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up to 40% of their total body weight and has high mito-

chondrial capacity. Homologs of UCP1 have been identi-

fied: UCP2 occurs in many tissues including white adipose

tissue, whereas UCP3 occurs in brown adipose tissue, white

adipose tissue, and muscle. Leptin has been shown to up-

regulate UCP2. However, it has yet to be demonstrated

that UCP3 in muscle participates in diet-induced thermo-

genesis. ATP-hydrolyzing substrate cycles such as that be-

tween fatty acids and triacylglycerol in adipose tissue (Sec-

tion 27-2C) may also be involved.

H. Did Leptin Evolve as a Thrifty Gene?

The unusual behavior of leptin, which serves to control

weight in normal-weight individuals while its concentration

continues to climb without apparent effect in obese individu-

als, has led to the proposal that leptin evolved as a “thrifty

gene.” In hunter-gatherer societies,it was a distinct advantage

to be able to survive intermittent famines. In order to do this,

fat must be stored in adipose tissue in times of plenty, making

short-term obesity advantageous. However, the accumula-

tion of fatty acids and lipids in non-adipose tissue results in

coronary artery disease, insulin resistance,and diabetes (Sec-

tion 27-4B). Leptin, by directly stimulating the oxidation of

fatty acids as well as inhibiting the accumulation of lipids in

non-adipose tissue, is thought to protect against these dis-

eases during short-term obesity, thereby providing an evolu-

tionary advantage. However, in recent times in industrialized

nations, the unprecedented availability of food and lack of

famine has made obesity a long-term rather than a short-

term condition, which is a liability rather than a benefit.

4 METABOLIC ADAPTATION

In this section we consider the body’s responses to two

metabolically abnormal situations: (1) starvation and (2) the

disease diabetes mellitus.

A. Starvation

Glucose is the metabolite of choice of both brain and

working muscle. Yet, the body stores less than a day’s sup-

ply of carbohydrate (Table 27-1).Thus, the low blood sugar

caused by even an overnight fast results, through an in-

crease in glucagon secretion and a decrease in insulin se-

cretion, in the mobilization of fatty acids from adipose tis-

sue (Section 25-5). The diminished insulin level also

inhibits glucose uptake by muscle tissue. Muscles therefore

switch from glucose to fatty acid metabolism for energy

production. The brain, however, still remains heavily de-

pendent on glucose.

In animals, glucose cannot be synthesized from fatty

acids. This is because neither pyruvate nor oxaloacetate,

the precursors of glucose in gluconeogenesis (Section 23-1),

can be synthesized from acetyl-CoA (the oxaloacetate in

the citric acid cycle is derived from acetyl-CoA but the

cyclic nature of this process requires that the oxaloacetate

be consumed as fast as it is synthesized; Section 21-1A).

During starvation, glucose must therefore be synthesized

from the glycerol product of triacylglycerol breakdown

and, more importantly, from the amino acids derived from

the proteolytic degradation of proteins, the major source of

which is muscle.Thus, after a 40-hour fast, gluconeogenesis

supplies ⬃96% of the glucose produced by the liver. How-

ever,the continued breakdown of muscle during prolonged

starvation would ensure that this process became irre-

versible since a large muscle mass is essential for an animal

to move about in search of food.The organism must there-

fore make alternate metabolic arrangements.

After several days of starvation, gluconeogenesis has so

depleted the liver’s oxaloacetate supply that this organ’s

ability to metabolize acetyl-CoA via the citric acid cycle is

greatly diminished. Rather, the liver converts the acetyl-

CoA to ketone bodies (Section 25-3), which it releases into

the blood.The brain gradually adapts to using ketone bod-

ies as fuel through the synthesis of the appropriate en-

zymes: After a 3-day fast, only about one-third of the

brain’s energy requirements are satisfied by ketone bodies

but after 40 days of starvation, ⬃70% of its energy needs

are so met. The rate of muscle breakdown during pro-

longed starvation consequently decreases to ⬃25% of its

rate after a several-day fast.The survival time of a starving

individual is therefore much more dependent on the size of

his or her fat reserves than it is on his or her muscle mass.

Indeed, highly obese individuals can survive for over a year

without eating (and have occasionally done so in clinically

supervised weight reduction programs).

a. Caloric Restriction May Increase Longevity

Caloric restriction is a modified form of starvation

whereby energy intake is reduced 30–40%, while micronu-

trient (vitamin and mineral) levels are maintained. Rodents

kept on such a diet live up to 50% longer than rodents on

normal diets and exhibit fewer of the debilitating symptoms

of old age.The life spans of a large range of organisms from

yeast to primates are similarly extended. Considerable

research effort is being expended to determine the bio-

chemical basis of these observations.

Section 27-4. Metabolic Adaptation 1101

Table 27-1 Fuel Reserves for a Normal 70-kg Man

Fuel Mass (kg) Calories

Tissues

Fat (adipose triacyglycerols) 15 141,000

Protein (mainly muscle) 6 24,000

Glycogen (muscle) 0.150 600

Glycogen (liver) 0.075 300

Circulating fuels

Glucose (extracellular fluid) 0.020 80

Free fatty acids (plasma) 0.0003 3

Triacylglycerols (plasma) 0.003 30

Total 166,000

One (dieter’s) Calorie ⫽ 1 kcal ⫽ 4.184 kJ.

