normally, as fat deposits accumulate in adipocytes, more and more lep- tin is produced in these cells and spewed into the bloodstream. Leptin levels in the blood communicate the status of triacylglycerol levels in the adipocytes to the cen- tral nervous system so that appropriate changes in appetite take place. If leptin lev- els are low (“starvation”), appetite increases; if leptin levels are high (“overfeeding”), appetite is suppressed. Leptin also regulates fat metabolism in adipocytes, inhibiting fatty acid biosynthesis and stimulating fat metabolism. In the latter case, leptin in- duces synthesis of the enzymes in the fatty acid oxidation pathway and increases ex- pression of uncoupling protein 2 (UCP2), a mitochondrial protein that uncouples oxi- dation from phosphorylation so that the energy of oxidation is lost as heat (thermogenesis). Leptin binding to leptin receptors in the hypothalamus inhibits re- lease of NPY. Because NPY is a potent orexic (appetite-stimulating) peptide hormone, leptin is therefore an anorexic (appetite-suppressing) agent. Functional leptin recep- tors are also essential for pituitary function, growth hormone secretion, and normal puberty. When body fat stores decline, the circulating levels of leptin and insulin also decline. Hypothalamic neurons sense this decline and act to increase appetite to re- store body fat levels. Intermediate regulation of eating behavior is accomplished by the gut hormone PYY 3-36 . PYY 3-36 is produced in endocrine cells found in distal regions of the small in- testine, areas that receive ingested food some time after a meal is eaten. PYY 3-36 inhibits eating for many hours after a meal by acting on the NPY/AgRP-producing neurons in the arcuate nucleus. Clearly, the regulatory controls that govern eating are complex and layered. Some believe that defects in these controls are common and biased in favor of overeating, an advantageous evolutionary strategy that may have unforeseen consequences in these bountiful times. HUMAN BIOCHEMISTRY The Metabolic Effects of Alcohol Consumption Ethanol metabolism alters the NAD ϩ /NADH ratio. Ethanol is metabolized to acetate in the liver by alcohol dehydrogenase and aldehyde dehydrogenase: CH 3 CH 2 OH ϩ NAD ϩ 34 CH 3 CHO ϩ NADH ϩ H ϩ CH 3 CHO ϩ NAD ϩ ϩ H 2 O 34 CH 3 COO Ϫ ϩ NADH ϩ 2H ϩ Acetate is then converted to acetyl-CoA. Excessive conversion of available NAD ϩ to NADH impairs NAD ϩ -requiring reactions, such as the citric acid cycle, gluconeogenesis, and fatty acid oxidation. Accumulation of acetyl-CoA favors fatty acid synthesis, which, along with blockage of fatty acid oxidation, causes elevated triacyl- glycerol levels in the liver. Over time, these triacylglycerols accu- mulate as fatty deposits, which ultimately contribute to cirrhosis of the liver. Impairment of gluconeogenesis leads to buildup of this pathway’s substrate, lactate. Lactic acid accumulation in the blood causes acidosis. A further consequence is that acetaldehyde can form adducts with protein ONH 2 groups, which may inhibit pro- tein function. Because gluconeogenesis is limited, alcohol con- sumption can cause hypoglycemia (low blood sugar) in someone who is undernourished. In turn, hypoglycemia can cause irre- versible damage to the central nervous system. 856 Chapter 27 Metabolic Integration and Organ Specialization AMPK Mediates Many of the Hypothalamic Responses to These Hormones The actions of leptin, ghrelin, and NPY converge at AMPK. Leptin inhibits AMPK activity in the arcuate nucleus, and this inhibition underlies the anorexic effects of leptin. Leptin action on AMPK depends on a particular melanocortin receptor type known as the melanocortin-4 receptor (MC4R). On the other hand, ghrelin and NPY activate hypothalamic AMPK, which stimulates food intake and leads, over time, to increased body weight. The effects of AMPK in the hypothalamus that lead to alterations in eating behavior may be mediated through changes in malonyl-CoA levels. Low [malonyl-CoA] in hypothalamic neurons is associated with increased food intake, and elevated malonyl-CoA levels are associated with suppression of eat- ing. The inhibition of acetyl-CoA carboxylase (and thus, malonyl-CoA synthesis) as a result of phosphorylation by AMPK plays an important part in the regulation of our eating behavior. 