Download Fat to the fire: the regulation of lipid oxidation

Comparative Biochemistry and Physiology, Part B 139 (2004) 443 – 460
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Review
Fat to the fire: the regulation of lipid oxidation with exercise
and environmental stress$
Grant B. McClelland*
Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON, Canada L8S 4K1
Received 23 March 2004; received in revised form 20 July 2004; accepted 20 July 2004
Abstract
Lipids are an important fuel for submaximal aerobic exercise. The ways in which lipid oxidation is regulated during locomotion is an
area of active investigation. Indeed, the integration between cellular regulation of lipid metabolism and whole-body exercise performance
is a fascinating but often overlooked research area. Additionally, the interaction between environmental stress, exercise, and lipid
oxidation has not been sufficiently examined. There are many functional and structural steps as fatty acids are mobilized, transported, and
oxidized in working muscle, which may serve either as regulatory points for responding to acute or chronic stimuli or as raw material for
natural selection. At the whole-animal level, the partitioning of lipids and carbohydrates across exercise intensities is remarkably similar
among mammals, which suggests that there is conservation in regulatory mechanisms. Conversely, the proportions of circulatory and
intramuscular fuels differ between species and across exercise intensities. Responses to acute and chronic environmental stress likely
involve the interaction of genetic and nongenetic changes in the fatty acid pathway. Determining which of these factors help regulate the
fatty acid pathway and what impact they have on whole-animal lipid oxidation and performance is an important area of future research.
Using an integrative approach to complete the information loop from gene to physiological function provides the most powerful mode of
analysis.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Carnitine palmitoyltransferase I; Exercise; Fatty acids; Hypoxia; Mitochondria; PPAR; Temperature; Vertebrates
Contents
1.
2.
3.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peter Hochachka and integrative exercise biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Whole-animal rates of lipid oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
444
444
445
Abbreviations: ACS, acyl-coenzyme A synthase; ACBP, acyl-coenzyme A binding protein; ACC, acetyl-coenzyme A carboxylase; AFABP, adipocyte fatty
acid binding protein; Alb, albumin; AlbR, albumin receptor; AMPK, AMP-kinase; CD36/FAT, fatty acid translocase; CHO, carbohydrates; CPT, carnitine
palmitoyltransferase; CAT, carnitine acyl-transferase; FATP, fatty acid transport protein; FABPpm, fatty acid binding protein plasma membrane; FABP, fatty
acid binding protein; FA-CoA, fatty acyl-coenzyme A; FA-Carn, fatty acyl-carnitine; FARE, fatty acid response element; FFA, free fatty acids; HA, high
altitude; HOAD, h-hydroxyacyl-coenzyme A dehydrogenase; HSL, hormone-sensitive lipase; MCD, malonyl-coenzyme A decarboxylase; PPAR, peroxisome
proliferator-activated receptor; PPRE, PPAR response element; PGC-1, PPAR gamma cofactor-1; RXR, retinoic acid receptor; SL, sea level; Sp1, specificity
protein-1; TAG, triacylglycerol; UCP, uncoupling protein; VO2max, maximum oxygen consumption; XCAD, acyl-coenzyme A dehydrogenase (long- or
medium-chain).
$
The papers in this volume are dedicated to the memory of our friend and mentor, Peter W. Hochachka, whose intelligence, curiosity, enthusiasm, and
encouragement catalyzed research in diverse areas of comparative physiology and biochemistry and taught us that following one’s curiosity in science can be
both productive and fun.
* Tel.: +1 905 525 9140x24266; fax: +1 905 522 6066.
E-mail address: [email protected].
1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2004.07.003
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G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
4.
5.
6.
7.
Conserved aspects of lipid oxidation. . . . . . . . . . . . . . . . . . . . . . .
Mechanisms of conserved lipid oxidation patterns . . . . . . . . . . . . . . . .
Partitioning of circulatory vs. intramuscular fuel sources during locomotion . .
The fatty acid oxidation pathway—cellular components . . . . . . . . . . . . .
7.1. Mobilization and transport of lipid stores. . . . . . . . . . . . . . . . .
7.2. Muscle uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3. Mitochondrial oxidation . . . . . . . . . . . . . . . . . . . . . . . . .
8. Regulation of cellular fatty acid oxidation: genetic and nongenetic mechanisms
8.1. Acute regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2. Chronic regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3. Nongenetic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9. Changes in lipid metabolism with environmental stress . . . . . . . . . . . . .
9.1. Hypoxia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2. Temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2.1. Acute effects . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2.2. Chronic effects . . . . . . . . . . . . . . . . . . . . . . . . .
10. Evolutionary variation in fatty acid oxidation . . . . . . . . . . . . . . . . .
11. Models for studying the evolution of lipid metabolism . . . . . . . . . . . . .
11.1. Developmental differences in lipid oxidation . . . . . . . . . . . . . .
11.2. Targeted alterations in gene expression . . . . . . . . . . . . . . . . .
12. Conclusions and future directions. . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Lipids are an exciting area of current research due to
their many roles as signaling molecules, membrane
components, and energy sources both for resting metabolism and low-intensity exercise. Biomedical researchers
are particularly interested in the regulation of fat
oxidation, which is pertinent to the treatment of obesity,
diabetes, and heart disease. Other groups are actively
investigating the way in which fats are used during
exercise and their effects on human performance
(reviewed in Spriet and Watt, 2003; Brooks, 1998). The
integration between cellular regulation of lipid metabolism and whole-body exercise performance is a fascinating but often overlooked area of research. Additionally,
the interaction between environmental stress, exercise,
and lipid oxidation has not been sufficiently examined.
Using an integrative approach to complete the information loop from gene to physiological function will be
necessary to resolve these important questions.
2. Peter Hochachka and integrative exercise
biochemistry
Although he was often categorized as a comparative
biochemist, a physiologist, or a zoologist, Peter
Hochachka was above all an integrative biologist. Having
spent his early career examining enzyme kinetics, one
would think that he would have a reductionist view of
biological problems. On the contrary, he abhorred bstamp
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collectingQ and vehemently cautioned against overly
reductionist approaches. He instilled this philosophy in
his students as soon as they arrived in the lab. bThink
big!Q was Peter’s directive when I joined his laboratory as
a graduate student in 1992. This simple phrase exemplified his approach to life and science. bThink big!Q meant
to challenge yourself and think beyond obvious mechanistic details. Think big and you might just discover
something fundamental about animal physiology. Peter
always pursued fundamentals in all areas of his research
and this extended to his interest in exercise biochemistry
and muscle metabolism.
Peter realized that exercise is one of the most
energetically costly activities performed by animals and
thus numerous metabolic pathways are designed to meet
its demands. He was among the first to examine exercise
biochemistry and muscle metabolism in a wide variety of
animals including those living in extreme environments.
His research examined the biochemical properties that
allow animals to demonstrate tremendous variation in
exercise ability and recovery therefrom. Animals studied
included fish (carp, tuna, trout, salmon, lungfish), squid,
thoroughbred horses, greyhounds, seals, llamas, rats,
humans, and hummingbirds (see Hochachka and Somero,
2002 for details). Peter’s enduring interest in oxygen led
him to wonder how some species maintain suitable levels
of locomotion when faced with high altitude or with
breath-hold-induced hypoxia. Peter realized that noninvasive measurement techniques provided powerful tools
for studying muscle metabolism and he was one of the
few comparative physiologists to use nuclear magnetic
G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
resonance (NMR or MRI) and positron emission tomography (PET) to examine responses to environmental
stress. Always looking for better ways to study an
animal in its own environment, Peter used computerassisted backpacks to take serial blood samples in diving
and exercising seals (Guppy et al., 1986). Peter was
continuously looking at the bigger picture, integrating
muscle biochemistry and whole-animal physiology within
the context of the environment in which the animal lives.