Source: Cahill, G.F., Jr., New Engl. J. Med. 282, 669 (1970).

JWCL281_c27_1088-1106.qxd 6/8/10 8:49 AM Page 1101

B. Diabetes Mellitus

The polypeptide hormone insulin acts mainly on muscle,

liver, and adipose tissue cells to stimulate the synthesis of

glycogen, fats, and proteins while inhibiting the breakdown

of these metabolic fuels. In addition, insulin stimulates the

uptake of glucose by most cells, with the notable exception

of brain and liver cells. Together with glucagon, which has

largely opposite effects, insulin acts to maintain the proper

level of blood glucose.

In the disease diabetes mellitus, which is the third leading

cause of death in the United States after heart disease and

cancer, insulin either is not secreted in sufficient amounts or

does not efficiently stimulate its target cells. As a conse-

quence, blood glucose levels become so elevated that the

glucose “spills over” into the urine, providing a convenient

diagnostic test for the disease. Yet, despite these high blood

glucose levels, cells “starve” since insulin-stimulated glucose

entry into cells is impaired. Triacylglycerol hydrolysis, fatty

acid oxidation, gluconeogenesis, and ketone body formation

are accelerated and, in a condition termed ketoacidosis, ke-

tone body levels in the blood become abnormally high.Since

ketone bodies are acids, their high concentration puts a

strain on the buffering capacity of the blood and on the kid-

ney, which controls blood pH by excreting the excess H

⫹

into the urine (Section 27-2E). This unusually high excess

⫹

excretion is accompanied by NH

⫹

,Na

⫹

, and H

excretion, causing severe dehydration (which compounds

the dehydration resulting from the osmotic effect of the high

glucose concentration in the blood; excessive thirst is a clas-

sic symptom of diabetes) and a decrease in blood volume—

ultimately life-threatening situations.

There are two major forms of diabetes mellitus:

1. Insulin-dependent, type 1, or juvenile-onset diabetes

mellitus, which most often strikes suddenly in childhood.

2. Noninsulin-dependent, type 2, or maturity-onset

diabetes mellitus, which usually develops rather gradually

after the age of 40.

a. Insulin-Dependent Diabetes Is Caused by a

Deficiency of Pancreatic ␤ Cells

In insulin-dependent (type 1) diabetes mellitus, insulin

is absent or nearly so because the pancreas lacks or has

defective ␤ cells. This condition results, in genetically sus-

ceptible individuals (see below), from an autoimmune re-

sponse that selectively destroys their ␤ cells. Individuals

with insulin-dependent diabetes, as Frederick Banting and

George Best first demonstrated in 1921, require daily

insulin injections to survive and must follow carefully bal-

anced diet and exercise regimens. Their life spans are,

nevertheless, reduced by up to one-third as a result of de-

generative complications such as kidney malfunction,

nerve impairment, and cardiovascular disease, which ap-

parently arise from the imprecise metabolic control pro-

vided by periodic insulin injections. The hyperglycemia

(high blood [glucose]) of diabetes mellitus also leads to

blindness through retinal degeneration and the glucosyla-

tion of lens proteins, which causes cataracts (Fig. 27-9).

Perhaps newly developed systems that monitor blood glu-

cose levels and continuously deliver insulin in the required

amounts will rectify this situation.

The usually rapid onset of the symptoms of insulin-

dependent diabetes had suggested that the autoimmune at-

tack on the pancreatic ␤ cells responsible for this disease is

one of short duration. Typically, however, the disease

“brews” for several years as the aberrantly aroused immune

system slowly destroys the ␤ cells. Only when ⬎80% of these

cells have been eliminated do the classic symptoms of dia-

betes suddenly emerge. Consequently, one of the most suc-

cessful treatments for insulin-dependent diabetes is a ␤-cell

transplant, a procedure that became possible with the devel-

opment of relatively benign immunosuppressive drugs.

Why does the immune system attack the pancreatic ␤

cells? It has long been known that certain alleles (genetic

variants) of the Class II major histocompatibility complex

(MHC) proteins are particularly common in insulin-

dependent diabetics [MHC proteins are highly polymorphic

(variable within a species) immune system components to

which cell-generated antigens such as viral proteins must

bind in order to be recognized as foreign; Sections 35-2Aa

and 35-2E]. It is thought that autoimmunity against ␤ cells is

induced in a susceptible individual by a foreign antigen, per-

haps a virus, which immunologically resembles some ␤ cell

component. The Class II MHC protein that binds this anti-

gen does so with such tenacity that it stimulates the immune

system to launch an unusually vigorous and prolonged at-

tack on the antigen. Some of the activated immune system

cells eventually make their way to the pancreas, where they

initiate an attack on the ␤ cells due to the close resemblance

of the ␤ cell component to the foreign antigen.

b. Noninsulin-Dependent Diabetes Is

Characterized by Insulin Resistance as

Well as Impaired Insulin Secretion

Noninsulin-dependent (type 2) diabetes mellitus

(NIDDM), which accounts for over 90% of the diagnosed

cases of diabetes and affects 18% of the population over

65 years of age, usually occurs in obese individuals with a

genetic predisposition for this condition (although one

that differs from that associated with insulin-dependent

1102 Chapter 27. Energy Metabolism: Integration and Organ Specialization

Figure 27-9 Photo of a diabetic cataract. The accumulation of

glucose in the lens leads to swelling and precipitation of lens

proteins.The resulting opacification causes blurred vision and

ultimately complete loss of sight. [© Sue Ford/Photo Researchers.]