27.7 Can You Really Live Longer by Eating Less? Caloric Restriction Leads to Longevity Nutritional studies published in 1935 showed that rats fed a low-calorie, but balanced and nutritious diet lived nearly twice as long as rats with unlimited access to food (2.4 years versus 1.3 years). Subsequent research over the ensuing 70 years has shown that the relationship between diet and longevity is a general one for organisms from yeast to mammals. To achieve this effect of caloric restriction (CR), animals are given a level of food that amounts to 60% to 70% of what they would eat if they were al- lowed free access to food. In animals, CR results in lower blood glucose levels, de- clines in glycogen and fat stores, enhanced responsiveness to insulin, lower body temperature, and diminished reproductive capacity. The extended life span given by CR offers a definite evolutionary advantage: Any animal that could slow the aging process and postpone reproduction in times of food scarcity and then resume re- production when food became available would out-compete animals without such ability. Another remarkable feature of CR is that it diminishes the likelihood for develop- ment of many age-related diseases, such as cancer, diabetes, and atherosclerosis. Is this benefit simply the result of lowered caloric intake, or does CR lead to significant reg- ulatory changes that affect many aspects of an organism’s physiology? The answer to this and other questions is emerging from vigorous research efforts toward under- standing CR. Mutations in the SIR2 Gene Decrease Life Span Many important clues came from genetic investigations. Deletion of a gene termed SIR2 (for silent information regulator 2) abolished the ability of CR to lengthen life span in yeast and roundworms, implicating the SIR2 gene product in longevity. SIR2 originally was discovered through its ability to silence the transcription of genes that encode rRNA. SIR2-related genes are found in some prokaryotes and virtually all eukaryotes, including yeast, nematodes, fruit flies, and humans. The human gene is designated SIRT1, for sirtuin 1; sirtuin is the generic name for proteins encoded by SIR2 genes. Sirtuins are NAD ϩ -dependent protein deacetylases (Figure 27.13). Cleav- age of acetyl groups from proteins is an exergonic reaction, as is cleavage of the N-glycosidic bond in NAD ϩ (⌬G°ЈϭϪ34 kJ/mol). Thus, involvement of NAD ϩ in the reaction is not a thermodynamic necessity. However, NAD + involvement does couple the reaction to an important signal of metabolic status, namely, the NAD ϩ /NADH ratio. Furthermore, both nicotinamide and NADH are potent in- hibitors of the deacelylase reaction. Thus, the NAD ϩ /NADH ratio controls sirtuin protein deacetylase activity, so oxidative metabolism, which drives conversion of 27.7 Can You Really Live Longer by Eating Less? 857 NADH to NAD ϩ , enhances sirtuin activity. One adaptive response found with CR is increased mitochondrial biogenesis in liver, fat, and muscle, which is a response that would raise the NAD ϩ /NADH ratio. Sirtuin-catalyzed removal of acetyl groups from lysine residues of histones, the core proteins of nucleosomes, allows the nucleosomes to interact more strongly with DNA, making transcription more difficult (see Chapter 29 for a discussion of the relationship between histone acetylation and transcriptional activity of genes). SIRT1 Is a Key Regulator in Caloric Restriction As a key regulator in CR, the human sirtuin protein SIRT1 connects nutrient avail- ability to the expression of metabolic genes. A striking feature of CR is the loss of fat stores and reduction in white adipose tissue (WAT). SIRT1 participates in the tran- scriptional regulation of adipogenesis through interaction with PPAR␥ (peroxisome proliferator-activator receptor-␥), a nuclear hormone receptor that activates tran- scription of genes involved in adipogenesis and fat storage. SIRT1 binding to PPAR␥ represses transcription of these genes, leading to loss of fat stores. Because adipose tissue functions as an endocrine organ, this loss of fat has significant hormonal con- sequences for energy metabolism. In liver, SIRT1 interacts with and deacetylates PGC-1 (peroxisome proliferator- activator receptor-␥ coactivator-1), a transcriptional regulator of genes involved in glucose production. Thus, CR leads to increased transcription of the genes encod- ing the enzymes of gluconeogenesis and repression of genes encoding glycolytic en- zymes. Acting in these roles, SIRT1 connects nutrient availability to the regulation of major pathways of energy storage (glycogen and fat) and fuel utilization. Resveratrol, a Compound Found in Red Wine, Is a Potent Activator of Sirtuin Activity Resveratrol (trans-3,4Ј,5-trihydroxystilbene, Figure 27.14) is a phytoalexin. Phyto- alexins are compounds produced by plants in response to stress, injury, or fungal infection. Resveratrol is abundant in wine grape skins as a result of common envi- ronmental stresses, such as infection by Botrytis cinerea, a fungus important in mak- ing certain wines. Because the skins are retained when grapes are processed to + ++ O HO OH H 2 C NH 2 O C O PPO N + NH 2 C O N + OH 3 C C NH Peptide Acetyl-peptide NAD + Deacylated- peptide Nicotinamide NH 2 Peptide O OOH O HO2Ј-O-acetyl-ADP- ribose OH H 2 CHO O P O – POO NH 2 N N N N O CH 3 C O O – O O – O O – O O O HO OH CH 2 CH 2 NH 2 N N N N FIGURE 27.13 The NAD ϩ -dependent protein deacetyl- ase reaction of sirtuins. Acetylated peptides include N-acetyl lysine side chains of histone H3 and H4 and acetylated p53 (p53 is the protein product of the p53 tumor suppressor gene). FIGURE 27.14 Resveratrol, a phytoalexin, is a member of the polyphenol class of natural products. As a polyphe- nol, resveratrol is a good free-radical scavenger, which may account for its cancer