He asked probing questions about the role of exercise
and conflicts with other biological functions. For example, he questioned how seals deal with the conflict of
conserving vs. consuming oxygen during diving and
exercise (Hochachka, 1986). Peter’s bright mind shone a
light on connections that others failed to see. He drew
information from a diversity of biological fields including
biochemistry, evolution, ecology, physiology, cell biology,
genetics, biophysics, exercise physiology, and biomechanics. From these connections, he would synthesize
provocative hypotheses that will occupy many of us for
years to come.
As one of the reviewers of this manuscript wondered:
why do many of Peter’s former students now work on
lipids—an area in which he did little work? Although Peter
would roll his eyes at the mention of any particular fatty
acid, he was interested in the integration and interaction of
metabolic pathways which occurs between lipids and
carbohydrates (CHO) during exercise. Addressing questions
of how lipid oxidation is regulated from the cellular level to
whole-body fuel selection patterns fits well with his
integrative view of exercise biochemistry.
3. Whole-animal rates of lipid oxidation
Whole-animal rates of lipid oxidation are the product of
oxidation rates in all tissues and from all available stores of
445
lipid fuel. To appreciate why an integrative approach is
needed in the study of lipid oxidation it is important to
realize the complex nature of the pathway from storage
depot to working muscle. Fig. 1 is a generalized depiction of
the pathway for lipids from storage sites to muscle
mitochondria. To be oxidized, lipid stores must first be
mobilized (M) from triacylglycerides (TAG) into free fatty
acids (FFA). Due to their low solubility in aqueous medium,
FFA are bound to proteins for transport (T) both in the
cytosol and in the circulation. It is also necessary for FFA to
pass through cellular membranes via specific transporter
proteins (TR) into the circulation from adipocytes and,
subsequently, for uptake into myocytes. The rate at which
FFA leave the adipocyte is, in part, dependent on the rate of
binding site delivery and the binding capacity of the
transporter protein (Weber, 1992). Similarly, the rate of
FFA delivery to the myocyte is dependent on plasma flow,
the product of cardiac output ( Q) and proportion of the
blood volume that is plasma [ Q plasma=Q (1 Hematocrit)],
because FFA are not transported in red blood cells. Once at
the myocyte, the rate of FFA uptake is dependent on many
factors including transporter density, myocyte cytosolic
protein binding capacity, and activation of the FFA to
acyl-CoAs to maintain a concentration gradient favorable to
FFA uptake. Finally, FFA must cross the mitochondrial
membrane before undergoing h-oxidation to generate
acetyl-CoA.
Each of these steps in the fatty acid oxidation pathway
represents a potential site for plastic responses to endurance
training or to environmental stress. These steps may also
serve as targets for natural selection in response to selective
pressure for changes in fatty acid oxidation rates. It is not
clear if the pathway is regulated as an entire unit or if
modification of specific steps in the pathway results in a
change in overall fatty acid flux rates. The variation in the
fatty acid pathway between species has received little
attention. However, current data on gene knockout, trans-
Fig. 1. As fatty acids (filled circles) move from storage sites to muscle mitochondria to provide ATP for contraction, they must be mobilized (M) from storage
forms, pass through specific membrane transporters (TR), and undergo either circulatory or cytosol transport (T) steps. Entry into the mitochondria is through
specific transporters and FFA undergo h-oxidation (h-ox) with each pass through the reaction spiral that yields acetyl-CoA. Acetyl-CoA then enters the
tricarboxylic acid cycle (TCA) producing reducing equivalents for the production of ATP and the subsequent generation of CO2. Figure adapted from Weibel et
al. (1996) and Weber (1992).
446
G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
genic models, and intertissue differences in the fatty acid
pathway provide clues and represent potential research
strategies to determine the underlying mechanisms that
result in interspecies variation in lipid oxidation.
4. Conserved aspects of lipid oxidation
Because animals display a wide variety of locomotory
abilities and occupy diverse environments, is it reasonable
to suggest that there are conserved aspects in their fuel
selection patterns? Dick Taylor, Jean-Michel Weber, and
Ewald Weibel systematically examined the matching of
oxygen and fuel delivery using dogs and goats as models
of adaptive variation (see J. Exp. Biol., 199 (8);
McClelland et al., 1994). As the result of this work
and other studies (McClelland et al., 1998, 1999, 2001),
it is apparent that one of the most conserved aspects of
fuel selection in mammals is the proportion of lipids and
carbohydrates used during locomotion relative to their
aerobic maximum (VO2max). Exercise data taken across
taxa, body size, varying aerobic capacities and after
acclimation to chronic hypoxia all show surprisingly
similar patterns of fuel selection relative to exercise
intensity (Fig. 2). As early as the 1930s, Edwards et al.
(1934) at the Harvard Fatigue Laboratory made the
observation that there was an increased reliance on
glycolysis with increasing exercise intensity in humans.
This phenomenon has recently been described and refined
in the human exercise physiology literature as the
bCrossover ConceptQ of Brooks and Mercier (1994). This
concept has its greatest utility in demonstrating the
effects of exercise intensity and exercise training on the
balance of lipid and carbohydrate use in humans. Indeed,
human training data have been included in Fig. 2 to
illustrate this point. However, the fact that relationships
between exercise intensity and fuel selection may be
generalized to mammals has been important for the
construction of general synthetic models of fuel selection
during locomotion (Fig. 2).
Maximum lipid oxidation rates occur at low exercise
intensity in mammals (Edwards et al., 1934; Bergman and
Brooks, 1999; Roberts et al., 1996c, McClelland et al.,
1994) and decrease as exercise intensity increases. This is
partly because power output achievable with lipids as the
sole fuel source is lower than with carbohydrates
(reviewed in Hochachka, 1986; Hochachka and Somero,
2002). In fact, in elite endurance runners, power output
was found to drop to 50% of maximum at a point when
glycogen stores were thought to be fully depleted and the
reliance on lipids thus increased (Frayn, 1996). This
phenomenon may be specific to mammals as birds and
insects can sustain high power outputs on lipids alone
(Suarez et al., 1986; O’Brien and Suarez, 2001). Duration
also plays an important role in lipid oxidation rates (Felig
and Wahren, 1975; Watt et al., 2002a). Depending on the
intensity, fatty acid oxidation can increase progressively
over several hours of exercise in humans (Wolfe et al.,
1990; Watt et al., 2002a).
The trend depicted in Fig. 2 provides researchers with
a model to test predictions about fuel use. For example,
one can predict that endurance-adapted animals will rely
to a greater extent on lipids during exercise to take
advantage of large lipid stores, a high ATP yield per mole
fuel oxidized and a way to conserve valuable muscle and
liver glycogen. Conversely, high-altitude animals may use
Fig. 2. The relationship (r 2=0.83, not including altitude data) between relative exercise intensity (% VO2max) and percentage of total fuel oxidation (%VO2)
supplied by (A) lipids and (B) carbohydrates (CHO) in animals of the same (dogs and goats) or different (rats and humans) sizes but all with different aerobic
capacities. Data are from dogs and goats (McClelland et al., 1994); trained and untrained rats (Brooks and Donovan, 1983); trained men (Bergman and Brooks,
1999); trained and untrained women (Friedlander et al., 1998); altitude acclimated (HA); and sea level (SL) rats (McClelland et al., 1998, 1999). Figure adapted
from Roberts et al. (1996c).
G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
more carbohydrates because of the favorable ATP yield
per mole of oxygen. Based on these predictions lipid and
carbohydrate oxidation use during exercise in these
animals should deviate from the relationship depicted in
Fig. 2. However, the opposite is true, endurance-adapted
and high-altitude acclimated mammals use the same
proportions of lipids and carbohydrates as their sedentary
and sea-level (SL) counterparts (see Weber et al., 1996;
McClelland et al., 1998). The fact that global patterns
emerge in Fig. 2 is remarkable considering that the data
come from many differences studies. Some variation in
the data exists due to differences in respirometry measurement techniques, nutritional status of the subjects, species
strain (in the case of rats), duration of exercise, and
accurate determination of VO2max used to determine
submaximal running speeds. Indeed, it would be preferable to construct a model from a single study on
animals that demonstrate a wide range of aerobic
capacities. In the absence of such a study, Fig. 2 provides
us with the best working hypothesis on the relationship
between exercise intensity and fuel selection. This framework allows not only for meaningful comparisons
between treatment groups or between species but also
for the identification of bexceptions to the rule,Q i.e.,
situations where lipid oxidation is either lower or higher
than would be predicted from Fig. 2. Some of these
exceptions include prolonged high-fat diets (Jansson and
Kaijer, 1982), women vs. men (Horton et al., 1998), as
well as birds and insects (Suarez et al., 1986; O’Brien and
Suarez, 2001). Currently, there are no clear explanations
for underlying mechanisms that explain either the
conserved patterns of fuel selection or the deviations
therefrom.