JWCL281_c27_1088-1106.qxd 4/21/10 9:42 AM Page 1102

diabetes). These individuals may have normal or even

greatly elevated insulin levels. Their symptoms arise from

insulin resistance, an apparent lack of sensitivity to insulin

in normally insulin-responsive cells.

The hyperglycemia that accompanies insulin resistance

induces the pancreatic ␤ cells to increase their production

of insulin. Yet the high basal level of insulin secretion di-

minishes the ability of the ␤ cells to respond to further in-

creases in blood glucose. Consequently, the hyperglycemia

and its attendant complications tend to worsen over time.

A small percentage of cases of type II diabetes result

from mutations in the insulin receptor that affect its

insulin-binding ability or tyrosine kinase activity. However,

a clear genetic cause has not been identified in the vast ma-

jority of cases. It is therefore likely that many factors play a

role in the development of this disease. For example, the in-

creased insulin production resulting from overeating may

eventually suppress the synthesis of insulin receptors. This

hypothesis accounts for the observation that diet alone is

often sufficient to control this type of diabetes.

Insulin resistance, which may precede NIDDM by as

much as 10 to 20 years, appears to be caused by an inter-

ruption in the insulin signaling pathway (Section 19-4F).

Gerald Shulman has proposed that this interruption is

caused by a Ser/Thr kinase cascade that phosphorylates

proteins known as insulin receptor substrates (IRSs; Sec-

tion 19-3Cg) so as to decrease their ability to be phospho-

rylated on their Tyr residues by activated insulin receptor.

Tyrosine phosphorylation is required for IRS activation

and communication with phosphoinositide 3-kinase (PI3K;

Section 19-4D), which subsequently activates the translo-

cation of GLUT4-containing vesicles to the cell surface for

increased glucose transport into cells (Section 20-2Ec).The

original Ser/Thr kinase cascade is triggered by the activa-

tion of an isoform of protein kinase C (PKC; Section 19-4C)

caused by an increase in fatty acyl-CoA,diacylglycerol,and

ceramides (Section 12-1D) resulting from elevated free

fatty acids (Fig. 27-10). The failure to activate IRSs de-

creases the cell’s response to insulin (Fig. 27-11).

c. Substances That Activate AMPK Attenuate the

Symptoms of Noninsulin-Dependent Diabetes

Other treatments for noninsulin-dependent diabetes

are drugs such as metformin and the thiazolidinediones

(TZDs),

which decrease insulin resistance by either suppressing

glucose release by the liver (metformin) or promoting

A thiazolidinedione (TZD)

Metformin

NHN

Section 27-4. Metabolic Adaptation 1103

Figure 27-10 The mechanism through which high concentra-

tions of free fatty acids cause insulin resistance. Elevated concen-

trations of free fatty acids in the blood diffuse into muscle cells

where they are converted to fatty acyl-CoA, diacylglycerols, and

ceramides.These lipotoxic substances activate an isoform of

protein kinase C (PKC), triggering a Ser/Thr kinase cascade that

results in the phosphorylation of IRS-1 and IRS-2.This

phosphorylation inhibits the Tyr phosphorylation required for

transmission of the insulin signal, thereby decreasing the

activation of PI3K, which decreases the rate of fusion of

GLUT4-containing vesicles with the plasma membrane and

hence the amount of glucose entering the cell. [Modified from

Shulman, G.I., J. Clin. Invest. 106, 173 (2000).]

Figure 27-11 Twenty-four-hour plasma glucose profiles in nor-

mal and noninsulin-dependent diabetic subjects. The basal level

of glucose and the peaks following meals are higher in the

diabetic individuals. [After Bell, G.I., Pilkis, S.J., Weber, I.T., and

Polonsky, K.S., Annu. Rev. Physiol. 58, 178 (1996).]

Plasma

Glucose

Insulin receptor

Insulin

IRS-1/IRS-2 Ser/Thr phosphorylation

PI3K

IRS-1/IRS-2 Tyr phosphorylation

Fatty acyl-CoA,

diacylglycerols,

ceramides

Fatty acids

Ser/Thr kinase

cascade

PKC

GLUT4

240

480

720

960

1200

1440

Plasma [Glucose] (mM)

Time (min)

Non-insulin-dependent

diabetes

Normal

breakfast

lunch dinner

JWCL281_c27_1088-1106.qxd 4/21/10 9:42 AM Page 1103

insulin-stimulated glucose disposal in muscle (TZDs).

These drugs act by increasing AMPK activity but by differ-

ent mechanisms.TZDs cause a large increase in the AMP to

ATP ratio in muscle cells, with the expected concomitant in-

crease in AMPK phosphorylation and activity. Metformin,

however, stimulates LKB1 to phosphorylate and hence ac-

tivate AMPK (LKB1 knockout mice are insensitive to met-

formin). In both cases, the increase in AMPK activity de-

creases gluconeogenesis in liver and increases glucose

utilization in muscle (Fig. 27-4). In addition, the TZDs de-

crease insulin resistance by binding to and activating a

transcription factor known as a peroxisome proliferator–

activated receptor ␥ (PPAR␥), primarily in adipose tissue.