preventative properties. OH OH HO Resveratrol (trans-3,4,5-trihydroxystilbene) 858 Chapter 27 Metabolic Integration and Organ Specialization make red wines, red wine is an excellent source of resveratrol. Resveratrol might be the basis of the French paradox—the fact that the French people enjoy longevity and relative freedom from heart disease despite a high-fat diet. When resveratrol is given to yeast cultures, roundworms (Caenorhabditis elegans), or fruit flies (Drosophila melanogaster), it has the same life-extending effects as CR. Resveratrol increased the replicative life span (the number of times a cell can divide before dying) of yeast by 70%. Resveratrol activates SIRT1 NAD ϩ -dependent deacetylase activity. Further- more, resveratrol activates AMPK in the brain and in cultured neurons. Because AMPK is a key energy sensor, resveratrol’s influences on longevity may arise through its effects on caloric homeostasis. Because the effects of CR and resveratrol on longevity are not additive, a reason- able conclusion is that they operate via a common mechanism. If this is so, then if you want to enjoy longevity, the appropriate advice would be “Eat less or drink red wine,” not “Eat less and drink red wine”! SUMMARY 27.1 Can Systems Analysis Simplify the Complexity of Metabolism? Cells are in a dynamic steady state maintained by considerable metabolic flux. The metabolism going on in even a single cell is so complex that it defies meaningful quantitative description. Nevertheless, overall relation- ships become more obvious by a systems analysis approach to intermedi- ary metabolism. The metabolism of a typical heterotrophic cell can be summarized in three interconnected functional blocks: (1) catabolism, (2) anabolism, and (3) macromolecular synthesis and growth. Photo- trophic cells require a fourth block: photosynthesis. Only a few metabolic intermediates connect these systems, and ATP and NADPH serve as the carriers of chemical energy and reducing power, respectively, between these various blocks. 27.2 What Underlying Principle Relates ATP Coupling to the Thermo- dynamics of Metabolism? ATP coupling determines the thermody- namics of metabolic sequences. The ATP coupling stoichiometry can- not be predicted from chemical considerations; instead it is a quantity selected by evolution and the fundamental need for metabolic se- quences to be emphatically favorable from a thermodynamic perspec- tive. Catabolic sequences generate ATP with an overall favorable K eq (and hence, a negative ⌬G) and anabolic sequences consume this en- ergy with an overall favorable K eq , even though such sequences may span the same starting and end points (as in fatty acid oxidation of palmitoyl-CoA to 8 acetyl-CoA versus synthesis of palmitoyl-CoA from 8 acetyl-CoA). ATP has two metabolic roles: a stoichiometric role in rendering metabolic sequences thermodynamically favorable and a reg- ulatory role as an allosteric effector. 27.3 Is There a Good Index of Cellular Energy Status? The level of phosphoric anhydride bonds in the adenylate system of ATP, ADP, and AMP can be expressed in terms of the energy charge equation: Energy charge ϭ ᎏ 1 2 ᎏ More revealing of the potential for an ATP-dependent reaction to occur is ⌫, the phosphorylation potential: ⌫ϭ[ATP]/([ADP][P i ]. 27.4 How Is Overall Energy Balance Regulated in Cells? AMP-activated protein kinase (AMPK) is the cellular energy sensor. When cellular energy levels are high, as signaled by high ATP concentrations, AMPK is inactive. When cellular energy levels are depleted, as signaled by high [AMP], AMPK is allosterically activated and phosphorylates many enzymes involved in cellular energy production and consumption. Competition between AMP and ATP for binding to the AMPK allosteric sites determines AMPK activity. AMPK activation leads to elevated energy production and diminished energy consumption. AMPK is an ␣␥-heterotrimer. The ␣-subunit is the catalytic subunit. AMP or ATP 2 [ATP] ϩ [ADP] ᎏᎏᎏ [ATP] ϩ [ADP] ϩ [AMP] binding to the ␥-subunit determines AMPK activity, with AMP binding increasing activity by more than 1000-fold. Activation of AMPK leads to phosphorylation of key enzymes in en- ergy metabolism, activating those involved in ATP production and inac- tivating those in ATP consumption. In addition, AMPK phosphorylation of various transcription factors leads to elevated expression of catabolic genes and diminished expression of genes encoding biosynthetic en- zymes. Beyond these cellular effects, AMPK plays a central role in en- ergy balance in multicellular organisms, because its activity is responsive to hormones that govern eating behavior and energy homeostasis. 