Fat adaptation in well-trained athletes: Effects on cell metabolism

Abstract

The performance of prolonged (>90 min), continuous, endurance exercise is limited by endogenous carbohydrate (CHO) stores. Accordingly, for many decades, sports nutritionists and exercise physiologists have proposed a number of diet-training strategies that have the potential to increase fatty acid availability and rates of lipid oxidation and thereby attenuate the rate of glycogen utilization during exercise. Because the acute ingestion of exogenous substrates (primarily CHO) during exercise has little effect on the rates of muscle glycogenolysis, recent studies have focused on short-term (<1-2 weeks) diet-training interventions that increase endogenous substrate stores (i.e., muscle glycogen and lipids) and alter patterns of substrate utilization during exercise. One such strategy is "fat adaptation", an intervention in which well-trained endurance athletes consume a high-fat, low-CHO diet for up to 2 weeks while undertaking their normal training and then immediately follow this by CHO restoration (consuming a high-CHO diet and tapering for 1-3 days before a major endurance event). Compared with an isoenergetic CHO diet for the same intervention period, this "dietary periodization" protocol increases the rate of whole-body and muscle fat oxidation while attenuating the rate of muscle glycogenolysis during submaximal exercise. Of note is that these metabolic perturbations favouring the oxidation of fat persist even in the face of restored endogenous CHO stores and increased exogenous CHO availability. Here we review the current knowledge of some of the potential mechanisms by which skeletal muscle sustains high rates of fat oxidation in the face of high exogenous and endogenous CHO availability.

Full text

REVIEW / SYNTHE
`
SE
Fat adaptation in well-trained athletes: effects on
cell metabolism
Wee Kian Yeo, Andrew L. Carey, Louise Burke, Lawrence L. Spriet, and
John A. Hawley
Abstract: The performance of prolonged (>90 min), continuous, endurance exercise is limited by endogenous carbohy-
drate (CHO) stores. Accordingly, for many decades, sports nutritionists and exercise physiologists have proposed a number
of diet-training strategies that have the potential to increase fatty acid availability and rates of lipid oxidation and thereby
attenuate the rate of glycogen utilization during exercise. Because the acute ingestion of exogenous substrates (primarily
CHO) during exercise has little effect on the rates of muscle glycogenolysis, recent studies have focused on short-term
(<1–2 weeks) diet-training interventions that increase endogenous substrate stores (i.e., muscle glycogen and lipids) and al-
ter patterns of substrate utilization during exercise. One such strategy is ‘‘fat adaptation’’, an intervention in which well-
trained endurance athletes consume a high-fat, low-CHO diet for up to 2 weeks while undertaking their normal training
and then immediately follow this by CHO restoration (consuming a high-CHO diet and tapering for 1–3 days before a ma-
jor endurance event). Compared with an isoenergetic CHO diet for the same intervention period, this ‘‘dietary periodiza-
tion’’ protocol increases the rate of whole-body and muscle fat oxidation while attenuating the rate of muscle
glycogenolysis during submaximal exercise. Of note is that these metabolic perturbations favouring the oxidation of fat
persist even in the face of restored endogenous CHO stores and increased exogenous CHO availability. Here we review
the current knowledge of some of the potential mechanisms by which skeletal muscle sustains high rates of fat oxidation
in the face of high exogenous and endogenous CHO availability.
Key words: AMP-activated protein kinase, b-hydroxyacyl-CoA-dehydrogenase, carnitine palmitoyl transferase, fatty acid
translocase, glycogen, intramuscular triglyceride, pyruvate dehydrogenase.
