Professional Documents
Culture Documents
of Physical Exercise
Livio Luzi
Editor
Cellular Physiology
and Metabolism
of Physical Exercise
123
Editor
Livio Luzi
Department of Sport Sciences, Nutrition and Health
University of Milan
Milan, Italy
The contents of the book are partially based on Biologia cellulare nell’esercizio fisico. Livio Luzi
© Springer-Verlag Italia 2010
DOI 10.1007/978-88-470-2418-2
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Cellular Physiology and Metabolism of Physical Exercise deals with several areas
of science, including evolution. Physical inactivity is one of the leading causes of
death in industrialized countries. Accordingly, intensive research has been devot-
ed to studies of the regulation of muscle physiology and contraction. In this vol-
ume, these topics are particularly up to date and thoroughly debated. The aim of
this book is to furnish both a basic and an advanced scientific portrait of the cel-
lular physiology of skeletal muscle cells. Basic information includes an overview
of muscle cell morphology, biochemistry, molecular biology, and physiology, with
special emphasis on the cell membrane, energy metabolism, and cell contraction.
Particularly innovative are the chapters dealing with methodologies to study, both
invasively (muscle biopsies) and non-invasively (NMR-spectroscopy, mathemati-
cal modeling), intracellular metabolism and physiology. A specific chapter is ded-
icated to a new frontier of research in the field of sport sciences, namely, the pos-
sibility of correlating specific DNA polymorphisms and athletic performance. The
micro environment of a contractile cell is of pivotal relevance to nutritional status.
For this reason, three chapters are dedicated to “cellular feeding” and related is-
sues. In many countries, the practice of sport is encouraged to prevent and treat
most chronic degenerative diseases. Nonetheless, excess physical activity may al-
so cause health problems. The common mechanism underlining both (positive
and negative) effects is inflammation, which is also treated in a chapter.
Inflammation, along with immune tolerance, is also a relevant issue in the host vs.
graft reaction, the basis of transplant rejection. Whether patients who have under-
gone organ transplantation benefit from exercise is a matter of debate that is treat-
ed herein. Hyperactivity is also profoundly related to disorders of alimentation,
such as anorexia, whose metabolic features are addressed in this book as well. The
non-human primate model is often used in biomedical research to test new drugs.
Modern concepts in suggesting an exercise program consider physical exercise as
a drug, introducing the necessity of studying patterns of physical exercise in an an-
imal model closest to the genus Homo (primates). To do so requires fundamental
knowledge of the basics of exercise physiology in primates. The last chapter of the
book is centered on the fundamentals of exercise physiology in primates, which
necessitated a discussion of how (and, possibly, why) the genus Homo developed
VII
VIII Preface
from Australopithecines some 1.5 million years ago. In conclusion, I believe this
work provides a complete manual for scientists interested in understanding the ba-
sic physiology and clinical relevance of physical exercise. The book’s realization
was made possible by the proactive and factual interaction of the authors (most of
them are or at some time were co-workers of mine), to whom I convey my most
sincere appreciation and acknowledgment.
IX
Contents
XI
XII Contents
11 The HPA Axis and the Regulation of Energy Balance .................... 109
Francesca Frigerio
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
11.2 Anatomy of the HPA Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
11.3 Physiology of the HPA Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
11.4 Molecular Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
11.5 HPA Axis and Energy Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
11.6 The HPA Axis and Non-homeostatic Energy Intake Regulation . 115
11.7 The HPA Axis and Energy Expenditure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
11.8 The Role of Glucocorticoids on Peripheral Organs . . . . . . . . . . . . . . . . . . 116
11.9 HPA Axis and Physical Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
11.10 Glucocorticoids and Doping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
XIV Contents
XV
XVI Contributors
Born to Run was the third album produced by the American singer-songwriter Bruce
Springsteen. It was released by Columbia Records on August 25, 1975. The same ti-
tle was used in the following decades for: at least one novel, an episode in the TV se-
ries Terminator, a book on a Mexican tribe of extreme runners, and it even appeared
on the cover page of Nature, in November 2004. The common denominator of all the
uses of Born to Run is the recognition of the need of humans to run in order to sur-
vive.
The present atmosphere of the Earth is composed of 21% oxygen. The remaining
gases are nitrogen (78%), argon (0.9%), carbon dioxide and other trace elements
(0.012%). About 5 billion years ago, at the birth of our planet, the atmosphere con-
tained virtually no oxygen. The advent of the first forms of life on Earth (prokary-
otes, primordial unicellular bacteria) was crucial for the change in composition of the
gas content of the atmosphere. Primordial bacteria were able to carry out photosyn-
thesis, utilizing hydrogen, obtained from water, and CO2 to release oxygen.
Therefore the development of life on Earth was determined by the appearance of or-
ganisms capable of surviving in the absence of oxygen, with their survival exclusive-
ly founded on anaerobic metabolism. The increasing amount of oxygen released by
prokaryotes into the primordial atmosphere favored the development of oxidative re-
actions to produce energy for life, a much more efficient method than anaerobic me-
L. Luzi ()
Department of Sport Sciences, Nutrition and Health
University of Milan
Milan, Italy
e-mail: livio.luzi@unimi.it
tabolism. Some 1500 million years ago, the first eukaryotes capable of producing en-
ergy with oxidative metabolism appeared on Earth. Millions of years were then
necessary for the development of multicellular eukaryotes. It is relevant for evolution
in general and for human evolution in particular that in parallel with the appearance
of more complex multicellular organisms much of the Earth’s ecosystem was altered
by dramatic geologic events [1]. The volcanic eruptions, continent shifts, and mete-
oric collisions forced major evolutionary leaps, as only organisms capable of adapt-
ing to the new environment survived. One such adaptation is described by the en-
dosymbiotic theory. Endosymbiosis means “cohabiting within” and in this case
refers to the postulated collaboration/interaction between organisms with different
metabolic capabilities and dimensions, both of which gain an evolutionary advantage
by merging their living environments. As stated, not all organisms were able to tol-
erate an oxidant atmosphere (i.e., an atmosphere increasingly rich in oxygen pro-
duced by photosynthesis). According to endosymbiotic theory, primordial eukaryotes
were able to survive due to their incorporation of prokaryotes bearing much-needed
complementary skills. Peroxisomes and mitochondria are thought to be remnants of
prokaryotes that eventually became eukaryotic organelles, conferring upon their
hosts the cellular machinery needed for oxygen detoxification and energy production
in aerobiosis [2].
Roughly 1.5 million years ago, Homo erectus appeared on the Earth. Our present
genes are similar to those of Homo erectus, Homo habilis, and the first Homo sapi-
ens (200,000 to 100,000 years ago) [3]. Australopithecines were the ancestors of
Homo erectus and their evolution was driven by an important change in the ecosys-
tem: the replacement of woodlands by grasslands and savannas in central Africa [4].
The expansion of savannas caused a fundamental change in the way hominids for-
aged and, consequently, in the quality and caloric content of food as well as the
amount of physical activity required to gather food. In fact, the disappearance of
woodlands induced hominids to cover longer distances in savannas, prompting the
natural selection of individuals with longer lower limbs, the ability to run, better ther-
moregulatory capacity, and with a higher resting and total daily energy expenditure.
Evolutionarily, longer lower limbs and bipedalism facilitated foraging behavior in the
new ecosystem, determining a strong association between changes in body size (and
metabolism) and ranging/foraging patterns [5, 6]. Therefore, the earliest representa-
tive of the human genus, considered to be the African Homo erectus, was indeeed
“born to run,” that is, to cope with an environment strikingly different from the
woodlands where previous hominids had gathered food. Several musculoskeletal
adaptations are representative of the genus Homo, including a large cranial vault, a
prominent nose, a thin mandible, a chin, small teeth, a modified hip joint, and a light
skeleton. These anatomic changes allowed our ancestors to walk and run for long
distances and times, as their bodies were specialized for endurance and physical ac-
tivity [7]. Indeed, humans are specifically adapted to engage in prolonged strenuous
1 Human Evolution and Physical Exercise: The Concept of Being “Born to Run” 3
ing; (2) many more calorie-rich and refined foods are available, in virtually unlim-
ited supply; (3) food deprivation and starvation, except during religious fasts, are un-
known (in contrast to the winters and other periods of food scarcity faced by Homo
erectus). As a matter of fact, we are currently benefiting from a major ecosystem
change that started 10,000 years ago, with the agricultural revolution (when popula-
tions of hunters/gatherers settled down and began to raise grains and conserve food
for the winter), and reached its apex at the beginning of the 20th century, with the in-
dustrial revolution and the introduction of machines to help humans perform labor-
intensive and energy-demanding tasks. Therefore, due to the mismatch between our
genetic background (what we are predisposed for) and our new environment (what
we are actually doing), the incidence of diseases such as obesity, type 2 diabetes,
metabolic syndrome, hypertension, cardiovascular events, and some forms of cancer
has increased dramatically, especially in recent decades [9, 10].
The metabolic mechanism utilized by our body to store rather than to burn calo-
ries is insulin resistance. Insulin sensitivity (the opposite of insulin resistance) is de-
fined as the ability of insulin to metabolize a load of glucose (and other energy sub-
strates such as free fatty acids). An impairment of the body’s capacity to metabolize
a glucose load protects the individual from periods of food scarcity, starvation, or a
deficit in carbohydrate or fat intake. Obviously, if evolution selected insulin-resist-
ant humans based on their ability to survive periods of famine, the above-described
changes in the 20th century ecosystem have made modern humans susceptible to hy-
perglycemia, hyperlipidemia, and their pathological consequences, namely diabetes,
obesity, and atherosclerotic disease. In principle, more insulin-sensitive individuals
should be favored today, as they are able to dispose of regular, high-calorie loads in
less time whereas during life on the African savanna they would have been con-
demned to extinction [9]!
The maintenance of normal glycemia is obtained by the balance between insulin
secretion and insulin action, a relationship known as glucose tolerance. In normal in-
dividuals, there is a hyperbolic relationship between insulin secretion and insulin ac-
tion (Fig. 1.1); accordingly, normal glucose tolerance can be obtained over a wide
range of secretory capacity and insulin action. It is also well established that an im-
balance between insulin secretion and insulin action causes hyperglycemia. The se-
cretion of insulin must therefore be considered along with its action in order to de-
termine “the metabolic wellness” of an individual. It is a common belief that today’s
marathon runners are the closest modern humans come to Homo erectus in terms of
lifestyle and metabolism. Marathon runners maintain a normal glucose tolerance by
means of relatively efficient insulin action, tempered by relatively low levels of in-
sulin secretion. In this scenario, hunters/gatherers should have benefited from a very
high level of insulin action matched by a low secretory capacity of the hormone.
There is an apparent discrepancy between the predisposition of our genes to store en-
ergy (the “thrifty genotype” hypothesis [9] and the highly efficient insulin action of
marathon runners (and, probably, of Homo erectus). Thus, an organism predisposed
to saving and storing energy needs constant physical exercise to maintain normal in-
sulin action and proper substrate utilization. Accordingly, a healthy lifestyle is de-
fined by regular physical exercise along with appropriate dietary habits. In other
1 Human Evolution and Physical Exercise: The Concept of Being “Born to Run” 5
Fig 1.1 The relationship between insulin sensitivity and beta-cell secretion is well-described by a
hyperbolic function, such that the product of insulin sensitivity times beta-cell secretion tends to re-
main constant. Physical exercise is known to enhance insulin sensitivity. Since less insulin is re-
quired to metabolize glucose, a concomitant reduction in beta-cell secretion takes place. The over-
all effect is as follows: a subject undertaking physical training slides along the hyperbola achieving
a position characterized by elevated insulin sensitivity and low circulating insulin levels
words, the lack of a physical exercise program renders vain all dietary interventions
(this is basically the clinical “on the field” experience of most physicians).
It is worth noting that it is not only the total amount but also the pattern of insulin
secretion that determines glucose disposal and the effective clearance a glucose
load. First-phase insulin release has been shown to have a consistent role in inhibit-
ing endogenous glucose production following a meal. Early stages of diabetes and
obesity are characterized by a loss of first-phase insulin release and thus by post-
prandial hyperglycemia and a reduction of the thermogenic effect of food. The com-
bination of the two defects leads to diabetes and obesity, respectively (or a combina-
tion thereof). Similar to insulin action, the "blindness" of the β-cell to glucose is
overcome by amino acid administration via a high-protein diet, indicating that pro-
tein homeostasis is the metabolic domain best protected by evolution. In fact, on the
one hand, in most conditions (with the notable exception of obesity) insulin’s action
on protein metabolism is spared (despite a marked impairment of its action on car-
bohydrate and lipid metabolism). On the other hand, amino acids/high-protein diets
are able to restore a normal secretory pattern of insulin secretion, thus overcoming
β-cell blindness to glucose during the early stages of type 2 diabetes mellitus.
Based on these considerations there are two possibilities. One is that current evo-
lutionary pressure will select one or a few protective genes/features of the sedentary
Homo sapiens that will allow humans to evolve such that the insulin sensitivity of fu-
ture generations is much higher that that conferred by our present genes. In other
words, presumably, only individuals with a higher capacity to burn calories and dis-
6 L. Luzi
pose of nutrient loads (without needing to perform physical exercise) will be select-
ed for survival by evolution. In this case, we have no choice but to passively wait for
evolution to find a solution (as our ancestors did!).
The other possibility is to change our behavior such that it mimics our ancestors’
way of life in terms of patterns of physical activity and the diet of hunters/gatherers.
That lifestyle was characterized by three cornerstones. First, physical exercise was
performed several hours a day, with different modalities and intensities. In Homo
erectus, walking and running were frequent forms of physical exercise. The behav-
ior of contemporary species of primates has been studied to deduce the physical ex-
ercise patterns and total daily energy expenditure of our ancestors. Although this kind
of information is difficult to extrapolate, based on a total energy expenditure of
2,500–3,500 kcal per day, physical exercise, ranging from active to strenuous, was
likely performed for between 1 and 4 hours daily. Moreover, even during periods of
daily rest and over the year, the average energy expenditure was higher than the pres-
ent-day value, reflecting non-shivering thermogenesis secondary to cold-temperature
exposure. Second, the diet of hunters/gatherers contained a much lower (up to 30%
less) percentage of complex carbohydrates than is consumed today, a higher protein
content (both vegetable and animal protein), and a total fat content similar to today’s
level, with the notable prevalence of mono- and polyunsaturated fats over saturated
fats. Third, of particular relevance was the modality of caloric intake of Homo erec-
tus, characterized by periods of forced starvation (presumably ranging from 1 day to
longer periods). Therefore, periodic fasting was a constant for hunters/gatherers
whereas, unless voluntarily performed, periods of food deprivation are for the most
part completely unknown in modern Western societies. Interestingly, a metabolic
model of fasting is provided by the initial stage of mental anorexia. Patients with this
disease voluntarily reduce their caloric intake while engaging in physical exercise for
several hours a day. Consequently, body weight, total daily energy expenditure, and
blood concentrations of glucose, lipids, and amino acids (with respect to matched
controls) are reduced, resulting in a clinical picture that is the mirror image of type
2 diabetes and metabolic syndrome. This clinical model suggests that our genes pre-
dispose us with the ability to well resist long periods of reduced caloric intake. If we
succeed in changing our lifestyle accordingly, we will eradicate diabetes, obesity, hy-
pertension, metabolic syndrome, cardiovascular disease, and even some forms of
cancer.
References
1. Kasting JF, Siefert JL (2002) Life and the evolution of Earth’s atmosphere. Science 10:1066-
1068
2. Alberts B, Johnson A, Lewis J et al (2002) Molecular biology of the cell. New York, Garland
Science
3. Wood B, Collar M (1999) The human genus. Science 284:65-71
4. Cerling TE (1992) Development of grasslands and savannas in East Africa during the neogene.
Paleogeog Paleoclimatol Paleoecol 97:241-247
5. Leonard WR, Robertson ML (1997) Comparative primate energetics and hominid evolution.
1 Human Evolution and Physical Exercise: The Concept of Being “Born to Run” 7
2.1 Introduction
A. Maestroni ()
Complications of Diabetes Unit,
Division of Metabolic and Cardiovascular Sciences
Istituto Scientifico San Raffaele, Milan, Italy
e-mail: maestroni.anna@hsr.it
The muscles are covered externally by connective tissue referred to as the epimy-
sium, which surrounds the entire muscle, holding it together. Inside the epimysi-
um are fiber bundles, the fasciculi, wrapped in a sheath of connective tissue. The
connective tissue sheath surrounding each fasciculus is called the perimysium.
Finally, within the perimysium are the muscle fibers, which are the individual
muscle cells. The endomysium, another sheath of connective tissue, surrounds
each muscle fiber. The epimysium, perimysium, and endomysium are connective
structures that together form the tendon (Fig. 2.1).
Muscle fibers range in length from 1 mm to a maximum of 12 cm in the sarto-
rius muscle. Their diameter ranges from a minimum of 10 μm to a maximum of
100-105 μm (average: 10–50 μm). These cellular elements are derived from the fu-
sion of progenitor cells called myoblasts and thus form syncytia. Skeletal muscle
fibers are cylindrical in shape and contain many nuclei (even hundreds) located
near the sarcolemma (the cell membrane of muscle cells). However, the defining
characteristic of muscle fibers is the cross-striations seen on light microscopy.
A gelatin-like substance fills the spaces between the myofibrils. This is the sar-
coplasm and it comprises the cytoplasm of muscle fiber. The sarcoplasm differs
from true cytoplasm in that it contains a large quantity of stored glycogen as well
as the oxygen-binding compound myoglobin, which is quite similar to hemoglo-
bin (Fig. 2.2).
Another special structure of the muscle fiber is the sarcoplasmic reticulum,
which is the smooth endoplasmic reticulum. Its distinct shape can be recognized
in every sarcomere. The sarcoplasmic reticulum is structured as follows: at the
junction between the A and I bands are the terminal cisternae, along which
tion microscopy. Finally, inside the H-zone is a thin M-line (from the German mit-
tel, middle of the sarcomere) (Fig. 2.4).
Each myosin filament typically comprises about 200 myosin molecules, lined
up end to end and side by side. The myosin molecule is composed of two identi-
cal heavy (larger) chains and two pairs of light (smaller) chains. The heavy protein
chains intertwine to form a tail end, a rigid spiral, and two globular heads. One of
the two light protein chains is associated with one of the heavy-chain heads. The
globular heads of the myosin cross-bridges mediate the interaction with thin actin
filaments during muscle contraction. The myosin filaments are connected from the
M-line to the Z-disc by tinin.
Actin is a globular protein (G-actin) that combines to form long, thin chains (F-
actin). Two F-actin strands form a helical twist, much like two strands of pearls
twisted together. Each actin molecule has an active binding site that serves as the
point of contact with the myosin filament.
In addition to actin, the thin filaments of the sarcomere are composed of
tropomyosin and troponin. Tropomyosin is a tubular protein that twists the actin
strands while troponin is a more complex protein made up three subunits (TnC,
TnI, and TnT) and attached at regular intervals to both the actin strands and to
tropomyosin. When calcium is bound to specific sites on TnC, tropomyosin rolls
out of the way of the actin filament’s active sites, thus allowing myosin to attach
to the thin filament and to subsequently produce force and/or movement. In the ab-
sence of calcium, tropomyosin interferes with the action of myosin such that the
muscles remain relaxed.
The individual subunits of troponin serve different functions in muscle contrac-
tion: troponin C binds to calcium ions to produce a conformational change in TnI;
troponin T binds to tropomyosin to form a troponin-tropomyosin complex; and tro-
ponin I binds to actin in thin myofilaments to hold the troponin-tropomyosin com-
plex in place.
Tropomyosin and troponin require the presence of calcium ions to maintain re-
laxation or to initiate contraction of the myofibril, which we examine later in this
chapter. In addition, actin and myosin interactions are regulated by another protein,
nebulin, which serves as an anchoring protein for actin (Fig. 2.5).
The events that trigger a muscle fiber are complex. The process is initiated by a mo-
tor nerve impulse from the brain or spinal cord. An action potential originating in
the CNS reaches an alpha motor neuron, which then transmits the action potential
down its own axon. The action potential is propagated by the activation of sodium-
dependent channels along the axon toward the synaptic cleft. An influx of Ca2+
causes vesicles containing the neurotransmitter acetylcholine to fuse with the plas-
ma membrane, releasing acetylcholine into the extracellular space between the mo-
tor neuron terminal and the motor end plate of the skeletal muscle fiber.
14 A. Maestroni
Acetylcholine diffuses across the synapse and binds to and activates acetylcholine
receptors on the motor end plate of the muscle cell. Activation of the acetylcholine
receptor opens its intrinsic sodium/potassium channel, causing sodium to rush in
and potassium to trickle out. Since the channel is more permeable to sodium, the
membranes of the muscle fibers becomes more positively charged, triggering an ac-
tion potential. The action potential spreads through the muscle fiber’s network of T-
tubules, depolarizing the inner portion of the muscle fiber. Depolarization acti-
vates voltage-dependent calcium channels in the T-tubule membrane, which are in
close proximity to calcium-release channels in the adjacent sarcoplasmic reticulum.
Activated voltage-gated calcium channels physically interact with and thereby
activate calcium-release channels, causing the sarcoplasmic reticulum to release cal-
cium. The released calcium binds to the troponin C present on the actin thin fila-
ments of the myofibrils. Troponin then allosterically modulates tropomyosin.
Normally, tropomyosin sterically obstructs myosin-binding sites on the thin fila-
ment; however, once calcium binds to troponin C and causes an allosteric change in
the protein, troponin T allows tropomyosin to move, unblocking the binding sites.
Myosin has ADP and inorganic phosphate bound to its nucleotide-binding pock-
et and is in an active state. In this form, it binds to the newly uncovered binding sites
on the thin filament in a process very tightly coupled to inorganic phosphate release,
in which actin serves as a cofactor. Myosin is now strongly bound to actin, with the
release of ADP and inorganic phosphate tightly coupled to the power stroke. During
the latter, the Z-bands are pulled towards each other, thus shortening the sarcomere
and the I-band.
Conversely, ATP binding to myosin allows it to release actin and to remain in
a weak binding state (a lack of ATP makes this step impossible, resulting in the rig-
or state characteristic of rigor mortis). Myosin then hydrolyzes the ATP and uses
the energy to move into the “cocked back” conformation. In vivo studies have con-
firmed model-based predictions regarding movement of the myosin head of skele-
tal muscle: during each power stroke the myosin head moves 10–12 nm; however,
there is also in vitro evidence of variations (smaller and larger) in this range of
movement that are specific to the myosin isoform (Fig. 2.6).
Sliding of the filaments occurs as long as ATP is available and calcium is pres-
ent. During the above-described steps, calcium is actively pumped back into the
sarcoplasmic reticulum, which creates a deficiency in the environment around the
myofibrils. As a result, calcium ions are removed from troponin such that the
tropomyosin reverts to its previous state, forming a complex with troponin and
again blocking myosin-binding sites. Myosin is thus unable to bind to the thin fil-
aments, and contraction ceases.
Suggested Reading
Macintosh BR, Gardiner PF, McComas AJ (2006) Skeletal muscle: form and function. Human
Kinetics, Leeds
Lieber RL (2002) Skeletal muscle structure, function, and plasticity. Lippincott Williams &
Wilkins, Baltimore
The Cell Membrane
of the Contractile Unit 3
Gianpaolo Zerbini
The cell membrane was initially considered only as a barrier delimiting the cytoplasm
from the extracellular environment but further research revealed that the cell membrane
has a number of functions that are essential to the cell [1]. Structurally, the cell mem-
brane is formed by a lipid bilayer (Fig. 3.1), with each of the two layers composed of
molecules called phospholipids. The lipid component is, by definition, water-repellent,
while the phosphate component is hydrophilic. The membrane is formed as the phos-
phate moves toward the outer surface of the cell, attracted by the aqueous environment,
which the inwards-oriented lipids seek to escape. An additional and very important
component of this double lipid structure consists of the membrane proteins.