Among other things, PPAR␥ activation induces the synthe-

sis of adiponectin (Section 27-3B), which leads to an in-

crease in AMPK activity. In adipose tissue, AMPK action

leads to a decrease in lipolysis and fatty acid export, de-

creasing the concentration of free fatty acids in the blood

and therefore decreasing insulin resistance (see above).

Intriguingly, Ronald Evans has shown that transgenic

mice expressing an activated form of PPAR␥ in their skele-

tal muscles can run around twice the distance of wild-type

mice and are resistant to weight gain, even on a high fat

diet.This activated PPAR␥ induces an increase in the num-

ber of the aerobic and hence fatty acid–oxidizing slow-

twitch (Type I) muscle fibers (Section 17-3Ca) relative to

the largely anaerobic and hence less energy-efficient fast-

twitch (Type II) muscle fibers.

Rodent adipocytes secrete a 108-residue polypeptide

hormone called resistin. The hormone is named for its abil-

ity to block the action of insulin on adipocytes. In fact, re-

sistin production is decreased by TZDs, a phenomenon

that led to the discovery of resistin. Overproduction of

resistin was proposed to contribute to the development of

noninsulin-dependent diabetes. An interesting difference

between rodents and humans is that in humans, resistin is

produced by macrophages, a divergence whose evolution-

ary and functional implications are unclear.

d. Obesity Is a Contributing Factor

in Metabolic Syndrome

Metabolic syndrome is a disturbance in metabolism

characterized by insulin resistance, inflammation, and a

predisposition to several disorders including type 2 dia-

betes, hypertension, and atherosclerosis. These disorders

are accompanied by an increase in coronary heart disease.

Obesity, physical inactivity, and possibly genetic determi-

nants have been implicated in its occurrence, which affects

as many as 65 million people in the United States alone.

Exercise, calorie/weight reduction, adiponectin, leptin,

metformin, and TZDs have all been successfully used to

treat metabolic syndrome. Similarly, the PPAR␥ agonist

known as GW1516

GW1516

O COOH

1104 Chapter 27. Energy Metabolism: Integration and Organ Specialization

alleviates the symptoms of metabolic syndrome in obese

men, probably by stimulating fatty acid oxidation.

Evans has shown that GW1516 greatly increases exer-

cise endurance in mice, particularly when it is administered

together with the AMPK agonist 5-aminoimidazole-4-

carboxamide ribotide [AICAR];

which is also a product of histidine biosynthesis (Section

26-5Be) and an intermediate in purine ribonucleotide

biosynthesis (Section 28-1A)]. This treatment mimics the

effects of the expression of activated PPAR␥, which sug-

gests that the administration of GW1516 and AICAR can

confer some of the benefits of exercise without actually ex-

ercising. Indeed, the World Anti-Doping Agency has

placed these compounds on the list of performance-

enhancing drugs that athletes are forbidden from taking.

e. DNA Chip Technology Permits the Integrated

Study of Metabolic Regulation

Our ability to understand the integrated nature of me-

tabolism and its genetic regulation in health and disease

has taken a giant step forward with the advent of DNA

chips (microarrays; Section 7-6B). For example, Ronald

Kahn has used this technology to study the genetic basis of

the metabolic abnormalities underlying both obesity and

diabetes. To do so, he isolated the mRNA from the skeletal

muscle of normal, diabetic, and insulin-treated diabetic

mice and reverse-transcribed it to cDNA (Section 5-5Fa),

which was then hydridized to oligonucleotide microarrays

that represented 14,288 mouse genes. Thereby, 129 up-

regulated and 106 down-regulated genes were identified in

diabetic mice. Not surprisingly, the expression of the mRNAs

encoding enzymes of the fatty acid ␤-oxidation pathway

were increased, whereas those for GLUT4, glucokinase,

the E1 component of the pyruvate dehydrogenase multien-

zyme complex, and the subunits of all four mitochondrial

electron-transport chain complexes were coordinately de-

creased. Intriguingly, only about half of these changes in

gene expression could be reversed by insulin treatment.

Thus, the post-genomic era will almost certainly witness an

explosion in our knowledge of metabolic regulation that

should yield major health benefits. Nevertheless, our ability

to sensibly interpret this huge influx of information may

prove to be the greatest challenge.

CCH

2–

OH OH

5-Aminoimidazole-4-carboxamide ribotide (AICAR)

JWCL281_c27_1088-1106.qxd 4/21/10 9:42 AM Page 1104

References 1105

1 Major Pathways and Strategies of Energy Metabolism:

A Summary

The complex network of processes involved in

energy metabolism are distributed among different compart-

ments within cells and in different organs of the body. These

processes function to generate ATP “on demand,” to generate

and store glucose, triacylglycerols, and proteins in times of

plenty for use when needed, and to keep the concentration of

glucose in the blood at the proper level for use by organs such

as the brain, whose sole fuel source, under normal conditions,

is glucose.The major energy metabolism pathways include gly-

colysis, glycogen degradation and synthesis, gluconeogenesis,

the pentose phosphate pathway, and triacylglycerol and fatty

acid synthesis, which are cytosolically based, and fatty acid ox-

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which are confined to the mitochondrion. Amino acid degra-

dation occurs, in part, in both compartments. The mediated

membrane transport of metabolites therefore also plays an es-

sential metabolic role.