5. Mechanisms of conserved lipid oxidation patterns
At what biological level and by what means are the
conserved patterns of fuel selection regulated? Due, in
part, to the overall complexity of fuel selection, it is
difficult to assess the regulatory basis of conserved and
malleable metabolic features to overall fuel selection.
Regulation may involve (1) muscle recruitment patterns;
(2) hormone concentration changes (specifically: norepinephrine, epinephrine, insulin, glucagon, cortisol and
leptin); (3) substrate and futile cycles [e.g., glucose–fatty
acid, Cori, triacylglycerol–fatty acid (TAG-FA) cycle,
lactate shuttle]; (4) oxygen; and lastly (5) coordinated
regulation of enzyme and transporter activities and content.
Some of these regulators may in fact be regulated
themselves. Therefore, the intricate interactions between
these factors make the interpretation of experimental
results difficult (Miyoshi et al., 1988) and very few of
these factors have been studied thoroughly. An intriguing
observation is that the general pattern of fuel selection is a
reflection of how muscle fibers are progressively recruited
447
as work rate goes up (Roberts et al., 1996c). At low
exercise intensities, slow oxidative (type I) muscle fibers
with a high capacity for fat oxidation (Jackman and Willis,
1996), are recruited. As intensity increases, faster (type II)
fibers with higher glycolytic capacity are then activated
(Armstrong and Laughlin, 1985). This parallels changes in
fuel selection. The biochemical properties of these muscles
may be similar between species but with a corresponding
upregulation or downregulation of the fatty acid oxidation
pathway. For example, at low intensities, all mammals may
recruit muscle fibers with a high capacity for lipid
oxidation but perhaps more aerobic species have higher
levels of transporters and enzymes than less aerobic
species. Hormonal regulation of fuel kinetics certainly
plays some role, but the degree to which each hormone
contributes to the regulation of exercise fuel selection is
uncertain. For instance, plasma concentration of norepinephrine correlates with the rate of glucose appearance (R a
glucose) and rises with %VO2max (Romijn et al., 1993;
Mazzeo et al., 1995), but it has been found that
manipulations of catecholamines at high exercise intensities in humans does not greatly alter fuel selection
(Chesley et al., 1994). It is also thought that the observed
increased lipid use in humans at the same power output
after training is due to a suppression of sympathetic
nervous system activity (Brooks and Mercier, 1994). The
implications of this observation to interspecies differences
in lipid oxidation are not known.
Much work has been dedicated to understanding the role
of metabolic cycles in the control of overall fuel selection in
humans and rats (Van Der Wusse and Reneman, 1996). As
reviewed recently by Spriet and Watt (2003), the regulation
of exercise fuel selection involves interactions between the
lipid and carbohydrate pathways. As described by the
classic glucose–fatty acid or Randle cycle (Randle et al.,
1963), intermediates of fatty acid oxidation, namely, citrate
and acetyl-CoA, inhibit carbohydrate oxidation through
pyruvate dehydrogenase (PDH) and phosphofructokinase
(PFK) inactivation. However, this may only occur in resting
muscle. Current evidence suggests that increased circulating
free fatty acids do decrease glycogenolysis during intense
aerobic exercise but not by classic Randle mechanisms
(Dyck et al., 1993). Conversely, increased glucose and
glycogen availability can decrease fatty acid oxidation, but
it remains to be seen if this cycle operates during exercise.
Modern metabolic control theory suggests that the interactions between lipids and carbohydrates will take place at
multiple points in these pathways rather than simply at socalled rate-limiting steps.
The mobilization and transport of fatty acids is a relatively
slow process, especially when compared to other fuel
sources. One of the roles of the TAG-FA cycle, which was
first demonstrated in humans by Wolfe and Peters (1987), is
to make fatty acids quickly available during the transition
from rest to exercise. At rest, 70% of FFA released are
recycled back to TAG via intracellular and/or extracellular
448
G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
routes (Rasmussen and Wolfe, 1999), but this drops to about
25% during moderate exercise, making more FFA available.
Moreover, at the end of exercise, the rate of recycling jumps
to nearly 90%, thus protecting against extremely large
increases in plasma FFA in recovery (Wolfe et al., 1990).
Although this cycle has now been measured in humans, rats,
fish, and rabbits (McClelland et al., 2001; Reidy and Weber,
2002; Bernard et al., 1999), there is little information
regarding its role in regulating lipid oxidation across exercise
intensities and with exposure to environmental stress.
Exposure to chronic hypoxia can increase the TAG-FA cycle
during exercise in rats. However, there was little effect on
whole-animal lipid oxidation rates although glycerol and
FFA kinetics were greatly effected (McClelland et al., 2001).
The cycle itself is regulated hormonally (e.g., epinephrine
and leptin; Miyoshi et al., 1988; Reidy and Weber, 2002),
which points to the complexity of interaction between many
factors that contribute to exercise lipid oxidation rates. The
relative role of these factors in regulating whole-animal lipid
oxidation and how they may contribute to the conserved
patterns of fuel selection (Fig. 2) remains to be seen.
6. Partitioning of circulatory vs. intramuscular fuel
sources during locomotion
During exercise lipids and carbohydrates are drawn
upon both from outside the myocyte (circulatory glucose
and FFA) and from inside the myocyte (muscle glycogen
and TAG). At a moderate exercise intensity of 60%
VO2max, rats use a mixture of all four fuel stores (Fig.
3). How this fuel partitioning changes with exercise
intensity, exercise duration, and across species is not well
known. The size of the fuel store and how rapidly it can
be mobilized during exercise may play important roles in
its use at any given intensity. Of the lipids available for
exercise, adipose tissue stores represent the most abundant
Fig. 3. The partitioning of different fuel sources in rats exercising at 60%
VO2max. Whole-animal carbohydrate (CHO) oxidation can be divided into
muscle glycogen and circulatory glucose oxidation. Whole-animal lipid
oxidation can be divided into muscle triacylglycerol (TAG) and circulatory
free fatty acid (FFA) oxidation. Circulatory FFA oxidation represents a
maximum value based on 100% of FFA tissue uptake being oxidized. Data
from McClelland et al. (1998, 1999).
depot to support locomotion. A typical 70kg man stores
approximately 16 kg or 600,000 kJ of energy in adipose
tissue TAG (Newsholme, 1982). Moreover, a typical fatty
acid provides 104 mol of ATP per mole of fuel compared
to 31 mol of ATP for the complete oxidation of a mole of
glucose (Brand, 1994). It is, therefore, surprising that
endurance training studies on humans have not found a
significant increase in flux rates of free fatty acids
(Holloszy et al., 1986; Martin et al., 1993). The same is
true when examining the proportion of total energy
supplied by circulatory FFA in endurance mammals
compared with more sedentary species (McClelland et
al., 1994; Weber et al., 1996). This suggests that there are
limitations to circulatory FFA supply to muscle in these
circumstances. At low exercise intensities, fatty acid
oxidation has been found to increase with plasma FFA
concentration (Hagenfeldt, 1975; Paul and Issekutz, 1967).