The organization of the cell membrane is therefore referred to as a “fluid mosaic,”
in which the hydrophobic and hydrophilic components interact with each other in
such a way that membrane fragments are able to detach from the main structure
without creating permanent holes. The membrane surrounding internal organelles,
such as the endoplasmic reticulum, the Golgi apparatus, lysosomes, and vacuoles, in-
teracts with these structures and is crucial to their function [2].
3.2.1 Lipids
Lipids are retained on the internal aspect of the cell membrane because of their wa-
ter repellency. Although they may also bond with oxygen molecules, lipids mainly
G. Zerbini ()
Complications of Diabetes Unit
Division of Metabolic and Cardiovascular Sciences
Istituto Scientifico San Raffaele, Milan, Italy
e-mail: g.zerbini@hsr.it
consist of hydrocarbons. The three main classes of lipids that make up the cell
membrane are fats, phospholipids, and steroids.
Fats (triacylglycerols) are not true polymers but are nonetheless composed of
large molecules formed from a number of smaller molecules; these are held togeth-
er due to their water-repellent properties. Fat consists essentially of two molecules:
glycerol and fatty acids. Glycerol belongs to the class of alcohols, while fatty acids
are composed of 16–18 carbon atoms. One end of the fatty acid is the carboxylic
group, which is joined to a long hydrocarbon tail. The C-H bonds of the fatty-acid
tail account for the hydrophobicity of fat.
Fat is formed by the binding of three fatty acids to a glycerol molecule, giving
rise to a bond between the hydroxyl group and the carboxylic group. The fat mol-
ecule thus generated is called triacylglycerol or triglyceride. The fatty acids com-
prising the fat molecule can be identical or different. The length, number, and loca-
tion of the double bonds present in a fatty acid define its physical and chemical
characteristics. Fat may be saturated or unsaturated depending on the structure of
the fatty acids that make up the hydrocarbon tail. The fluidity of the membrane
tends to change according to the prevalence of saturated or unsaturated fats within
the cell membrane [3].
Phospholipids are the major component of the cell membrane. They are structural-
ly similar to fat but contain only two fatty acids instead of three. The third hydroxyl
group of glycerol is in this case attached to a negatively charged phosphate group,
which is usually linked to small hydrophilic molecules. Different types of phospho-
lipids can be generated based on the nature of the molecule bound to the phosphate.
Phospholipids contain both a hydrophobic and a hydrophilic region and are thus de-
fined as amphipathic molecules. The hydrophobic component is the hydrocarbon tail,
while the hydrophilic head is formed by the phosphate group and its attachments. The
morphology of phospholipids is such that once they are in contact with water they or-
ganize themselves in clusters in which the hydrophilic side is exposed toward the
aqueous extracellular milieu while the hydrophobic part is aligned inwards. This struc-
ture is called a micelle and it is the main structural component of the phospholipid bi-
layer, comprising the semi-permeable structure characteristic of any cell membrane.
Steroids consist of cholesterol and include several hormones. The carbon skele-
ton of steroids is arranged in four concentric rings. Cholesterol in particular is a key
element of animal cell membranes and is essential to their stability. All steroids are
formed from a cholesterol precursor. In the cell membrane, cholesterol molecules
are incorporated into the phospholipid bilayer [4].
3.2.2 Proteins
Proteins alone account for > 50% of a single cell’s dry weight. Although the cell
membrane contains tens of thousands of proteins, each protein can be considered as
a polymer organized from the different sequential arrangements of 20 amino acids.
Membrane proteins may be integral or peripheral. Integral proteins are generally
transmembrane proteins in which the hydrophobic part traverses the cell mem-
brane between its extracellular and intracellular aspects, while the hydrophilic ends
3 The Cell Membrane of the Contractile Unit 19
of the protein emerge on either side. Within the membrane, integral proteins are
larger than lipids; some of them diffuse very slowly in this environment while oth-
ers are anchored to the cytoskeleton. Peripheral proteins, as their name implies, are
not located inside the cell membrane but are instead weakly anchored to its outer
surface, often in contact with the external portions of integral membrane proteins.
3.2.3 Carbohydrates
Membranes are exposed to the extracellular milieu and to the cytoplasm; according-
ly, they have different internal and external surfaces. Since the two lipid layers dif-
fer in their composition, membrane proteins also assume different spatial arrange-
ments. However, carbohydrates are found only on the outer surface of the mem-
brane.
3.3.1 Transport
The cell membrane allows the internal and external passage of material. Transport
of the various molecules may be energy-independent or coupled to an energy-de-
pendent reaction or process.
3.3.2 Diffusion
Many small molecules are able to cross the cell membrane simply by moving
across a gradient from an area of higher to one of lower concentration.
Only molecules small enough to pass through the small pores within the mem-
brane are transported by diffusion. Since no energy is involved to move these mol-
ecules, diffusion tends to be a slow process. The transition of the molecules
through the membrane is also influenced by whether they are lipid-soluble or wa-
ter-soluble.
Some membrane proteins can form channels that allow water-soluble molecules to
pass through the hydrophobic lipid layer inside the membrane. This is the mecha-
20 G. Zerbini
nism by which important molecules such as glucose, which supplies energy to the
cell, pass through the membrane. The protein channels allow these molecules to
pass from areas of higher to those of lower concentration.
For some molecules, a higher concentration must be maintained on one side of the
membrane than on the other. To maintain this concentration gradient requires ener-
gy. Perhaps the best studied model of active transport is the sodium-potassium
pump, but minerals are also moved by this mechanism.
Nerve cells use pumps to transport ions in order to ultimately transmit their chem-
ical messages.
Sometimes the cell must allow the entry of molecules that are too large to pass
through the normal channels of the plasma membrane. In this case, the membrane
surrounds the molecule of interest, forming a vesicle that can be easily transported
inside the cell. Phagocytosis and pinocytosis refer to the vesicle-mediated transport
of solid and liquid molecules, respectively.
The proteins that make up the cell membrane are obviously very important for the
immune system. Some of them form channels or transporters, but other are need-
ed for the identification and characterization of the cell. The recognition of self re-
lies on the presence of proteins and glycoproteins on the cell surface. An organ that
is transplanted from one individual to another will be recognized as foreign if the
membrane proteins differ from those of the recipient organism; in such cases, un-
less so-called immunosuppressive drugs are administered to the host, the trans-
planted organ will be rejected. The same mechanism underlies autoimmune dis-
eases such as rheumatoid arthritis and diseases of the thyroid; in both cases, mem-
brane proteins of the human body are mistakenly recognized as foreign and then
rejected.
complex followed by either on-site degradation of the complex itself or its internal-
ization with further activity inside the cell.
The sarcolemma is the cell membrane of a muscle cell (skeletal, cardiac, and
smooth muscle). It consists of the typical plasma membrane but also an outer coat
made up of a thin layer of polysaccharide material containing thin collagen fibrils.
At each end of the muscle fiber, the surface layer of the sarcolemma combines with
a tendon fiber. The tendon fibers finally collect into bundles to form the muscle ten-
don, which inserts into bones. The sarcolemma is specialized to receive and conduct
stimuli. Dysfunctions in the stability of the sarcolemma membrane and its repair
system underlie diseases such as muscular dystrophy [2].
References
1. Hollán S (1996) Membrane fluidity of blood cells. Haematologia 27:109-27
2. Jacobson K, Sheets ED, Simson R (1995) Revisiting the fluid mosaic model of membranes.
Science 268:1441-2
3. Singer SJ (2004) Some early history of membrane molecular biology. Annu Rev Physiol
66:1-27
4. Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure of cell membranes.
Science 175:720-31
Gene Polymorphisms and Athletic
Performance 4
Ileana Terruzzi
4.1 Introduction
Researchers have long worked to identify and describe the morphologic, anthropo-
metric, physiologic, and functional characteristics of athletes who have reached
high levels in various sports. But year after year, athletic records are broken and the
limits of human performance are continuously redefined.
Despite these increasingly high performance levels, all living organisms are in
a state of homeostasis, in which the body is maintained in a state of biochemical
balance even when subjected to strong environmental stimuli. This adaptive abil-
ity is a primary defense mechanism that the body exploits to protect itself from
changes in the external environment and/or from systematic repetition of stress-
ful physical changes. Since training and exercise in general are stress factors
with demands on the metabolism of protein and energy, the supply of oxygen in
the blood, and all other homeostatic control systems, the body has evolved a
state of readiness allowing it to react even to the demands of extreme physical
performance.
Physical effort, if sufficiently intense, causes a fatigue process that after an ad-
equate and necessary recovery phase prevents the return of energy reserves, protein
synthesis, and numerous regulatory mechanisms to their initial, pre-loading state
but instead brings about a level that is significantly higher, resulting in greater per-
formance capabilities (Fig. 4.1). In fact, during the mandatory recovery phase, not
only is the energy consumed offset but reserves above the initial level are built up
according to a mechanism called “super-compensation”. The ability to adapt to dif-
ferent situations and to different environmental circumstances related to physical
activity is an amazing feature typical of living beings. If the human body were not
able to respond positively to all the demands it encounters, such as cold, heat, oxy-
I. Terruzzi ()
Division of Metabolic and Cardiovascular Sciences
Istituto Scientifico San Raffaele, Milan, Italy
e-mail: terruzzi.ileana@hsr.it
gen deprivation, manual work, inactivity, or disease—in other words, all those
sometimes difficult circumstances that life entails—it would face certain death.
The human body is a marvelous machine that will improve or worsen its per-
formance depending on the type, amount, and frequency of the stimuli with which
it is confronted and it will adapt its skills to cope with substantial workloads.
Muscular work involves a coordinated series of intracellular changes that lead to
movements of muscle fibers and, consequently, of the muscles themselves. The hu-
man body’s ability to adapt to muscular work means that its muscles can be trained
to carry out this work and thus to reach a degree of contraction different from the
resting state, resulting in improved neuromuscular response and increased resist-
ance. In fact, muscle development is the natural adaptation of the body to increas-
ing physical activity, with a very complex set of changes. Consequently, the body
is equipped to deal with a stressful event of greater magnitude, as the duly stimu-
lated muscles which periodically undergo effort become stronger each time.
Therefore, systematic training induces the body to successfully confront in-
creasingly higher levels of fatigue through the development of morphologic and
functional changes that are stable over time and depend on the type, intensity, and
duration of the exercise but also on the physiologic characteristics of the individual.
Progress in the body’s performance occurs in response to a training stimulus
that produces an improvement in the starting conditions. Moreover, improvement
requires that the training stimulus consists of a steady and gradual increase based
on a person’s individual organic capability and without interruptions, to avoid
losing the adaptations thus far achieved. Accordingly, a new state of homeostasis
is achieved. In contrast, low-intensity and inconsistent training do not alter either
the quality or the metabolic performance of an athlete (Fig. 4.2).
In recent years, much attention has been paid to the type and amplitude of the
changes that develop with physical exercise, at the cellular and molecular levels,
4 Gene Polymorphisms and Athletic Performance 25
in order to assess whether there is a correlation between them and the body’s
adaptability and ability to perform.
A series of tests can be used to investigate the physiologic factors that deter-
mine an athlete’s physical and sports performance. For example, the measure-
ment of blood lactate is an indicator of lactic acid metabolism under stress, allow-
ing training loads and recovery to then be modulated accordingly. The determina-
tion of maximal and submaximal O2 consumption is a good indicator of perform-
ance, while the evaluation of slow muscle fiber composition reflects the amount of
muscle strength. All of these tests are very effective for periodic monitoring, which
is extremely important for an athlete in order to assess the results of his or her
training program. A through analysis of the results allows performance to be relat-
ed to training strategies, thus creating a successful training program that provides
optimal results.
However, the chosen indicator serves only to measure that particular parame-
ter, such that a related improvement or deterioration in performance can only be in-
directly inferred. Instead, measurements of the complex processes of exercise-in-
duced stress adaptation are necessary to make the appropriate choice of exercise
and to decide upon the duration and characteristics of its execution, in order to pro-
vide the athlete with the right support and guarantee improved performance. But
can these parameters, which show a significant correlation with performance and
allow estimations of adaptability and performance capabilities, be used to identi-
fy an athlete a priori?
These types of tests are able to measure retrospectively how an athlete re-
sponds to the training stimulus and to determine the effect of that training, but not
26 I. Terruzzi
to predict an individual’s response to the stimulus. Will the tested athlete have the
talent to be among the elite? Will he have the skills to better respond to the kind of
training in question? Will she merely be one of the many competitors or will she
be a winner?
Sports performance and motor ability have always shown a large degree of varia-
tion even between individuals who use the same training protocols. This variabil-
ity can be seen in Fig. 4.3, which shows the running times of the athletes who par-
ticipated in the Vancouver marathon in 1999 (Fig. 4.3, left panel). The distribution
of the arrival times can be explained by a variety of factors—extrinsic and intrin-
sic—that affect the performance of each runner.
Age (Fig. 4.3, center panel) and sex (Fig. 4.3 right panel) are certainly among
the factors able to determine the different performance responses of each individ-
ual athlete. Figure 4.3 show that, on average, women are slower than men, al-
though it is not clear whether this is reflects anatomic differences between the sex-
es or social and cultural influences. The environment is certainly one of the most
relevant extrinsic factors influencing the development of athletic potential, but it is
equally certain that potential is innate and determined by an individual’s genetic
heritage.
Each of us owes our uniqueness to the information contained in our DNA, the
genetic code that we inherited from our parents and which we pass on to our chil-
dren. What is written in that code determines not only phenotypic traits, such as
hair, eyes, skin color and other physical features, but also our character, our sus-
ceptibility to disease, and our ability to react to stimuli. Of course, the environment
and our life experiences greatly affect the manifestation of this information such
that, depending on the type and amount of stimuli we receive, our response will re-
flect the adaptability with which our DNA has equipped us (Fig. 4.4).
The way we progress as a result of training is certainly due to the presence of
a stimulus that acts by placing our body under stress, but our response to that stim-
ulus is dictated by the instructions written in our DNA and it is these instructions
that generate different responses to equivalent stimuli. Physical activity induces a
wide variety of biochemical and biophysical responses that act on the organism
and determine a broad range of phenotypic adaptations. The results in terms of per-
formance vary and this variability is particularly observed in athletes, in whom al-
most no measurable differences in performance can distinguish the winner from
his or her competitors. It is clear that some athletes possess an innate talent that
distinguishes them from other competitors who show the same strength of will, the
same effort, and the same perseverance in training: genetics provide the competi-
tors with the opportunity to participate and the winner with the ability to excel.
Sir Roger Bannister was the first person to run the mile in less than four min-
utes, but he was also the first to become aware of “the obvious but overlooked fact
that black sprinters, and all black athletes in general, have natural anatomic advan-
tages.” If we consider the twenty best runners of all time in the distances from 800
m to the marathon, more than half have been from Kenya. Does this depend on the
high-altitude highlands of their country, on their nutrition, on their body structure,
or on the fact that many Kenyan children run for miles every day? However, while
East Africans reign over long distances, athletes with roots in West Africa, as is the
case for most Afro-Americans, dominate the sprint. This leads to the question: is
there a genetic selection of talent?
Just what do genes really tell us about athletic ability? If genes determine the po-
tential of each person and the environment acts on them by ensuring their optimal
expression, then will identifying those genes and studying their function allow us
to predict the performance of each athlete? What can our genes truly reveal about
28 I. Terruzzi
our athletic potential? Surely if we were able to distinguish the specific genes that
contribute to performance, muscle strength, and maximal aerobic capacity, a genet-
ic test would be able to reveal to a parent whether his or her child will excel in a
particular sport. In actual fact, genetics shape us in many ways, including our po-
tential to excel in sports. However, the relationship between genes and physical
and sports performance is still an open field of investigation, although in recent
years there have been significant advances.
Searching for the effects of an individual genetic variant in a complex and en-
vironmentally influenced activity such as sports performance is extremely difficult.
There is no direct relationship between a gene and the characteristics of perform-
ance; instead, multiple genes are responsible for defining a performance, even the
simplest one imaginable.
It is also very difficult to quantify the performance of a specific sport, for ex-
ample, the marathon. Athletes who have reached high competitive levels have a
combination of different genotypes favorable for physical performance. In fact,
performance represents a trait controlled by multiple genes and a single gene can-
not be responsible for performance; rather, it may only increase or decrease a
person’s physical abilities. For this reason, modulating the expression of a single
gene may not result in substantial changes in sports performance.
In June 2001, the first human gene map linked to performance was described
by Rankinen. Since then, the number of genes potentially associated with physical
performance has increased yearly (Table 4.1). In the human gene map updated in
2005, the number of such genes had expanded to about 190.
Approximately 20 000 genes make up the genetic heritage that defines each of us
as human. However, despite our common heritage, substantial variations exist be-
tween individual human genomes, including alterations in gene sequences (copy
number variation, tandem repeats) and changes in individual base pairs (mutations
if < 1% frequency and single nucleotide polymorphisms if > 1% frequency).
The genetic code written in our DNA is specified by four nucleotides referred
to by their first letters: A (adenine), T (thymine), C (cytosine), and G (guanine).
The particular sequence of these nucleotides is specific for every single gene and
determines its specific function. A gene consists of a promoter, which indicates the
starting point of transcription and determines when and how frequently a gene is
expressed, as well as a specific coding sequence of nucleotides, which determines
the amino acid sequence of the protein encoded by the mRNA transcript.
Sometimes, variations occur within the original sequence of nucleotides such
that, for example, an A replaces one of the other three nucleotides (C, G, or T).
These small genetic changes are called single nucleotide polymorphisms (SNP).
They occur once in every 300 nucleotides on average, which means that there are
roughly 10 million SNPs in the human genome. Most commonly, these variations
are found in the intronic regions of the DNA (around 26% of the genome) and in
regions that separate adjacent genes, i.e., stretches of non-coding DNA that, ac-
cording to our current knowledge, are without function. When SNPs occur within
a gene or in its regulatory region, they may directly influence its function. For ex-
ample, the substitution of a base in the coding sequence of a gene can alter the cor-
responding protein, by the insertion of the wrong amino acid, or even cause its pre-
mature termination, by the insertion of a “stop” signal instead of an amino acid.
Such proteins are very often non-functional.
SNP is the most common type of “genetic variation” and each person’s genet-
ic material contains a unique SNP pattern that is made up of many different genet-
ic variations. Although more than 99% of human DNA sequences are the same,
variations in DNA due to the presence of SNPs can have a major impact on how a
person responds to environmental stimuli. In this light, SNP profile studies may
help to predict an individual’s response to certain stimuli (such as physical exer-
cise) in addition to being used to track the inheritance of predisposing genes with-
in families. Because SNPs occur normally throughout DNA and are evolutionari-
ly stable, with few changes from generation to generation, they represent excellent
biological markers (segments of DNA with an identifiable physical location that
30 I. Terruzzi
can be easily tracked) in studies of the relationship between particular gene vari-
ations and their respective, potentially modified proteins. Given this crucial abili-
ty to use such genetic variations in gene identification, together with recent tech-
nological advances, the discovery and detection of SNPs has become an important
field of genetic research.
Athletes show an altered muscle phenotype and enhanced performance that are due
to physical exercise, which is accompanied by a continuous and constant training
stimulus leading to new metabolically and morphologically adaptive goals. The
mechanism by which exercise stimulates these actions in athletes is poorly under-
stood. While, as extensively detailed in this chapter, it is clear that environmental
influences such as training and diet are important, nonetheless, genetic back-
ground is strongly related to performance. This aspect suggests that athletes pos-
sess a genetic advantages predisposing them to better sport performances than
achieved by non-athletes. Hundreds of genes have been studied in relation to per-
formance in an attempt to unravel the complex relationship between genetic ex-
pression and physical performance in athletes.
Specific mechanisms (Fig. 4.5) take part in controlling gene expression, in
particular the adequate supply of methyl groups to the DNA and therefore the spe-
cific enzymes responsible for the proper functioning of DNA methylation. The role
Suggested Reading
1. Terruzzi I, Senesi P, Montesano A et al (2011) Genetic polymorphisms of the enzymes invol-
ved in DNA methylation and synthesis in elite athletes. Physiol Genomics 43:965-973
2. Watson JD, Baker TA, Bell SP et al (2008) Molecular biology of the gene. Benjamin
Cummings, 6th edn. New York
3. Bompa TO, Haff G (2009) Periodization: theory and methodology of training. Human
Kinetics, 5th edn. Chaimpaign, IL
4. Rankinen T, Pérusse L, Rauramaa R et al (2001) The human gene map for performance and
health-related fitness phenotypes. Med Sci Sports Exerc 33:855-867
Nutrients and Whole-Body Energy
Metabolism: The Impact of Physical Exercise 5
Stefano Benedini
5.1 Introduction
Energy represents the capacity of a physical system to perform work. When ap-
plied to nutrition, the term energy refers to the chemical energy locked in food-
stuffs because of the chemical bonds present in nutrients. In order to produce en-
ergy, the body converts foods to glucose, fatty acids, and amino acids; it is these
products that reach the cells, where they react with oxygen to form carbon dioxide
and water.
S. Benedini ()
Department of Sport Sciences, Nutrition and Health
University of Milan, Milan, Italy
Research Center of Metabolism
IRCCS Policlinico San Donato Milanese, Milan, Italy
stefano.benedini@unimi.it
These series of reactions proceed through numerous steps whose rates are con-
trolled by the respective enzymes (proteins that increase the rates of chemical re-
actions in the human body). The energy produced is used to create adenosine
triphosphate (ATP), a nucleotide composed of adenine (nitrogen base), ribose
(pentose sugar), and three phosphate radicals. The last two phosphate radicals in
ATP are attached through chemical bonds that contain relatively high amounts of
energy. ATP can release its energy instantly for use in a wide range of functions es-
sential to the body: mechanical work (muscle contraction), membrane transport,
and the synthesis of chemical compounds. During this energy-releasing process,
ADP (adenosine diphosphate) or AMP (adenosine monophosphate) is formed.
Either one can be rephosphorylated to ATP by oxidative reactions, which is a con-
tinuous cellular process. Creatine phosphate (CP) is another energy-rich com-
pound. It is considered the “reservoir” of high-energy phosphate because it is
stored in the body in larger quantities than ATP [4].
Mitochondria are described as “cellular power plants” because they generate
most of the cell’s pool of ATP [4]. In addition to supplying cellular energy, mito-
chondria are involved in a variety of other processes, such as signaling, cellular dif-
ferentiation, cell death, and the control of the cell cycle and cell growth. [5].
However, the dominant mitochondrial function is the production of ATP, as evi-
Fig 5.1 Excess free fatty acids in the plasma cause major impairments in muscle mitochondrial
function, associated with decreased glucose oxidation. In particular, excess fatty acids result in de-
creased glucose intake by muscle cells and simultaneously block the availability of pyruvate in the
mitochondria by inhibiting the Krebs cycle. GLUT1 Insulin stimulated glucose transporter 1, HK II
hexokinase II, PDH pyruvate dehydrogenase, NAD nicotinamide adenine dinucleotide, GS glyco-
gen synthase
5 Nutrients and Whole-Body Energy Metabolism: The Impact of Physical Exercise 35
denced by the large number of proteins in the inner membrane. The synthesis of
ATP requires the oxidation of the major products glucose, pyruvate, and NADH,
which are produced in the cytosol.
This process of cellular respiration, also known as aerobic respiration, is de-
pendent on the presence of oxygen. When oxygen supplies are limited, the gly-
colytic products are metabolized to produce ATP by anaerobic respiration, a
process that is independent of the mitochondria [6]. ATP production from glucose
has an approximately 13-fold higher yield during aerobic than during anaerobic
respiration [7].
Beta oxidation of fatty acids largely takes place in the mitochondrial matrix and
must be activated by coenzyme A in order to form a fatty acyl-CoA thioester.
Short- and medium-chain fatty acids undergo this reaction within the mitochondria
(Fig. 5.1) whereas long-chain fatty acids are unable to cross the mitochondrial
membrane; thus, instead, the thioester reaction occurs at the outer mitochondrial
membrane and the product is then carried by carnitine across the inner mitochon-
drial membrane.