2 Organ Specialization The brain normally consumes

large amounts of glucose. Muscle, under intense ATP demand

such as in sprinting, degrades glucose and glycogen anaerobi-

cally, thereby producing lactate, which is exported via the

blood to the liver for reconversion to glucose through gluco-

neogenesis. During moderate activity, muscle generates ATP

by oxidizing glucose from glycogen, fatty acids, and ketone

bodies completely to CO

and H

O via the citric acid cycle and

oxidative phosphorylation. Adipose tissue stores triacylglyc-

erols and releases fatty acids into the bloodstream in response

to the organism’s metabolic needs. These metabolic needs are

communicated to adipose tissue by means of the hormones in-

sulin, which indicates a fed state in which storage is appropri-

ate, and glucagon, epinephrine, and norepinephrine,which sig-

nal a need for fatty acid release to provide fuel for other

tissues. The liver, the body’s central metabolic clearinghouse,

maintains blood glucose concentrations by storing glucose as

glycogen in times of plenty and releasing glucose in times of

need both by glycogen breakdown and by gluconeogenesis. It

also converts fatty acids to ketone bodies for use by peripheral

tissues. During a fast, it breaks down amino acids resulting

from protein degradation to metabolic intermediates that can

be used to generate glucose. The kidney filters out urea from

the blood, recovers important metabolites, and maintains pH

balance. To do so, glutamine is broken down to produce NH

⫹

for H

⫹

excretion.The resulting ␣-ketoglutarate product is con-

verted to CO

to resupply HCO

⫺

to the blood to maintain

its buffering capacity. During starvation, the kidney uses the

␣-ketoglutarate from glutamine breakdown for gluconeo-

genesis.

3 Metabolic Homeostasis: Regulation of Appetite,

Energy Expenditure, and Body Weight

AMP-dependent

protein kinase (AMPK), the cell’s fuel gauge, senses the cell’s

need for ATP and activates metabolic breakdown pathways

while inhibiting biosynthetic pathways. Adiponectin, an

adipocyte hormone that increases insulin sensitivity, acts by

activating AMPK. Appetite is suppressed by the actions of

leptin, a hormone produced by adipose tissue, insulin, pro-

duced by the ␤ cells of the pancreas, and PYY

3–36

, produced by

the gastrointestinal tract, which act in the hypothalamus to in-

hibit the secretion of neuropeptide Y (NPY) and stimulate the

secretion of ␣-MSH and CART. This decreases the appetite

and hence food intake. Ghrelin, a hormone secreted by the

empty stomach, opposes the actions of leptin, insulin, and

PYY

3–36

, stimulating appetite and food intake. Leptin also acts

in peripheral tissues to stimulate energy expenditure by fatty

acid oxidation and thermogenesis.

4 Metabolic Adaptation During prolonged starvation,

the brain slowly adapts from the use of glucose as its sole fuel

source to the use of ketone bodies, thereby shifting the meta-

bolic burden from protein breakdown to fat breakdown. Dia-

betes mellitus is a disease in which insulin either is not se-

creted or does not efficiently stimulate its target tissues,

leading to high concentrations of glucose in the blood and

urine. Cells “starve” in the midst of plenty since they cannot

absorb blood glucose and their hormonal signals remain those

of starvation.Abnormally high production of ketone bodies is

one of the most dangerous effects of uncontrolled diabetes.

Metabolic syndrome is caused by obesity, physical inactivity,

and possibly genetic determinants. It symptoms can be re-

lieved by substances that activate AMPK.

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1. Describe the metabolic effects of liver failure.

2. What is the basis of the hypothesis that athletes’ muscles

are more heavily buffered than those of normal individuals?

3. Experienced runners know that it is poor practice to ingest

large amounts of glucose prior to running a long-distance race

such as a marathon. What is the metabolic basis of this apparent

paradox?

4. Explain why urea output is vastly decreased during

starvation.

5. Explain why people survive longer by total fasting than on

a diet consisting only of carbohydrates.

6. Explain why the breath of an untreated diabetic smells of

acetone.

7. Among the many eat-all-you-want-and-lose-weight diets

that have been popular for a time is one that eliminates all carbo-

hydrates but permits the consumption of all the protein and fat

desired.Would such a diet be effective? (Hint: Individuals on such

a diet often complain that they have bad breath.)

8. Pancreatic ␤ cells express a receptor for fatty acids. Fatty

acid binding to this protein appears to stimulate insulin secretion.

(a) Does this phenomenon make metabolic sense? (b) Fatty acids

appear to stimulate insulin secretion to a much greater extent

when glucose is also present.Why is this significant?

9. High concentrations of free fatty acids in the blood are

known to cause insulin resistance in muscle, but only after

5 hours. This suggests that a metabolite of these fatty acids may

be responsible for this phenomenon. It is also known that an iso-

form of protein kinase C is activated during the process and that

high concentrations of free fatty acids result in intramuscular

accumulation of triacylglycerols. With this information, review

the mechanism of activation of PKC and the pathway of triacyl-

glycerol biosynthesis and suggest a metabolite that may be

responsible for PKC activation.