However, this is not always the case and studies on
training in humans have shown a decrease in plasma FFA
associated with an increase in fat oxidation during
exercise (Klein et al., 1994; Martin et al., 1993). Although
circulatory FFA are important fuels for low-intensity
exercise (Romijn et al., 1993; Weber et al., 1996) and
oxidation rates increase with exercise duration (Felig and
Wahren, 1975; Bulow, 1988; Wolfe et al., 1990), the
importance of this fuel decreases as exercise intensity
increases. This decrease in oxidation with intensity may
be in part due to inhibition of long-chain fatty entry into
the mitochondria (Sidossis et al., 1997) or into the
myocyte itself. Muscle cell membrane uptake appears to
limit circulatory FFA oxidation since the rates of
oxidation cannot be significantly increased at high
exercise intensities by increasing FFA availability through
experimentally induced increases in circulatory FFA
(Hargreaves et al., 1991; Romijn et al., 1995). Maximal
circulatory FFA oxidation rates may also be limited by the
transport of FFA in plasma from adipose tissue to the
muscle. Plasma FFA transport rate is dependent on the
concentration of albumin (Alb), the number of binding
sites available for FFA, and Q plasma (Fig. 1; McClelland et
al., 1994). More aerobic species have lower plasma
volumes due to increased hematocrit (for increased O2
transport). This decrease in plasma volume can be
partially compensated for by higher Q, resulting in similar
Q plasma between sedentary and aerobic species (McClelland
et al., 1994). A greater FFA binding capacity of their
albumin helps aerobic species increase plasma transport, but
it only partially compensates for low Q plasma (McClelland et
al., 1994). As a result, circulatory delivery of FFA in more
aerobic species is not increased to the extent that total lipid
oxidation rates are in relation to sedentary species. It
appears that the design of the circulatory system is set
principally by oxygen delivery needs and not that of fuel
transport (McClelland et al., 1994).
Circulatory transport limitations for FFA result in the use
of a greater proportion of intramuscular lipids during exercise
G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
in both trained humans and highly aerobic animals (Martin et
al., 1993; Hurley et al., 1986; Weber et al., 1996). Additionally, the use of intramuscular TAG increases as exercise
intensity increases (Weber et al., 1996) although this has not
been studied in detail in many mammals. In fact, some
controversy exists as to whether muscle TAG are used to any
great extent during exercise (reviewed by Watt et al., 2002b).
It has been suggested that their main role is to power glycogen
repletion during recovery from an exercise bout (Kiens and
Richter, 1998). The inability to detect muscle TAG depletion
during exercise may be due to the variability in biopsy
measurements that are often greater than the expected TAG
utilization. Studies using less variable techniques involving
isotopes or 1H MRS show significant TAG utilization during
exercise in humans (Watt et al., 2002b). In addition, highly
aerobic mammals and trained humans store more lipid in
muscle than sedentary mammals do (Vock et al., 1996), and
more active fish store lipid within muscle rather than in
adipose tissue or liver (Weber and Zwingelstein, 1995). This
does suggest that intramuscular TAG plays an important role
in the upregulation of maximal lipid oxidation rates. It is also
important to realize that there are adipocytes interlaced on the
outside of myocytes (Van Der Wusse and Reneman, 1996),
but the contribution of this lipid pool to exercise fuel selection
is currently uncertain. Clearly, much work is needed to fully
elucidate the exercise conditions in which different fuel stores
449
are used as well as how the regulation of the fatty acid
pathway contributes to this partitioning.
7. The fatty acid oxidation pathway—cellular
components
In the last few years, many of the specific cellular
components of the fatty acid oxidation pathway have been
identified (Fig. 4). Together, they form the raw material for
regulation of flux through this pathway.
7.1. Mobilization and transport of lipid stores
The mobilization of FFA from adipocyte and myocyte
TAG stores occurs via hydrolysis by hormone-sensitive
lipase (HSL), liberating FFA and glycerol. Adipose fatty
acid binding proteins (AFABP, also known as aP2) serve to
solubilize the FFA liberated and transport them to the cell
membrane for release (Fruhbeck et al., 2001). The
mechanisms of adipose FFA release are not fully understood
but probably involve the transmembrane protein fatty acid
translocase (CD36/FAT). The rate of release of FFA into the
circulation depends on membrane transporter density and
the rate of albumin binding site delivery to the adipocyte
(Bulow et al., 1985).
Fig. 4. Many of the enzymes and transporters involved in the fatty acid pathway are known. Mobilization of free fatty acids (FFA) from adipose tissue and
muscle stores occurs via hormone sensitive lipase (HSL), which promotes the release of free fatty acids from triacylglycerol (TAG) storage form. Transport of
fatty acids in and out of adipocytes is thought to occur via the CD36/FAT membrane transporter. Once in the circulation, FFA are bound to albumin which is
thought to bind to an albumin receptor (AlbR) on the muscle cell membrane. Muscle uptake of FFA may occur through CD36/FAT, fatty acid binding protein
plasma membrane (FABPpm) or fatty acid transport protein (FATP). Once in the myocyte cytosol, FFA are bound to fatty acid binding proteins (FAPB). FFA
are then converted to fatty acyl-CoAs through the actions of acyl-CoA synthase (ACS) and potentially FATP. This FA-CoA is bound to a acyl-CoA binding
protein (ACBP) and converted to FA-carnitine via carnitine palmitoyltransferase (CPT)I. FA-carnitine is transported into the mitochondria by carnitine
acyltransferase (CAT) and converted back to FA-CoA by CPTII on the inner mitochondrial membrane. FA-CoA then enters the h-oxidation pathway which
consists of four enzymes: (1) long- or medium-chain acyl-CoA dehydrogenase (LCAD or MCAD, collectively XCAD); (2) enoyl-CoA hydratase; (3) hhydroxyacyl-CoA dehydrogenase (HOAD); and (4) acyl-CoA acetyltransferase (ketothiolase). The product of every pass through the h-oxidation spiral is
acetyl-CoA which either enters the tricarboxylic acid cycle (TCA) or is transported out of the mitochondria as acetyl-carnitine and converted to malonyl-CoA
via acetyl-CoA carboxylase (ACC) to allosterically regulate CPTI. ACC is regulated covalently via AMP-kinase (AMPK). Figure adapted from Jeukendrup
(2002), Kerner and Hoppel (2000), and Schaffer (2002).
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7.2. Muscle uptake
After transport through the circulation, FFA cross the
endothelium, interstitial space, the plasmalemma, and
finally the cytosol of the myocyte. The events involved in
myocyte uptake of FFA are the subject of much debate.
Indeed, there is some question of which specific proteins are
involved or even if transporters are needed for FFA uptake.
Some suggest that FFA uptake occurs by a spontaneous
transfer of FFA across the membrane by a transporterindependent dflip-flopT mechanism (Hamilton, 1998).
Although this mechanism is fairly rapid, evidence from
gene knockout studies (Febbraio et al., 1999; Coburn et al.,
2000) suggests that muscle FFA uptake is transporter
mediated (Schaffer, 2002). Once albumin has reached the
myocyte it is thought to bind to an albumin receptor (AlbR)
on the muscle membrane (Reed and Burrington, 1989),
which may accelerate FFA transfer (Van Der Wusse and
Reneman, 1996). FFA are then transferred to and pass
through one of three putative membrane transporters. There
is evidence indicating that the fatty acid transport protein
(FATP), the 88 kDa CD36/FAT or the fatty acid binding
protein plasma membrane (FABPpm) (Stahl et al., 2001;
Luiken et al., 2002; Schaffer, 2002) are involved. Currently,
the CD36/FAT transport mechanism has received the most
attention as the major transport protein. Once inside the cell
FFA are bound to fatty acid binding proteins (FABP), which
increase FFA solubility by 700-fold. The whole complex
moves rather than just the FFA moving from one FABP
molecule to another and this association is thought to
increase the diffusion through the cytosol by up to 17-fold
(Van Der Wusse and Reneman, 1996). FABP also tend to
aggregate close to mitochondria, with the majority bound to
the cytosolic side of the outer mitochondrial membrane.