The long-chain fatty acid is transported across the membrane by a translocase
Fig 5.2 L-Carnitine (LC) is essential for the entry of activated long-chain fatty acids from the cy-
tosol into mitochondria and for the transport of activated medium- and short-chain organic acids
from peroxisomes to mitochondria. The acetyl-coenzyme A (AcCoA) to coenzyme A (CoA) ratio is
maintained by LC, which functions as a pool of activated acetyl units. The toxic effects of poorly
metabolized acetyl groups can be lowered through the transesterification of CoA and the excretion
of acetyl-LC (ALC) esters by carnitine acetyltransferase (CAT) and palmitoyltransferases (CPT I
and CPT II). Carnitine–acylcarnitine translocase (CT) enables the trafficking of short-chain ALC es-
ters in and out of mitochondria. OMM Outer mitochondrial membrane, IMM inner mitochondrial
membrane
36 S. Benedini
and then passed to carnitine acyltransferase II on the matrix side. This enzyme
places the fatty acyl group back on CoA thereby restoring the original fatty acyl-
CoA (Fig. 5.2).
Nutrients that are consumed, digested, and absorbed need to be delivered to the
correct tissues so as to meet their metabolic needs. However, many factors can in-
hibit the normal metabolism of nutrients, including nutrient-nutrient interactions,
drug-nutrient interactions, and excess alcohol consumption. In fact, regular alco-
hol consumption is the greatest antagonist of performance in athletes.
Nutrient intake and availability clearly impact energy metabolism; even the de-
ficiency of a single nutrient component will corrupt the normal metabolic path-
ways for energy utilization and thus affect athletic performance. It is therefore im-
portant that athletes are informed of the potential hazards of inadequate nutrient in-
take, poor digestion, malabsorption, and altered metabolic processes caused by
drug or alcohol ingestion. While the body is able to compensate an unbalanced nu-
trient supply in the short term, chronic insults from nutrient deficiency, heavy al-
cohol abuse, or untreated illness will eventually have a negative impact on health,
and on athletic performance. Consequently, nutrition must be an important concern
not only of athletes but also of their coaches.
Physical activity increases the demand for fuel and therefore the metabolic
processes involved in its utilization. Anything that limits the supply of adequate
calories to support cellular requirements or alters the cells’ capacity to metabolize
the provided fuel will reduce performance. Some factors are under the athlete’s
control, including adequate food consumption, careful consumption of medications
and supplements, and avoidance of regular alcohol consumption; others, however,
are not, such as disease status, which may alter food intake or food absorption.
It is not the aim of this book to discuss how best to acquire the fuels needed to
successfully pursue athletic endeavors. It suffices to say that athletes should not
hesitate to seek medical advice because of the importance of good health for per-
formance [8].
Food intake is widely regulated by the CNS and involves many areas of the brain,
as feeding is a complex behavior. The hypothalamus is known to control body
weight and appetite, but the underlying mechanisms are not entirely clear. Recent
findings suggest that hypothalamic neurons directly sense circulating nutrient lev-
els. The CNS is thought to play a key role in the control of glucose metabolism via
central neural pathways that overlap with those controlling food intake and body
weight. The brain is an insulin-sensitive organ. Insulin along with another hor-
mone, leptin, provides afferent inputs to the CNS, informing the body of the suf-
5 Nutrients and Whole-Body Energy Metabolism: The Impact of Physical Exercise 37
ficiency of fat stores. Insulin receptors are concentrated in hypothalamic areas. The
intracerebroventricular administration to baboons of low-dose insulin was demon-
strated to reduce food intake and body weight. Most of the gastrointestinal hor-
mones affect not only food intake and glucose metabolism but also the CNS, the
latter via peripheral and/or central mechanisms.
The long-term homeostasis of the body’s energy supply is a multifactorial bi-
ological process achieved by numerous complementary mechanisms that imply fu-
el-availability sensors at peripheral and central locations. The hypothalamus is a
primary site of integration of nutritional information, including neural inputs and
circulating metabolic signals, i.e., glucose and fatty acids. Moreover, the hypothal-
amus elicits appropriate behaviors and metabolic responses to counterbalance any
changes in energy homeostasis.
Previous studies have already shown that an overload of circulating lipids
stimulates the activity of specific hypothalamic neurons and modulates the expres-
sion of neuropeptides, the key effectors of the hypothalamus, in turn leading to
changes in peripheral metabolism. In addition, the chronic elevation of lipids may
alter hepatic sensitivity to insulin, through direct effects on the CNS. Recent find-
ings indicate that circulating lipids directly act as signaling molecules and thus in-
form the hypothalamus about the body’s metabolic status. The intracerebroventric-
ular administration of long-chain fatty acids has been shown to inhibit food intake
and to stimulate peripheral energy storage.
Of particular importance is the role of cellular metabolism in hypothalamic nu-
trient sensing. For example, elevated levels of cytosolic long-chain fatty
acid–CoAs in the arcuate nucleus of the hypothalamus result in a decrease in food
intake and in whole-body glucose production. Furthermore, defects in the cellular
metabolism of fatty acids in the brain prevent hypothalamic esterification of long-
chain fatty acids and can disrupt peripheral glucose homeostasis. Controlling the
abundance of the intracellular malonyl-CoA pool is another way that the body
monitors fuel availability, and a persistent decrease in hypothalamic malonyl-CoA
is sufficient to stimulate food intake and to induce obesity.
A recent study performed on an animal model determined that lipid overload
stimulates a reactive oxygen species (ROS)-dependent signaling pathway within
the hypothalamus in order to regulate energy homeostasis. Thus, ROS act as intra-
cellular messengers between the mitochondria and the cytosol in response to nu-
trient influx.
demonstration, that the profound hyperphagia and obesity of ob/ob mice are due to
autosomal recessive mutation of the gene encoding leptin, a hormone secreted by
adipocytes, provided compelling evidence of a second adiposity signal. Subsequent
studies demonstrated that both insulin and leptin fulfill the criteria that define an
adiposity signal. Both hormones circulate at levels proportional to body fat content
and enter the CNS in proportion to their plasma levels. Leptin and insulin recep-
tors are expressed by brain neurons involved in energy intake. The direct admin-
istration of these peptides signals the brain to reduce food intake while a deficien-
cy of these hormones has the opposite effect [9].
As discussed in greater detail below, in obese patients insulin resistance is a key
factor in the development of metabolic syndrome and overt diabetes. While altered
leptin levels in adipose tissue cause leptin resistance, the mechanisms involved in
leptin secretion and resistance are quite different. The rate of insulin-stimulated
glucose utilization in adipocytes provides the essential link between leptin secre-
tion and body fat mass. Although not completely understood, the underlying mech-
anism may involve glucose flux through the hexosamine pathway. During acute
changes in energy balance, adipocyte glucose metabolism is markedly altered and
leptin secretion can become transiently dissociated from levels of total body fat.
For example, food deprivation very rapidly lowers plasma leptin concentrations in
both rodents and humans to a greater extent than would be expected simply from
the decrease in body fat content. This exaggerated early decline of leptin levels
may activate compensatory responses before energy stores become substantially
depleted.
Leptin binding to its receptor (Ob-Rb) induces the activation of Janus kinase
(JAK), receptor dimerization, and JAK-mediated phosphorylation of the intracel-
lular part of the receptor, followed by phosphorylation and activation of the signal
transducer and activator of transcription-3 (STAT3). Activated STAT3 dimerizes,
translocates to the nucleus, and trans-activates target genes, including suppressor
of cytokine signaling-3 (SOCS3), neuropeptide Y (NPY), and pro-opiome-
lanocortin (POMC). Sahu [10] suggested that leptin also activates phosphatidyli-
nositol 3-kinase (PI3K) and phosphodiesterase 3B (PDE3B) and reduces cAMP
levels in the hypothalamus. It was postulated that the interaction between
PI3K–PDE3B–cAMP and JAK2–STAT3 pathways constitutes a critical component
of leptin signaling in the hypothalamus. Accordingly, the defects in either one or
both signaling pathways may be responsible for the leptin resistance seen in obe-
sity [10], as diagrammed in Fig. 5.3. For the sake of simplicity and because these
pathways are not fully understood, the figure omits other potential signaling path-
ways, including the SHP2–GRB2–Ras– Raf–MAPK/ERK pathway and PTP1B,
regulating leptin action in the hypothalamus. Also, the role of cofactors and co-ac-
tivators, such as p300/CBP and NCoA/SRC1a, in STAT3 transcriptional activity in
the hypothalamus has yet to be established.
The expansion of fat depots, particularly omental fat, decreases insulin sensi-
tivity and increase insulin secretion. In contrast, when lean mass is enhanced (for
example, after fitness training), insulin sensitivity increases and the production of
insulin by pancreatic beta cells decreases [11].
5 Nutrients and Whole-Body Energy Metabolism: The Impact of Physical Exercise 39
Fig 5.2 Leptin induces the activation of Janus kinase (JAK), receptor dimerization, and JAK-me-
diated phosphorylation of the intracellular part of the receptor, followed by phosphorylation and ac-
tivation of signal transducer and activators of transcription-3 (STAT3). Activated STAT3 dimerizes,
translocates to the nucleus, and trans-activates target genes, including suppressor of cytokine sig-
naling-3 (SOCS3), neuropeptide Y (NPY), and pro-opiomelanocortin (POMC). The POMC recep-
tor (MCR4) is a central system that determine the increase of glucoseneogenesis, inhibition of glu-
cose oxidation, and the increased deposition of visceral fat
5.6 Exercise
physical activities. In fact, there was a tendency for non-exercise physical activi-
ty to increase. This finding was consistent with the decreases in body mass and fat
mass observed in all three exercise groups cited in the article, which could not have
occurred if the increased exercise energy expenditure had been compensated by re-
ductions in other physical activities [16]. In our opinion, the 8-month specific ex-
ercise-training program principally differentiates the STRRIDE study from stud-
ies demonstrating no change in total PAEE as the longer training program better
replicates the long-term effects of regular exercise. Accordingly, we propose that
the effects of an exercise program on total and non-exercise PAEE depend on the
duration of that program.
Skeletal muscle is a metabolically active tissue that is critical to whole-body
homeostasis, in part through its important role in fatty acid oxidation (FAO). Under
resting conditions, lipid oxidation contributes significantly to overall energy needs,
with most of the energy requirements of muscle being delivered via FAO, which is
quantitatively relevant for maintaining muscle mass. Factors that elicit a decrement
in the ability of skeletal muscle to oxidize lipid evoke far reaching changes in
whole-body lipid and fat mass homeostasis.
5.7 Obesity
Limited prospective data indicate that a propensity for weight gain is associated with
a low rate of lipid oxidation in skeletal muscle. In Pima Indians, Zurlo et al. [17] re-
ported a low capacity for fat oxidation (measured with whole-body indirect calorime-
try) which in turn is associated with an increased propensity for weight gain. Similar
findings were reported by Marra et al. [18], who observed that weight gain in lean
women was associated with a low rate of whole-body fat oxidation. Other research
supports a relationship between a low rate of fat oxidation and weight gain in both
lean and obese individuals [19].
Studies examining the effect of weight loss on substrate utilization in obese in-
dividuals obtained similar results. Larson et al. [20] assessed a group of previous-
ly obese individuals who had lost an average of 57 kg via energy restriction and
found that fat oxidation (determined with indirect calorimetry) was significantly
lower in the weight-loss (post-obese) group than in weight-matched controls.
Similarly, Kelley et al. [21] examined substrate utilization across a skeletal mus-
cle bed before and after weight loss and reported no change in the capacity of
obese individuals to oxidize fat.
Insulin resistance is a major health problem in obese patients. The coexistence
of insulin resistance and reduced FAO in skeletal muscle is often determined in the
obese. However, the mechanistic connection between FAO and insulin resistance
in the skeletal muscle of these individuals is not well understood. There is evidence
suggesting that the accumulation of lipids in the skeletal muscle of obese individ-
uals induces insulin resistance[22]. Other researchers have suggested that metabo-
lites such as long-chain acyl CoA, diacylglycerol, and ceramide accumulate in the
cytosol of skeletal muscle of the obese [23]. Directly or indirectly these interme-
5 Nutrients and Whole-Body Energy Metabolism: The Impact of Physical Exercise 41
diates impair insulin signal transduction and/or the activity of enzymes involved in
glucose utilization, which in turn induces insulin resistance. The accumulation of
these metabolically active lipid intermediates could reflect disturbances in mito-
chondrial functions, specifically, the ability to completely oxidize fatty acids to
acetyl-CoA. Moreover, the accumulation of incompletely oxidized fatty acid
metabolites such as ceramide interferes with insulin signaling and leads to insulin
resistance.
5.8 Conclusions
In summary, it is clear that an adequate level of exercise can lead to substantial de-
creases in body weight, total body fat, and visceral fat. Additionally, evidence now
supports a dose–response relationship between the amount of exercise and body
weight, body fat, and visceral fat. A number of important cardiometabolic risk fac-
tors seem to be affected by moderate vs. vigorous exercise but this relationship re-
mains to be confirmed. Nonetheless, it is well-established that in sedentary middle-
aged men and women even relatively short periods of physical inactivity lead to
significant weight gain, substantial increases in visceral fat, and, consequently,
metabolic alterations. Exercise training, by contrast, induces changes in mito-
chondrial oxidative capacity, especially in skeletal muscle, and appears to im-
prove insulin action by reducing the accumulation of incompletely oxidized fatty
acids [24].
References
1. Ziegler EE, Filer Jr LJ (1996) Present knowledge in nutrition. ILSi, Washington, DC
2. Willett W (1998) Nutritional epidemiology. Oxford University Press, NY
3. Krause MV, Maham LK (1984) Food, nutrition and diet therapy. W.B. Saunders, Philadelphia
4. Campbell NA, Williamson B, Heyden RJ (2006) Biology: exploring life. Pearson Prentice
Hall, Boston, Massachusetts
5. McBride HM, Neuspiel M, Wasiak S (2006) Mitochondria: more than just a powerhouse.
Curr Biol 16 (14):R551
6. Voet, D, Voet JG, Pratt CW (2006). Fundamentals of biochemistry, 2nd edn. Wiley, Hoboken NJ
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31 (6):1095–105
8. Benardot D (2006)Advanced sports nutrition. Human Kinetics, Champaign, IL
9. Benedini S (2009) The hypothalamus and energy balance. Sport Sci Health 5(2)45-53
10. Sahu A (2003) Leptin signaling in the hypothalamus: emphasis on energy homeostasis and
leptin resistance. Front Neuroendocrinol 24(4):225-53
11. Sahu A (2011) Intracellular leptin-signaling pathways in hypothalamic neurons: the emerging
role of phosphatidylinositol-3 kinase-phosphodiesterase-3B-cAMP pathway. Neuroendocrino-
logy. [Epub ahead of print]. doi: 10.1159/000326785
12. Meijer EP, Westerterp KR, Verstappen FT (1999) Effect of exercise training on total daily
physical activity in elderly humans. Eur J Appl Physiol Occup Physiol 80:16–21
13. Goran MI, Poehlman ET (1992) Endurance training does not enhance total energy expendi-
ture in healthy elderly persons. Am J Physiol263:950–957
14. Meijer G, Jannssen G, Westerterp K et al (1991) The effect of a 5-month endurance training
42 S. Benedini
6.1 Introduction
Energy is required to perform any kind of mechanical work. In living organisms, the
energy for all biological functions is provided chemically by the hydrolysis of
adenosine triphosphate (ATP). ATP supplies the energy required to synthesize cel-
lular components and to maintain cell viability, by donating one or two phosphate
groups, leaving adenosine diphosphate (ADP) or adenosine monophosphate (AMP),
respectively. However, energy storage in the form of ATP is limited such that ATP
must be resynthesized continuously in order to meet cellular energy demands. The
generation or replenishment of ATP depends upon key metabolic pathways, glycol-
ysis, glycogenolysis, and oxidative phosphorylation, which interact to regulate the
rate of ATP metabolism and to direct cellular bioenergetics toward a defined home-
ostasis.
The different mechanisms involved in the breakdown and resynthesis of ATP
may be summarized as follows:
1. ATP is broken down enzymatically to ADP and inorganic phosphate (Pi), yield-
ing energy for muscle activity.
2. Phosphocreatine (PCr) is broken down enzymatically to creatine and phos-
phate, with the latter transferred to ADP thereby yielding ATP.
3. Glucose 6-phosphate, derived from muscle glycogen or blood-borne glucose, is
converted to lactate through anaerobic glycolysis and produces ATP by sub-
strate-level phosphorylation reactions.
4. The products of carbohydrates, lipid, protein, and alcohol metabolism can enter
the mitochondrial tricarboxylic acid (TCA or Krebs’) cycle, where they are ox-
idized to carbon dioxide and water. This process is known as oxidative phospho-
R. Codella ()
Department of Sport Sciences, Nutrition and Health
University of Milan
Milan, Italy
e-mail: roberto.codella@unimi.it
rylation and it produces energy for the synthesis of ATP. Some of this ATP is
used for the resynthesis of PCr, which becomes depleted during exercise.
Bioenergetics and metabolism are essential to maintaining health and may be
disturbed in disease. Both can be studied with in vivo magnetic resonance spec-
troscopy (MRS), which is uniquely suited to quantitatively measure cellular ATP-
generating activities in vivo. Intramuscular storage and the turnover of important
nutrients (e.g., glycogen) can be monitored non-invasively by MRS as well. This
technique has therefore made a substantial contribution to our understanding of
mammalian cell energy metabolism, its control, and its alteration by disease. Thus,
in vivo MRS represents a promising method to investigate human metabolism,
with further developments and applications likely to ensure its continued use.
In vivo MRS is a non-invasive, safe technique that enables a unique, innovative per-
spective on tissue biochemistry in that it allows: (a) assessment of cellular metabo-
lite concentrations and their alterations; (b) monitoring of the intracellular fate of in-
fused labeled substrates; (c) measurements of chemical exchange processes under
steady state or equilibrium conditions.
The most MR-sensitive nuclei and those most commonly used are 1H, 31P, and
13C, each of which generates specific information on distinct metabolic and physi-
6.2.1 1H-MRS
1H is the most sensitive nucleus for MR studies because of its high relative sensi-
6.2.2 13C-MRS
6.2.3 31P-MRS
Fig 6.1 a 31P spectrum obtained from a hindlimb mouse muscle. b Principle underlying the meas-
urement of ATP synthesis rate by 31P MR saturation transfer as applied to the exchange between Pi
and ATP. In blue, the symmetric saturation spectrum: the saturation pulse is applied symmetrical-
ly to γ-ATP, but on the downfield side of Pi. In red, the spectrum obtained with selective γ-ATP ir-
radiation (NS=128). Subtraction of the spectra yields the fraction of Pi involved in the synthesis of
ATP by the reaction: ADP + Pi → ATP [22]
imal and human energy metabolism, its control, and its modulation by different fac-
tors, including mitochondrial dysfunction [25] and a number of chronic diseases.
Several approaches to quantitatively analyze and interpret 31P-MRS measurements
of energy balance in muscle during and after several types of exercise have been
proposed [13]. For example, it is possible to estimate the rates of glycogenolytic and
aerobic ATP synthesis, i.e., oxidative capacity, according to distinct protocols ad-
dressing various types of exercise, such as, ischemic exercise, pure aerobic exercise
under steady-state conditions or during work jumps, and mixed exercise. Chance et
al. [14] carried out graded, steady-state, non-exhaustive metabolic investigations
that identified ADP as a major control element of oxidative metabolism in human
skeletal muscle under these conditions. The authors showed that maximum veloc-
ity could serve as a measure of oxidative capacity. Dynamic measurements of the
initial rate of PCr depletion during pure aerobic work jumps [15] yielded an esti-
mate of the total rate of ATP synthesis (typically, at low workloads glycogenolysis
can be neglected). The recovery rate of PCr immediately after exercise appears to
reflect mitochondrial capacity. Other protocols based on ischemic exercise were
used to calculate glycogenolytic ATP production [16] and to measure the rate of ox-
idative metabolism under non-steady-state exercise conditions. Also, an additional
and important piece of information retrievable from 31P spectra, albeit indirectly, is
intracellular pH, as tissue pH can be deduced from the chemical shift of Pi.
During mixed exercise, both glycogenolytic and oxidative ATP synthesis can be
estimated by a calculation that includes total proton production and total ATP
6 Mitochondrial and Non-mitochondrial Studies of ATP Synthesis 47
turnover; the latter can be determined in different ways: (a) by calibration from is-
chemic exercise at the same power, (b) from the non-oxidative ATP synthesis rate
in the first exercise interval, when oxidative ATP production is still small; and (c)
from very early changes in the PCr concentration alone (neglecting both
glycogenolytic and oxidative ATP synthesis). In other words, during ischemic ex-
ercise glycogenolytic ATP production can be directly calculated from changes in
pH, corrected for the number of protons consumed by PCr hydrolysis as calculat-
ed based on changes in the concentration of PCr and the rate at which the cell
buffers protons, assuming the buffer capacity is known [14] (see Sect. 6.5.1).
In another remarkable application of 31P-MRS, the measured signal strength can
be sensitized to the rate of metabolite turnover even under steady-state conditions,
making use of magnetization transfer between nuclei linked by chemical exchange
[26]. Magnetization transfer can be studied by selectively perturbing the equilibrium
magnetization of one of the nuclei involved in the exchange process and then meas-
uring the effect on the signal strength from its exchange partner (see Sect. 6.3).
in vivo by magnetization transfer. This latter technique is based on MRS and has the
capability to non-invasively measure the reaction kinetics of enzymes in situ when
the reaction rates involved are relatively fast. It involves perturbing the magnetiza-
tion of a nuclear spin system in a particular compound and then monitoring how this
perturbation influences the nuclear magnetization of this spin system in another
compound in chemical exchange with the first [26].
Saturation is a particular kind of magnetization transfer in which the com-
pounds under discussion are, respectively, γ-ATP and Pi. In 31P-MRS saturation
transfer studies, the rates of mitochondrial phosphorylation are assessed: the unidi-
rectional rates of ATP synthesis are measured with the MR-saturation transfer
method applied to the exchange between Pi and ATP (i.e., the kinetics of Pi → ATP).
The steady-state intramyocellular Pi magnetization is determined during selective
irradiation of the γ resonance of ATP and compared with the magnetization of Pi at
equilibrium in a control spectrum (without saturation of the γ resonance of ATP).
This reduction of Pi magnetization yields the fraction of Pi involved in ATP synthe-
sis (VATP) (Fig. 6.1b). Hence, under appropriate conditions (i.e., resting, steady-
state) the magnetic transfer technique is capable of measuring mitochondrial oxida-
tive phosphorylation catalyzed by mitochondrial ATPase.
In MRS studies conducted on animal models in vivo, muscular contraction was in-
duced by electrical stimulation [17]. Using 31P MRS, the rate of oxidative phospho-
rylation was modeled from the recovery of PCr after exercise [18]. The PCr recov-
ery rate can be expressed as a recovery-time constant and is thought to reflect max-
imal ATP generation from oxidative metabolism via the creatine kinase (CK) reac-
tion (also called the Lohman reaction):
The calculation assumes that the ATP concentration is constant during recovery and
that ATP production from glycolysis is discontinued with the cessation of exercise
[16]. The results are often described as being independent of work level providing
that the acidic change in pH is not large [18]. This workload independence or insen-
sitivity is assumed to have a practical advantage in that measurements of force or
work are not required. This allows the evaluation of a broad range of subjects (in-
cluding the elderly) without the need for them to perform sub-maximal exercise
tests. Moreover, during pure aerobic work jumps, dynamic measurements of the ini-
tial rate of PCr depletion yield an estimate of the total rate of ATP synthesis.