10. Discuss, in molecular terms, how physical inactivity might

lead to insulin resistance.

PROBLEMS

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Glutamine amide

Formate

spartate

amine

HCO

–

Glycine

Formate

1107

CHAPTER 28

Nucleotide

Metabolism

1 Synthesis of Purine Ribonucleotides

A. Synthesis of Inosine Monophosphate

B. Synthesis of Adenine and Guanine Ribonucleotides

C. Regulation of Purine Nucleotide Biosynthesis

D. Salvage of Purines

2 Synthesis of Pyrimidine Ribonucleotides

A. Synthesis of UMP

B. Synthesis of UTP and CTP

C. Regulation of Pyrimidine Nucleotide Biosynthesis

3 Formation of Deoxyribonucleotides

A. Production of Deoxyribose Residues

B. Origin of Thymine

4 Nucleotide Degradation

A. Catabolism of Purines

B. Fate of Uric Acid

C. Catabolism of Pyrimidines

5 Biosynthesis of Nucleotide Coenzymes

A. Nicotinamide Coenzymes

B. Flavin Coenzymes

C. Coenzyme A

Nucleotides, as we have seen, are biologically ubiquitous

substances that participate in nearly all biochemical

processes: They are the monomeric units of DNA and

RNA; the hydrolysis of ATP and GTP drives many free

energy–requiring processes; the levels of ATP, ADP, and

AMP regulate numerous metabolic pathways; cAMP and

cGMP mediate hormonal signals; and NAD



, NADP



FMN, FAD, and coenzyme A are essential coenzymes in a

great variety of enzymatic reactions. The importance of

nucleotides in cellular metabolism is indicated by the ob-

servation that nearly all cells can synthesize them both

de novo (anew) and from the degradation products of nu-

cleic acids. In this chapter, we consider the nature of these

biosynthetic pathways. In doing so, we shall examine how

they are regulated and the consequences of their blockade,

both by genetic defects and through the administration of

chemotherapeutic agents. We then discuss how nucleotides

are degraded. Finally, we outline the biosynthesis of the

nucleotide coenzymes.

1 SYNTHESIS OF PURINE

RIBONUCLEOTIDES

In this section we commence our considerations of how

nucleic acids and their components are synthesized by

describing the synthesis of purine ribonucleotides. In 1948,

John Buchanan obtained the first clues as to how this

process occurs de novo by feeding a variety of isotopically

labeled compounds to pigeons and chemically determining

the positions of the labeled atoms in their excreted uric

acid (a purine).

He used birds in these experiments because they ex-

crete waste nitrogen almost entirely as uric acid, a water-

insoluble and therefore easily isolated substance. The results

of his studies, which are summarized in Fig. 28-1, demon-

strated that N1 of purines arises from the amine group of

aspartate; C2 and C8 originate from formate; N3 and N9

are contributed by the amide group of glutamine; C4, C5,

and N7 are derived from glycine (strongly suggesting that

this molecule is wholly incorporated into the purine ring);

and C6 comes from HCO



(CO

Uric acid

Figure 28-1 The biosynthetic origins of purine ring atoms.

Note that C4, C5, and N7 come from a single glycine molecule

but each of the other atoms is derived from an independent

precursor.

JWCL281_c28_1107-1142.qxd 4/22/10 9:16 AM Page 1107

The actual pathway by which these precursors are incor-

porated into the purine ring, the subject of Section 28-1A,

was elucidated in subsequent investigations performed

largely by Buchanan and by G. Robert Greenberg. These

investigations showed that the initially synthesized purine

derivative is inosine monophosphate (IMP),

the nucleotide of the base hypoxanthine. AMP and GMP

are subsequently synthesized from this intermediate via

separate pathways (Section 28-1B).Thus, contrary to naive

expectation, purines are initially formed as ribonucleotides

rather than as free bases. Additional studies have demon-

strated that such widely divergent organisms as E. coli,

yeast, pigeons, and humans have virtually identical path-

ways for the biosynthesis of purine nucleotides, thereby

further demonstrating the biochemical unity of life.

A. Synthesis of Inosine Monophosphate

IMP is synthesized in a pathway comprising 11 reactions

(Fig. 28-2):

1. Activation of ribose-5-phosphate. The starting mate-

rial for purine biosynthesis is -

D-ribose-5-phosphate

(R5P), a product of the pentose phosphate pathway (Sec-

tion 23-4). In the first step of de novo purine biosynthesis,

ribose phosphate pyrophosphokinase (also known as

phosphoribosylpyrophosphate synthetase) activates R5P

by reacting it with ATP to form 5-phosphoribosyl--

pyrophosphate (PRPP). This reaction, which occurs via the

nucleophilic attack of the R5P’s C1¬H group on the P



ATP, is unusual in that a pyrophosphoryl group is directly

transferred from ATP to C1 of R5P and that the product

has the  anomeric configuration. PRPP is also a precursor

in the biosynthesis of pyrimidines (Section 28-2A) and the

amino acids tryptophan and histidine (Section 26-5Bd,e).