Release of FFA from FABP is increased by a drop in
intracellular pH. This may be an important mechanism for
increasing intracellular FFA availability during exercise
(Van Der Wusse and Reneman, 1996). FFA entering the
muscle are converted to fatty acyl-CoA (FA-CoA) to ensure
that a concentration gradient for FFA is maintained between
the blood and the cytosol (Van der Vusse and Roemen,
1995). This reaction is catalyzed via acyl-CoA synthase
(ACS), but the fatty acid transport protein may possess
synthase activity as well (Hall et al., 2003). The newly
formed FA-CoA are bound to the 10 kDa acyl-CoA binding
protein (ACBP) and transported to the mitochondrial
membrane. Since the outer mitochondrial porin restricts
entry of proteins over 5000 Da, ACBP do not transport FACoA into the intermembrane space (Zammit, 1999).
considered the controlling step in mitochondrial fatty acid
oxidation (Kerner and Hoppel, 2000). FA-Carn is transferred into the mitochondrial matrix by carnitine acyltransferase (CAT) and converted back to FA-CoA by
CPTII on the inner surface of the inner mitochondrial
membrane. CPTI and CPTII are enriched at bcontact sitesQ
of the mitochondrial inner and outer membranes (Zammit,
1999), which may be important sites for regulation of FFA
entry. FA-CoA in the mitochondrial matrix then enters the
four reactions of h-oxidation catalyzed by (1) long- or
medium-chain acyl-CoA dehydrogenase (LCAD or
MCAD, collectively XCAD in Fig. 4); (2) enoyl-CoA
hydratase; (3) h-hydroxyacyl-CoA dehydrogenase (HOAD);
and (4) acyl-CoA acetyltransferase (ketothiolase). Each
passage through the h-oxidation spiral shortens the FA by
2 carbons and produces acetyl-CoA. Acetyl-CoA condenses with oxaloacetate to form citrate in the first reaction
of the tricarboxylic acid cycle (TCA). Reducing equivalents formed in the TCA are transferred to the electron
transport system for the formation of ATP. These components of the fatty acid pathway have been well worked out
for mammals. Currently, many homologous cellular components have been identified in other species, suggesting
that the regulation of the fatty acid pathway is similar
across species.
8. Regulation of cellular fatty acid oxidation: genetic and
nongenetic mechanisms
Cellular fatty acid oxidation can be regulated at the level
of membrane transporters, at cytosolic FABP, and at fatty
acid entry points into the mitochondria. The mechanisms of
regulation depend on the time available to mount a
response. If demand for increased fatty acid oxidation
occurs acutely, existing machinery can undergo metabolic
regulation, which can occur either via allosteric or covalent
regulation of enzymes and transporters. There may also be
the translocation of enzymes and transporters within the cell
or organelle to sites of fatty acid transport and/or oxidation.
If the demand for fatty acid oxidation occurs chronically,
then either levels of enzymes and transporters or isozyme
composition can be modified by changes in transcription
and/or synthesis–degradation pathways for protein or RNA.
Given multiple generations and the appropriate selection
pressure, new enzymes with altered kinetics can occur or the
constitutive or induced expression of genes can change due
to changes in the properties of gene promoter regions
(Schulte, 2001).
7.3. Mitochondrial oxidation
8.1. Acute regulation
Entry into the mitochondria begins with the conversion
of FA-CoA to a fatty acyl-carnitine (FA-Carn in Fig. 4) by
carnitine palmitoyltransferase (CPT)I on the inner side of
the outer mitochondrial membrane. This is generally
Acute regulation of cellular fatty acid uptake occurs via
the translocation of CD36/FAT to the cell membrane
(Luiken et al., 2002), possibly controlled by IP-3 kinase
signaling (Jeukendrup, 2002). Rapid adjustment in mito-
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chondrial fatty acid oxidation chiefly occurs via allosteric
regulation of CPTI by malonyl-CoA, which is the product in
the first committed step in fat synthesis from acetyl-CoA by
the enzyme acetyl-CoA carboxylase (ACC). ACC activity is
controlled covalently via phosphorylation by AMP-kinase
(AMPK) (Winder, 2001). Since significant fat synthesis
does not occur in muscle, the major function of this system
is thought to be the control of mitochondrial lipid oxidation.
Mammalian CPTI consists of liver- and muscle-type isoforms, which are encoded by the separate genes CPTIa and
h, respectively. They differ in their affinity for the
cosubstrate carnitine and in their degree of inhibition by
malonyl-CoA (Moore et al., 2001). The liver isoform
(CPTIa) has a lower sensitivity to malonyl-CoA that is
further lowered by fasting (Bremer, 1981). The muscle
isoform, CPTIh, is more sensitive to malonyl-CoA inhibition (McGarry et al., 1983) and malonyl-CoA concentration
is highest in muscle at rest. During low-intensity exercise,
fatty acid oxidation increases as malonyl-CoA concentration
falls both in rats (Winder et al., 1990) and in trout (Richards
et al., 2002), but not in humans (Odland et al., 1998). This
suggests that there are species differences and alternate
regulatory mechanisms. Interestingly, recent evidence suggests that CPTI activity can be negatively affected by
interaction with the cytoskeleton in vivo but not in vitro
(Velasco et al., 1998). CPTI activity is also sensitive to
changes in muscle pH in humans (40% inhibition with a
decrease in pH of 0.2 units; Bezaire et al., 2004). The
regulation of h-oxidation enzymes is not thought to play a
large role in controlling the overall rates of fatty acid
oxidation since intermediates of this pathway change little
under most conditions. However, there is evidence that high
NADH/NAD+ ratios are inhibitory to HOAD and high
acetyl-CoA/CoASH ratios to thiolase (Eaton, 2002).
451
PPAR gamma cofactor-1 (PGC-1, Lemberger et al., 1996).
Responsive to cellular metabolism, PPAR can be activated by
increases in either monounsaturated (MUFA) or polyunsaturated fatty acids (PUFA) (Clarke, 2001). Fine tuning of this
transcriptional activity can be either enhanced or repressed by
kinase activity. For example, a-adrenergic agonist stimulated
hypertrophy decreases PPAR expression but also inactivates
its activity through the ERK-MAPK pathway (Barger et al.,
2000). Conversely, p38-MAPK activation has been found to
increase PPAR activity (Barger et al., 2001). PPAR has been
found to regulate many genes in the fatty acid oxidation
pathway (Zhang et al., 2004). It is known that h-oxidation
8.2. Chronic regulation
Altered fatty acid oxidation rates in response to chronic
stimuli may involve changes in enzyme and/or transporter
expression. FABPpm and CD36/FAT content, for instance,
are responsive to changes in energy demand (Bonen et al.,
1999). There is also evidence that both of these proteins are
regulated transcriptionally in mammals. CPTI isoforms are
tissue-specific and their contents are also regulated transcriptionally in mammals (Van der Leij et al., 2002). Since the
genes CPTIa and h lack sequences called TATA boxes,
which are commonly used for basal expression in other genes,
they rely on transcription factor specificity protein-1(Sp1) for
baseline transcriptional regulation (Steffen et al., 1999). The
induction of CPT and many other genes in the fatty acid
pathway in response to stress (Fig. 5) occurs via the fatty acid
response element (FARE) or peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) that bind
transcription factor PPAR. PPARa is the most common
isoform found in cardiac and skeletal muscle. DNA binding is
enhanced by the two factors: retinoic acid receptor (RXR) and
Fig. 5. The genetic regulation of fatty acid genes occurs principally by the
transcription factor PPAR. PPAR is a nuclear receptor that can be activated
by fatty acids (FA), which binds to response elements (PPRE) on the FA
gene. PPAR interacts with binding partner retinoid-X-receptor (RXR)
forming a heterodimer and DNA binding, and interaction with PGC-1 can
be affected by ligands FA and 9-cis retinoic acid. Genes that have been
found to contain PPRE in their promoter region include CPTI, medium
chain acyl-CoA dehydrogenase (MCAD), Acyl-CoA synthase (ACS),
malonyl-CoA decarboxylase (MCD), fatty acid binding protein (FABP),
and uncoupling protein (UCP)1. Figure adapted from Van Bilsen et al.
(1998), Barger and Kelly (2000a), and Barrero et al. (2003).