Pure aerobic exercise also allows the maximal mitochondrial ATP synthesis
flux of muscle to be estimated, by following the methodology reported by Chance
et al. [14] with MRS measurements of energy balance at multiple workloads in a
ramp protocol. In fact, from this dataset, the kinetic transfer function of power-out-
put and an index of the cytosolic ADP concentration can be derived. The 31P spec-
tra do not yield a discernibile ADP signal; thus, to assess ADP concentrations inves-
tigators have assumed CK to be at equilibrium and have used the observables in the
31P spectra (PCr, ATP, P and pH) to calculate ADP activity, which has accordingly
i
been found to significantly exceed the activity determined in biochemical assays. In
other words, the ADP concentration can be calculated from the PCr/Pi value with
appropriate assumptions [14].
position. Since α-ketoglutarate is in relatively rapid exchange with glutamate, the lat-
ter will be likewise 13C-enriched at position C-4. Incorporation of the 13C label from
plasma [2-13C]-acetate into the muscle [4-13C]-glutamate pool is essential for the de-
termination of TCA cycle flux (VTCA). In fact, as the TCA cycle proceeds, in the sec-
ond turn the labeling will involve the [2-13C]- and [3-13C]-glutamate pools.
Sophisticated mathematical models can be used to infer the rates of TCA cycle
flux from the time course of 13C isotopic enrichment of plasma acetate and muscle
glutamate C-2 and C-4, by iterative fitting of metabolic simulations to the data us-
ing the program Cwave. The CWave model consists of isotopic mass-balance equa-
tions that describe the metabolic fate of plasma [2-13C]-acetate. The isotopic enrich-
ment and concentrations are used as input drivers. Based on these input parameters,
flux rates are determined that give the best fit to the observed time course of [2-
13C]- and [4-13C]-glutamate enrichment (Fig. 6.2).
tons attached to 13C nuclei are detected (this MR technique is called POCE, “pro-
ton-observed carbon-edited”) [1].
Two separate systems are available that permit the generation of energy in muscle
without oxygen, namely, the phosphagen or “high-energy phosphate system” (intra-
muscular stores of ATP and PCr) and the glycolytic system (Fig. 6.3). The total ca-
pacity of the glycolytic system for producing energy in the form of ATP is larger
50 R. Codella
ADP
ATP
ATP reserve
Lactate + H+ ATP
Table 6.1 Capacity and power of anaerobic systems for the production of ATPa
Capacity Power
(mmol ATP kg dm-1) (mmol ATP kg dm-1 s-1)
Phosphagen system 55-95 9
Glycolytic system 190-300 4.5
Combined 250-370 11
aValues are expressed per kg dry mass (dm) of muscle and are based on estimates of ATP provision du-
ring high-intensity exercise of human vastus lateralis muscle.
than that of the phosphagen system, although the rate at which it can produce ATP
is lower (Table 6.1).
At the start of the energy challenge, hydrolyzed ATP is resynthesized from the
breakdown of PCr and, depending on conditions, from anaerobic glycogenolysis.
Anaerobic energy production is essential for the maintenance of high-intensity ex-
ercise, when the demand for ATP is greater than what can be provided aerobically.
In fact, the approximate contribution of anaerobic and aerobic sources to overall
ATP production during high-intensity exercising lasting ∼3 min are: 80/20% in the
initial 30 s, 45/55% from 60 to 90 s, and 30/70% from 120 to 180 s [27].
To assess anaerobic ATP provision, such as during high-intensity exercise, key
substrates and metabolites must be repeatedly measured. For years, this was accom-
plished through biopsy sampling of contracting muscle at frequent intervals during
high-intensity exercise. The advent of MRS has several advantages over the former
technique: (a) it is non-invasive, allowing repeated measurements of metabolite con-
centrations over time; (b) it involves stables isotopes (no ionizing radiation); and (c)
it yields chemical information such that the intracellular fate of a labeled molecule
can be monitored as it is metabolized.
Localized 31P MRS can be used to calculate the glycolytic rate based on changes in
pH during high-intensity exercise, after correcting for the buffering of protons by
6 Mitochondrial and Non-mitochondrial Studies of ATP Synthesis 51
changes in PCr and ATP concentrations, the rate of aerobic ATP utilization, the ap-
parent muscle buffer capacity, and proton efflux [19]. Conley et al. studied the
regulation of glycolysis using 31P MRS during ischemic stimulation of the human
forearm [20]. They showed that the glycolytic rate is proportional to the muscle
stimulation frequency and does not depend on metabolite levels and intracellular
pH. This result is consistent with the dominant control of glycolysis by factors oth-
er than the products of ATP hydrolysis that scale with nerve-firing frequency (e.g.,
the free calcium concentration).
The rate of ATP synthesis from the net breakdown of PCr (ATPCK) via the CK re-
action (see Sect. 6.3.1) can be determined as during 31P MRS, i.e., from the change
in PCr during each muscular contraction. Since the synthesis of ATP is stoichiomet-
ric with the hydrolysis of PCr in the CK reaction, calculation of the latter takes the
simple form [12]:
ATPCK = dPCr/dt
6.5.3 Glycogen
Glycogen is readily available in muscle and can quickly be used to fuel glycolysis.
Following the infusion of [1-13C]-glucose, [1-13C]-glycogen can be detected in
muscle. While the 13C MRS detection of glycogen is relatively straightforward, the
detection of 1H glycogen remains elusive.
During exercise, muscle glycogen levels decrease according to: (a) the degree
of exercise, reaching lower levels with increasing workloads, and (b) the rate of
muscle contraction. Muscle glycogen can be monitored during different types of
exercise (also involving different and isolated muscle groups) by means of 13C
MRS, which enables studies of muscle glucose metabolism, including the effects
of acute exercise. For example, through MRS studies it has been possible to
demonstrate that, when exercise ceases, the exercised muscles resynthesize glyco-
gen at a rate that is influenced by the post-exercise concentration of intracellular
glycogen. Also, between rapid muscle contractions, the PCr pool is replenished
mainly by glycogenolysis after PCr breakdown [21]. However, the metabolic roles
of muscle glycogen in exercise are currently not known. High glycogen levels im-
prove endurance, whereas glycogen depletion is often associated with the onset of
fatigue [21].
With the advent of in vivo MRS, new techniques have emerged with which to inves-
tigate reaction kinetics directly. In probing energy regulation and assessing the
above-described ATP-generating pathways, in vivo MRS might be preferred over
52 R. Codella
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A, Del Maschio A, and Luzi L (2003) Insulin resistance, intramyocellular lipid content, and
plasma adiponectin in patients with type 1 diabetes. Am J Physiol Endocrinol Metab
285(6):E1174-1181
3. Hsu AC, Dawson MJ (2000) Accuracy of 1H and 31P MRS analyses of lactate in skeletal mus-
cle. Magn Reson Med 44(3):418-26
4. Kruiskamp M.J, de Graaf RA, van Vliet G, Nicolay K (1999) Magnetic coupling of
creatine/phosphocreatine protons in rat skeletal muscle, as studied by (1)H-magnetization
transfer MRS. Magn Reson Med 42(4):665-72
5. Nicolay K, Braun KP, Graaf RA, Dijkhuizen RM, Kruiskamp MJ (2001) Diffusion NMR
spectroscopy, NMR Biomed, 14(2):94-111
6. Richardson RS, Duteil S, Wary C, Wray D.W, Hoff J, Carlier PG (2006) Human skeletal mus-
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7. Carlier PG, Bertoldi D, Baligand C, Wary C, Fromes Y (2006) Muscle blood flow and oxyge-
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Excessive Nutrients and Regional
Energy Metabolism 7
Gianluca Perseghin
7.1 Introduction
There is general agreement that type 2 diabetes is the consequence of insulin resist-
ance, defined as an impaired ability of insulin to control hepatic and peripheral glu-
cose metabolism, and of compromised pancreatic β-cell function such that in-
sulin secretion is insufficient to compensate the degree of insulin resistance [1]. The
pivotal role of insulin resistance is confirmed by the fact that it is a consistent find-
ing in patients with type 2 diabetes. Indeed, insulin resistance may be detected
10–20 years before the onset of overt hyperglycemia and prospective studies have
demonstrated that it is the best predictor of whether an individual will later become
diabetic [2].
However, while this “glucocentric” view represents the traditional explanation
of the metabolic derangements of diabetes, a more “lipocentric” vision of the
metabolic problem has been proposed to explain simultaneously (a) the impair-
ment of insulin action in skeletal muscle, liver, heart, and adipose tissue and (b) the
impaired β-cell function [3, 4] as a consequence of chronically high circulating
free fatty acid (FFA) concentrations, i.e., lipotoxicity. Increased plasma FFA con-
centrations are associated with many insulin-resistant states, including obesity
and type 2 diabetes mellitus [5]. In a cross-sectional study of the young, normal-
weight offspring of type 2 diabetic patients, we found an inverse relationship be-
tween fasting plasma FFA concentrations and insulin sensitivity, consistent with
the hypothesis that altered FFA metabolism contributes to insulin resistance in pa-
tients with type 2 diabetes [6]. In addition, the deleterious effects of chronically el-
evated FFA on muscle glucose metabolism in healthy individuals [7] are similar to
the established abnormality of glucose metabolism affecting the skeletal muscle of
G. Perseghin ()
Division of Metabolic and Cardiovascular Sciences
Istituto Scientifico San Raffaele Milan, Italy
Department of Sport Sciences, Nutrition and Health, University of Milan, Milan, Italy
e-mail: perseghin.gianluca@hsr.it
patients with overt diabetes [8] and of their non-diabetic insulin-resistant off-
spring [9]. In this chapter, we focus on some of the recent advances in our under-
standing of human insulin resistance with respect to its relationships to excessive
ectopic fat accumulation in skeletal muscle, heart, and liver.
Ectopic fat accumulation within skeletal muscle has been proposed as the patho-
genic event in the development of peripheral insulin resistance [3]. An increased
intramyocellular lipid content (IMCL) has been reported in association with in-
sulin resistance in normal humans [10], individuals with an increased risk of devel-
oping type 2 diabetes [11, 12], and in patients with overt type 2 diabetes [13].
Convincing evidence of the association between IMCL content and whole-body in-
sulin sensitivity also derives from an interventional study in which the effects of
biliopancreatic diversion, which induces lipid malabsorption, and of a hypocaloric
diet in the treatment of patients with morbid obesity were compared [14]. The sur-
gical approach determined a drop of body weight in association with a selective
depletion of the IMCL content (assessed by means of quantitative histochemistry
of quadriceps-muscle biopsy specimens) paralleled by full reversion of the insulin
resistance state. The same results were not obtained with the hypocaloric treat-
ment, in which a less significant drop of body weight was associated with a small-
er reduction of IMCL content and less improvement of insulin sensitivity. This
work was of particular interest also because it showed that 6 months after surgical
treatment a full normalization of whole-body insulin sensitivity and quadriceps
IMCL content were achieved despite the persistence of a body mass index (BMI)
still in the obese range.
Although the association between insulin resistance and IMCL content may be
considered a classical finding in humans, several discrepancies in the above-men-
tioned literature question this assumption. Specifically, when the anthropometric
parameters of the study populations are more rigorously controlled there is less
certainty of a causative association between increased IMCL content and the de-
velopment of insulin resistance. For example, in a study of non-diabetic, normal-
weight men and women matched for peripheral insulin sensitivity as assessed by
the clamp technique, the amount of total body fat and of soleus IMCL content was
higher in women than in men [15], suggesting that the gender-dependent hormon-
al milieu modulates the interaction between IMCL content and insulin sensitivity.
Moreover, obesity and aerobic fitness mutually interact regarding their impact on
IMCL content: while IMCL content and insulin sensitivity were correlated in un-
trained subjects, in endurance-trained subjects, by contrast, high IMCL content
predicted high insulin sensitivity [16]. Also, individuals with a primary muscular
disease (myotonic dystrophy type 1) do not develop insulin resistance associated
7 Excessive Nutrients and Regional Energy Metabolism 57
with glucose and lipid metabolism despite a markedly higher IMCL content in
both the soleus and tibialis anterior muscles [17], suggesting a difference in the
subcellular localization of fat in muscle and its metabolic influence. In fact, in con-
trast with other forms of muscular dystrophy, the replacement of muscle by fat and
fibrous tissue is not typical of myotonic dystrophy type 1; rather, ultrastructural al-
terations are common and they may have an impact on fat localization around the
mitochondria (rapid disposal pool) or within lysosomes.
7.2.2 Heart
7.2.3 Liver
An association between ectopic fat accumulation within the liver and impaired glu-
cose metabolism has been reported. Both insulin-stimulated glucose metabolism
and the suppression of endogenous glucose production were found to be impaired
in individuals with non-alcoholic fatty liver disease (NAFLD) [24]. In addition,
58 G. Perseghin
NAFLD is common in obese patients with type 2 diabetes [25] and is considered
to be the hepatic component of the metabolic syndrome [26, 27]. The development
of fatty liver appears to be peculiarly associated with hepatic insulin resistance; in
fact, moderate weight reduction in obese patients with type 2 diabetes is associat-
ed with reduced intra-hepatic fat content and improved hepatic insulin sensitivity
although insulin-stimulated peripheral glucose metabolism remains unchanged
[28]. Recently, intra-hepatic fat content was assessed as a continuous variable by
means of 1H-magnetic resonance spectroscopy (MRS) and was reported to be as-
sociated with hepatic insulin resistance also in non-diabetic individuals [29] and
obese adolescents [30].
plays a relevant role in patients with fatty liver. Using tracer methodologies, it was
found that in patients with NAFLD 60% of liver triglycerides arise from FFA in the
fasting state [36]. Importantly, in the same study, 26% of liver triglycerides were
shown to have been the product of de novo lipogenesis [36]. An apparent in-
creased contribution of de novo lipogenesis vs. FFA reesterification was also re-
ported in patients with NAFLD [37, 38].
7.4.2 Heart
31P-MRS has proven to be an essential tool in the in vivo study of cardiac high-en-
ectopic fat accumulation, on the other, has not, as stated above, been established.
To pursue the hypothesis that the increased flow of FFA towards the heart has
detrimental metabolic effects linked to their disposal via oxidative pathways we
initiated a study of individuals with heart failure. The results showed that the pro-
longed (3 months) administration of a partial inhibitor of FFA oxidation (trimetazi-
dine) improved the study patients’ functional class and their LV function. These ef-
fects were associated with an increase in the PCr/ATP ratio in the resting state, in-
dicating the preservation of myocardial HEP levels [67]. Moreover, we were able
to confirm the beneficial effects of inhibiting FFA oxidative disposal not only
over the intermediate term but also within a few hours in patients with coronary
disease. In these patients, the administration of trimetazidine 24 h before stress
treadmill exercise testing (according to the Bruce protocol) after an 8-h fast, after
a high-fat meal, or after a high-carbohydrate meal improved the time to 1-mm ST-
segment depression (time to 1 mm) and the stress wall motion score index (WM-
SI) compared to patients taking placebo. Furthermore, this improvement was
achieved regardless of the meal composition [68]. Taken together, these data sug-
gest that the functional improvement and better LV energy homeostasis observed
in these patients reflected the better glucose handling induced by the inhibition of
FFA oxidative disposal. Also, the administration of perhexiline maleate, an an-
tianginal drug that potently inhibits the mitochondrial FFA uptake enzymes carni-
tine palmitoyl transferase-1 (CPT-1) and CPT-2, thereby shifting substrate uti-
lization from FFA toward glucose, improved VO2max, LV ejection fraction, symp-
toms, and resting and peak stress myocardial function in patients with heart fail-
ure [69]. A direct deleterious effect of FFA metabolism on cardiac energy metab-
olism is still controversial and deserves further investigation. For example, based
on the same working hypothesis, other authors [70] performed the reverse exper-
iment in patients with heart failure. In that study, acute administration of the lipol-
ysis inhibitor Acipimox resulted in a depletion of circulating FFA. Accordingly, an
improvement in the parameters of myocardial efficiency (as measured by positron
emission tomography and [15O]H2O, [11C]acetate, and [11C]palmitate administra-
tion) was expected. However, the myocardial efficiency of these patients deterio-
rated, suggesting that failing hearts are unexpectedly more dependent than healthy
hearts on FFA availability. It was therefore proposed that both glucose and fatty
acid oxidation are required for the optimal function of the failing heart.
7.4.3 Liver
While defective FFA oxidative disposal may take place also in the liver, the data
are controversial. We and others have recently shown that in patients with type 1
diabetes [71], in obese adolescents [30], and in lean patients with NAFLD [72] dif-
ferent patterns of whole-body lipid oxidation may be associated with the intra-he-
patic fat content. We have also shown that habitual physical activity is associated
with intra-hepatic fat content regardless of insulin resistance, suggesting an effect
of exercise per se on hepatic lipid disposal [73]. These studies provide only indi-
62 G. Perseghin
rect evidence because they were mostly performed using indirect calorimetry;
thus, lipid oxidation reflected events taking place in the whole body rather than
specifically in the liver. Liver-specific data were recently reported or are in the
process of being evaluated and no conclusions have been drawn. Iozzo et al. report-
ed that in patients with impaired glucose tolerance who have all the features of
metabolic syndrome (overweight, high plasma triglyceride and low high-density
lipoprotein cholesterol levels, hyperinsulinemia, insulin resistance, and a slight in-
crease in blood pressure) the liver’s ability to extract FFA from the circulation is
impaired [74]. Those authors speculated that in the fasting state beta-oxidation is
the primary route for intracellular FFA utilization, with FFA uptake therefore de-
pendent on the efficiency of this metabolic pathway. A consequence of this rela-
tionship is that defective liver FFA oxidative disposal is at the basis of the impaired
liver FFA uptake in individuals with reduced glucose tolerance. Contrary to this
finding, Misu et al. [75] recently reported that genes involved in mitochondrial ox-
idative phosphorylation (OXPHOS) are up-regulated in the liver of patients with
type 2 diabetes. This finding suggests that the regulation of OXPHOS genes in the
liver of patients with type 2 diabetes, despite the presence of steatosis, is a mirror
image of that in the skeletal muscle and heart of type 2 diabetics, in whom the
genes involved in OXPHOS appear to be down-regulated. It is possible that the liv-
er compensates for steatosis by increasing fatty acid β-oxidation and activating
OXPHOS even if the increased hepatic oxidative capacity is not enough to stop he-
patic steatosis. Further studies are obviously necessary to elucidate the significance
of oxidative fatty acids metabolism in the development of ectopic fat accumulation
in the liver.
7.5 Conclusion
Excessive ectopic fat accumulation may have direct relevance to the altered regula-
tion of insulin-mediated metabolic pathways and the impaired function of different
organs and tissues in diabetes and related diseases. An excessive FFA flux towards
the peripheral tissues (skeletal muscle, heart, liver, and beta-cells) has been pro-
posed as a pivotal event responsible for the high-level accumulation of intracellu-
lar triglycerides such as occurs in conditions of insulin resistance, i.e., obesity,
type 2 diabetes, and metabolic syndrome. If this is indeed the case, then local
acutely or chronically determined alterations of energy metabolism (oxidative phos-
phorylation and/or creatine phosphate and ATP metabolism) as well as oxidative
FFA disposal also may be involved. So far, the spillover of FFA flux has yet to be
confirmed; instead, efforts to treat fat-induced insulin resistance have focused on
improving insulin sensitivity by reducing the levels of this hormone using, most
commonly, thiazolidinediones, which are oral hypoglycemic agents. However, the
demonstration of abnormalities of energy metabolism would offer new strategies to
treat the deleterious metabolic effects of excessive ectopic fat accumulation. One
approach would be to develop and implement novel therapeutic tools aimed at reg-
ulating the FFA oxidative potential of organs and tissues before the development of
7 Excessive Nutrients and Regional Energy Metabolism 63
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Muscle Biopsy To Investigate Mitochondrial
Turnover 8
Rocco Barazzoni
R. Barazzoni ()
Department of Medical, Surgical and Health Sciences
University of Trieste
Trieste, Italy
e-mail: barazzon@units.it
and function were early and obvious targets for diagnostic and research applica-
tions of muscle biopsy. Needle biopsy has been further extensively applied in the
study of exercise physiology and pathophysiology, with the goal of investigating
the regulation of mitochondrial function and substrate oxidation. In recent years,
studies in the fields of obesity and diabetes have also focused on muscle mitochon-
drial function, and muscle biopsies have become increasingly common in human
metabolic assessments. This chapter provides an overview of mitochondrial func-
tion and substrate utilization while addressing the major concepts emerging from
basic studies on mitochondrial regulation. Available data from human skeletal
muscle biopsy studies are reviewed, with particular emphasis placed on the effects
of nutritional state, diet, and exercise and their potential interactions with insulin
resistance and disease state.
sistance, and type 2 diabetes, thereby opening novel and exciting fields for mito-
chondrial research.
ATP consumption leads to adenosin di- and mono-phosphate (ADP and AMP) pro-
duction. The ratios between ADP and AMP concentrations and the ATP concentra-
tion are sensitive markers of tissue energy state and they play an important role in
the regulation of mitochondrial oxidative metabolism (Fig. 8.2). In the presence of
70 R. Barazzoni
Fig 8.2 AMP-activated protein kinase (AMPK) activation favors changes in energy metabolism in-
volving mitochondrial biogenesis and enhanced ATP production
low energy availability, a higher ADP and AMP/ATP ratio enhances mitochondr-
ial function and ultimately potentially restores energy homeostasis. In full agree-
ment with this concept, AMP-activated protein kinase (AMPK) has emerged in the
last decade as a paradigm for energy sensing, mediating multiple responses aimed
at lowering energy expenditure while enhancing ATP production to restore ener-
gy balance. An increase in tissue AMP concentration and the AMP/ATP ratio
leads to AMPK activation through mechanisms that include direct allosteric mod-
ification by AMP, which favors protein kinase-mediated activating phosphoryla-
tion [7, 8]. AMPK-activated mitochondrial biogenesis involves phosphorylation of
transcription factors that coordinate the expression of mitochondrial and nuclear
genes such as peroxisome proliferator-activated receptor gamma-coactivator 1α
(PGC-1α) [7, 8]. In recent years, the network of signalling molecules involved in
AMPK-mediated activation of mitochondrial biogenesis has extended to sirtuins,
especially SIRT1 [9-11]. These deacetylating enzymes were originally described
as mediators of the positive effects of caloric restriction on energy metabolism
(see: below), and acetylated PGC1α is also a key SIRT1 target [9-11]. Coordinated
AMPK effects are also aimed at enhancing substrate availability through stimula-
tion of glucose uptake, fatty acid uptake, and fatty acid oxidative metabolism
through the inhibition of acetylCoA utilization for lipogenesis [7. 8]. Physical ex-
ercise and altered nutrient availability are among the major conditions involving
physiological adaptive changes in AMPK activation to modulate mitochondrial en-
ergy production.
8 Muscle Biopsy To Investigate Mitochondrial Turnover 71
8.4.2 Exercise
Exercise is one key area for the study of skeletal muscle mitochondria. Metabolic
changes induced by exercise are discussed in detail elsewhere in this textbook.
Overall, contraction-induced phosphocreatine and ATP consumption is a major
cause for the activation of coordinated signals to enhance mitochondrial function.
As outlined above, one metabolic signal of substrate and energy depletion is indeed
muscle AMPK activation, and its coordinated effects on SIRT1 and PGC1α have
been confirmed also following exercise in human and experimental models [12, 13].
A specific association with tissue metabolic needs is supported by elegant studies
in which AMPK activation was measured during exercise protocols performed at
different muscle glycogen levels [14]. Under the above conditions, AMPK activa-
tion was strongly enhanced in the presence of low glycogen stores while mainte-
nance of an adequate glycogen supply prevented substantial AMPK phosphoryla-
tion [14]. At variance with AMPK, calcium-dependent signaling represents a para-
digm of contraction-associated signaling for mitochondrial biogenesis. Following
contraction-induced calcium release from the sarcoplasmic reticulum (12), calcium
increments activate mitochondrial biogenesis through pathways involving protein
kinase C and Ca-calmodulin-activated protein kinase [15] while also leading to tran-
scriptional activation of PGC-1α, playing a pivotal role in orchestrating coordinat-
ed nuclear and mitochondrial gene expression [9, 16].