Re
´
sume
´
: La performance au cours d’un exercice continu d’endurance (>90 min) est limite
´
e par les re
´
serves endoge
`
nes de
sucres (CHO). Par conse
´
quent les nutritionnistes du sport et les physiologistes de l’activite
´
physique proposent depuis des de
´
-
cennies des programmes d’entraı
ˆ
nement combine
´
sa
`
des re
´
gimes alimentaires afin d’accroı
ˆ
tre la disponibilite
´
des acides gras
et le degre
´
d’oxydation des lipides, et ce faisant, de restreindre l’utilisation du glycoge
`
ne au cours de l’effort. Du fait que
l’apport de substrats exoge
`
nes (surtout les CHO) au cours d’un exercice a peu d’effet sur le taux musculaire de la glycoge
´
no-
lyse, des e
´
tudes re
´
centes proposent des programmes de courte dure
´
e (<1–2 semaines) combinant entraı
ˆ
nement et al.imenta-
tion pour accroı
ˆ
tre les re
´
serves endoge
`
nes de substrats (glycoge
`
ne musculaire et lipides) et pour modifier l’utilisation des
substrats au cours de l’effort. « L’adaptation aux graisses » constitue un tel programme au cours duquel l’athle
`
te d’endurance
consomme des aliments riches en gras et pauvres en sucres sur une pe
´
riode ne de
´
passant pas 2 semaines et ce, tout en mainte-
nant un re
´
gime d’entraı
ˆ
nement normal; tout de suite apre
`
s, il passe a
`
la phase de restauration des CHO en consommant des
aliments riches en sucres et en diminuant l’apport au cours des 3 jours pre
´
ce
´
dant la compe
´
tition d’importance. Comparative-
ment a
`
un re
´
gime isoe
´
nerge
´
tique de CHO sur une me
ˆ
me dure
´
e, la « pe
´
riodisation alimentaire » augmente les taux corporel et
musculaire d’oxydation des graisses tout en diminuant le taux de la glycoge
´
nolyse musculaire au cours de l’effort sous-maxi-
mal. Fait notable, ces modifications du me
´
tabolisme en faveur de l’oxydation des graisses demeurent me
ˆ
me en pre
´
sence de
la restauration des re
´
serves endoge
`
nes de CHO et d’une plus grande disponibilite
´
des CHO exoge
`
nes. Maintenant, nous vous
pre
´
sentons les connaissances courantes au sujet des me
´
canismes potentiels permettant au muscle squelettique d’entretenir un
Received 24 August 2010. Accepted 6 October 2010. Published on the NRC Research Press Web site at apnm.nrc.ca on 13 January 2011.
W.K. Yeo. Health Innovations Research Institute, School of Medical Sciences, RMIT University, P.O. Box 71, Bundoora, Victoria
3083, Australia; National Sports Institute of Malaysia, Bukit Jalil, Kuala Lumpur, Malaysia.
A.L. Carey. Health Innovations Research Institute, School of Medical Sciences, RMIT University, P.O. Box 71, Bundoora, Victoria
3083, Australia; Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia.
L. Burke. Department of Sports Nutrition, Australian Institute of Sport, Belconnen, ACT 2616, Australia.
L.L. Spriet. Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada.
J.A. Hawley.
1
Health Innovations Research Institute, School of Medical Sciences, RMIT University, P.O. Box 71, Bundoora, Victoria
3083, Australia.
1
Corresponding author (e-mail: john.hawley@rmit.edu.au).
12
Appl. Physiol. Nutr. Metab. 36: 12–22 (2011) doi:10.1139/H10-089 Published by NRC Research Press

haut taux d’oxydation des graisses malgre
´
la disponibilite
´
de beaucoup de CHO exoge
`
nes et endoge
`
nes.
Mots-cle
´
s:prote
´
ine kinase active
´
e par l’AMP, be
ˆ
ta-hydroxyacyl-CoA de
´
shydroge
´
nase, carnitine palmitoyl transfe
´
rase,
translocase des acides gras, glycoge
`
ne, triglyce
´
rides intramusculaires, pyruvate de
´
shydroge
´
nase.