Thus, as is expected for an enzyme at such an important

biosynthetic crossroads, the activity of ribose phosphate py-

rophosphokinase varies with the concentrations of numer-

ous metabolites, including PP

and 2,3-bisphosphoglycerate,

which are activators, and ADP and GDP, which are mixed

inhibitors (Section 14-3C). The regulation of purine nu-

cleotide biosynthesis is further discussed in Section 28-1C.

2. Acquisition of purine atom N9. Amidophosphoribo-

syltransferase (alternatively, glutamine PRPP aminotrans-

ferse or PurF; the latter being named for the E. coli gene

encoding it, purF) catalyzes the displacement of PRPP’s

Inosine monophosphate (IMP)

Hypoxanthine

base

OHOH

–

pyrophosphate group by glutamine’s amide nitrogen to

yield -5-phosphoribosylamine (PRA). This is the first re-

action in the pathway that is unique to de novo purine

biosynthesis (and hence some sources refer to it as the first

reaction of the pathway, which is then said to consist of 10

reactions).This process occurs in two consecutive reactions

that take place on separate active sites on the enzyme:

Step 1 is catalyzed by a member of the N-terminal nucle-

ophile (Ntn) amidotransferase family (Section 26-5Aa).

Step 2 occurs with inversion of configuration about ribose

C1 and hence establishes the anomeric form of the future

nucleotide. The NH

passes between the two active sites

through a 20-Å-long tunnel that is lined with conserved

 PRPP S PRA  PP

Glutamine  H

O S glutamic acid  NH

1108 Chapter 28. Nucleotide Metabolism

Figure 28-2 (Opposite) The metabolic pathway for the de

novo biosynthesis of IMP. The purine residue is built up on a

ribose ring in 11 enzymatically catalyzed reactions.The X-ray

structures for all enzymes are shown to the outside of the

corresponding reaction arrow. The peptide chains of monomeric

enzymes are colored in rainbow order from their N-termini

(blue) to their C-termini (red).The oligomeric enzymes, all of

which consist of identical polypeptide chains, are viewed along a

rotation axis with their various chains differently colored. Bound

ligands are shown in space-filling form. Enzyme 1, determined by

Sine Larsen, University of Copenhagen, Denmark, is a D

hexamer from B. subtilis that binds ,-methylene-ADP at its

catalytic (red) and allosteric (blue) sites; PDBid 1DKU. Enzyme

2, determined by Janet Smith, Purdue University, is a D

tetramer

from B. subtilis that binds GMP (blue), ADP (red), and a

[4Fe–4S] cluster (orange, which appears to have a regulatory

rather than a redox function); PDBid 1AO0. Enzymes 3 and 6,

both from E. coli, were determined by JoAnne Stubbe, MIT, and

Steven Ealick, Cornell University; PDBids 1GSO and 1CLI.

Enzyme 4, from E. coli, determined by Robert Almassy, Agouron

Pharmaceuticals, San Diego, California, binds GAR (cyan) and

5-deazatetrahydrofolate (red); PDBid 1CDE. Enzyme 5, from

Thermatoga maritima, was determined by Ian Wilson, Scripps

Research Institute, La Jolla, California; PDBid 1VK3. Reaction

7, in E. coli, is catalyzed by two sequentially acting enzymes,

Class I PurE (above) and PurK (below). Class I PurE,

determined by JoAnne Stubbe, MIT, and Steven Ealick, Cornell

University, is a D

octamer that binds AIR (red); PDBid 1D7A.

PurK, determined by JoAnne Stubbe, MIT, and Hazel Holden,

University of Wisconsin, is a C

dimer that binds ADP (red);

PDBid 1B6S. Enzyme 8, from yeast, was determined by Victor

Lamzin,Academy of Sciences, Moscow, Russia, and Keith

Wilson, EMBL, Hamburg, Germany; PDBid 1A48. Enzyme 9,

from Thermatoga maritima, determined by Todd Yeates, UCLA,

is a D

tetramer; PDBid 1C3U. Reactions 10 and 11 in chicken

are catalyzed by a bifunctional enzyme that was determined by

Stephen Benkovic, Pennsylvania State University, and Ian

Wilson, The Scripps Research Institute, La Jolla, California. It

forms a C

dimer shown with its AICAR transformylase function

above and its IMP cyclohydrolase function, which binds GMP

(purple), below; PDBid 1G8M.

See the Animated Figures

JWCL281_c28_1107-1142.qxd 4/22/10 9:16 AM Page 1108

H OH

OH OH

–2

α-D-Ribose-5-phosphate (R5P)

H O

OH OH

–2

5-Phosphoribosyl-

α-pyrophosphate (PRPP)

–

PPO

–

OH OH

-5-Phosphoribosylamine (PRA)

OH OH

Glycinamide ribotide (GAR)

Ribose-5-phosphate

Formylglycinamide ribotide (FGAR)

Ribose-5-phosphate

Formylglycinamidine ribotide (FGAM)

Ribose-5-phosphate

5-Aminoimidazole ribotide (AIR)

Ribose-5-phosphate

Carboxyaminoimidazole ribotide (CAIR)