27.5 How Is Metabolism Integrated in a Multicellular Organism? Organ systems in complex multicellular organisms carry out specific physiologi- cal functions, with each expressing those metabolic pathways appropriate to its physiological purpose. Essentially all cells in animals carry out the central pathways of intermediary metabolism, especially the reactions of ATP synthesis. Nevertheless, organs differ in the metabolic fuels they pre- fer as substrates for energy production. The major fuel depots in animals are glycogen in liver and muscle; triacylglycerols (fats) in adipose tissue; and protein, most of which is in skeletal muscle. The order of preference for the use of these fuels is glycogen Ͼ triacylglycerol Ͼ protein. Never- theless, the tissues of the body work together to maintain caloric home- ostasis: Constant availability of fuels in the blood. The major organ systems have specialized metabolic roles within the organism. The brain has a strong reliance on glucose as fuel. Muscle at rest pri- marily relies on fatty acids, but under conditions of strenuous contrac- tion when O 2 is limiting, muscle shifts to glycogen as its primary fuel. The heart is a completely aerobic organ, rich in mitochondria, with a preference for fatty acids as fuel under normal operating conditions. The liver is the body’s metabolic processing center, taking in nutrients and sending out products such as glucose, fatty acids, and ketone bod- ies. Adipose tissue takes up glucose and, to a lesser extent, fatty acids for the synthesis and storage of triacylglycerols. 27.6 What Regulates Our Eating Behavior? Appetite and weight reg- ulation are governed by hormones produced in the stomach, small in- testines, pancreas, adipose tissue, and central nervous system. These hormones act on neurons within the arcuate nucleus region of the hy- pothalamus that control pathways involved in eating (food intake) and energy expenditure. Hormones that regulate eating behavior can be di- vided into short-term regulators that determine individual meals and long-term regulators that act to stabilize the levels of body fat deposits. Short-term regulators of eating include ghrelin and cholecystokinin. Ghrelin is produced when the stomach is empty, but ghrelin levels fall quickly once food is consumed. Cholecystokinin is released from the gastrointestinal tract during eating. In contrast to ghrelin, chole- Problems 859 cysokinin signals satiety (the sense of fullness) and tends to curtail fur- ther eating. Together, ghrelin and cholecystokinin constitute a meal-to- meal control system that regulates the onset and end of eating behavior. Long-term regulators include insulin and leptin, both of which in- hibit eating and promote energy expenditure. Blood insulin levels cor- relate with body fat amounts. Insulin also stimulates fat cells to make leptin. Leptin is produced principally in adipocytes. As fat accumulates in adipocytes, more leptin is released into the bloodstream to commu- nicate the status of adipocyte fat to the central nervous system. If leptin levels are low (“starvation”), appetite increases; if leptin levels are high (“overfeeding”), appetite is suppressed. Leptin binding to its receptors in the hypothalamus inhibits release of NPY. NPY is a potent orexic hor- mone; therefore, leptin is an anorexic agent. When body fat stores de- cline, the circulating levels of leptin and insulin also decline. Hypothal- amic neurons sense this decline and act to increase appetite to restore body fat levels. Intermediate regulation of eating behavior is accomplished by the gut hormone PYY 3Ϫ36 . Produced in distal regions of the intestines, PYY 3Ϫ36 delays eating for many hours after a meal by inhibiting the NPY/AgRP-producing neurons in the arcuate nucleus. The regulatory controls that govern eating are complex and layered. 27.7 Can You Really Live Longer by Eating Less? Caloric restriction (CR) prolongs the longevity of organisms from yeast to mammals. CR results in lower blood glucose levels, decline in glycogen and fat stores, enhanced responsiveness to insulin, lower body temperature, and di- minished reproductive capacity. CR also diminishes the likelihood for development of many age-related diseases, such as cancer, diabetes, and atherosclerosis. Genetic investigations revealed that mutations in the SIR2 gene abolish the extension of life span by CR. The human SIR2 gene equivalent is SIRT1. SIRT genes encode sirtuins, a family of NAD ϩ - dependent protein deacetylases. The NAD ϩ /NADH ratio controls sir- tuin protein deacetylase activity, so oxidative metabolism, which drives conversion of NADH to NAD ϩ , enhances sirtuin action. CR increases mitochondrial biogenesis in liver, fat, and muscle, a response that would raise the NAD ϩ /NADH ratio. SIRT1 is a key regulator in CR. The phys- iological responses caused by CR are the result of a tightly regulated program that connects nutrient availability to the expression of meta- bolic genes. A striking feature of CR is the loss of fat stores and reduc- tion in white adipose tissue. SIRT1 binding to PPAR␥, a nuclear hor- mone receptor that activates transcription of genes involved in adipogenesis and fat storage, represses transcription of these genes, leading to the loss of fat stores. Because adipose tissue functions as an endocrine organ, this loss of fat has significant hormonal consequences for energy metabolism. In liver, SIRT1 interacts with and deacetylates PGC-1, a transcriptional