[Traduit par la Re
´
daction]
_______________________________________________________________________________________
Introduction
Low muscle glycogen concentrations (both diet and exer-
cise induced) are associated with fatigue during prolonged,
submaximal (<85% of peak oxygen uptake (
_
VO
2 peak
)) en-
durance exercise lasting >2 h (Bergstro
¨
m et al. 1967). Gly-
cogen depletion is a function of the initial (pre-exercise)
glycogen concentration and its rate of utilization during a
work bout. Therefore, nutritional strategies to enhance en-
durance performance have typically focused on ways to in-
crease carbohydrate (CHO) availability by maximizing
CHO storage in muscle and liver in the days and hours be-
fore, and while consuming additional CHO during, an event
(Burke 2003). However, the rate of muscle glycogen utiliza-
tion during exercise is a function of the initial glycogen con-
centration (Hargreaves et al. 1995; Helge 2000). In addition,
consuming CHO immediately before and (or) during exer-
cise increases rates of whole-body CHO oxidation (Coyle et
al. 1986; Arkinstall et al. 2001). Therefore, an alternative
strategy to delay fatigue and (or) improve endurance per-
formance would be to increase the availability and (or) ca-
pacity to oxidize fat, while concomitantly reducing the rate
of muscle glycogen utilization. Indeed, exercise scientists
have long been interested in interventions that enhance fat
oxidation while simultaneously attenuating rates of muscle
glycogenolysis during prolonged submaximal exercise com-
menced with elevated glycogen stores (Hawley et al. 1998).
It is well known that endurance training results in meta-
bolic adaptations that increase rates of fat oxidation and de-
crease muscle glycogen utilization during submaximal
exercise (60%–85%
_
VO
2 peak
) (Holloszy and Coyle 1984;
Romijn et al. 1993). The increased reliance on fat oxidation
is generally attributed to increased mitochondrial volume,
along with increased mitochondrial enzymatic adaptations
to use fat, coupled with a reduction in the signals (free
[ADP] and [AMP] that activate the major enzymes that me-
tabolize CHO (glycogen phosphorylase (PHOS), phospho-
fructokinase (PFK), and pyruvate dehydrogenase (PDH)).
Perhaps less appreciated is that short-term, high-fat diets
also increase the rates of fat oxidation and ‘‘spare’’ muscle
glycogen during submaximal exercise. ‘‘Fat adaptation’’ is a
protocol in which endurance athletes consume a high-fat,
low-CHO diet for up to 14 days while undertaking their nor-
mal training (both high volume and high intensity). Fat
adaptation can be undertaken as a stand-alone dietary strat-
egy (Phinney et al. 1983; Lambert et al. 1994) or can be fol-
lowed immediately by a period of CHO restoration,
achieved by consuming a high-CHO diet and tapering for
1–3 days (Burke et al. 2000; Carey et al. 2001). When com-
pared with an isoenergetic CHO diet for the same duration,
both fat adaptation and the combined ‘‘dietary periodiza-
tion’’ of fat adaptation–CHO restoration increase whole-
body rates of fat oxidation and attenuate the rate of muscle
glycogen utilization during subsequent exercise (Phinney et
al. 1983; Lambert et al. 1994; Burke et al. 2000; Carey et
al. 2001). What is interesting about the ability of the fat-
adaptation protocol to increase rates of fat oxidation is that
it occurs without an increase in mitochondrial volume, im-
plying that a different mechanism may be responsible for
the adaptation compared with traditional exercise training
from an untrained to trained state. In this review, we focus
on several of the potential mechanisms that may be respon-
sible for the increases in whole-body rates of fat oxidation
measured after high-fat diets. In addition, we consider the
causes and outcomes of the intriguing observation that these
perturbations persist, at least in the short term, in the face of
high CHO availability (Burke et al. 2000). Because (i) this
protocol is designed only for the benefit of endurance per-
formance, (ii) consumption of high-fat diets might be detri-
mental to health (Burke et al. 2000; Helge 2000; Hennig et
al. 2001), and (iii) well-trained endurance subjects tolerate
the effects of a high-fat diet better than untrained individuals
during exercise (Erlenbusch et al. 2005), we refer only to
studies that have been undertaken in well-trained humans.