8.4.3 Nutrition
Fatty Acids
Several in vitro studies using muscle cell preparations including C2C12 myotubes
or primary cultures from skeletal muscle cells have found a negative impact of free
fatty acids on the expression levels and function of several mitochondrial genes, as
reflected by mitochondrial DNA, enzyme activities, and ATP production [24-26].
The saturated fatty acid palmitate has been mostly, although not exclusively, used
in experimental protocols and it appears that differential, less negative effects may
be observed with unsaturated fatty acids [27]. In one study, however, the enhance-
ment of AMPK activation despite the concomitant impairment of measured mito-
chondrial parameters was reported [25]. In addition, fatty acids were shown to en-
hance transcriptional expression of the muscle isoform of the rate-limiting enzyme
for fatty acid oxidation (CPT-I) in cultured cardiomyocytes [28]. The above obser-
vations are consistent with potential stimulatory effects by fatty acids on muscle mi-
tochondrial biogenesis and their own oxidative metabolism.
In vivo reports on the impact of substrate availability on muscle mitochondrial func-
tion in experimental models provide the most controversial results. Studies on excess
dietary fat are mostly based on high-fat feeding for several weeks, leading to diet-in-
duced obesity. Short-term studies are also available that avoid the impact of substan-
tial changes in body weight and fat content, although they also prevent the onset of
potential adaptive metabolic muscle changes. A negative impact of high-fat feeding
on several mitochondrial parameters, with particular regard to mitochondrial enzyme
activities and oxygen consumption, has been observed in some studies [29, 30].
Additional investigations in rodent models, however, reported mitochondrial stimu-
latory effects of dietary fat at the level of protein content and oxidative capacity
[31,32]. A positive role of free fatty acids was specifically confirmed in one paper in
which free fatty acid elevation was induced by heparin treatment in the context of fat-
induced obesity [32]. A potential stimulatory effect of fatty acids on mitochondrial
biogenesis could also be mediated through activation of peroxisome proliferator-ac-
tivated receptor δ (PPARδ) which can in turn enhance PGC1α expression at a post-
transcriptional level [12]. Short-term treatment with high-fat diets, also in humans,
leads to a negative modulation of muscle PDH activity through stimulation of PDH
kinase [33]. This effect indicates one potential mechanism whereby enhancement of
fat oxidative utilization may be associated with insulin resistance through substrate
competition and impaired glucose oxidation.
8 Muscle Biopsy To Investigate Mitochondrial Turnover 73
Glucose
The direct impact of hyperglycemia on mitochondrial parameters in muscle cell
preparations has been less extensively studied. Available reports suggest a potential
negative impact of sustained glucose elevations on substrate oxidation and mito-
chondrial functions in both skeletal and cardiac muscle preparations [34, 35]. With
regard to glucose levels, diabetic models are often associated with skeletal muscle
mitochondrial dysfunction. As stated above, it is difficult to dissect the potential role
of glucose per se from that of concomitant profound hormonal and metabolic dis-
turbances. One interesting observation from the insulin-deprived, markedly hyper-
glycemic streptozotocin-diabetic rodent model reported a lack of muscle mito-
chondrial dysfunction in untreated insulinopenic animals [36]. A seemingly para-
doxical impairment of mitochondrial function following glucose lowering by in-
sulin replacement was in turn reported, thereby confirming the complexity of meta-
bolic interactions in these models [36].
Oxidative Stress
Production of reactive oxygen species (ROS) from incompletely reduced oxygen
molecules is inevitably associated with oxidative substrate metabolism. Antioxidant
systems eliminate ROS and maintain their tissue concentrations within physiolog-
ical levels. Excess ROS production may however overcome antioxidant capacity,
thereby leading to oxidative stress, with damage to cell and tissue molecules, and
potential disease. Importantly, oxidative stress has been long postulated to cause ag-
ing through mitochondrial damage, in the mitochondrial or oxidative theory of ag-
ing [22]. Recent studies have demonstrated that exposure to high levels of fat sub-
strates (fatty acids in vitro and high-fat or high-calorie feeding in vivo) enhances
ROS production in muscle cell preparations [37] or in muscle tissue [38, 39]. In one
paper [38] diet-induced oxidative stress in skeletal muscle was directly reported to
cause tissue mitochondrial dysfunction, suggesting that excess lipid substrates in-
duce mitochondrial alterations at least in part by altering tissue redox state. Chronic
and acute glucose elevation has been also reported to induce oxidative stress, with
a relevant role in the onset of diabetic complications [40] and a potential negative
impact on tissue energy metabolism.
Inflammation
Both local and systemic inflammation result from imbalanced production of proin-
flammatory and antiinflammatory cytokines. While acute inflammation represents
an adaptive mechanism contributing to limit and reverse specific infectious or trau-
74 R. Barazzoni
8.5.1 Exercise
Aerobic exercise has been almost invariably reported to enhance muscle mitochon-
drial oxidative capacity and to activate mitochondrial biogenesis in humans. Several
more recent studies have confirmed the role of PDH kinase in the regulation of sub-
strate selection and fat in glucose-derived acetylCoA utilization [47]. The regulato-
ry network of AMPK and SIRT1 activation has been fully confirmed also in human
skeletal muscle as a key regulator of exercise-induced mitochondrial biogenesis in-
volving PGC1α activation [13, 48-50]. Besides the sustained effect of regular train-
ing, acute bouts of exercise have been also shown to enhance PGC1α expression
and activity [49, 50], indicating its involvement also in the short-term regulation of
mitochondrial energy metabolism. It is important to point out that the positive im-
pact of aerobic exercise on muscle mitochondrial biogenesis and oxidative capaci-
ty has been confirmed under several pathophysiological conditions characterized by
altered aerobic capacity and impaired mitochondrial function. These groups in-
clude obese, insulin-resistant, and type 2 diabetic patients, aging subjects and pa-
8 Muscle Biopsy To Investigate Mitochondrial Turnover 75
tients with chronic wasting diseases and loss of lean muscle mass. These effects will
be discussed below.
In the last decade, it has become clear that altered muscle lipid metabolism plays a
key role in the onset of insulin resistance in obesity and type 2 diabetes [51].
Muscle lipid accumulation may lead to impaired insulin signaling, with reported in-
volvement of diglycerides and ceramides [52], whereas the potential direct role of
triglyceride accumulation is still under debate [53]. It has been hypothesized that al-
tered mitochondrial function contributes to lipid accumulation, and thereby to in-
sulin resistance in obese and diabetic skeletal muscle [54]. The hypothesis is plau-
sible also in the light of the favorable mitochondrial effects reported from experi-
mental studies for caloric restriction and exercise training, i.e., the two corner-
stones of lifestyle changes recommended as treatment of obesity and type 2 dia-
betes. In the following sections, the in vivo evidence obtained through muscle
biopsy studies in favor and against a causal role of muscle mitochondrial dysfunc-
tion in the onset of insulin resistance are summarized.
On the other hand, short-term reduction of circulating free fatty acid concentra-
tion following administration of the inhibitor of lipolysis acipimox resulted in a
paradoxical decrease of PGC1α and energy metabolism gene expression in insulin-
resistant subjects [64]. In agreement with these findings, lack of negative effects on
muscle mitochondrial ATP production and membrane potential was reported in an-
other study in healthy humans following acute fatty acid elevation [65]. Negative
effects have been reported for acute marked hyperglycemia on mitochondrial res-
piration in type 2 diabetic patients [66], although sustained changes in glucose
control did not appear to affect muscle mitochondrial function in the same report
and in other studies on type 2 diabetic muscle [66].
Dietary Treatment
Long-term studies of controlled dietary manipulation are difficult in humans and
muscle biopsy studies of mitochondrial function following sustained low-calorie di-
etary treatment in obese and insulin-resistant patients are scarce. Data from select-
ed groups of healthy individuals voluntarily undergoing long-term strict hypocaloric
dietary regimens have confirmed its substantial positive impact on several car-
diometabolic risk factors including circulating lipids and systemic inflammation
markers [18]. The potential positive impact on muscle energy metabolism has how-
ever not been measured under these conditions. Overall, evidence indicates that
weight loss obtained in the absence of exercise training over a period of several
weeks to a few months does not substantially stimulate muscle mitochondrial ener-
gy metabolism [67] despite concomitant increments in insulin sensitivity.
Exercise
Muscle mitochondrial effects of aerobic exercise training for usually 12-16 weeks
have been extensively investigated in humans and the beneficial impact has been
confirmed by most studies also in human obesity and insulin resistance [68-71].
Aerobic training was indeed reported to enhance mitochondrial enzyme activities,
respiration and lipid oxidative capacity [68-71] also in obese insulin-resistant patients,
and studies further demonstrated a positive impact of exercise training with dietary in-
tervention on fatigue and aerobic performance in diabetic patients [72, 73].
function after 3 days of fasting, that was attributed by the authors to acute muscle
insulin resistance due to high fatty acid mobilization and availability [76].
Fig 8.3 Potential interactions between skeletal muscle mitochondrial dysfunction and insulin resist-
ance, and the putative role of oxidative stress and inflammation
ations. The oxidative or mitochondrial theory of aging postulated decades ago that
age-related tissue dysfunctions are due to progressive accumulation of oxidative
damage specifically to mitochondria, where oxidative reactions generate high lev-
els of reactive oxygen species [22]. A high prevalence of mitochondrial DNA mu-
tations and deletions has been reported also in human tissues, providing general
support for the hypothesis [82]. Importantly, biopsy studies have confirmed a gen-
eral decline in all steps of mitochondrial gene expression in skeletal muscle in oth-
erwise healthy aging humans, including low mitochondrial transcript levels, protein
synthetic rate, enzyme activities and ATP production [83, 84]. Based on current
knowledge and the above consideration, the pathogenesis of mitochondrial dysfunc-
tion in aging muscle is multifactorial and likely due to a combination of oxidative
stress, inflammation as well as sedentary lifestyle. Indeed studies in which aging
and young participants were matched for sedentary or active lifestyle indicate that
a lack of physical activity rather than aging per se is a major determinant of mito-
chondrial dysfunction [13]. Since insulin resistance may impair muscle mitochon-
drial anabolism, it is plausible that age-related insulin resistance also contributes to
these changes.
Similar to aging, several chronic and acute wasting conditions characterized by
loss of muscle mass are often also characterized by skeletal muscle mitochondrial
alterations. Impaired mitochondrial oxidative capacity in terms of citrate synthase
and respiratory chain enzyme activities has been reported in muscle biopsies from
chronic kidney disease, chronic heart failure and chronic obstructive pulmonary dis-
ease patients [85-88]. Critically ill patients with multiple organ failure also show an
impairment of skeletal muscle mitochondrial enzyme activities [89]. It is possible
to hypothesize that these alterations are due at least in part to a combination of low-
grade inflammation, oxidative stress, insulin resistance and low physical activity
which often characterize the disease condition. In chronic kidney disease patients
undergoing conservative treatment, a decline in muscle mitochondrial protein syn-
thesis has been specifically reported, associated with declining synthesis rates of
8 Muscle Biopsy To Investigate Mitochondrial Turnover 79
mixed proteins, mainly contractile ones [90]. It is important to point out that ATP
is required for the maintenance of muscle proteins mass, such that impaired muscle
mitochondrial turnover and function may contribute to muscle wasting under sev-
eral chronic disease conditions.
8.6 Conclusions
Skeletal muscle strongly relies on a constant, adequate energy supply. Mitochon-
drial oxidative phosphorylation provides adequate amounts of ATP under physio-
logical conditions, playing a major role in glucose and lipid substrate utilization and
contributing to preserve muscle protein anabolism. Muscle mitochondrial dysfunc-
tion occurs in obesity, insulin resistance and chronic diseases associated with meta-
bolic abnormalities as well as impaired muscle mass and strength. Inflammation,
oxidative stress and insulin resistance likely contribute to disease-associated mito-
chondrial changes, and they can be induced and modulated by changes in nutrient
intake and nutritional status. Exercise training represents a major stimulator of mi-
tochondrial biogenesis and a powerful therapeutic tool for disease conditions in-
volving mitochondrial-related alterations.
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Introduction to the Tracer-Based Study
of Metabolism In Vivo 9
Andrea Caumo and Livio Luzi
9.1 Introduction
The term homeostasis was coined around 1930 by the American physiologist
Walter Bradford Cannon and popularized in his book The Wisdom of the Body.
Homeostasis refers to the attempts of living organisms to maintain certain physi-
ological variables (temperature, acid-base balance, blood glucose, etc.) within
narrow margins of variation.
Cannon developed the concept of homeostasis, expanding on Claude Bernard’s
idea of the milieu interieu, that is, the body’s internal environment, whose stabil-
ity is a prerequisite for the maintenance of life. Around 1935, Rudolph
Schoenheimer refined this insight in a theoretical framework that quantitatively de-
scribed the dynamic state of body constituents.
The main idea is that the concentration of a substance in the body is a function
of three processes that occur simultaneously: production/secretion, distribution/ex-
change between the blood and other body fluids, and utilization/disposal. The
continuous renewal of the circulating levels of a substance is called turnover.
Schoenheimer was a pioneer in the use of radioactive and stable isotopes to study
the turnover of proteins and lipids in animals, and his work exerted tremendous in-
fluence on subsequent generations of biochemists.
Indeed, the dynamic state of body constituents has become a paradigm of bio-
medical research. In this chapter, we introduce the reader to the fundamental
tracer-based methods used to measure the turnover of a particular substance.
A. Caumo ()
Department of Sport Sciences, Nutrition and Health
University of Milan, Milan, Italy
e-mail: andrea.caumo@unimi.it
The fundamental concepts underlying tracer-based methods can be more easily un-
derstood by describing the metabolic system under study with a compartmental
model in which there are a finite number of compartments with specified intercon-
nections among them. Each compartment is an idealized store of the substance of
interest that behaves like a distinct, homogenous, well-mixed amount of material.
Each interconnection represents the flux of the substance of interest, which in
physiological terms represents transport from one location to another, chemical
transformation, or both. In the model, we must distinguish between compartments
that are accessible for measurement and those that are nonaccessible. Usually,
there is only one accessible compartment (the blood) where one can measure the
concentration of the substance, while the other, nonaccessible compartments rep-
resent the organs and tissues in which the substance is distributed. In the example
shown in Fig. 9.1, the accessible compartment is indicated by a dashed line and a
bullet, while the nonaccessible portion of the system is denoted by a gray area. The
nonaccessible portion consists of three interconnected compartments, two of which
exchange with the accessible pool. Arrows connecting the compartments represent
fluxes of the substance from one compartment to another. It can be seen that the
production of the substance, denoted as P, is a flux that enters the accessible com-
partment directly. This corresponds to the very common situation in which the sub-
stance, once produced or secreted, is released directly into the bloodstream. We can
also observe that there are fluxes leaving the system not only from the accessible
compartment but also from two nonaccessible compartments. Such outfluxes may
represent utilization, elimination, or degradation. The presence of outfluxes in
nonaccessible compartments is common and is well-exemplified by glucose
turnover. Glucose is produced endogenously in the liver and enters the blood cir-
culation. Two sites of glucose utilization are, for example, the brain and the mus-
cular system, which are distinct from the blood.
The fluxes of the substance between one compartment and another, as well as the
masses of the substance in various compartments of the metabolic system, are gov-
erned by the mass-balance principle. Consider a generic compartment within the
body. The mass-balance principle states that at any point in time the rate at which
the substance’s mass changes within the compartment is the difference between the
mass entering the compartment and the mass leaving the compartment. The intu-
itive explanation is that each molecule of the substance entering the compartment
has only two options: either leave the compartment or contribute to increasing the
mass within the compartment. Let us call Fin the sum of the fluxes entering the
compartment and Fout the sum of all fluxes leaving the compartment (Fig. 9.2). The
relationship between input and output fluxes and the mass of the substance in the
compartment, q(t), is governed by the mass-balance equation:
dq(t)
= Fin(t) - Fout (t) (1)
dt
At Fin > Fout the mass in the compartment increases; at Fin < Fout it decreases,
and at Fin= Fout it remains constant.
We can apply the mass-balance principle to any of the compartments of the
where q1 denotes the mass of the substance in the accessible compartment. Let us
now turn our attention to the metabolic system at the whole-body level. In this
case, we must consider both the accessible compartment and the nonaccessible
compartments. As far as the whole system is concerned, P denotes the endogenous
production flux and comprises all de novo fluxes of the substance entering the
metabolic system, while U denotes whole-body utilization and comprises all flux-
es irreversibly leaving the metabolic system. P and U are related by the mass-bal-
ance principle applied to the entire metabolic system:
dqT (t)
= P(t) - U(t) (3)
dt
where qT denotes the total mass of the substance in the metabolic system.
What is the relationship between the accessible pool fluxes Ra and Rd and the
whole-body fluxes P and U? In the following we assume, for the sake of simplic-
ity, that the newly produced substance (the de novo entry) appears directly in the
accessible compartment from which blood samples are drawn. In this case, P co-
incides with Ra (minus any exogenous component). As far as utilization is con-
cerned, U does not necessarily coincide with Rd because the substance is typical-
ly utilized by tissues in the accessible compartment and in the nonaccessible com-
partments.
the drain-pipe represent the production and utilization fluxes, respectively, and the
water level in the tank represents the plasma concentration of the substance.
Suppose that the tank is initially empty. When the faucet is opened, the water
level in the tank will rise until a constant level is attained. In this state of equilib-
rium, the flow of water leaving the tank balances the flow of water entering the
tank. If the faucet is now closed, the water level will start decreasing such that
eventually the tank remains empty. In this “thought experiment”, at any point in
time the water level in the tank is the result of the dynamic balance between the
flow of water entering the tank from the faucet and the flow of water leaving it
through the drain-pipe.
The hydraulic analogy allow us to naturally approach the concepts of steady state
and turnover. The tank is in steady state when the water level does not change in
time and the flows of water entering the tank and leaving it are constant and equal.
Analogously, a metabolic system is in steady state with respect to a particular sub-
stance when the concentration of the substance remains constant in time and the
utilization and production fluxes are constant and equal. At steady state, the mass-
balance principle applied to the whole metabolic system yields the following rela-
tionship:
dqT(t)
= 0; P = U (5)
dt
90 A. Caumo and L. Luzi
Thus, in steady state the total mass in the system does not change in time (its
time derivative is 0) and P is equal to U. Also, under this condition the mass-bal-
ance principle applied to the accessible compartment yields the following relation-
ship:
dq1(t)
= 0; Ra = Rd (6)
dt
Fig. 9.4 Block diagram of the glucose-insulin system and time courses of the plasma concentrations
of glucose and insulin during a meal. Glucose is produced endogenously by the liver and is utilized
by insulin-independent tissues such as the central nervous system and by insulin-dependent tissues,
including muscle and fat. Insulin is secreted by the beta-cells of the pancreas and is degraded main-
ly in the kidneys and liver. In the fasting steady state, glucose and insulin concentrations are con-
stant in time and have values of around 85 mg/dl (and 7 μU/ml), respectively. The oral ingestion of
a meal shifts the system out of steady state and into a non-steady state. The increase in glycemia
stimulates insulin secretion by the pancreas, with the circulating insulin exerting its hypoglycemic
effect by inhibiting glucose endogenous production and stimulating peripheral glucose uptake by
the insulin-dependent tissues. This chain of events acts as a feedback loop, returning the metabol-
ic system to its pre-existing steady state
The clearance rate (CR) is usually defined in relation to the accessible compart-
ment and is a measure of the rate at which a substance is removed from that com-
partment. It is calculated as the ratio between the substance’s rate of disappearance
from the accessible pool and its plasma concentration:
Rd
CR = (7)
c
92 A. Caumo and L. Luzi
Fig. 9.5 Hydraulic analogy illustrating the necessity of a tracer experiment to quantitate the turnover
of a substance. The water level in the two tanks is the same but the input and output flows of tank
A differ from those of tank B. This exemplifies the notion that the simple measurement of the plas-
ma concentration of a substance in steady state is necessary but not sufficient to quantitate the
turnover of that substance
9 Introduction to the Tracer-Based Study of Metabolism In Vivo 93
Analogously, it may happen that two people have the same plasma concentration
of the substance but different turnover rates. In summary, knowledge of the
steady-state plasma concentration of the substance is valuable but it is not suffi-
cient to permit assessment of the turnover. To overcome this difficulty, it is nec-
essary to resort to an experiment entailing the administration of a tracer. A trac-
er is a substance (labeled with a radioactive or stable isotope) having the same
metabolic behavior of the substance under study (the tracee). A tracer is thus an
exogenous indicator that is administered to trace the metabolic pathways of the
tracee. The tracer is administered into the accessible compartment and its plasma
concentration is measured by withdrawing blood samples from this compart-
ment. The rationale for the use of the tracer takes advantage of the asymmetric in-
formation concerning the tracer and the tracee. Whereas the tracee input (i.e., the
turnover rate) is unknown, the input tracer flux (i.e., the tracer administration rate)
is known. The tracer administration rate, when coupled with the tracer concentra-
tion measurements, allows the investigator to calculate the tracer disappearance
rate (using the mass-balance principle applied to the tracer). This in turn allows
calculation of the tracer CR. Since we are assuming that the tracer behaves kinet-
ically like the tracee, the tracer CR coincides with that of the tracee. Once the
tracee CR is known, the turnover of the tracee can be easily computed using Eq.
7. In the following section, we provide a formal description of the characteristics
of a tracer, illustrating the most common formats of tracer administration em-
ployed to quantitate the turnover and CR of a substance.
An ideal tracer has the following characteristics: (a) it has the same metabolic be-
havior as the tracee (this is known as the tracer-tracee indistinguishability princi-
ple); (b) it is distinguishable from the tracee; (c) administration of the tracer does
not perturb the metabolic system. Real tracers satisfy such conditions to different
extents. For the sake of simplicity, hereafter we assume that we are working with
an ideal tracer. A tracer is injected into the body with the purpose of quantitating
the unknown fluxes of the tracee. The design of a tracer experiment entails choic-
es regarding the format of tracer administration (how much tracer and for how
long) and the sampling schedule, i.e., the time points at which blood samples
must be collected in order to measure the plasma tracer concentration. In the fol-
lowing, we describe the two most common tracer experimental protocols used to
quantitate tracer turnover: the constant-infusion technique and the single-injection
technique.
Fig. 9.6 Time courses of plasma tracer concentration during a constant-infusion (a) and a single-in-
jection (b) protocol. The solid lines represent the theoretically predicted time courses of tracer con-
centration; the black dots represent the actual tracer concentrations measured in the blood samples
collected during the experiment. The actual experimental data do not coincide with the theoretical
prediction due to the unavoidable presence of experimental noise
tracer has achieved steady state, at which its rate of disappearance equals its rate
of appearance:
R*a = R*d (8)
At this point, both the tracee and the tracer are in steady state. As a result, ac-
cording to the tracer-tracee indistinguishability principle, the tracer and tracee
concentrations in plasma are proportional to their respective rates of entry into the
system:
R d : R *d = c : c (9)
We can thus calculate the turnover, that is the rate of appearance/disappearance
of the tracee:
Ra = Rd = R*a . c (10)
c*
9 Introduction to the Tracer-Based Study of Metabolism In Vivo 95
In the single-injection technique, a dose of tracer is rapidly injected into a vein, fol-
lowed by frequent sampling to measure the tracer concentration. This format of
tracer administration results in a tracer concentration decay curve (Fig. 9.6b) that
can be well described by a sum of decaying exponentials:
n
where c* is the plasma tracer concentration and (Ai) and (λi) are the coefficients
and eigenvalues , respectively, of the multi-exponential function. In the following,
for the sake of simplicity, let us suppose that the disappearance curve can be de-
scribed by a single exponential term:
By taking the natural logarithm of both sides of Eq. 14, we obtain the follow-
ing relationship between t1/2 and λ:
loge(2) 0.69 (16)
t1/2 = =
λ λ
D* D* . λ (17)
CR = ⴥ =
[AUC] 0 A
where D* is the tracer dose injected at time 0 and AUC (area under the curve) is
the area under the tracer-disappearance curve. When the CR becomes available,
turnover can be calculated by multiplying the CR by the steady-state concentration
of the tracee. All in all, the issue of determining both CR and turnover is solved
once the parameters A and λ of the mono-exponential function are estimated from
the tracer concentration data. This can be accomplished using any software capa-
ble of performing nonlinear regression.