regulator of glucose production. Transcription of genes encoding the enzymes of gluconeogenesis and repression of genes encoding glycolytic enzymes are increased upon PGC-1 deacety- lation. Thus, SIRT1 connects nutrient availability to the regulation of major pathways of energy storage and fuel use. Resveratrol, a phytoalexin, has the same life-extending effects as CR. Resveratrol activates both SIRT1 activity and brain AMPK, a key energy sensor. The influences of resveratrol on longevity may arise through its effects on caloric homeostasis. PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. (Integrates with Chapters 3, 18, and 22.) The conversion of PEP to pyruvate by pyruvate kinase (glycolysis) and the reverse reac- tion to form PEP from pyruvate by pyruvate carboxylase and PEP carboxykinase (gluconeog enesis) represent a so-called substrate cycle. The direction of net conversion is determined by the rela- tive concentrations of allosteric regulators that exert kinetic con- trol over pyruvate kinase, pyruvate carboxylase, and PEP car- boxykinase. Recall that the last step in glycolysis is catalyzed by pyruvate kinase: PEP ϩ ADP 34 pyruvate ϩ ATP The standard free energy change is Ϫ31.7 kJ/mol. a. Calculate the equilibrium constant for this reaction. b. If [ATP] ϭ [ADP], by what factor must [pyruvate] exceed [PEP] for this reaction to proceed in the reverse direction? The reversal of this reaction in eukaryotic cells is essential to gluco- neogenesis and proceeds in two steps, each requiring an equivalent of nucleoside triphosphate energy: Pyruvate carboxylase Pyruvate ϩ CO 2 ϩ ATP ⎯⎯→ oxaloacetate ϩ ADP ϩ P i PEP carboxykinase Oxaloacetate ϩ GTP ⎯⎯→ PEP ϩ CO 2 ϩ GDP Net: Pyruvate ϩ ATP ϩ GTP ⎯⎯→ PEP ϩ ADP ϩ GDP ϩ P i c. The ⌬G°Ј for the overall reaction is ϩ0.8 kJ/mol. What is the value of K eq ? d. Assuming [ATP] ϭ [ADP], [GTP] ϭ [GDP], and P i ϭ 1 mM when this reaction reaches equilibrium, what is the ratio of [PEP]/[pyruvate]? e. Are both directions in the substrate cycle likely to be strongly favored under physiological conditions? 2. (Integrates with Chapter 3.) Assume the following intracellular con- centrations in muscle tissue: ATP ϭ 8 mM, ADP ϭ 0.9 mM, AMP ϭ 0.04 mM, P i ϭ 8 mM. What is the energy charge in muscle? What is the phosphorylation potential? 3. Strenuous muscle exertion (as in the 100-meter dash) rapidly depletes ATP levels. How long will 8 mM ATP last if 1 gram of muscle consumes 300 mol of ATP per minute? (Assume muscle is 70% water.) Muscle contains phosphocreatine as a reserve of phosphorylation potential. Assuming [phosphocreatine] ϭ 40 mM, [creatine] ϭ 4 mM, and ⌬G°Ј (phosphocreatine ϩ H 2 O 34 creatine ϩ P i ) ϭϪ43.3 kJ/mol, how low must [ATP] become before it can be replenished by the reaction: phosphocreatine ϩ ADP 34 ATP ϩ creatine? [Remember, ⌬G°Ј (ATP hydrolysis) ϭϪ30.5 kJ/mol.] 4. (Integrates with Chapter 20.) The standard reduction potentials for the (NAD ϩ /NADH) and (NADP ϩ /NADPH) couples are identical, namely, Ϫ320 mV. Assuming the in vivo concentration ratios NAD ϩ / NADH ϭ 20 and NADP ϩ /NADPH ϭ 0.1, what is ⌬G for the follow- ing reaction? NADPH ϩ NAD ϩ 34 NADP ϩ ϩ NADH Assuming standard state conditions for the reaction, ADP ϩ P i ⎯→ ATP ϩ H 2 O, calculate how many ATP equivalents can be formed from ADP ϩ P i by the energy released in this reaction. 5. (Integrates with Chapter 3.) Assume the total intracellular pool of adenylates (ATP ϩ ADP ϩ AMP) ϭ 8 mM, 90% of which is ATP. What are [ADP] and [AMP] if the adenylate kinase reaction is at equilibrium? Suppose [ATP] drops suddenly by 10%. What are the concentrations now for ADP and AMP, assuming that the adenylate kinase reaction is at equilibrium? By what factor has the AMP con- centration changed? 6. (Integrates with Chapters 18 and 22.) The reactions catalyzed by PFK and FBPase constitute another substrate cycle. PFK is AMP ac- 860 Chapter 27 Metabolic Integration and Organ Specialization tivated; FBPase is AMP inhibited. In muscle, the maximal activity of PFK (mmol of substrate transformed per minute) is ten times greater than FBPase activity. If the increase in [AMP] described in problem 5 raised PFK activity from 10% to 90% of its maximal value but lowered FBPase activity from 90% to 10% of its maximal value, by what factor is the flux of fructose-6-P through the glycolytic path- way changed? (Hint: Let PFK maximal activity ϭ 10, FBPase maxi- mal activity ϭ 1; calculate the relative activities of the two enzymes at low [AMP] and at high [AMP]; let J, the flux of F-6-P through the substrate cycle under any condition, equal the velocity of the PFK reaction minus the velocity of the FBPase reaction.) 7. (Integrates with Chapters 23 and 24.) Leptin not only induces syn- thesis of fatty acid oxidation enzymes and uncoupling protein 2 in adipocytes, but it also causes inhibition of acetyl-CoA carboxylase, resulting in a decline in fatty acid biosynthesis. This effect on acetyl- CoA carboxylase, as an additional consequence, enhances fatty acid oxidation. Explain how leptin-induced inhibition of acetyl-CoA car- boxylase might promote fatty acid oxidation. 