We focus only on investigations that have short-to-medium
(up to 28 days) fat-adaptation protocols because interven-
tions of less than 3 days lower resting muscle glycogen lev-
els and impair endurance performance (Starling et al. 1997;
Pitsiladis and Maughan 1999). Readers are directed to ear-
lier reviews pertaining to the effects of short-term (Burke
and Hawley 2002) and long-term (Helge 2002) fat adapta-
tion on metabolism and performance in both trained and un-
trained humans, as well as discussions of some of the
regulatory mechanisms in the interaction between CHO and
lipid oxidation during exercise (Spriet and Watt 2003; Spriet
and Hargreaves 2006).
Metabolic adaptations in response to
endurance training
Endurance training increases the fatigue resistance of con-
tracting skeletal muscle during prolonged submaximal exer-
cise (Holloszy and Coyle 1984; Hawley 2002). This is
achieved via increases in capillary density (Hermansen and
Wachtlova 1971) and substrate transport proteins (e.g., glu-
cose transporter 4 (GLUT4) and plasma membrane fatty
acid (FA)-transport proteins) (Andersen et al. 1993; Talanian
et al. 2010) as well as augmentation of mitochondrial vol-
ume, FA transport proteins, and representative enzymes of
the major metabolic pathways (e.g., citrate synthase,
b-hydroacyl-CoA-dehydrogenase, cytochrome c oxidase IV,
aspartate aminotransferase, and PDH) (Gollnick et al. 1972;
Hoppeler et al. 1973; Chi et al. 1983; Perry et al. 2008;
Talanian et al. 2010). Such adaptations enhance the main-
tenance of metabolic control (i.e., match ATP production
with ATP hydrolysis via oxidative mechanisms) during ex-
ercise at high submaximal work rates (Hawley 2002). In
addition, endurance training increases the storage capacity
for various fuels in skeletal muscle, with both intramyo-
cellular glycogen and lipid concentrations being higher in
Yeo et al. 13
Published by NRC Research Press

well-trained compared with untrained humans (Gollnick et
al. 1972; Hoppeler et al. 1973). Collectively, these adapta-
tions alter the pattern of fuel utilization during submaximal
exercise, whereby whole-body rates of fat oxidation are in-
creased while the rate of CHO oxidation is decreased, prin-
cipally through the sparing of muscle glycogen, at both the
same absolute and relative work rates as before training
(Holloszy et al. 1998; Hawley and Stepto 2001; Hawley
2002). This is achieved by the increased mitochondrial vol-
ume, which enables greater fat oxidation, reduces the mag-
nitude of exercise-induced increases in free ADP and AMP
(decreased perturbation of the energy state of the cell) at
any exercise intensity, and decreases the activation of
CHO metabolism (PHOS, PFK, and PDH activities).
Fat adaptation: rationale and background
Whereas glycogen storage in human skeletal muscle and
liver is limited, lipid storage in muscle, and particularly adi-
pose tissue, is abundant. In well-trained humans, the energy
storage of muscle triglyceride (TG) approaches or equals the
energy equivalence of the muscle CHO store, whereas the
absolute amounts of fat in humans are sufficient for many
hours or days of continuous exercise even in the leanest of
athletes (Burke and Hawley 2002). Logically, one would as-
sume that any training method that increases rates of fat ox-
idation during exercise above those levels already attained