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enzymes LCAD, MCAD and HOAD have PPREs (Gulick et
al., 1994). FATP and ACS are also regulated by PPAR in a
coordinated fashion (Martin et al., 1997). CD36/FAT transcriptional activation is indirectly dependent on PPAR, with a
true PPRE being absent from its promoter region (Sato et al.,
2002). Although PPAR and its binding partners and cofactors
play a central role, other transcription factors may be
important for the regulation of genes involved in fatty acid
oxidation. For example, the orphan nuclear receptor chicken
ovalbumin upstream promoter-transcription factor (COUPTF) has been found to suppress expression of many genes for
fat oxidation (Disch et al., 1996).
8.3. Nongenetic factors
Probably of equal importance to gene expression changes
in regulating lipid oxidation are the changes in cellular
milieu in which the fatty acid pathway functions. Changes
in cellular pH, phosphorylation potential, and membrane
properties are just a few examples of nongenetic factors that
affect fatty acid oxidation. Studies of mammalian CPTI
show that experimentally induced changes in membrane
fluidity can affect the sensitivity of the enzyme to malonylCoA (McGarry and Brown, 2000; Kolodziej and Zammit,
1990; Zammit et al., 1998). Membranes are highly ordered
and the mitochondrial outer membrane contains roughly
equal amounts of lipid and proteins (Hochachka and
Somero, 2002). Proper functioning of these proteins
depends on their ability to change conformations, a product
of membrane physical state. CPTI is thought to undergo
large conformational changes during interaction with
malonyl-CoA (Zammit et al., 1998); thus, membrane order
may be very important to its sensitivity to this effector.
Changes in the physical state of membranes can occur by
variation in lipid: (1) types: phospholipids (PL) and
cholesterol; (2) classes: PLs phosphatidylethanolamine
(PE) and phosphatidylcholine (PC); and (3) species: acyl
chain composition of PL. Other membrane properties may
also be important for the proper functioning of membrane
proteins such as CPTI. Lipid rafts (Zehmer and Hazel, 2003)
and local contact sites (Hoppel et al., 2001) are two such
properties that may have a great impact on not only
membrane fluidity but also localized membrane effects on
protein function. For example, in 48-h fasted rats and in
diabetic rats, the sensitivity of liver CPTI to malonyl-CoA
decreased and this correlated to changes in fluidity of the
mitochondrial membrane core but not the periphery
(Zammit et al., 1998). This makes sense since CPTI spans
the outer mitochondrial membrane with two highly hydrophobic segments (Zammit et al., 1998). Thus, there can be
little change in the general lipid characteristics of the
membrane, but changes in specific microdomains may be
important for the regulation of enzymes like CPTI. This is
nicely demonstrated in experiments where cardiolipin is
added to isolated rat liver mitochondria, which results in an
increase in CPTI malonyl-CoA sensitivity (reviewed in
Kerner and Hoppel, 2000). Cardiolipin content is higher in
membrane contact sites, which are known to contain high
levels of CPTI (Kerner and Hoppel, 2000). This change in
CPTI sensitivity has been seen in mammalian liver but has
not yet been observed in heart or skeletal muscle. The
mechanisms responsible for changed in regulation of
mammalian CPTI is an active area of research.
9. Changes in lipid metabolism with environmental
stress
Most of the work done to date on the cellular components
of the fatty acid pathway and their regulation have focused
on mammals. Moreover, this work is mainly focused on
elucidating the mechanisms responsible for human disease
or how to improve exercise performance. Few studies have
examined how lipid oxidation is affected by environmental
stress or how animals maintain locomotory ability under
conditions of hypoxia or temperature extremes.
9.1. Hypoxia
For many years, the prediction has been that carbohydrates are preferred over lipids under hypoxic conditions.
Animals acclimated or adapted to chronic hypoxia should
take advantage of the greater ATP yield per mole oxygen
with carbohydrates. This increase in ATP yield may be
greater than the 15% theoretical difference based on
comparing phosphate to oxygen ratios for the two fuels
(Brand, 1994) due to the so-called boxygen-wasting effectQ
of lipids (Korvald et al., 2000). Mostly studied in the heart,
the observed oxygen consumption difference of FFA vs.
pyruvate can be greater than 30%. Potential mechanisms for
the FFA oxygen wasting effects include (1) uncoupling of
mitochondrial oxidation from ATP production, (2) increased
TAG-FA cycling, and (3) altered Ca2+ handling (Korvald et
al., 2000). It is true that specific tissues have a preference for
carbohydrate oxidation under hypoxic conditions. In a series
of exquisite and technically challenging studies, Hochachka
et al. (1996) showed that the hearts of high-altitude native
humans are geared towards higher carbohydrate use. Indeed,
several studies on high-altitude acclimation in humans have
shown an increased reliance on carbohydrate by working
muscles during exercise at altitude (Roberts et al., 1996a,b;
Young et al., 1992; Brooks et al., 1991). Conversely, when
mammalian plasma FFA have been measured at altitude,
they are always elevated (McClelland et al., 1999; Jones et
al., 1972; Klain and Hannon, 1968; Whitten and Janoski,
1969), and this has often been attributed to an increased
reliance on lipid metabolism at altitude to conserve valuable
carbohydrate stores (Young et al., 1982). However, the only
measurement to date has found that FFA turnover is
decreased after altitude acclimation despite increased plasma
FFA (McClelland et al., 1999). Another confounding factor
is that the majority of high-altitude studies have used the
G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
same work rate when comparing sea-level and high-altitude
measurements of exercise fuel utilization. Undoubtedly,
valuable data are obtained from comparing groups at the
same ATP turnover rate, but because exercise intensity has
such an overriding effect on fuel selection (see Fig. 2), many
high-altitude studies may be masking important information
regarding fuel use. Maximum aerobic metabolic rate is
greatly affected by hypoxia, and after acclimation to
moderate high altitude (HA, 4300 m), there is a 20–30%
decrease in VO2max. As a consequence of this decrease, the
same absolute work rate at altitude represents a higher
percentage of VO2max than it does at sea level. Given the
relationship between exercise intensity and fuel use, past
studies may have found a greater reliance on carbohydrates
due to differences in exercise intensity and not due to altitude
acclimation. Recent work on rats and humans has shown that
this is in fact the case. When exercising at the same
%VO2max, both rats and humans acclimated to altitude
(simulated and natural) use similar proportions of lipid and
CHO to power aerobic exercise (McClelland et al., 1998,
1999; Braun et al., 2000). This is despite some reports of
changes in muscle biochemistry for both the carbohydrate
(Hoppeler et al., 2003) and the lipid pathways (Kennedy et
al., 2001) and demonstrates the overriding effect of exercise
intensity on fuel selection patterns.
Resting and exercise lipid oxidation and lipid transport
kinetics data on nonmammalian vertebrates are scarce,
especially in regards to the effects of environmental stress.
Existing data do suggest that at rest fish acutely exposed to
hypoxia respond with a decrease in FFA turnover (Haman et
al., 1997). Whether this translates to decreased fatty acid
oxidation at rest or during exercise is currently not known.
Moreover, anoxia and hypoxia cause opposite effects on
plasma FFA in tolerant and intolerant species (Van Raaij et
al., 1996). It has also been suggested that fatty acid
synthesis and chain elongation via what is essentially a
reversal of h-oxidation, can help regulate cellular reduction–
oxidation during anoxia and severe hypoxia (Hochachka,
1980; Van Raaij et al., 1994). This process occurs in
mammalian heart mitochondria so perhaps it occurs in
locomotory muscle as well. How this strategy impacts on
the locomotory ability of these animals due to the
potentially conflicting roles of h-oxidation needs to be
addressed.
Changes in muscle metabolic properties in response to
environmental stress may impact overall fuel selection
during exercise. Mammals exposed to acute hypoxia show
changes in gene expression for the glycolytic pathway via
the hypoxia inducible factor (HIF)-1a and this may affect
whole-animal rates of fatty acid and glucose oxidation
(Hoppeler et al., 2003). Moreover, gene expression patterns
differ under acute and chronic hypoxia conditions but the
consequences of this to exercise metabolism have not been
studied in detail. For example, rats exposed to chronic
hypoxia for several weeks do not show profound changes to
either their muscle morphometry or biochemistry (Abdel-
453
malki et al., 1996). Nonmammalian vertebrate muscle is
also sensitive to acute hypoxia. Zebrafish embryos show
changes in expression of many metabolic genes demonstrating the sensitivity of fish gene expression to hypoxia
(Ton et al., 2003). However, more work is needed to
determine what effect hypoxia has on overall muscle
metabolism and whole-animal fuel oxidation rates in these
species.