8. (Integrates with Chapters 19 and 20.) Acetate produced in ethanol metabolism can be transformed into acetyl-CoA by the acetyl thio- kinase reaction: Acetate ϩ ATP ϩ CoASH ⎯⎯→ acetyl-CoA ϩ AMP ϩ PP i Acetyl-CoA then can enter the citric acid cycle and undergo oxida- tion to 2 CO 2 . How many ATP equivalents can be generated in a liver cell from the oxidation of one molecule of ethanol to 2 CO 2 by this route, assuming oxidative phosphorylation is part of the process? (Assume all reactions prior to acetyl-CoA entering the cit- ric acid cycle occur outside the mitochondrion.) Per carbon atom, which is a better metabolic fuel, ethanol or glucose? That is, how many ATP equivalents per carbon atom are generated by combus- tion of glucose versus ethanol to CO 2 ? 9. (Integrates with Chapter 23.) Assuming each NADH is worth 3 ATP, each FADH 2 is worth 2 ATP, and each NADPH is worth 4 ATP: How many ATP equivalents are produced when one molecule of palmitoyl-CoA is oxidized to 8 molecules of acetyl-CoA by the fatty acid -oxidation pathway? How many ATP equivalents are con- sumed when 8 molecules of acetyl-CoA are transformed into one molecule of palmitoyl-CoA by the fatty acid biosynthetic pathway? Can both of these metabolic sequences be metabolically favorable at the same time if ⌬G for ATP synthesis is ϩ50 kJ/mol? 10. (Integrates with Chapters 18–21.) If each NADH is worth 3 ATP, each FADH 2 is worth 2 ATP, and each NADPH is worth 4 ATP, cal- culate the equilibrium constant for cellular respiration, assuming synthesis of each ATP costs 50 kJ/mol of energy. Calculate the equi- librium constant for CO 2 fixation under the same conditions, ex- cept here ATP will be hydrolyzed to ADP ϩ P i with the release of 50 kJ/mol. Comment on whether these reactions are thermody- namically favorable under such conditions. 11. (Integrates with Chapter 22.) In type 2 diabetics, glucose produc- tion in the liver is not appropriately regulated, so glucose is over- produced. One strategy to treat this disease focuses on the devel- opment of drugs targeted against regulated steps in glycogenolysis and gluconeogenesis, the pathways by which liver produces glucose for release into the blood. Which enzymes would you select for as potential targets for such drugs? 12. As chief scientist for drug development at PhatFarmaceuticals, Inc., you want to create a series of new diet drugs. You have a grand plan to design drugs that might limit production of some hormones or promote the production of others. Which hormones are on your “limit production” list and which are on your “raise levels” list? 13. The existence of leptin was revealed when the ob/ob genetically obese strain of mice was discovered. These mice have a defective leptin gene. Predict the effects of daily leptin injections into ob/ob mice on food intake, fatty acid oxidation, and body weight. Similar clinical trials have been conducted on humans, with limited success. Suggest a reason why this therapy might not be a miracle cure for overweight individuals. 14. Would it be appropriate to call neuropeptide Y (NPY) the obesity- promoting hormone? What would be the phenotype of a mouse whose melanocortin-producing neurons failed to produce melano- cortin? What would be the phenotype of a mouse lacking a func- tional MC3R gene? What would be the phenotype of a mouse lack- ing a functional leptin receptor g ene? 15. The Human Biochemistry box, The Metabolic Effects of Alcohol Consumption, points out that ethanol is metabolized to acetate in the liver by alcohol dehydrogenase and aldehyde dehydrogenase: CH 3 CH 2 OH ϩ NAD ϩ 34 CH 3 CHO ϩ NADH ϩ H ϩ CH 3 CHO ϩ NAD ϩ ϩ H 2 O 34 CH 3 COO Ϫ ϩ NADH ϩ 2H ϩ These reactions alter the NAD ϩ /NADH ratio in liver cells. From your knowledge of glycolysis, gluconeogenesis, and fatty acid oxida- tion, what might be the effect of an altered NAD ϩ /NADH ratio on these pathways? What is the basis of this effect? 16. A T172D mutant of the AMPK is locked in a permanently active state. Explain. 17. a. Some scientists support the “malonyl-CoA hypothesis,” which suggests that malonyl-CoA is a key indicator of nutrient avail- ability and the brain uses its abundance to assess whole-body en- ergy homeostasis. Others have pointed out that malonyl-CoA is a significant inhibitor of carnitine acyltransferase-1 (see Figure 24.16). Thus, malonyl-CoA may be influencing the levels of an- other metabolite whose concentration is more important as a sig- nal of energy status. What metabolite might that be? b. Another test of the malonyl-CoA hypothesis was conducted through the creation of a transgenic strain of mice that lacked functional hypothalamic fatty acid synthase (see Chapter 24). Pre- dict the effect of this genetic modification on cellular malonyl- CoA levels in the hypothalamus, the eating behavior of these transgenic mice, their body fat content, and their physical activity levels. Defend your predictions. 