by endurance training would enhance performance during
prolonged exercise in which CHO availability is a limiting
Table 1. Summary of studies investigating fat adaptation with and without carbohydrate restoration on whole-body metabolism, skeletal
Study Subjects Protocol
Phinney et. al. 1983 n = 5 M; well-trained cyclists (>65 mLkg
–1
min
–1
) 1-wk eucaloric balanced diet followed by 4-wk keto-
genic diet (<20 g CHOd
–1
)
Fisher et. al. 1983 n = 5 M; well-trained cyclists (5.1 Lmin
–1
) 1-wk eucaloric balanced diet followed by 4-wk keto-
genic diet (<20 g CHOd
–1
)
Muoio et. al. 1994 n = 6 M; well-trained runners (63.7 mL kg
–1
min
–1
) Cont: Normal (6.5 gkg
–1
CHO, 1.1 gkg
–1
fat)
Expt: HCHO (9.7 gkg
–1
CHO, 0.9 gkg
–1
fat)
Expt: HFAT (6.7 gkg
–1
CHO, 2.2 gkg
–1
fat)
Lambert et. al. 1994 n = 5 M; trained cyclists (4.2 Lmin
–1
) Cont: 2-wk HCHO
Expt: 2-wk HFAT
Goedecke et. al. 1999 n = 16 M; trained cyclists (63.5 mLkg
–1
min
–1
) Cont: Habitual (5.6 gkg
–1
CHO, 1.4 gkg
–1
fat)
Expt: HFAT (2.6 gkg
–1
CHO, 4.1 gkg
–1
fat)
Burke et. al. 2000 n = 8 M; well-trained cyclists (64.4 mLkg
–1
min
–1
) Cont: 6-d HCHO (9.6 gkg
–1
CHO, 0.7 gkg
–1
fat)
Expt: 5-d HFAT (2.4 gkg
–1
CHO, 4.0 gkg
–1
fat) + 1-d
HCHO
Venkatraman et al. 2001 n = 12 M, 13 F; recreationally trained
runners (F, 50 mLkg
–1
min
–1
;M,58mLkg
–1
min
–1
)
Cont: 4-wk low-fat diet (0.5 gkg
–1
F, 0.5 gkg
–1
M)
Expt: 4-wk moderate-fat diet (1.2 gkg
–1
BM F,
1.4 gkg
–1
BM M)
Expt: 4-wk HFAT (1.8 gkg
–1
BM F, 2.1 gkg
–1
BM M)
Carey et. al. 2001 n = 7 M; well-trained cyclists (5.06 Lmin
–1
) Cont: 7-d HCHO (9.0 gkg
–1
CHO, 1.8 gkg
–1
fat)
Expt: 6-d HFAT (2.5 gkg
–1
CHO, 4.6 gkg
–1
fat) + 1-d
HCHO
Lambert et. al. 2001 n = 5 M; well-trained cyclists (4.9 Lmin
–1
) Cont: 10-d habitual diet + 3-d HCHO
Expt: 10-d HFAT + 3-d HCHO
Rowlands and Hopkins 2002 n = 7 M; well-trained (72 mLkg
–1
min
–1
) Cont: 14-d HCHO (9.1 gkg
–1
CHO, 0.9 gkg
–1
fat)
Expt 1: 14-d HFAT (2.4 gkg
–1
CHO, 4.7 gkg
–1
fat)
Expt 2: 11.5-d HFAT + 2.5-d HCHO
Burke et. al. 2002 n = 8 M; well-trained cyclists (68.6 mLkg
–1
min
–1
) Cont: 6-d HCHO (9.3 gkg
–1
CHO, 1.1gkg
–1
fat)
Expt: 5-d HFAT (2.5 gkg
–1
CHO, 4.3 gkg
–1
fat) + 1-d
HCHO
Cameron-Smith et al. 2003 n = 14 M; well-trained (67 mLkg
–1
min
–1
) Cont: 5-d HCHO (9.6 gkg
–1
CHO, 0.7 gkg
–1
fat)
Expt: 5-d HFAT (2.4 gkg
–1
CHO, 4.0 gkg
–1
fat)
Havemann et. al. 2006 n = 8 M; well-trained (57.8 mLkg
–1
min
–1
) Cont: 7-d HCHO (7.5 gkg
–1
CHO, 0.8 gkg
–1
fat)
Expt: 6-d HFAT (1.9 gkg
–1
CHO, 3.3 gkg
–1
fat) + 1-d
HCHO
Stellingwerff et al. 2006 n = 7 M; well-trained cyclists (60.7 mLkg
–1
min
–1
) Cont: 6-d HCHO (2.5 gkg
–1
CHO, 4.6 gkg
–1
fat)
Expt: 5-d HFAT (10.3 gkg
–1
CHO, 1.0 gkg
–1
fat) +
1-d HCHO
Yeo et al. 2008 n = 8 M; well-trained cyclists (61.5 mLkg
–1
min
–1
) Cont: 6-d HCHO (10.3 gkg
–1
CHO, 1.0 gkg
–1
fat)
Expt: 5-d HFAT (2.5 gkg
–1
CHO, 4.6 gkg
–1
fat) + 1-d
HCHO
Note: Cont, Control group; M, male; CHO, carbohydrate; :, increase; TTE, time to exhaustion;
_
V
O
2max
, maximal oxygen consumption; $,unchanged; ;, decrease;
output; CS, citrate synthase; b-HAD, b-hydroxyacyl-CoA-dehydrogenase; TT, time trial; F, female; FAT/CD36, fatty acid translocase; EMG, electromyogram;
14 Appl. Physiol. Nutr. Metab. Vol. 36, 2011
Published by NRC Research Press

muscle adaptation, and performance.