Both the continuous infusion protocol and the single-injection protocol allow the
investigator to quantify the CR and the turnover of a substance. The benefits and
shortcomings of the two experimental approaches are symmetrical. The continu-
ous-infusion protocol requires an infusion pump and is rather invasive and labor-
intensive. On the other hand, the blood samples that the investigator needs to
withdraw are very few because the calculations for CR and turnover are based ex-
clusively on the plateau (i.e., steady state) level of the tracer. Likewise, the single-
injection approach is simple and requires no special equipment. However, calcu-
lations for CR and turnover hinge on the evaluation of the area under the tracer-dis-
appearance curve, which requires that blood samples are drawn frequently enough
and that the experiment extends for a time sufficiently long so as to ensure accu-
rate estimates of parameters A and λ.
The two protocols may also be performed simultaneously. The resulting exper-
imental protocol is called primed, continuous infusion. When the dose of the trac-
er injection (i.e., the prime) is chosen appropriately, the plasma tracer concentra-
tion quickly reaches a plateau. When the tracer plateau has been reached, CR and
9 Introduction to the Tracer-Based Study of Metabolism In Vivo 97
Suggested Reading
1. Jacquez JA (1992) Theory of production rate calculations in steady and non-steady states and
its applications to glucose metabolism. Am J Physiol 262:E779-E790
2. Cobelli C, Caumo A (1998) Using what is accessible to measure that which is not: necessi-
ty of model of system. Metabolism 47:1009-1035
3. Zierler K (1999) Whole body glucose metabolism. Am J Physiol 276:E409-E426
4. Carson and Cobelli (2001) Modeling methodology for physiology and medicine. Academic
Press, San Diego
Physical Activity and Inflammation
10
Raffaele Di Fenza and Paolo Fiorina
The exact etiology of T2D is currently unknown, as the pathogenesis of the charac-
teristic insulin resistance and/or impaired insulin secretion is unclear. However, fol-
lowing up on the hypothesis formulated by Pickup et al. in 1997 and 1998, recent
studies have shown that innate immunity, stress and acute-phase responses, and
more specifically inflammation play leading roles in the development of obesity-re-
lated insulin resistance in T2D [5-9]. Indeed, compared to T1D patients, those with
T2D generally test positive for serum pro-inflammatory cytokines such as C-reactive
protein (CRP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α [9]. For exam-
ple, studies directly linking these pro-inflammatory cytokines to T2D have shown
that in obese rodents TNF-α levels are elevated in adipose tissues and blood samples
and that neutralization of TNF-α can greatly improve insulin sensitivity in these an-
imals [9, 10]. TNF-α causes insulin resistance by indirectly phosphorylating in-
sulin receptor substrate (IRS)-1 and IRS-2, thus inhibiting insulin signaling [11, 12].
It has been proposed that lipid accumulation in adipocytes, inducing a state of cel-
lular stress marked by activation of JNK and NF-κB, leads to an increase in the se-
cretion of TNF-α and other pro-inflammatory cytokines [13]. The pro-inflammato-
ry cytokines IL-6 and CRP are also capable of decreasing insulin sensitivity, either
by degrading the peroxisome proliferator-activated receptor γ (PPARγ), a key regu-
lator of normal insulin sensitivity, or by inducing the suppressor of cytokine signal-
ing proteins (SOC), which targets IRS for degradation [11, 12, 14].
The pro-inflammatory response in T2D is activated via the JNK/activator protein
1 (AP1) and IKK/NF-κB signaling pathways in adipose tissue [15]. This can, in turn,
lead to cell death or to the establishment of a characteristic inflammatory response
that involves the recruitment of macrophages. The subsequent release of pro-in-
flammatory cytokines by macrophages results in enhanced activation of the JNK1
and IKK/NF-κB pathways [16]. In a positive feed-forward loop, the activation of
these pathways induces chemokine release, which again recruits macrophages to adi-
pose tissue. Finally, a pro-inflammatory site is established that causes insulin resist-
ance also in neighboring adipocytes via paracrine effects [9, 16]. The pro-inflamma-
tory role of adipocytes is also a consequence of their secretion of free fatty acids
(FFAs) and adipokines such as leptin and adiponectin, both of which promote insulin
sensitivity [9, 17].
The pancreatic islets of patients with T2D undergo apoptosis due a severe process of
inflammation and functional exhaustion. Among the factors that mediate islet de-
struction are leptin, IL-1β, TNF-α and lipoproteins. Thus, leptin is not only in-
volved in insulin secretion; it is also capable of inducing pancreatic β-cell apoptosis
by enhancing the release of IL-1β and diminishing that of IL-1 receptor antagonist
in human islets [18]. The release into the blood stream of FFAs and lipoproteins,
which is usually a direct consequence of obesity in T2D patients, is detrimental to β-
cells as it provokes a reduction in insulin content, abnormally elevated insulin release
in the absence of stimuli, and a diminished capacity of these cells to secrete insulin
in response to glucose [19-21]. Moreover, some fatty acids, such as palmitate, are ca-
pable of inducing β-cell apoptosis [22, 24]. Studies carried out by Solinas et al. and
Arkan et al., utilizing IKKβ− or JNK1-knockout mice, showed that either strain of
knockout mice was resistant to induced glucose intolerance, hyperinsulinemia, and
insulin resistance in adipose and skeletal muscle tissue [25, 26]. Given that these
mice developed the same degree of obesity as their wild-type counterparts, it seems
that obesity itself cannot cause insulin resistance without a functional inflammato-
ry component. The active role of IL-1β in β-cell impairment and apoptosis led to the
10 Physical Activity and Inflammation 101
concept that IL-1β is a potential target to protect β-cells in patients with T2D.
Indeed, in a double-blind randomized controlled study the administration of recom-
binant human IL-1ra (Anakinra) led to improvements in glycemia and β-cell secre-
tory function as well as a reduction in the markers of systemic inflammation [27].
tive (Fig. 10.1) [34]. In another cross-sectional study, circulating levels of sTNF-R1,
sTNF-R2, IL-6, and CRP were measured in 859 individuals. All of these markers
were significantly decreased in those who were more physically active. However, ad-
justment for body mass index (BMI) and leptin diminished the association’s strength,
suggesting a role of fat tissue loss in the beneficial effect of exercise on inflamma-
tion [35]. Nonetheless, the results do not always point in the same direction. In
Verdaet’s study, a correction for BMI negated the correlation between inflammation
and exercise [36]. In a sample of 2833 individuals, aerobic exercise and CRP levels
were statistically correlated even after correcting for BMI, although there was no cor-
relation at all between exercise and CRP levels in women [37].
Longitudinal studies have the advantage of allowing the testing of lifestyle in-
tervention effects on the study parameters. In one such study, 39 patients with
coronary artery disease (CAD) underwent percutaneous coronary intervention (PCI)
and were then divided into two groups. Patients receiving PCI only had a significant
decrease in IL-6 levels, while those undergoing a 14-week rehabilitation program
had significant decreases in IL-6, TNF-α, and PCR levels (Fig. 10.1) [38]. Another
study of CAD patients, in this case undergoing 12 weeks of endurance training,
showed that exercise significantly decreases MMP-8 and IL-8 levels but has no sig-
nificant effect on IL-6 or CRP [39]. Two studies on overweight adolescents gave
contradictory outcomes concerning CRP levels: a 12-week aerobic training program
showed a decrease (together with a decrease in body fat and insulin resistance) but
an 8-week program showed no such reduction [40, 41]. In Okita’s study on 199
women with 2 months of exercise training, there was an association between weight
loss and CRP decrease [42]. Weight loss appears to be a determinant in decreasing
CRP levels. After a 4-month exercise period, serum CRP levels were measured in
a study group made up of middle-aged, overweight, insulin-resistant individuals.
After correcting for various confounding factors, no association could be observed
between fitness, expressed as VO2max, and exercise itself [43]. In 43 study partici-
pants exercising regularly during a 6-month period, there was a significant decline
in IFN-γ and TNF-a production, while the levels of IL-4, IL-10, and TGF-β signif-
icantly increased (Fig. 10.1) [44]. A 12-week period of aerobic exercise resulted in
a decrease in pro-inflammatory cytokines such as IL-1, IL-6, IFN-γ, and CRP in 28
patients with stable CAD [45].
Various tissues and cell populations have been studied to determine the mecha-
nisms by which exercise decreases inflammation. Based on its secretion of several
hormones and inflammatory mediators, adipose tissue can be considered as an en-
docrine organ [46]. The proliferation of this tissue is associated with hypoxia, which
causes the HIF-1 mediated release of IL-6 and MIF [47]. By contrast, adiponectin,
a unique anti-inflammatory adipokine whose levels inversely correlate with those of
CRP and with insulinemia [48], increases with exercise, as was shown in a study of
middle-school children [49]. White adipose tissue is also the site of macrophage ac-
cumulation in humans and rodents, with significant amount of circulating TNF-α
coming from these cells [50, 51]. This population of macrophages contributes to the
low-level chronic inflammation associated with excess body fat [52] and is attract-
ed to the tissue by MCP-1 production. Weight loss has been shown to decrease
10 Physical Activity and Inflammation 103
MCP-1 expression as well as peripheral levels of MCP-1, leptin, and insulin [53],
while exercise alters cytokine production by PBMCs [44].
Skeletal muscle is also considered as an endocrine organ; in fact, it is the largest
in the human body, secreting mediators (myokines) that have the ability to reduce or
enhance systemic low-level inflammation. The main myokine is IL-6. Muscle is its
major source and during acute exercise its secretion correlates with exercise inten-
sity, with circulating levels rising by up to 100-fold and then in the post-exercise pe-
riod declining [54]. Baseline levels of IL-6 are lower in individuals engaged in
physical activity than in those who are sedentary, as demonstrated in a randomized
trial on obese premenopausal women [55]. Despite being an acute-phase protein,
studies have suggested anti-inflammatory roles for IL-6 as well [30]. In Starkie’s
study, IL-6 administration reduced the inflammatory response to LPS [32]. In diabet-
ics given rhIL-6, insulinemia improved, with insulin levels reaching those of age-
and BMI-matched healthy people [56]. Controversially, IL-6 levels were found to be
higher in patients with impaired glucose tolerance than in healthy (insulin secretion
blocked) controls and the higher plasma levels lasted longer [57].
high fat/high sucrose diet (HFD), TNF-α, MCP-1, PAI-1, and Ikkβ (all inflammato-
ry cytokines) were higher than in control mice fed a balanced diet [63]. In HFD-fed
mice that performed a 4-week exercise session, expression levels of the same cy-
tokines were still high but there was improved glucose tolerance and insulin sensi-
tivity [63]. These results suggest that exercise can partially mitigate adiposity, reverse
insulin resistance, and decrease adipose tissue inflammation in mice with diet-in-
duced obesity [63].
Physical activity has also been associated with modification of the adaptive re-
sponse of skeletal muscles [64]. Individuals with insulin resistance and/or T2D tend
to have more glycolytic type IIx skeletal muscle fibers than healthy individuals,
and fiber distribution has been linked to insulin resistance [64, 65]. A relationship
was determined between exercise and increased AMP-activated protein kinase
(AMPK), which, according to Rockl et al. is an important mediator of changes in
muscle fiber type; this, in turn, led the authors to speculate about the enzyme’s role
in exercise-induced insulin sensitivity in skeletal muscle fibers [64]. In another
work, by Kivela et al., exercise was shown to induce vascular endothelial growth fac-
tor-A (VEGF-A), a major angiogenesis factor [66]. In a post-exercise comparison of
the capillary mRNA values of diabetic and healthy mice, only in the latter were
VEGF-A values markedly higher than in the sedentary control groups. Indeed, in di-
abetic mice, while there was no significant post-exercise increase in the levels of
VEGF-A, this was not the case for thrombospondin-I (TSP-I), a known angiogene-
sis inhibitor [66]. By contrast, serum TSP-I was not increased in healthy exercised
mice. This study, in conjunction with the knowledge that diabetes impairs cardiac
and skeletal muscle angiogenesis, provides novel data about the distinct responses of
healthy and diabetic capillaries to exercise [66].
10.5 Conclusion
Awareness of the positive effects of exercise on inflammation is evolving.
Historically, exercise was not indicated in patients with inflammatory muscle dis-
eases but this is no longer the case [68]. The primary therapeutic strategy in patients
with T2D includes diet, physical exercise, and the anti-diabetic drug metformin
[69]. Although small-scale interventional studies have provided evidence that exer-
cise training diminishes inflammation, data from large randomized controlled trials
designed to definitively test the effects of exercise on inflammation are limited and
their results are inconclusive [70].
Further studies are needed to fully understand the effect of exercise on systemic
low-grade inflammation and to establish an optimal exercise training protocol that
significantly interferes with low-grade chronic inflammation and its role in chronic
diseases such as T2D.
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The HPA Axis and the Regulation
of Energy Balance 11
Francesca Frigerio
11.1 Introduction
The hypothalamic-pituitary-adrenal (HPA) axis is a neuroendocrine complex compris-
ing the hypothalamus, hypophysis, and peripheral adrenal glands, all of which are
joined in a feedback-loop-generating network of neural and chemical communica-
tions. The HPA is fundamental for the regulation of basic functions such as eating,
drinking, reproduction, locomotion, and response to stress, thereby assuring individ-
ual and species survival.
The hypothalamus lies at the base of the brain, in an area corresponding to the second-
ary portion of the ventral prosencephalon, ventral to the thalamus and above the third
cerebral ventricle. Sagittally, it extends from the rostral limit of the optic chiasma and
the caudal limit of the mammillary bodies.
The specialized cells of the hypothalamus have neural and secretory activity and
are referred to as endocrine neurons. These are of various dimensions and are either
organized in well defined structures called nuclei or scattered within the nervous tis-
sue. Distinct nuclei and neuronal populations carry out different physiological func-
tions. The parvocellular system is characterized by small-diameter neurons and is lo-
calized in the periventricular zone; the middle zone is composed of large-diameter
neurons comprising the magnocellular secretory system [1].
F. Frigerio ()
Novartis Farma S.p.A.
Saronno (Varese), Italy
francesca.frigerio@novartis.com
The hypophysis, or pituitary gland, lies at the base of the skull, lodged in the sella tur-
cica, a depression within the sphenoid bone. It is divided into three functionally rele-
vant lobes. The anterior lobe (adenohypophysis) accounts for 80% of the total volume
and is formed by hormone-producing glandular cells. The adenohypophysis is connect-
ed to the hypothalamic parvocellular neurosecretory system, receiving projections
principally from the periventricular hypothalamic nucleus. The posterior lobe (neuro-
hypophysis) is made up of neural cells with secretory function, glial cells (pituicytes),
and the axonal endings of neurons whose cell bodies reside in the supraoptic and par-
aventricular nuclei of the hypothalamus. The posterior and anterior lobes are connect-
ed by a third one, small and scarcely vascularized, called the pars intermedia.
The hypothalamus and pituitary gland are strictly anatomically and functionally relat-
ed through connections made up of neuronal and endocrine components, both en-
closed in the pituitary stalk, a hollow tube arising from the ventromedial hypothala-
mus. The latter has three functional portions: vascular, neuronal, and glandular [1].
The hypothalamic-pituitary system is one of the most vascularized areas in the
brain and its blood supply derives from the superior and inferior hypophyseal arter-
ies, branches of the internal carotids. The superior hypophyseal arteries run through
the median eminence and separate into a widespread capillary plexus within the ade-
nohypophysis. This is fundamental to convey hypothalamic parvocellular signals.
By contrast, the posterior part of the pituitary gland (neurohypophysis) receives blood
directly from the inferior hypophyseal arteries and communicates with the hypothal-
amus through the neuronal component of the pituitary stalk. The neuronal processes
of the latter are supported by the glial-like pituicytes. Axons projecting from magno-
cellular nuclei reach, first, the infundibular stalk. Once in the neurohypophysis, the ax-
ons terminate in the perivascular or extracellular space, in contact with fenestrated
capillaries. Hormones cross the perivascular basement to enter the blood stream [1].
The adrenal glands are located at the upper poles of the kidneys. They are subdivid-
ed into cortex (about 90% in humans) and medulla. The adrenal cortex is composed
of three histological zones: zona glomerulosa, zona fasciculata, and zona reticularis.
Cortisol is mainly produced in the zona fasciculata and, to a lesser extent, in the zona
reticularis. The effect exerted by ACTH on the adrenal cortex is significant. Within a
few minutes of its release, ACTH increases blood flow in the adrenal gland; after 2–3
h, it increases the weight of the gland, potentially doubling its size. Prolonged ACTH
stimulation provokes hypertrophy in the zonae fasciculata and reticularis, while a de-
ficiency of the hormone leads to gland atrophy and cellular apoptosis [2].
11 The HPA Axis and the Regulation of Energy Balance 111
The relation between the HPA axis and energy intake is highly complex. A stress re-
sponse, in fact, can either increase or decrease food intake, contributing to anorexia or
obesity. Therefore, studies in human and animal models are often difficult to interpret.
Moreover, the HPA axis comprises several components that play different roles in en-
ergy intake. Together with circadian regulation, HPA axis activity is determined by the
body’s energy levels and, in particular, by brain glucose availability.
It is now generally accepted that circulating ACTH and glucocorticoids induce
11 The HPA Axis and the Regulation of Energy Balance 113
orexigenic effects. In humans, excess glucocorticoids induce obesity while their de-
pletion leads to anorexia. Specifically, glucocorticoids induce nutrient-specific hunger,
preferentially encouraging carbohydrate intake and, in humans, small snacks. The glu-
cocorticoid-induced preference for small and highly energetic nutrients is intuitively
connected to the prompt energy demands required in stress situations, when arousal
of the sympathetic nervous system represses appetite. The orexigenic effect cannot be
ascribed to a peripheral role of glucocorticoids, but instead to their central effects.
CRH, the first component in the HPA axis cascade, is localized in the PVN of the hy-
pothalamus, one of the most important centers for energy intake control. CRH nega-
tively regulates the NPY neuronal pathway whereas glucocorticoids inhibit CRH se-
cretion, releasing NPY to exert its orexigenic effect. Glucocorticoids stimulate NPY
gene expression in a mechanism involving, among others, the mTOR pathway and
they potentiate NPY activity. In addition, the positive effects of ghrelin and insulin on
NPY gene expression occur only during the permissive action of glucocorticoids.
CRH and glucocorticoids play opposite roles in food intake, since cortisol increases
NPY-mediated NPY-Y2 receptor expression at the level of abdominal fat, contribut-
ing to adipogenesis. Physiologically, the conflicting effects induced by CRH and its
downstream products (glucocorticoids) can be explained by the chronological events
occurring during the stress response. Initially, the “fight or flight” response requires
a certain amount of readily available energy in the absence of the ability of the organ-
ism to search for food. Later, secreted glucocorticoids act to replenish the expended
energy stores, increasing food intake and inhibiting CRH release [7]. The induction
of satiety follows two different routes depending on whether CRF1 or CRF2 is acti-
vated. CRF1 induces a short-onset and abbreviated anorexic response, while CRF2
stimulation provokes a delayed response and has been implicated in the modulation
of gastric motility.
As mentioned above, 11β-HSD1 is responsible for glucocorticoid activation at the
tissue level. The enzyme’s expression has been extensively linked to obesity and
metabolic syndrome and is thought to play a role in the central regulation of feeding.
In obese (Zucker) rats, for example, 11β-HSD1 expression in the hippocampus is re-
duced, which implies lower central corticosterone activation and thus a reduced neg-
ative feedback of CRH release.
The interaction between glucocorticoids and the noradrenergic system has been
determined in several studies. Central administration of norepinephrine (NE) increas-
es carbohydrate-like food intake through the activation of α2-adrenergic receptors in
the PVN. Immunocytochemical studies within the PVN revealed that glucocorticoid
type II receptors receive inputs from noradrenergic fibers and that NE injection in the
PVN induces an augmented release of corticosteroids. Moreover, the stimulatory ef-
fect on food intake exerted by NE is abolished upon adrenalectomy.
are localized on several types of neurons, including those that produce NPY, ACTH,
CRH, and POMC. Leptin is also synthesized and stored in pituitary corticotroph
cells. The small amount of leptin production at the central level does not influence pe-
ripheral leptinemia; instead, it is thought to modulate the secretion of other pituitary
hormones. Moreover, it is now clear that leptin and the HPA axis are strongly corre-
lated, mutually counterbalancing each other’s effects.
Studies on rodents revealed that glucocorticoids enhance leptin mRNA levels and
secretion. In vivo, glucocorticoid infusion causes an increase in leptin production by
adipocytes through the direct stimulation of the glucocorticoid responsive element
(GRE) located in the leptin promoter [8]. Concomitantly, glucocorticoids reduce lep-
tin efficacy on food intake inhibition, acting on leptin sensitivity in the melanocortin
system. In humans, pharmacological doses of dexamethasone increase leptin mRNA
and plasma levels, although chronically glucocorticoids act also at physiological dos-
es and in a BMI-independent manner.
Conversely, leptin is able to reverse stress or starvation-induced ACTH and/or
corticosterone levels, most probably by inhibiting CRH- and ACTH-releasing neu-
rons at a central level. Normally, there is an inverse relation between plasma ACTH
and leptin concentration. Furthermore, leptin acts directly on the adrenal glands, in-
hibiting steroidogenesis at the transcriptional level [9]. While acutely leptin stimu-
lates CRH expression [10] and inhibits that of NPY, in a chronic setting it prevents
HPA axis activation. Interestingly, CRH lowers the body weight threshold at which
starving rats start to store food within their cages and participates in the central ac-
tion of leptin.
described are strongly influenced by the simultaneous levels of insulin. For example,
the lipolytic effect is more important when insulin levels are low and adrenalin levels
are high. The lipolytic effect is also regulated by catecholaminergic activity.
Glucocorticoid activity is indispensable for full adipocyte differentiation through the
repression of β1 and β3 receptor expression and the enhancement of β2 receptor ex-
pression. At the same time, β1/3 suppression inhibits both the thermogenic effect and
lipolysis.
The liver is the main target of glucocorticoid action. It is the organ responsible for
gluconeogenesis, glucose output, and glycogen synthesis and storage. As in the oth-
er organs described, glucocorticoids limit PI3 kinase activity and reduce glucose uti-
lization. Suppression of glycolytic and oxidative pathways involves FOXO-mediated
activation of PDK4. Importantly, glucocorticoid treatment augments the expression of
PEPCK and G6Pase, key enzymes in glucose synthesis. This action is accomplished
through glucocorticoid-mediated PPARα activation. The modifications in glucose
metabolism are paralleled by an increase in lipid storage and a decrease in lipolysis.
In vitro evidence describes a glucocorticoid-mediated increase in VLDL synthesis and
secretion, together with augmented expression of the enzymes responsible for triglyc-
eride synthesis. In vitro experiments have demonstrated glucocorticoid induction of
β-oxidation.
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Physical Exercise in Obesity and Anorexia
Nervosa 12
Alberto Battezzati e Simona Bertoli
A more sedentary lifestyle and reduced energy expenditure are frequently proposed
as causes of the obesity pandemics in westernized societies. This hypothesis is
sound for a number of reasons, as recently reviewed by Popkin [1]. Briefly, rapid
changes in physical activity patterns are occurring worldwide due to: (1) occupation-
al changes from agriculture and labor-intensive jobs towards the tertiary sector of the
economy; (2) a reduction of the level of physical activity within each occupation; (3)
changes in transportation systems, substantially reducing the need for walking; (4)
changes in education and in leisure activity patterns; and (5) the introduction of la-
bor-saving domestic devices and of more convenient food sources and locations, de-
creasing the cost of all home production-related activities. These changes in physi-
cal activity are characteristic of the transition from a developing to an industrialized
society and are associated with a higher mean body mass index (BMI) and a greater
prevalence of obesity among people in those countries. Therefore, it seems natural to
assume that energy expenditure differs between populations at different stages of
their social and economic development and that these differences explain, at least in
part, different rates of obesity.