–

OOC

ADP

+ P

ATP

AMP

ribose phosphate

pyrophosphokinase

Glutamine

Glutamate

amidophosphoribosyl

transferase

Glycine + ATP

ADP

+ P

GAR synthetase

FGAM synthetase

-Formyl-THF

THF

GAR transformylase

ATP + Glutamine + H

ADP + Glutamate + P

AIR synthetase

ATP

ADP

+ P

AIR carboxylase

ATP + HCO

–

Ribose-5-phosphate

5-Aminoimidazole-4-(N-succinylocarboxamide)

ribotide (SAICAR)

COO

–

COO

–

Ribose-5-phosphate

5-Aminoimidazole-4-carboxamide ribotide (AICAR)

Ribose-5-phosphate

5-Formaminoimidazole-4-carboxamide

ribotide (FAICAR)

CHO

SAICAR synthetase

ADP

+ P

Aspartate

+ ATP

adenylosuccinate lyase

Fumarate

THF

AICAR

transformylase

-Formyl-THF

IMP

cyclohydrolase

OH OH

Inosine monophosphate (IMP)

–2

Section 28-1. Synthesis of Purine Ribonucleotides 1109

JWCL281_c28_1107-1142.qxd 4/22/10 9:16 AM Page 1109

1110 Chapter 28. Nucleotide Metabolism

attacked by an amine nitrogen atom to yield a tetrahedral

adduct that, in turn, expels P

to form product.

6. Formation of the purine imidazole ring. The purine

imidazole ring is closed in an ATP-requiring intramolecu-

lar condensation that yields 5-aminoimidazole ribotide

(AIR) in a reaction catalyzed by AIR synthetase (PurM).

nonpolar residues that lack hydrogen bonding groups and

hence do not impede the diffusion of the NH

[we have

seen that NH

generated by glutamine hydrolysis is simi-

larly channeled to the active site that uses it in carbamoyl

phosphate synthetase (Section 26-2Aa) and glutamate syn-

thetase (Section 26-5Aa)]. These reactions, which are

driven to completion by the subsequent hydrolysis of the

released PP

, constitute the pathway’s flux-generating step.

Not surprisingly, therefore, amidophosphoribosyltrans-

ferase is subject to feedback inhibition by purine nu-

cleotides (Section 28-1C).

3. Acquisition of purine atoms C4, C5, and N7.

Glycine’s carboxyl group forms an amide with the amino

group of PRA, yielding glycinamide ribotide (GAR) in a

reaction, catalyzed by GAR synthetase (PurD), that oc-

curs via the intermediate phosphorylation of glycine’s car-

boxyl group. The reaction, which is reversible despite its

concomitant hydrolysis of ATP to ADP  P

, is the only

step of the purine biosynthesis pathway in which more

than one purine ring atom is acquired. The observation

that PRA is chemically unstable (it is hydrolyzed to R5P

and NH

with a half-life of 5 s at 37°C) suggests that GAR

synthetase and amidophosphoribosyltransferase associate

in a way that channels PRA between them. Indeed, a ster-

ically and electrostatically plausible model of such a com-

plex has been built based on the X-ray structures of these

two enzymes.

4. Acquisition of purine atom C8. GAR’s primary -

amino group is formylated by GAR transformylase (PurN)

to yield formylglycinamide ribotide (FGAR). The formyl

donor in this reaction is N

-formyltetrahydrofolate

-formyl-THF), a cofactor that transfers C

units from

such donors as serine, glycine, and formate to various ac-

ceptors in biosynthetic reactions (Section 26-4D). Struc-

tural and enzymological studies indicate that the reaction

proceeds via the nucleophilic attack of the GAR primary

amine group on the formyl carbon of N

-formyl-THF to

yield a tetrahedral intermediate.

5. Acquisition of purine atom N3. The amide amino

group of a second glutamine is transferred to the growing

purine ring to form formylglycinamidine ribotide (FGAM).

This reaction, which is catalyzed by FGAM synthetase

(PurL), is driven by the coupled hydrolysis of ATP to

ADP  P

. It is thought to proceed by the mechanism dia-

grammed in Fig. 28-3. Here the oxygen of the FGAR

isoamide form reacts with ATP to yield a phosphoryl ester

intermediate. This intermediate then reacts with NH

(the

glutamine amide nitrogen as labilized through the tran-

sient formation of an enzyme thioester) to form a tetra-

hedral adduct. The adduct then eliminates P

to yield the

imine product, FGAM. Such reactions, in which a carbox-

amide oxygen is replaced by an imino group, are common

in the biosynthesis of nucleotides. For example, Reaction 6

of this pathway and the reactions converting IMP to AMP

(Section 28-1B) and UTP to CTP (Section 28-2B) follow

similar mechanisms, that is, conversion of a carboxamide

oxygen to a phosphoryl ester that is nucleophilically

–

CSE

SHE

–

2–

ADP

ATP

FGAR (isoamide form)

FGAR (amide form)

Glutamine

Glutamate

FGAM



Figure 28-3 The proposed mechanism of formylglycinamide

ribotide (FGAM) synthetase. The glutaminase domain of the

enzyme contains an active site Cys residue that catalyzes the

release of NH

with the transient formation of an enzyme

thioester (not shown) whose hydrolysis produces glutamate. The

isoamide form of FGAR is phosphorylated by ATP and then

reacts with “NH

” to form a tetrahedral intermediate whose

collapse yields FGAM  P

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