FAT
ox
(vs. Cont) Skeletal muscle adaptation (vs. Cont) Performance (vs. Cont)
: 3-fold ; in glucose oxidation; 4-fold ; in glycogen utlization
Cycle TTE (62%–64%
_
V
O
2 max
; *150 min) $
:;Glycogen content (resting); ; glycogen utilzation (exercise)
Cycle TTE (*60%
_
V
O
2 max
; *150 min) $
: CPT activities; ; HK
$
HFAT :
_
V
O
2max
and running TTE
$
$
; Glycogen content (resting); glycogen utilization (exercise) $
HFAT : TTE (60%
_
V
O
2max
; 80 vs. 43 min);
:
TTE $ (90%
_
V
O
2max
; 8–13 min)
Maximal PO $ (30-s Wingate test; 804–862 W)
; Estimated rates of glycogen oxidation 40-km TT $ (*65 min)
::CPT activities; CS and b-HAD $
; Glycogen content (resting) after 5-d HFAT but restored after
1-d HCHO; ; glycogen utlization (exercise)
TT performance (7 kJkg
–1
; 30–35 min) with 120-min preload
(*150 min in total) $
:
High- and moderate-fat diet : running TTE (F, 39–47 min; M, 44–
56 min)
:
60 min TT (km) with 4-h (65%
_
V
O
2 peak
) preload $
:
; Estimated rates of muscle glycogen and lactate oxidation Expt. : 20-km TT performance (*30 min) with 150-min preload at
70%
_
V
O
2 peak
(*180 min in total)
:
Expt. 1 and 2 attenuated the decline in power output
: During the last 5 km of a 100-km (*155-min) TT
:
Plasma glucose uptake $ TT performance (7 kJkg
–1
; *25 min) with 120-min preload
(*145 min in total) $
:
HFAT : FAT/CD36 (protein and mRNA) and b-HAD mRNA
:
Normalized EMG amplitude during 1-km sprint $ Expt. ; PO during 1-km sprint but not during 4-km; sprint at desig-
nated distances during a 100-km TT; 100-km TT performance
(100 km; *155 min) $
:
; PDH activity (resting and exercise); ; glycogenolysis (exercise)
:;Estimated substrate phosphorylation (exercise)
: Resting TG; : resting AMPKa1 and a2 activity;
::p-ACC postexercise
CPT, carnitine palmitoyl transferase; HK, hexokinase; Expt, Experimental group; HCHO, high-carbohydrate diet; HFAT, high-fat diet; PO, power
PDH, pyruvate dehydrogenase; TG, triglyceride; AMPK, 5’AMP-activated protein kinase; p-ACC, phosphorylation of acetyl-CoA-carboxylase.
factor for performance. Both acute and chronic modification
of dietary fat and CHO content have long been known to re-
sult in altered proportions of substrate oxidation both at rest
and during exercise (Krogh and Lindhard 1920). Against this
background, the concept of fat adaptation, or ‘‘fat loading’’
was formulated and refers to the strategy of consuming a
high-fat, low-CHO diet while undertaking an endurance-train-
ing program, to promote higher rates of fat utilization during
exercise (Hawley and Hopkins 1995). However, it is also im-
portant to note that fat-adaptation strategies represent as much
a low-CHO challenge (i.e., training in the face of low muscle
glycogen availability) as it is a high-fat challenge (i.e., train-
ing with high fat availability) to homeostasis as muscle glyco-
gen contents are reduced during this adaptation phase.