9.2. Temperature
Nonmammalian vertebrates, particularly ectotherms,
show profound changes in muscle properties with acute
and chronic changes in ambient temperature. In eurythermal
species these changes help maintain locomotory ability in
the face of declining body temperatures (see Guderley, 2004
this volume). Increases in mitochondrial mass with cold
acclimation are well documented in fish (e.g., St.-Pierre et
al., 1998) but changes in the fatty acid pathway and the
mechanistic stimulus for these changes have not been fully
elucidated (Pörtner, 2002).
9.2.1. Acute effects
Even baseline data on the acute regulation of lipid
oxidation in nonmammalian vertebrates such as fish are
scarce. We do know that fish rely on fatty acids to power
submaximal exercise (Kieffer et al., 1998), and one
regulator of this pathway, malonyl-CoA, decreases over an
exercise bout (Richards et al., 2002). However, unlike
mammals, exercise results in little change in plasma FFA
turnover (kinetics; Bernard et al., 1999), which suggests that
lipid kinetics differ among species. The oxidation of fatty
acids by isolated fish red muscle mitochondria is high
(Moyes et al., 1989; Suarez and Hochachka, 1981; Johnston
et al., 1998) and these mitochondria do contain CPTI
(Froyland et al., 1998; Rodnick and Sidell, 1994). Current
evidence suggests that trout express only one isoform of
CPTI, which has mammalian liver-type enzyme kinetics
(i.e., CPTIa in mammals) (Gutieres et al., 2003). Therefore,
one can deduce that acute regulation of mitochondrial fat
oxidation in fish may differ from that in mammals. It is
possible that acute change in temperature may exert its
greatest effect on lipid metabolism through temperaturerelated changes in pH on CPTI kinetics. Membrane properties change dramatically with acute temperature exposure
with tighter packing of acyl chains leading to a decrease in
membrane fluidity, but it is not clear what effect this has on
fatty acid oxidation.
9.2.2. Chronic effects
Chronically, environmental stress may exert its effects
both through changes in gene expression for the fatty acid
pathway and through changes in nongenetic factors that
contribute to the cellular milieu. Transcriptional regulation
of the fatty acid pathway in nonmammalian vertebrates has
not been studied in detail but CPTI activity is related to
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mRNA expression across trout tissues (Gutieres et al.,
2003). As well, PPARs do exist in fish (Ibabe et al., 2002)
and have a tissue isozyme distribution pattern that is similar
to humans. How transcription factors, such as PPAR and
PPAR gamma cofactor-1 (PGC-1), respond to environmental stress to affect mitochondrial biogenesis (reviewed in
(Moyes and Hood, 2003) or the expression of CPTI and
other genes is not well understood. Another unknown is the
mechanistic trigger responsible for muscle remodeling. Part
of the problem is that possible candidates, such as energy
deficiency and hypoxia, occur at both cold and warm
temperatures but result in both an increase or a decrease in
mitochondrial mass, respectively. Recent evidence suggests
that in mammals there is at least 1 splice variant of muscletype CPTIh that is malonyl-CoA insensitive (Kim et al.,
2002). It is yet to be determined if a similar strategy is
employed by nonmammalian vertebrates or how differential
expression of these splice variants contributes to muscle
plasticity. To date there are no data on changes in gene
expression for the fatty acid pathway with cold acclimation
in fish.
As mentioned, in ectothermic vertebrates such as fish,
cold acclimation results in profound changes in mitochondrial density (St.-Pierre et al., 1998). In addition, there is an
increase in CPTI activity and possibly its malonyl-CoA
sensitivity (Rodnick and Sidell, 1994) as well as an
increased capacity of mitochondria to oxidize acyl-carnitines (Guderley et al., 1997). Another well-characterized
response of ectotherms to cold acclimation are changes in
membrane composition (Cordier et al., 2002; Hazel and
Zerba, 1986) and fluidity (Logue et al., 2000; Zehmer and
Hazel, 2003). Since membrane order increases with acute
decreases in temperature, it is not surprising that animals
adapted or acclimated to low temperature increase the
fluidity (and decrease order) of cellular membranes by
changing lipid composition to preserve function at reduced
temperature. Cellular membrane composition in mammals is
not only responsive to changes in dietary fatty acid
composition but also to exercise (Kriketos et al., 1995;
Helge et al., 1999, 2001). Also, it differs between muscle
fiber types in fish (Leary et al., 2003). There is good
evidence from the mammal literature that membrane fluidity
can affect CPTI kinetics by changing its sensitivity to
malonyl-CoA (McGarry and Brown, 2000; Kolodziej and
Zammit, 1990; Zammit et al., 1998). Whether this is a
mechanism employed by ectotherms in response to cold
acclimation has not yet been demonstrated. In addition,
membrane microdomains may play an even greater role than
bulk membrane changes in determining mitochondrial fatty
acid oxidation (Zehmer and Hazel, 2003). Currently, there is
very little information regarding the effects of temperature
acclimation on changes in these domains or changes in
mitochondrial membrane components such as cardiolipin.
The response of fatty acid oxidation to environmental stress
must involve the interaction between these genetic and
nongenetic mechanisms.
10. Evolutionary variation in fatty acid oxidation
Interspecies variation in fatty acid oxidation can be great
(McClelland et al., 1994), but the underlying mechanisms
that explain this diversity are currently under explored.
Differences in absolute lipid oxidation rates exist across
taxa, across body size, and in response to environmental
stress. There are many steps in the mobilization, delivery,
and oxidation of fatty acids (Figs. 1 and 4) that represent
potential sites for selection. Many of these steps have not
been studied in detail as they relate to interspecies diversity
in fuel selection. However, changes in absolute fatty acid
oxidation rates associated with animals of increased aerobic
capacity include increases in lipolysis (Weber et al., 1993;
Shaw et al., 1975), in total plasma flow, and in binding
capacity of albumin for FFA (McClelland et al., 1994).
There appear to be upper limits to sarcolemma transport
rates which are the same between aerobic and sedentary
species (Vock et al., 1996). However, interspecies differences in glucose and fatty acid transporter densities have not
been directly quantified. More aerobic species rely to a
greater extent on intramuscular fuel supplies to power
exercise (Weber et al., 1996). Indeed, intramuscular stores
of glycogen and TAG are greater in more aerobic species
(Weibel et al., 1996). Examples of extreme endurance
performers, namely, migratory birds and fish, show the
ability to store large amounts of lipid (Blem, 1980; Van
Ginneken and van den Thillart, 2000) presumably for
increased lipid use during migration. This is mirrored by
changes in cellular components like FABP (Guglielmo et
al., 2002). Fish designed for increased activity store fat in
muscles, while more sedentary fish primarily store fat in
liver or adipose tissue (McClelland et al., 1995; Weber and
Zwingelstein, 1995). The breakdown of intramuscular lipids
is dependent on activities of HSL within muscle and HSL
content is correlated with TAG content across different
muscle fiber types (reviewed in Jeukendrup, 2002). This is
perhaps true between species and represents a potential
adaptation for increased lipid mobilization. Increases in lipid
oxidation associated with exercise training or high-fat
feeding (Jansson and Kaijer, 1982) involve increases in
enzymes for fatty acid oxidation. This suggests that
increases in enzyme content may help explain interspecies
differences in fatty acid oxidation.
There is a wide variation in muscle mitochondrial density
between species and it varies with body mass in the same
way as Vo2max (Hoppeler and Taylor, 1992). Less
information is available regarding the variation between
species in mitochondrial fatty acid oxidation rates or levels
of CPTI and other h-oxidation enzymes with body mass.