18. a. Leptin was discovered when a congenitally obese strain of mice (ob/ob mice) was found to lack both copies of a gene encoding a peptide hormone produced mainly by adipose tissue. The pep- tide hormone was named leptin. Leptin is an anorexic (appetite- suppressing) agent; its absence leads to obesity. Propose an ex- periment to test these ideas. b. A second strain of obese mice (db/db mice) produces leptin in abundance but fails to respond to it. Assuming the db mutation leads to loss of function in a protein, what protein is likely to be nonfunctional or absent? How might you test your idea? Preparing for the MCAT Exam 19. Consult Figure 27.7 and answer the following questions: Which or- gans use both fatty acids and glucose as a fuel in the well-fed state, which rely mostly on glucose, which rely mostly on fatty acids, which one never uses fatty acids, and which one produces lactate. 20. Figure 27.3 illustrates the response of R (ATP-regenerating) and U (ATP-utilizing) enzymes to energy charge. a. Would hexokinase be an R enzyme or a U enzyme? Would glu- tamineϺPRPP amidotransferase, the second enzyme in purine biosynthesis, be an R enzyme or a U enzyme? b. If energy charge ϭ 0.5: Is the activity of hexokinase high or low? Is ribose-5-P pyrophosphokinase activity high or low? c. If energy charge ϭ 0.95: Is the activity of hexokinase high or low? Is ribose-5-P pyrophosphokinase activity high or low? FURTHER READING Systems Analysis of Metabolism Brand, M. D., and Curtis, R. K., 2002. Simplifying metabolic complexity. Biochemical Society Transactions 30:25–30. ATP Coupling and the Thermodynamics of Metabolism Atkinson, D. F., 1977. Cellular Energy Metabolism and Its Regulation. New York: Academic Press. Newsholme, E. A., Challiss, R. A. J., and Crabtree, B., 1984. Substrate cycles: Their role in improving sensitivity in metabolic control. Trends in Biochemical Sciences 9:277–280. Newsholme, E. A., and Leech, A. R., 1983. Biochemistry for the Medical Sci- ences. New York: John Wiley & Sons. AMP-Activated Protein Kinase Hardie, D. G., 2007. AMP-activated/SNF 1 protein kinases: Conserved guardians of cellular energy. Nature Reviews Cell Molecular Biology 8:774–785. Hardie, D. G., Hawley, S. A., and Scott, J. W., 2007. AMP-activated pro- tein kinase: Development of the energy sensor concept. Journal of Physiology 574:7–15. McGee, S. L., and Hargreaves, M., 2008. AMPK and transcriptional reg- ulation. Frontiers in Bioscience 13:3022–3033. Metabolic Relationships Between Organ Systems Harris, R., and Crabb, D. W., 1997. Metabolic interrelationships. In Text- book of Biochemistry with Clinical Correlations, 4th ed., Devlin, T. M., ed. New York: Wiley-Liss. Sugden, M. C., Holness, M. J., and Palmer, T. N., 1989. Fuel selection and carbon flux during the starved-to-fed transition. Biochemical Journal 263:313–323. Creatine as a Nutritional Supplement Ekblom, B., 1999. Effects of creatine supplementation on performance. American Journal of Sports Medicine 24:S-38. Kreider, R., 1998. Creatine supplementation: Analysis of ergogenic value, medical safety, and concerns. Journal of Exercise Physiology 1, an international onnline journal available at http://www.css.edu/ users/tboone2/asep/jan3.htm Fat-Free Mice Gavrilova, O., et al., 2000. Surgical implantation of adipose tissue re- verses diabetes in lipoatrophic mice. Journal of Clinical Investigation 105:271–278. Moitra, J., et al., 1998. Life without white fat: A transgenic mouse. Genes and Development 12:3168–3181. Leptin and Hormonal Regulation of Eating Behavior Barinaga, M., 1995. “Obese” protein slims mice. Science 269: 475–476, and references therein. Buettner, C., 2007. Does FASing out new fat in the hypothalamus make you slim? Cell Metabolism 6:249–251. Clement, K., et al., 1998. A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392:398–401. Coll, A. P., Farooqi, S., and O’Rahilly, S., 2007. The hormonal control of food intake. Cell 129:251–262. Saper, C. B., Chou, T. C., and Elmquist, J. K., 2002. The need to feed: Homeostatic and hedonic control of eating. Neuron 36:199–211. Schwartz, M. W., and Morton, G. J., 2002. Obesity: Keeping hunger at bay. Nature 418:595–597. Vaisse, C., et al., 2000. Melanocortin-4 receptor mutations are a fre- quent and heterogeneous cause of morbid obesity. Journal of Clini- cal Investigation 106:253–262. Zhou, Y -T., et al., 1997. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation. Proceedings of the National Academy of Sciences U.S.A. 94:6386–6390. Caloric Restriction and Longevity Dasgupta, B., and Milbrandt, J., 2007. Resveratrol stimulates AMP kinase activity in neurons. Proceedings of the National Academy of Science U.S.A. 104:7217–7222. Denu, J. M., 2005. The Sir2 family of protein deacetylases. Current Opin- ion in Chemical Biology 9:431–440. Guarente, L., 2005. Caloric restriction and SIR2 genes—Towards a mechanism. Mechanisms of Aging and Development 126:923–928. Guarente, L., and Picard, F., 2005. Caloric restriction—The SIR2 con- nection. Cell 120:473–482. Michan, S., and Sinclair, D., 2007. Sirtuins in mammals: Insights into their biological function. Biochemical Journal 404:1–13. Milne, J. C., Lambert, P. D., Schenk, S., Carney, D. P., et al., 2007. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 450:712–716. Moynihan, K. A., and Imai, S-I., 2006. Sirt1 as a key regulator orches- trating the response to caloric restriction. Drug Discovery Today: Dis- ease Mechanisms 3:11–17. Further Reading 861 “Dawn of the Double Helix,” by Julie Newdoll 28 DNA Metabolism: Replication, Recombination, and Repair Heredity, which we can define generally as the tendency of an organism to pos- sess the characteristics of its parent(s), is clearly evident throughout nature and since the dawn of history has served to justify the classification of org anisms ac- cording to shared similarities. The basis of heredity, however, was a mystery. Early in the 20th century, geneticists demonstrated that genes, the elements or units carrying and transferring inherited characteristics from parent to offspring, are contained within the nuclei of cells in association with the chromosomes. Yet the chemical identity of genes remained unknown, and genetics was an abstract sci- ence. Even the realization that chromosomes are composed of proteins and nu- cleic acids did little to define the molecular nature of the gene because, at the time, no one understood either of these substances. The material of heredity must have certain properties. It must be very stable so that genetic information can be stored in it and transmitted countless times to sub- sequent generations. It must be capable of precise copying or replication so that its information is not lost or altered. And, although stable, it must also be subject to change in order to account, in the short term, for the appearance of mutant forms and, in the long term, for evolution. DNA is the material of heredity. 28.1 How Is DNA Replicated? Transfer of genetic information from generation to generation requires the faithful reproduction of the parental DNA. DNA reproduction produces two identical copies of the original DNA in a process termed DNA replication. The mechanism for DNA replication is strand separation followed by the copying of each strand. In the process, each separated strand acts as a template for the synthesis of a new com- plementary strand whose nucleotide sequence is fixed by the base-pairing rules Watson and Crick proposed (see Chapter 10). Strand separation is achieved by untwisting the double helix (Figure 28.1). Base pairing then dictates the proper sequence of nucleotide addition to achieve an accurate replication of each orig- inal strand. Thus, each original strand ends up paired with a new complementary partner, and two identical double-stranded DNA molecules are formed from one. This mode of DNA replication is referred to as semiconservative because one of the two original strands is conserved in each of the two progeny molecules. DNA Replication Is Bidirectional Replication of DNA molecules begins at one or more specific regions called the origin(s) of replication and, excepting certain bacteriophage chromosomes and plas- mids, proceeds in both directions from this origin (Figure 28.2). For example, repli- cation of E. coli DNA begins at oriC, a unique 245-bp chromosomal site that contains 11 GATC tetranucleotide sequences along its length. From oriC, replication ad- vances in both directions around the circular chromosome. That is, bidirectional Julie Newdoll’s painting “Dawn of the Double Helix” composes the DNA duplex as human figures. Her theme in this painting is “Life Forms:The basic struc- tures that make our existence possible.” Heredity I am the family face; Flesh perishes, I live on, Projecting trait and trace Through time to times anon, And leaping from place to place Over oblivion. The years-heired feature that can In curve and voice and eye Despise the human span Of durance—that is I; The eternal thing in man, That heeds no call to die. Thomas Hardy (in Moments of Vision and Miscellaneous Verses, 1917) KEY QUESTIONS 28.1 How Is DNA Replicated? 28.2 What Are the Properties of DNA Polymerases? 28.3 Why Are There So Many DNA Polymerases? 28.4 How Is DNA Replicated in Eukaryotic Cells? 28.5 How Are the Ends of Chromosomes Replicated? 28.6 How Are RNA Genomes Replicated? 28.7 How Is the Genetic Information Shuffled by Genetic Recombination? 28.8 Can DNA Be Repaired? 28.9 What Is the Molecular Basis of Mutation? 28.10 Do Proteins Ever Behave as Genetic Agents? Special Focus: Gene Rearrangements and Immunology—Is It Possible to Generate Protein Diversity Using Genetic Recombination? ESSENTIAL QUESTIONS DNA is the physical repository of genetic information in the cell and the material of heredity that is passed on to progeny. How is this genetic information in the form of DNA replicated, how is the infor- mation rearranged, and how is its integrity maintained in the face of damage? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. . W., 1997. Metabolic interrelationships. In Text- book of Biochemistry with Clinical Correlations, 4th ed., Devlin, T. M., ed. New York: Wiley-Liss. Sugden, M. C., Holness, M. J., and Palmer, T.