Proof of this hypothesis is currently lacking as it requires objective measurements
of daily energy expenditure in free-living individuals. Such measurements can, how-
ever, be obtained with the doubly labeled water technique [2], in which water labeled
with 2H and 18O is administered and the respective isotopic enrichments in the body
water are monitored during the following days. The rate of 18O elimination is faster
than that of 2H because the former is eliminated not only as water but also as carbon
dioxide, due to the reversible reactions that combine the two molecules to form carbon-
A. Battezzati ()
International Center for the Assessment of Nutritional Status (DiSTAM)
University of Milan, Milan, Italy
e-mail: alberto.battezzati@unimi.it
ic acid in the blood. From the difference in elimination rates of 2H and 18O, daily car-
bon dioxide elimination is calculated and the daily energy expenditure estimated.
Westerterp and Speakman [3] formally addressed the hypothesis that reduced lev-
els of physical activity and thus of energy expenditure have driven the obesity epi-
demics. Their approach involved three separate tests: first, the authors examined the
20-year trend in daily energy expenditure as measured in free-living individuals in
the Dutch town of Maastricht and across the USA. When possible, resting energy ex-
penditure and physical activity level were also calculated. Despite the increment in
the rates of obesity, physical activity levels during the observation period slightly in-
creased in Europe and in the United States. Second, they compared the data obtained
in developed countries with those from third-world rural communities. Quite surpris-
ingly, once the data were normalized for body weight, age, and gender, there was no
difference in daily energy expenditure among the two groups. Finally, they compared
the data on the daily energy expenditure of modern humans with those of wild ter-
restrial mammals studied with the same doubly labeled water technique. Again,
once the data were corrected for anthropometric and ambient temperature data, it be-
came apparent that the energy expenditure of humans is in line with that of other
mammals. In summary, this analysis suggests the unlikelihood that decreases in en-
ergy expenditure have fueled recent obesity epidemics, since there is no inverse
correlation between physical activity and obesity rates. Indeed, in terms of energy ex-
penditure, the results for humans are in line with those of wild mammals.
Very recently, doubly labeled water studies were subjected to a meta-analysis to
compare energy expenditure in developing and industrialized countries [4].
Currently, this is the most comprehensive set of data available, comprising 98 stud-
ies from countries ranked low, middle, and high on the human development index
(HDI). The weight and BMI of the participants in the 14 studies from low and mid-
dle HDI countries were, respectively, almost 20 and 10% lower than the correspon-
ding values of participants from high HDI countries, but there were no differences in
daily energy expenditure when corrected for weight, age, and gender. The physical
activity level tended to decrease with age but, again, there was no association with
HDI status. Therefore, while the pattern of physical activity clearly differs between
people in developing vs. industrialized countries, available data suggest that the dif-
ference is not quantitative.
It seems therefore that daily energy expenditure and physical activity levels
are strongly related to body size, age, and gender but not to industrialization.
Thus, the hypothesis that reduced physical activity is the driver of the current obe-
sity pandemics is certainly very reasonable but it is not supported by data at the
population level.
The question whether reduced physical activity is the cause of weight gain in obese
individuals has been addressed at the individual level as well; specifically, whether
12 Physical Exercise in Obesity and Anorexia Nervosa 125
obese individuals expend less energy for physical activities, and whether decreased
physical activity is prospectively related to obesity. Also in this case, pivotal conclu-
sions could be drawn using the doubly labeled water technique rather than activity
records or accelerometry. While either of the latter techniques are able to assess the
type, intensity, and duration of each activity, they do not allow the actual energetic
costs to be measured, such that the values would have been assumed.
The results of the first doubly labeled water study in obese people clearly demon-
strated that energy expenditure is higher in obese than in lean women [5]. This find-
ing was reproduced in actively growing adolescents [6], in whom the ratio between
basal and daily energy expenditure was similar among the lean and the obese, indi-
cating similar physical activity levels. In either case, energy expenditure was shown
to increase with body size, suggesting that physical activity costs would increase as
well. Therefore, as shown in a more recent study in adolescents [7], there may be no
reductions in the actual amount of energy consumed for physical activity even when
the duration and the intensity of physical activity are decreased.
Despite the inability to demonstrate lower energy expenditure for physical activ-
ities in westernized societies with a high prevalence of obesity or in groups of obese
individuals, it should be noted that individuals gaining 0.5 kg of body weight year-
ly (the average rate of weight gain for people becoming obese in most countries)
have a positive energy imbalance of < 3% with respect to energy exchange. On a dai-
ly basis, however, this is below the quantification limit of any of the available meth-
ods to measure energy intake and expenditure. Moreover, not only are such measures
likely to be inaccurate in their detection of small energy imbalances, they may also
be untimely because the body weight increment consequent to an energy imbalance
should in turn cause a rapid increase in both basal energy expenditure and the cost
of physical activity. Therefore, if an already obese subject is studied, an energy bal-
ance between intake and expenditure may be observed but at a higher level of ex-
change, consistent with the greater body mass. If the subject is studied when he or
she is in the process of becoming obese (few prospective data are available), the im-
balance would be too small to be detected.
Since observational data are presently of little help in resolving the primacy of re-
duced physical activity vs. increased intake in the development of obesity, experi-
mental work manipulating energy intake and physical activity in humans may pro-
vide significant clues. This topic has been recently reviewed [8] based on doubly la-
beled water studies evaluating the effects of overeating on physical activity, under-
eating on physical activity, exercise training on food intake, and reduced physical ac-
tivity on food intake. An experimental increase in energy intake of 50% did not pro-
duce appreciable changes in the level of physical activity, with the exception of one
study in which energy intake was doubled and a reduction in physical activity level
was accordingly measured. By contrast, experimental energy restriction in healthy or
126 A. Battezzati and S. Bertoli
obese subjects reduces energy expenditure for both basal metabolism and physical
activity. Interestingly, adding physical exercise programs to caloric restriction in ex-
cess-weight patients produces no additional benefits in terms of weight loss, because
a decrement in non-training activity offsets the benefits of training on energy bal-
ance. Furthermore, a few studies have shown that increasing physical activity alone
does not generally produce weight loss because energy intake is concomitantly stim-
ulated. The opposite is not true, however, because reductions in physical activity do
not induce proportional decrements in energy intake. This observation is particular-
ly relevant because energy expenditure related to physical activity decreases consis-
tently between the third and fourth decades of life, and the changes in physical ac-
tivity are in reasonable countertendency with the changes in weight. Possibly, the key
to understanding obesity is to consider that the effects of over- and undereating on
physical activity and of hyper- or hypoactivity on energy intake are not exactly re-
versible. Hyperactivity and undereating may be compensated in most conditions
whereas hypoactivity and overeating may not, producing an inevitable tendency to
gain weight if food is available at any moment.
Excess weight, however, is not the sole concern of westernized societies. Quite sur-
prisingly, in an era of exceptional food availability, there has been an alarming in-
crease in undernutrition, with the clinical picture of anorexia nervosa. Clearly, the
mechanisms ensuring energy balance can be strongly impaired also in excessive
weight loss, reflecting another aspect of the current lifestyle of westernized societies.
Anorexia nervosa (AN) is a common eating disorder affecting up to 1% of
young women in Western cultures and associated with a 4% mortality. It is diag-
nosed, according to the Manual of Mental Disorders (DSM IV) [9] based on the fol-
lowing criteria: (1) refusal to maintain a healthy weight range for age and height, or
failure to appropriately gain weight during periods of growth and physical develop-
ment, (2) fear of gaining weight or becoming fat, (3) distorted body image, and (4)
absence of menstrual periods (cycle) in women not using any external source of es-
trogen, e.g., oral contraceptives. Two types of anorexia are defined: the restricting
type, in which “weight loss” is achieved by severe caloric restrictions, and the
binge and purge type, in which episodes of binge eating are followed by purging be-
havior to avoid weight gain.
AN is associated with a number of somatic and psychopathological changes that
result from semistarvation. The somatic changes consist of hypothermia, reduced
heart rate, lowered blood pressure, reduced hematopoiesis (with the production of fe-
tal hemoglobin), and certain endocrine alterations. The latter include excess produc-
tion of the catechol estrogen 2-hydroxyestrone at the expense of estradiol, leading to
amenorrhea, which frequently occurs after caloric restriction has been initiated; a re-
duction in the ratio of androstendione/etiocholanolone, due to decreased hepatic
microsomal 5α-reductase activity, a subnormal plasma dehydroisoandrosterone to
12 Physical Exercise in Obesity and Anorexia Nervosa 127
cortisol ratio, attributable to reduced 17,20 lyase activity; a reduction in brain gly-
colytic activity, with consequences for brain function; and lowering of T3, with an in-
crease of 3, 3′, 5′-triiodothyronine (reverse T3) that is associated with low physiolog-
ical activity. These somatic symptoms can be viewed as a meaningful adaptation by
the body to the reduced energy intake (patients can survive with daily energy intakes
well below 1000 kcal). The psychopathological symptoms include depression, rigid-
ity, weight phobia, and preoccupation with thoughts related to food and eating.
“drive for activity and food restriction” in AN patients have been derived from the rat
model of activity-based anorexia (ABA), also called “semi-starvation-induced hyper-
activity,” which mimics several of the characteristics of hyperactive-AN patients and
is considered appropriate to understand the neuroendocrine pathways involved in
AN. With this model it was observed that when rats with access to a running wheel
are restricted in their food intake, they paradoxically become excessively active in al-
most direct proportion to this restriction, resulting in accelerated weight loss (at least
20%). ABA rats also show hypothermia, loss of the estrous cycle, and stomach ulcer-
ation and eventually die of emaciation. When food is again provided ad libitum, ABA
rats will quickly reduce running wheel activity, increase food intake, and therefore
gain weight [13]. Ad libitum fed control rats with continuous access to running
wheels show stable levels of activity and increased food intake to compensate for the
increased energy expenditure.
The behavior of the ABA rats can be explained by a failure of the part of the brain
involved in rest and activity regulation. The discovery of adipokines (leptin,
adiponectin, and resistin) and of neural circuits controlling feeding behavior, ener-
gy expenditure, body weight, and neuroendocrine function has furthered our under-
standing of the mechanisms contributing to AN hyperactivity. In addition, opioid re-
lease caused by physical exercise decreases food intake under starvation conditions,
adding another possible aspect to the relationship between AN and exercise.
The adipokine leptin informs the hypothalamus regarding whole-body adiposity
status. In the arcuate nucleus (ARC), two populations of neurons are present that ex-
press the long form of the leptin receptor (lepr), referred to as leprb. This isoform is
crucial for leptin’s effects and exhibits antagonistic actions in the control of feeding
behavior and energy expenditure. The binding of leptin to leprb inhibits neurons ex-
pressing orexigenic agouti-related protein (AgRP) and neuropeptide Y (NPY) and
stimulates neurons expressing pro-opiomelanocortin (POMC), which encodes the
anorexigenic α-melanocyte-stimulating hormone (α-MSH), and cocaine- and am-
phetamine-regulated transcript (CART).
In the ABA model, increasing running wheel activity decreases plasma leptin lev-
els whereas leptin administered intracerebroventricularly reduces the hyperactivity
associated with reduced food intake and increases thermogenesis (resulting in a
rapidly worsening condition); in contrast, ad libitum fed rats do not decrease their
running wheel activity. These behaviors have been interpreted by assuming that lep-
tin controls hyperactivity on the basis of the state of energy balance or by the actions
of its downstream targets (ARC, NPY) or corticotrophin releasing hormone (CRH):
thus, stressful situations, such as ABA, would increase CRH expression and release,
leading to the activation of dopamine neurons in the ventral tegmental area and of
noradrenergic neurons in the locus coeruleus, i.e., brain areas implicated in reward
mechanisms and selective attention. Leptin can bind lepr expressed on dopamine
neurons, silencing these neurons and reducing the rats’ motivation to run [14].
12 Physical Exercise in Obesity and Anorexia Nervosa 129
In AN patients, leptin levels measured in plasma and cerebrospinal fluid were shown
to be low, in agreement with the reduced body weight and fat mass, and to increase
during weight gain to the extent that they were disproportionately high compared
with the levels in healthy controls. These observations suggest that hypoleptinemia
is the major signal underlying both the somatic and the behavioral adaptations to
starvation. As in the ABA model, the level of physical activity was shown to be neg-
atively correlated with leptin levels whereas during treatment this relationship be-
comes positive [14].
A recent study focusing on the adipokines profiles of AN patients confirmed the
decrement of serum leptin levels and showed a specific adipocytokines profile de-
pending on the intensity of physical activity. Hyperactive AN patients were found to
have significantly higher serum leptin levels and lower serum resistin levels than
non-hyperactive AN patients, without a difference in serum adiponectin; this contra-
dicts the hypothesis that the low leptin levels in AN facilitate the motor restlessness
and intensive exercise behavior observed in the ABA model. However, in this study,
the relationship between serum leptin levels and physical activity followed an invert-
ed U-shape curve; physical activity levels were lower in severely undernourished AN
patients, leading to the hypothesis that the effect of hypoleptinemia on physical ac-
tivity levels declines with the severity of the undernutrition [15]. Several other sig-
nificant changes in the endocrine profiles of hyperactive-AN patients have been
demonstrated. For example, a recent clinical study showed that cortisol concentra-
tions in plasma were significantly increased in hyperactive-AN patients, suggesting
a possible treatment strategy (i.e., glucocorticoid antagonists) to reduce activity.
Further investigations are needed to understand the cause-effect relationship:
does physical activity produce excessive HPA (hypothalamus-pituitary-adrenal) ax-
is activation, or does over-activity of the HPA axis (e.g., increased CRH or cortisol
secretion) ‘’drive’’ restlessness and thereby increase physical activity?. Furthermore,
the role of psychological factors in reinforcing the hyperactivity behavior remains to
be determined [16].
References
1. Popkin BM (2005) Using research on the obesity pandemic as a guide to a unified vision of nu-
trition. Public Health Nutrition 8:724–729
2. Schoeller DA (2008) Insights into energy balance from doubly labeled water. Int J Obes 32
Suppl 7:S72-5
3. Westerterp KR, Speakman JR (2008) Physical activity energy expenditure has not declined sin-
ce the 1980s and matches energy expenditures of wild mammals. Int J Obes 32:1256-63
4. Dugas LR, Harders R, Merrill Set at (2011) Energy expenditure in adults living in developing
compared with industrialized countries: a meta-analysis of doubly labeled water studies. Am J
Clin Nutr 93:427-41
5. Prentice AM, Black AE, Coward WA et al (1986) High levels of energy expenditure in obese
women. Br Med J 292: 983–987
6. Bandini LG, Schoeller DA, Dietz WH (1990) Energy expenditure in obese and nonobese ado-
lescents. Pediatr Res 27:198–203
7. Ekelund U, Aman J, Yngve A et al (2002) Physical activity but not energy expenditure is redu-
ced in obese adolescents: a case-control study. Am J Clin Nutr 76:935-41
8. Westerterp KR (2010) Physical activity, food intake, and body weight regulation: insights
from doubly labeled water studies. Nutr Rev 68:148-54
9. American Psychiatry Association (2000) Diagnostic and statistical manual of mental disorders.
4th text revision Washington (DC).
10. Davis C, Katzman DK, Kirsh C (1999) Compulsive physical activity in adolescents with ano-
rexia nervosa: a psychobehavioral spiral of pathology. J Nerv Ment Dis 187:336-42
11. Shroff H, Reba L, Thornton LM et al (2006) Features associated with excessive exercise in wo-
men with eating disorders. Int J Eat Disord 39:454-61
12. Eisler I, Le Grange D (1990) Excessive exercise and anorexia nervosa. Int J Eat Disord 9:
377-86
12. Dixon DP, Ackert AM, Eckel LA (2003) Development of, and recovery from, activity-based
anorexia in female rats. Physiol Behav 80:273-9
13. Hillebrand JJ, Kas MJ, van Elburg AA et al (2008) Leptin’s effect on hyperactivity: potential
downstream effector mechanisms. Physiol Behav 94:689-95
14. Nogueira JP, Maraninchi M, Lorec AM et al (2010) Specific adipocytokines profiles in patients
with hyperactive and/or binge/purge form of anorexia nervosa. Eur J Clin Nutr 64:840-4
12 Physical Exercise in Obesity and Anorexia Nervosa 131
15. Klein DA, Mayer LE, Schebendach JE et al (2007) Physical activity and cortisol in anorexia
nervosa. Psychoneuroendocrinology 32:539-47
16. Steinhausen HC, Grigoroiu-Serbanescu M, Boyadjieva S et al (2008) Course and predictors of
rehospitalization in adolescent anorexia nervosa in a multisite study. Int J Eat Disord 41:29-36
17. Davies S, Parekh K, Etelapaa K et al (2008) The inpatient management of physical activity in
young people with anorexia nervosa. Eur Eat Disord Rev.6:334-40
Physical Exercise and Transplantation
13
Valentina Delmonte, Vincenzo Lauriola, Rodolfo Alejandro
and Camillo Ricordi
13.1 Introduction
The Global Observatory on Donation and Transplantation (a division of the World
Health Organization, WHO) in 2009 reported that an estimated 104,650 transplants
were performed in 89 countries (72,100 kidney, 21,175 liver, 5405 heart, 3650 lung,
2320 pancreas). This number represents an increase of 4% since 2008 but less than
10% of the global need.
Prolonged graft function and the improved clinical profiles, physical functioning,
and quality of life of patients undergoing solid-organ transplantation have provided
not only an economical advantage but also a much needed strategy to address the
shortage of organs available for transplants.
Transplant procedures are undergoing constant modifications, with transplant cli-
nicians and scientists aiming to achieve long-term host acceptance of transplanted or-
gans without the requirement of indefinite immunosuppression. Currently, however,
immunosuppression and drugs that counteract the side effects of immunosuppressive
drugs remain the pillars of treatment. Simultaneously, novel strategies, such as di-
etary intervention and exercise, are being investigated for their beneficial effects with
respect to all aspects of transplantation.
The advantages of physical activity are well documented in the American College
of Sport Medicine’s Position Stand and the U.S. Surgeon General’s Report on
Physical Activity and Health, Department of Health [1, 2]. Its benefits have been
confirmed also in transplanted individuals. Regular exercise decreases total choles-
terol, low density lipoprotein (LDL) cholesterol, and triglyceride levels and increas-
es the percentage of high density lipoprotein (HDL) cholesterol, thereby reducing the
number of coronary risk factors. Exercise, either as a function of its acute effects dur-
V. Delmonte ()
Diabetes Research Institute
University of Miami, Miller School of Medicine
Miami, USA
e-mail: VDelmonte@med.miami.edu
ing a single session or the cumulative effects of many sessions, increases insulin sen-
sitivity. This adjustment in patients with impaired glucose tolerance (IGT) or im-
paired fasting glucose (IFG) is reflected in better glucose tolerance and improved
glycemic control. The benefits of improving insulin sensitivity are not restricted to
glucometabolic aspects but extend to cardiovascular ones, given that hyperinsuline-
mia and insulin resistance are well-recognized direct cardiovascular risk factors.
Observational epidemiological studies and clinical trials have confirmed an asso-
ciation between physical activity and a lowering of blood pressure. A meta-analysis
of randomized, controlled trials conducted to determine the effect of aerobic exercise
on blood pressure found a significant reduction in mean systolic and diastolic blood
pressures in hypertensive, normotensive, overweight, and normal-weight individuals.
These benefits hold great significance for a population in which the prevalence of
cardiovascular disease is high.
Furthermore, participants in physical activity programs report an improvement in
their quality of life as well as an enhancement of their self-esteem and sense of well-
being. Exercise also correlates with behavioral characteristics that are felt to be pos-
itively related to health and disease prevention, such as reductions in smoking, alco-
hol consumption, and stress. Indeed, it can be stated that the role of physical activi-
ty, or exercise-related energy expenditure, as a cornerstone in the maintenance and
improvement of health is universally accepted [1]. Moreover, beneficial effects have
been proven in healthy as well as transplanted individuals.
The following statement from an article by Painter [3] summarizes the conclusion
reported in almost all the studies carried out in the last few decades: “In order to op-
timize functioning and overall health in organ transplant recipients, regular physical
activity should be prescribed and encouraged as a part of the routine post transplant
care”. The aim of this chapter is to highlight the importance of physical activity on
the physical condition of patients pre- and post-transplantation and to discuss the ef-
fect of training in patients who have undergone heart, lung, liver, kidney, pancreas,
or islet transplantation.
fy for transplantation. BMI, lipid profile, and blood pressure are perfect examples of
outcomes positively modifiable by regular exercise. Furthermore, it ensures that the
patient arrives at surgery in better physical condition, resulting in improved outcome
and a faster recovery from the transplant procedure.
Moderate physical activity in patients waiting for a transplant is also crucial in or-
der to maintain health markers such as VO2max, muscle strength, lipid profile, and
insulin sensitivity within their required ranges. In addition, cachexia, body-fat per-
centage, and hypertension diminish with exercise, while oxygen uptake increases and
fitness level may surpass the age-related expected values. In addition, regular exer-
cise can help the patient to attain a more favorable psychological profile.
It can therefore be concluded that before transplantation physical training im-
proves both exercise capacity and psychological state, while afterwards it increases
the likelihood of a better surgical outcome [4].
< 0.05) and psychological wellness (vitality score, SF-36 score p < 0.05) 1 year af-
ter therapy in renal transplant recipients treated with the immunosuppressive steroid
prednisone than in a control group receiving an interleukin-2 receptor inhibitor.
Prednisone use is also correlated with increased muscle lipid deposits, a reduction in
the number of capillaries, and a decreased myofibrillar volume. Further support for
these cause-effect relationships is highlighted by the partial reversal of the damage
upon suspension of the drug.
The second factor, also common to all transplants, is the patient’s physical con-
dition before and after surgery. The impaired level of physical fitness that develops
as a result of prolonged bed rest and inactivity leads to a decrease in the number of
myofibrils, the intracellular accumulation of glycogen and lipid deposits, thickened
capillary blood lamina, a decrease in the size of individual muscle fibers, and a re-
duced capillarization and oxidative capacity in muscle fibers. The prolonged state of
de-conditioning affects not only muscle tissue but also bone formation, in turn fur-
ther decreasing cardio-respiratory fitness and increasing metabolic risk [7].
Denervation of the transplanted organ is the most important organ-specific fac-
tor reducing physical work capacity. A lack of sympathetic nerves impairs the nor-
mal response of renin and insulin to exercise in kidney and pancreas transplant recip-
ients, respectively [4]. A reduction in chronotropic reserve, the impairment of dias-
tolic function, and a slower exercise-induced increase in heart rate are consequences
of denervation in heart transplant recipients [4, 8].
All allograft recipients experience a progressive increase in body fat in the first
year after transplantation, which is exacerbated by immunosuppressive drugs and
sedentarism. It is therefore not surprising that the post-transplantation condition of
the recipients particularly predisposes them to metabolic syndrome, defined as in-
sulin resistance, central obesity, inflammation, and endothelial dysfunction. In fact,
the increase in BMI post-transplantation is directly correlated with metabolic syn-
drome [3, 7, 9-11]. This relationship was emphasized by Sharif, who examined the
etiological and pathophysiological causes of metabolic syndrome after transplanta-
tion [11]. Dyslipidemia, kidney disease, hepatic gluconeogenesis, and aggravated in-
sulin resistance contribute to increasing the body-fat mass. Moreover, each of these
conditions can pose serious long-term threats to post-transplant survival; indeed, the
presence of metabolic syndrome one year after transplantation is a predictor of the
predisposition for diabetes and cardiovascular diseases, which are the most common
causes of death post-transplantation [10]. Diabetes mellitus occurs in 15–20% of sol-
id-organ transplant recipients [10, 12].