Circulatory glucose oxidation rates do scale with body size
in the same way as metabolic rate in mammals (Weber et al.,
1986). However, lactate turnover does not (Weber, 1991).
Plasma FFA turnover appears to change with body mass
(personal observation), but how this impacts on oxidation is
not clear due to potential interspecies differences in muscle
G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
FFA uptake. Finally, body size does not explain other
interspecies differences in fuel selection. Indeed, fish and
mammals of the same size have very different circulatory
glucose oxidation rates (Weber, 1991).
A dramatic difference in lipid metabolism exists between
the elasmobranchs (sharks and rays) and teleosts. The
elasmobranchs show non-detectible fatty acid oxidation and
almost undetectable levels of CPT in muscle (Moyes et al.,
1990). This group (principally the dogfish, Squalus acanthias) has been studied extensively regarding their metabolism (Moyes et al., 1990; Singer and Ballantyne, 1989;
Richards et al., 2003). Dogfish muscles rely on ketone
bodies, produced through liver fatty acid oxidation, to fuel
exercise and recovery (Richards et al., 2003; Ballantyne,
1997). Currently, there is no molecular explanation for why
elasmobranchs do not express CPT in muscle. This is
especially intriguing since they do express other h-oxidation
enzymes (Moyes et al., 1990), which are also regulated by
PPAR in mammals. Moreover, it is not known which
isoform of CPTI is expressed in dogfish liver. The metabolic
organization of the dogfish may represent the ancestral lipid
metabolism within the fishes.
11. Models for studying the evolution of lipid metabolism
Obviously, the best models to use for the study the
evolution of lipid metabolism are species that have clear
differences in rates of fat oxidation. However, they may not
be the most convenient due to problems of availability,
husbandry, and logistics regarding exercise. Moreover, there
is often a lack of background physiological, biochemical,
and genetic data. Alternatively, intertissue differences in fuel
selection and rodents with targeted alteration in components
of fatty acid oxidation can be used as windows into
interspecies differences in muscle metabolism.
455
1989), suggesting that these mitochondria are specialized
for higher rates fat oxidation. In fact, although mitochondrial oxygen consumption with pyruvate is similar
between these muscle types, rates of palmitoyl-carnitine
oxidation are higher in red muscle mitochondria (Moyes
et al., 1989). Higher total lipid content (Johnston, 1977)
likely helps support this higher lipid oxidation. There are
limited data on interspecies differences in mitochondrial
properties, but there is evidence that differences in
mitochondrial fuel selection properties do exist (Rasmussen et al., 2004). Intertissue variants in PPAR, PGC-1, and
RXR and other regulators of fatty acid pathway gene
expression might represent mechanisms for interspecies
differences in muscle mitochondrial content and quality
(reviewed in Moyes, 2003). Although PGC-1 appears to act
as a master controller for mitochondrial content, its role in
species-specific differences in muscle fuel selection remains
to be demonstrated.
11.2. Targeted alterations in gene expression
Biomedical research has provided a vast array of transgenic and gene knockout mice with targeted changes in
transporters and enzymes important for muscle fatty acid
oxidation. For example, mice lacking ACC had 30-fold
lower levels of muscle malonyl-CoA and a 30% higher rate
of fatty acid oxidation, which was insensitive to insulin
(Abu-Elheiga et al., 2001). Those lacking PPARa show
decreased rates of cardiac fatty acid oxidation, partly due to
lower activities of malonyl-CoA decarboxylase (MCD),
11.1. Developmental differences in lipid oxidation
Developmental differences have led to fuel selection
and biochemical property differences between tissue types.
Using these differences not only can be a powerful way
to examine the regulation of fatty acid oxidation but also
can be used as a window into interspecies variation in
lipid metabolism. Red and white muscles differ in their
biochemistry, mitochondrial profiles, and fuel selection
characteristics (Fig. 6). Since fish possess highly differentiated muscles, they can be use as a powerful
experimental tool for examining intertissue metabolic
differences. Fish red muscle possesses higher palmitate
transport rates (Richards et al., 2004), activities of FFA
oxidation enzymes CPT (CPTII) and HOAD (Moyes et
al., 1989) than fish white muscle. The two muscle types
also have qualitative and quantitative differences in their
mitochondria. Red muscle has a greater mitochondrial
density and proportionally more CPT (Moyes et al.,
Fig. 6. The ratio of red vs. white muscle values in fish for variables
involved muscle fatty acid oxidation. Palmitate uptake data from trout
(Richards et al., 2004); CPT (CPTII), HOAD, mitochondrial oxidation
measurements from carp (Moyes et al., 1989); and total lipid content from
carp (Johnston, 1977). Dashed line represents no difference between red
and white muscle.
456
G.B. McClelland / Comparative Biochemistry and Physiology, Part B 139 (2004) 443–460
resulting in elevated malonyl-CoA levels (Campbell et al.,
2002). The h-oxidation machinery was also downregulated
with LCAD expressed at lower levels (Aoyama et al., 1998).
PPAR overexpressing mice show increases in mRNA for
CPTI, an increase in CPTI activity, increased palmitate, and
decreased glucose oxidation in heart (Hopkins et al., 2003).
A null mutation in the CD36/FAT gene resulting in no
detectable CD36/FAT protein leads to significantly reduced
fatty acid uptake rates (Febbraio et al., 1999; Coburn et al.,
2000). A defective CD36/FAT transporter also seems to be
the mechanism of defective fatty acid uptake in hearts of
spontaneously hypertensive rats (Hajri et al., 2001). Alternatively, overexpression of CD36/FAT results in an increase
in muscle fatty acid oxidation (Ibrahimi et al., 1999). From
these data, it is evident that changes in specific transcription
factors and changes in specific enzymes and transporters can
lead to significant changes in muscle fatty acid oxidation.
However, none of these studies have looked at the wholeanimal lipid oxidation or exercise metabolism consequences
of these changes. It is not clear if the fatty acid pathway is
acted on as a whole by evolution. Current evidence suggests
that changes to single transporters or enzymes do not result
in corresponding changes in flux through the entire pathway. Upregulation of specific enzymes in glycolysis in yeast
does not result in a corresponding increase in pathway flux
(Schaaff et al., 1989), and overexpression of FABPpm does
increase membrane FFA transport but oxidation is not
markedly altered (Clarke et al., 2004). More integrated
approaches are needed to determine the functional consequences of targeted alterations in aspects of the fatty acid
pathway.
12. Conclusions and future directions
Currently, we have a good understanding of how
carbohydrate metabolism is regulated during exercise and
how interspecies variations and adaptations to extreme
environment occur. Hochachka and his coworkers contributed greatly to this understanding and much of what
we do now, as Somero (2002) described it, are truly
bfootnotes to Peter.Q Our understanding of lipids is less
complete, in part, due to their complex nature. Lipids are
stored in different forms throughout the body, have a
complex transport system and can be incorporated into
different biological structures. How animals regulate lipid
metabolism is an important part of the enantiostatic
mechanism (conserved function) used by animals in
response to stress. There are many functional and
structural steps as fatty acids are mobilized, transported,
and oxidized in working muscle that may serve as
regulatory points for responding to acute or chronic
stimuli or as raw material for natural selection. Determining which of these factors help regulate the fatty acid
pathway and what impact they have on whole-animal
lipid oxidation and performance is an important area of
future research. Peter was concerned but optimistic about
the future of comparative physiology (see Mangum and
Hochachka, 1998). He realized that comparative physiology needs to embrace current technologies like molecular
biology, but cautioned that as we emerge from a very
reductionist period in science, integrative approaches are
needed. For instance, more studies need to attempt to
integrate gene expression with physiological function,
especially in response to environmental stress. Certainly,
determining the genetic regulation of biological structures
and physiological function remains one of the big
challenges of the next decade.
Acknowledgements
This review is dedicated to the late Peter W. Hochachka.
Those of us in his last generation of students have missed
his guidance and friendship during these early stages of our
careers. Thanks to Peter and to Dick Taylor for teaching me
that there is no substitute for a good question. I also
sincerely thank the reviewers of this manuscript for their
helpful suggestions that have greatly improved the final
product.
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