In a cross-sectional study of 1791 patients, Nabipour et al. [13] determined a di-
rect association between metabolic syndrome and prior infection in transplanted
patients (including cytomegalovirus and herpes simplex type 1). Also, De Vries [14]
et al. reported a 63% incidence of metabolic syndrome in 606 renal allograft recip-
ients. Interestingly, similar results were found for all solid-transplant recipients.
Physical activity in conjunction with proper nutrition and hygiene is essential in
preventing and fighting post-transplant complications. Maximal exercise capacity is
positively correlated with physical activity and inversely correlated with the risk fac-
tors for metabolic syndrome. The ACSM recommends an exercise program consist-
13 Physical Exercise and Transplantation 137
ing of moderate physical activity in which 1000 kcal/week or 150 min/week of in-
tensity (3–5.9 metabolic equivalent tasks, METs) are expended [1]. An additional
benefit of training is that it helps to prevent some of the side effects of immunosup-
pression without interfering with the anti-rejection actions of these drugs.
Surgit et al. [15] showed that 8 weeks of aerobic training for 45 min 3 times/week
improved the aerobic capacity levels of transplant recipients on immunosuppressive
therapy by 11% (p < 0.01). Improvements were also seen in T-helper cell count,
CD4+ to CD8+, natural killer cell activity, IgG and IgM (p < 0.01), without negative
effects on the graft, during the 6-month exercise program. Painter [6] cited Zhao et
al., who demonstrated that after 6 weeks of SWEET training (4 min 50% maximal
tolerated power +1 min 100% MTP for 45 min) patients improved their maximal tol-
erate power, VO2max, anaerobic and ventilatory threshold (p < 0.05). No significant
differences were reported in the immunological profile, rate of rejection episodes, or
rate of infections within 4 months of training. Luzi et al. [16] showed an inverse re-
lationship between the autoimmune markers glutamic acid decarboxylase (GAD)
and tyrosine-phosphatase-like protein (IA) and weekly energy expenditure derived
from physical exercise, suggesting positive effects on immune system function even
at low intensity and duration. Given the powerful ability of physical activity to mod-
ulate oxidative stress and mitigate chronic inflammatory conditions, such as those
leading to autoimmune diseases, exercise training programs may prevent an au-
toimmune response. Moreover, it can be safely concluded that the immunomodula-
tion resulting from physical activity does not interfere with the immunosuppressive
action of the drugs administered to transplant patients, and there have been no reports
of patients showing symptoms of short-term (6 months) graft rejection or dysfunc-
tion due to an exercise program. Unfortunately, there are as yet no studies on the
long-term consequences of exercise in transplanted patients.
capacity is lower than normal. Blood pressure is generally higher than normal at rest
and increases regularly during exercise, but the peak is still below normal. A compro-
mised left ventricular diastolic function, in addition to an impaired stroke volume and
heart rate, leads to a lower cardiac output during exercise. After exercise, the heart
rate’s return to the basal level is prolonged as a result of catecholamine and adren-
ergic hypersensivity.
Another outcome seen in heart transplant recipients is compromised pulmonary
function and oxygen uptake kinetics as well as a decreased peak exercise VO2. This
leads to an excessive dependence on anaerobic metabolism and, consequently, a rise
in blood lactate [4].
According to the Agency for Health Care Policy and research guidelines on car-
diac rehabilitation, aerobic exercise and resistance training are recommended post-
transplantation [4, 8]. After 2–6 months of aerobic exercise, heart rate and blood
pressure shift towards normal levels; peak VO2 increases by an average of 24% and
mitochondrial oxidative capacity improves, while lactate production and fatigue de-
crease. The results documented after moderate resistance training suggest a change
in muscle wasting caused by steroid drugs (prednisone) and that the strength gained
after 8–10 weeks is between 25 and 50% [6].
bolic syndrome and atherosclerotic burden [10]. Thus it can be concluded that,
through physical training, kidney transplant recipients can improve strength, exercise
capacity (10–34%), VO2max (>30%), METs max (19–114%), body composition,
and bone density while reducing the risk of developing metabolic syndrome and in-
sulin resistance [4, 6, 10]. Painter et al. showed a positive effect on HDL cholesterol
as well [6].
A 44-year-old male patient with type 1 diabetes mellitus who underwent islet trans-
plantation was subsequently monitored longitudinally for autoimmune markers,
142 V. Delmonte et al.
per session at an intensity of 12–14 on the Borg scale) and inactive groups.
Cardiorespiratory fitness, percentage of body fat, and health-related quality of life
were evaluated based on peak oxygen uptake, skin fold measurements, and Medical
Outcomes Short Form Questionnaire, respectively. The active group achieved
101.1% of their age-predicted peak oxygen uptake compared to only 72.7% by the
inactive group.
Lower BMI, lower body fat percentage, and higher quality of life scores were de-
termined in the active group [3]. Even though these data do not represent the gener-
al transplant population, since the author selected a highly specific group, they do
suggest that after transplantation near-normal levels of physical functioning and
quality of life can be achieved.
Maurice Slapak ex-president of the WTG, rightly argued: “Paradoxically, these
handicaps make the benefit derived from sport greater and more pertinent than for
the ordinary individual”.
13.10 Conclusions
Science has made it clear that through regular exercise solid-organ or cell transplant
recipients can achieve better physical functioning and substantially diminish the
detrimental effects of surgery, hospitalization-related inactivity, and drug treatment.
Physical rehabilitation is already routinely implemented in heart- and lung-transplant
programs. By contrast, exercise regimes and counseling for a more active lifestyle
are still not established as standard practice for patients undergoing other organ
transplantations, such as kidney or liver.
Exercise is a key factor in the strategy to improve health markers such as
VO2max, heart rate, blood pressure, anaerobic threshold, and metabolism. It also im-
proves lipid and glucose profiles, bone density, and body fat, is an important contrib-
utor to the reduction of symptoms such as fatigue and metabolic syndrome, and is
correlated with better psychological profile and muscle strength.
The immune-modulating properties of exercise are, however, not fully under-
stood. The evidence shows a significant positive impact of exercise on the human im-
mune system, a decrease in auto-antibodies, and an increase in positive-acting pro-
inflammatory cytokines. This might also be considered a strong factor in prescribing
exercise to transplant patients.
In summary, transplant clinicians, transplant-related health care professionals,
and exercise physiologists are encouraged to advocate regular exercise as a standard
treatment to improve the overall health of patients in all steps of transplantation: be-
fore and during the waiting list period, after transplantation, and during the rehabil-
itation process, as it should result in the restoration of health and the reintroduction
of health-promoting habits.
References
22. ADA (2011) Executive summary: standards of medical care in diabetes-2011 Diabetes Care
34: S4-10
23. Codella R, Delmonte V, La Torre A, Luzi L (2011) Exercise in an Islet-transplanted non-pro
marathon-runner: Effects on Training, Autoimmunity and Metabolic Profile (in press)
The Baboon as a Primate Model To Study the
Physiology and Metabolic Effects of Exercise 14
Francesca Casiraghi, Alberto Omar Chavez, Nicholas Musi
and Franco Folli
F. Folli ()
Department of Medicine, Division of Diabetes
University of Texas Health Science Center
San Antonio, USA
e-mail: folli@uthscsa.edu
energy expenditure during free-living activities. Accelerometers [12, 13, 27, 28] are
useful to estimate energy expenditure in non-human primates as they do not inter-
fere significantly in the animals’ normal life by minimizing the need for external
agents, such as different cages and different procedures.
Classic techniques employed in non-human primates include indirect calorime-
try using a modified metabolic chamber for non-human primates, allowing the deter-
mination of gas exchange between O2 and CO2 during different types of exercises
[28-33] with additional calculation of the RQ [34]. Another study made use of the
DLW method [5].
In a recent study Papillion et al. [13] used an accelerometer placed in a collar to
detect activities that involve the whole body in rhesus monkeys living in cages. The
study demonstrated that the accelerometer is a useful tool for quantifying whole-body
movements in non-human primates but it does not register the amount of energy ex-
penditure for behaviors such as chewing and arm movements, as only the total ener-
gy expenditure is calculated with this device. Additional studies were performed in
rhesus monkeys [12, 28] and in marmosets [27].
Running on a treadmill and biking on a cycle-ergometer are very common meth-
ods in humans to evaluate the level of fitness and for training. In all the non-human
primate studies considered herein, researchers similarly evaluated energy expendi-
ture during running, biking, and climbing. For example, Edgerton et al. [35] trained
11 Senegal bushbabies (Galago senegalensis) for 6 months to run upright, rather than
on four limbs, on a treadmill, achieving 60 min of exercise at 43 m/min over four
grades of inclination. After the training sessions, the authors continued to determine
the differences in muscle properties and structure involved in the exercises. Rhyu et
al. [36], in an investigation on the effect of aerobic exercise training on cognitive
functions, studied 12 cynomolgus monkeys divided into three different groups, each
with a different workload, and trained four of them to run on a treadmill for 1 h a day,
5 days a week for 5 months at 80% of maximal aerobic power. In addition, Ivy et al.
[37] evaluated the adaptations by 18 baboons during low to moderate quadrupedal
walking exercise on a motorized treadmill.
Another type of exercise used to trained non-humans primates was developed by
Hohimer et al. [30, 31], involving a modified chair-ergometer with a special metabol-
ic chamber. The animals, placed in a restraining chair, performed dynamic leg exer-
cises. Various methods were used by the authors to determine regional blood flow
distribution in diverse regions of the animals’ bodies during the exercises.
In other studies, researchers used a different method to achieve the same effect of
aerobic training such as running or biking. Talan et al. [29] trained three Macaca mu-
latta to lift weights repeatedly. In a study addressing bone mass and bone cellular
variations, Bourrin et al. [38] trained five rhesus monkeys to execute a rope-climb-
ing exercise for 1 h a day for 5 months; the authors reported a decrease in bone vol-
ume and bone formation activity. Zerath et al. [39] also trained five male rhesus mon-
keys to practice climbing for 1 h a day for 5 months continuously to simulate en-
durance training; changes in bone mass in response to intense aerobic exercise were
demonstrated.
Different levels of physical activity are very important in every period of life to
14 The Baboon as a Primate Model To Study the Physiology and Metabolic Effects of Exercise 151
maintain a healthy body. It is therefore essential not only to establish the minimal
level of activity necessary for fitness but also to quantify the decline in physical ac-
tivity during aging and to identify the mechanism involved.
Ingram [40] and Sallis [41], in different works, analyzed the age-related decline in
physical activity using non-human primates as a model. An age-related decline in ac-
tivity is observed across a wide range of non-human species and it seems to be predic-
tive of lifespan [40, 41]. Epidemiological studies have noted a general decline in phys-
ical activity with advancing age and that it is generally greater in males than in females,
especially in the teen years [13-18]. Other studies have shown that age is inversely as-
sociated with physical activity in studies with children, adolescent, and adults.
To further understand these observations scientists have developed different an-
imal models for establishing dose-response relations for various physiological out-
comes that can then be further tested and validated in humans. Physical activity has
been demonstrated to produce benefits on human brain function, influencing brain
volume and cognitive performance, but the mechanisms involved cannot be easily
tested in humans; instead, rodent models have been established. Chronic exercise
was shown to increase the vascular volume fraction in different areas of the cortices
and striatum as well as blood flow in the cerebellum [36].
Similar questions have been asked in non-human primates. In one study,
cynomolgus monkeys were trained to run on a treadmill to improve their fitness and
the effect of exercise on the CNS was subsequently examined. Such models allow
scientists to standardize and control all of the different factors involved in the exer-
cise regimen, together with lifestyle factors, such as diet and stress exposure, that are
difficult to control in humans. Rhyu et al. [36] examined whether regular exercise
improves cognitive functions. The exercise regime was the same as the one proposed
for humans by the American College of Sports Medicine and the American Heart
Association: the subject should run at 80% of his or her own maximal aerobic capac-
ity. Starting at the 9th week of training, cognitive tests were performed to determine
the rate of learning and the variations of blood flow in the brain with regular exercise.
This study showed that a regular exercise program at a defined level improved learn-
ing and vascular density in the brains of non-human primates.
During exercise there are physiological changes in the blood flow distribution in
different parts of the body. Baboons, better than other animal models, such as dogs,
show interesting similarities to humans regarding the redistribution of cardiac output.
Hales et al. [42] tested the difference in blood flow to most of the major organs in
awake heart-stressed baboons. Despite small differences, the authors concluded that
splanchnic and renal vasoconstriction was of a similar magnitude in humans and
non-human primates during heart stress. Slightly different results were obtained by
Vatner et al. [43], in which non-uniform responses in blood redistribution with re-
spect to mesenteric and renal flow during exercise and excitement in non-human pri-
mates were found.
Hohimer et al. [31] studied the variation in renal blood flow during a mild dy-
namic leg exercise in 12 baboons, confirming previous findings of a decrease in re-
nal blood flow during exercise. In a follow-up study [30], the authors examined
blood redistribution in several organs and tissues. They concluded that baboons are
152 F. Casiraghi et al.
a very useful animal model to investigate the different responses of different tissues
during exercise.
Baboons are also a highly appropriate model to study osteoporosis because their
bone metabolism and endocrine physiology are similar to those of humans [7, 38,
39]. In a series of pilot studies, Aufdemorte et al. [8] used different techniques (ra-
diograph exams, dual X-ray absorptiometry, histomorphometric analysis, oral bone
exams) to analyze the differences in bone density between young and aged female
baboons. They found dramatic differences in bone mass and volume between the two
groups, concluding that osteoporosis and oral bone loss correlated with ovarian dys-
function. This linkage has also been established in humans. Zerath et al. [39] per-
formed a study in monkeys, in which changes in bone mass occurred in response to
intense aerobic exercise. Future studies involving genetic factors may help identify
genes that influence the development of osteopenias in humans [7].
Non-humans primates are a very good model for studies of gynecological phys-
iology [4, 12, 33]. Different physiological mechanisms can be affected by patholog-
ical conditions and behaviors, leading to reproductive dysfunction. For example,
Williams et al. [33] concluded that a key aspect of exercise-associated menstrual dis-
orders is the balance between energy intake and energy expenditure. In their study,
Macaca fasicularis were trained to run on a treadmill, progressing up to 30 min a
day, 7 days a week at 12 km/h, and the effect of very intense training on the onset of
amenorrhea was examined [33].
Hunnell et al. [12] searched for differences in physical activities over the course
of the menstrual cycle in seven adult rhesus monkeys. The level of activity over the
course of several days was recorded with an accelerometer, with the results showing
no changes as a function of the menstrual cycle.
Sullivan et al. [28] asked whether there is a correlation between weight gain in
adulthood and the level of activity. That study tested 18 adult female rhesus monkeys
during a 9-month period, controlling the important parameters of weight, food in-
take, level of physical activity, and resting metabolic rate. Throughout the study, the
activity level was measured with a 3-way accelerometer, which the animals wore as
a collar. Food intake was calculated for each meal and the monkeys’ weights record-
ed weekly. Resting metabolic rate was monitored in a metabolic chamber at the be-
ginning of the study and after 3 months. A very strong correlation was found be-
tween the individual activity level and the tendency to gain weight; the higher the
level of physical activity, the lower the weight gain. These results supported the da-
ta obtained in humans, in whom a high level of physical activity prevents or limits
weight and fat gain.
invasive monitor that allows estimations of energy expenditure during different ac-
tivities for prolonged periods of up to 7 days. It has been used in humans and vali-
dated in both adults [44-50] and children [51-56]. It also has been used to monitor
physical activity in patients with diseases such as obesity [50, 57], cancer and
Parkinson’s disease [58, 59], cystic fibrosis [60], and chronic kidney disease [61].
The SWA is usually worn on the upper right arm, over the triceps muscle at the mid-
point between the acromion and the olecranon process. Through five different sen-
sors (two-axis accelerometer, heat flux sensor, skin temperature sensor, near-body
ambient temperature sensor, and galvanic skin response sensor), the device pro-
vides data regarding the caloric cost of physical activities as determined with propri-
etary algorithms. The data are recorded continuously as long as the SWA is worn and
remains in contact with the skin.
One limitation of the SWA is that it cannot be placed on the upper right limb of
baboons because of their innate curiosity and tendency to remove the device.
Therefore, we conducted pilot studies to find alternative sites for localization in or-
der to analyze the reliability and consistency of the data. Our initial experiments
compared measurements in humans, in whom the device had been positioned at dif-
ferent sites: the upper right arm, the triceps (the conventional site), the lower back
(lumbar region), the abdominal area (around the umbilicus on the right side), the
right thigh (quadriceps muscle), and the right calf (gastrocnemius and soleus mus-
cles). The study participants were examined in two different resting positions and
following moderate physical activity (walking 5 min on a treadmill at 5 km/h). Our
initial results showed that consistent readings could be obtained with the SWA at the
various sites in the two resting positions (mean ± SE: 0.022 ± 0.002 kcal/kg/min
(Fig. 14.2a, c). However, readings during activity were higher with the SWA on the
leg than on the arm and trunk (0.11 ± 0.005 vs. 0.075 ± 0.002 and 0.08 ± 0.003
kcal/kg/min, p < 0.001) (Fig. 14.2b). Whether these results represent true differences
between the metabolic activities of the exercising muscles or suboptimal readings
secondary to movement was not determined, although we suspect the former (“walk-
ing” quadriceps vs. triceps).
Based on these data, the trunk was chosen, as it was the area in which SWA es-
timates of energy expenditure were the most similar to those obtained with the de-
vice placed on the arm. In order to verify that the results obtained using the SWA
were correct, it was placed on the arm and back of the animals for 30 min during
which energy expenditure was measured using indirect calorimetry, defined as the
gold standard for such measurements [47-49].
During the second experiment, we tested 19 human subjects to determine the re-
liability and accuracy of the data estimated by the SWA placed on the arm and
back, comparing the data with those provided by indirect calorimetry. There were no
statistical differences in the results obtained by the two methods (mean ± SE): arm,
0.015 ± 0.0003 kcal/kg/min; back, 0.014 ± 0.001 kcal/kg/min; and indirect calorime-
try, 0.014 ± 0.0006 kcal/kg/min (Fig. 14.3). Following these encouraging preliminary
results, we used the SWA in ten sedated baboons, likewise placing the device on the
arm and back (Fig. 14.4).
The SWA data provided from the two sites did not significantly differ (mean ±
154 F. Casiraghi et al.
SE): 0.019 ± 0.0003 vs. 0.019 ± 0.0003 kcal/kg/min for the arm and back, respective-
ly. Subsequently, we compared the results obtained with the SWA placed on both
sites (arm and back) with the same data obtained in humans during 30 min of rest-
ing. Although there are differences in the metabolic rates of humans and baboons
(the latter have higher basal metabolic activity ), the SWA recorded consistent read-
ings at the two sites in both species (Fig. 14.5).
Considering our previous results in humans, we chose the back as the best place
to attach the sensor for long-term studies in baboons, since it was difficult for the an-
imals to reach and thus to potentially disrupt the readings. Accordingly, we used a
specially designed metabolic jacket, modified from the one used in tethered ba-
boons [62]. The jacket has a slit in the back allowing placement of the SWA, which
was preset to register the animal’s body characteristics, and continuous and firm con-
tact with the skin of the baboon’s back (Fig. 14.6). In our initial studies, the baboons
156 F. Casiraghi et al.
a b
Fig. 14.6 Metabolic jacket for energy expenditure measurements in baboons. The device is cus-
tomized to fit a preset SWA in each baboon’s back in order to continuously record energy expen-
diture over a week (a). Installation of a metabolic jacket in baboons (b); to ensure continuous read-
ing and data recording, a direct and firm contact with the skin is critical
a b
Fig. 14.7 Metabolic jackets can be customized and adjusted to different sizes based on baboon gen-
der (a, female; b, male), size, and age
wore the jacket for one week, during which they were kept in individual cages with
enough space for free ranging movements and fed ad libitum, maintaining regular
sleeping and activity patterns. The metabolic jackets can be customized to different
sizes, accounting for differences in the animals’ weight and gender (Fig. 14.7). The
SWA data were downloaded and stored in electronic form during a once-weekly re-
moval of the device and then analyzed with the appropriate software.
Finally, baboons wearing the metabolic jacket were studied with respect to ener-
14 The Baboon as a Primate Model To Study the Physiology and Metabolic Effects of Exercise 157
gy expenditure, including differences between basal levels and during different reg-
imens of physical activity. Our initial results demonstrated the feasibility and consis-
tency of the obtained measurements, with a ~50% increment of metabolic rate and
energy expenditure measured under exercise conditions. Energy expenditure (mean
± SE) was 0.021 ± 0.0008 kcal/kg/min at rest and 0.031 ± 0.0028 kcal/kg/min dur-
ing physical activity (p < 0.03) (Fig. 14.8). These observations suggest a wide range
of possibilities in terms of correlating and integrating the metabolic data with proto-
cols designed to study the effects of pharmacological and non-pharmacological in-
terventions on regional blood flow, muscle-protein gene expression, inflammatory
markers, and molecular signaling during physical activity and exercise.
14.4 Summary
The baboon is a well established and valuable non-human primate model for the
study of multiple human chronic diseases, with the goal of identifying common un-
derlying mechanisms responsible for human metabolic pathologies and assessing
novel pharmacological interventions in the common intricate pathophysiology of
obesity, diabetes, and metabolic syndrome. The use and demand of non-human pri-
mates has increased in recent decades and several species, including baboons, have
been studied using a wide range of approaches.
Not surprisingly, accumulating research has shown that metabolic variables (at
rest and after exercise) as well as molecular signaling in adipose tissue and skeletal
muscle are highly similar in baboons and humans. Baboons therefore provide a nat-
ural model for the study of exercise and its effect on adipose tissue, heart, lungs,
bone, and skeletal muscle metabolism, as well as the fascinating interaction be-
tween the “fit” body and the brain.
With the recent identification of novel signaling pathways and of molecular tar-
gets (i.e. TLR4, NFkB, AMPK) [11, 63-66] modulated by exercise and the develop-
ment of innovative pharmacological agents designed to enhance the molecular sig-
naling exerted by physical activity, there is an increasing potential for the use of non-
human primates in research. The morphologic and metabolic characteristics of ba-
158 F. Casiraghi et al.
boons together with the availability of improved sensing devices for energy expen-
diture measurements make these animals an attractive and valuable model with
which to study molecular signaling, its integration in skeletal muscle and other
metabolically active tissues during physical activity, and its correlation with metabol-
ic variables obtained under real-life conditions.
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Subject Index
B F
Baboon, 147, 152, 157 Fatty acids, 18, 33-37, 41
β-oxidation, 62, 118 Free fatty acids, 69, 72, 100, 116, 118
C G
13C MR spectroscopy (13C MRS), 45, Genetic polymorphisms, 30
49, 51-52 Glucocorticoids, 112-120
cardiac steatosis, 58 Glucose, 4-6, 20, 33-36, 41, 43, 48, 51,
Carnitine acetyltransferase (CAT), 35 57, 61, 69-70, 72-73, 86, 88-91, 100,
Cell membrane, 17-21 116-118, 134, 139
Clearance rate 91 Glucose metabolism, 36-38, 51, 55,
Central Nervous System (CNS), 9, 13, 57-58, 69, 77, 115, 148,
36-37 Glucose tolerance, 4, 58-59, 62, 104, 141
Contractile unit, 17-21 Impaired glucose tolerance (IGT),
cortisol, 110, 112-113, 119, 127, 129 134, 141, 59, 62, 103
Corticotropin Releasing Hormone Mitochondrial glucose, 69
(CRH), 111-112, 115, 119, 128-129 Glycogen, 10, 43, 45, 51, 71, 116, 118,
136
D Glycogenolysis, 43, 46, 50-51, 117
Doping, 120 Glycolysis, 43, 48, 51, 68-69
Doubly labeled water technique, 123- Glycolytic flux, 50
125, 127
E H
Eating behavior, 114, 127-128 1H MR spectroscopy (1H MRS), 44, 58
Ectopic fat, 56-57, 58, 59-60, 62 H-band, 11
163
164 Subject Index