Of importance is the repeated observation that fat-
adaptation strategies dramatically increase whole-body rates
of fat oxidation during submaximal exercise in already
well-trained athletes above the rates typically induced by
endurance training alone (Table 1). However, despite this
augmented response and the concomitant glycogen sparing,
the effects of fat adaptation on a range of endurance-based
performance tasks have been equivocal: some studies have
reported benefits (Lambert et al. 1994; Muoio et al. 1994;
Venkatraman et al. 2001), whereas others have shown no
advantage to performance (Phinney et al. 1983). In addi-
tion, the responses of athletes to such diets appear to be
highly variable and can impair training capacity (Yeo et
al. 2008). It should be noted that such diets result in re-
Yeo et al. 15
Published by NRC Research Press

duced muscle glycogen content and that, regardless of the
level to which fat oxidation is elevated during exercise, it is
important for athletes to approach endurance sporting compet-
itions with maximized glycogen storage (Burke et al. 2004).
Fat adaptation and CHO restoration
To circumvent the problem of reduced precompetition
glycogen storage resulting from high-fat, low-CHO diets,
we (Hawley and Hopkins 1995; Burke et al. 2000, 2002;
Carey et al. 2001; Stellingwerff et al. 2006; Yeo et al.
2008) and others (Lambert et al. 2001; Rowlands and Hop-
kins 2002) proposed a ‘‘dietary periodization’’ model involv-
ing fat adaptation followed by CHO restoration; such a
protocol would result, in theory, in optimization of both
rates of fat oxidation and pre-event glycogen storage. This
model incorporates a period of fat adaptation (*5–14 days,
*70% energy from fat and *15% energy from CHO) fol-
lowed by a short-term (1–3 days, *15% energy from fat
and *70% energy from CHO) CHO restoration phase
(Fig. 1). We hypothesized that the short phase of CHO re-
storation would be sufficient time to replenish muscle (and
liver) glycogen but that at least part of the elevated fat oxi-
dative response to fat adaptation would remain, even in the
face of higher glycogen content (Burke et al. 2000). This
was critical to the concept, because after a ‘‘standard’’ high-
CHO diet, elevated resting muscle glycogen concentration
coincides with a higher rate of utilization during subsequent ex-
ercise. Consequently, physiological adaptations that permit an
increased rate of fat oxidation and ‘‘sparing’’ of glycogen in
the face of elevated pre-exercise muscle glycogen levels repre-
sent the ideal scenario for maximizing endurance capacity.
In general, fat-adaptation and CHO-restoration protocols
have included a cross-over design, whereby a control diet
consisting of a high-CHO diet (containing the same macro-
nutrient composition as CHO restoration) is compared with
the fat adaptation and CHO restoration diet (Fig. 1). Further-
more, in these studies, subjects were usually required to
maintain their regular training program, including high-
intensity training sessions, throughout the intervention pe-
riod. Interestingly, even though the duration of various
studies has ranged from 1–3 days with respect to the resto-
ration phase, we have shown consistently that only 1 day
of CHO restoration is necessary to restore muscle glycogen
concentration in endurance-trained athletes (Burke et al.
2000; Stellingwerff et al. 2006; Yeo et al. 2008). The re-
sults from all these studies demonstrate that although CHO
restoration suppresses fat oxidation during submaximal ex-
ercise relative to values seen prior to restoration (i.e., after
only fat adaptation), the values remain significantly higher
than at baseline (pre-fat adaptation) (Burke et al. 2000;
Stellingwerff et al. 2006; Yeo et al. 2008). The robustness
of these changes was demonstrated by their persistence de-
spite high CHO availability achieved by consuming a high-
CHO breakfast prior to exercise (Carey et al. 2001) and
high-CHO drinks during the session (Carey et al. 2001;
Burke et al. 2002). Results from further experiments re-
vealed that the lower rates of CHO oxidation could be ex-
plained by a reduction in muscle glycogen utilization,
despite higher pre-exercise muscle glycogen stores (Burke
et al. 2000; Lambert et al. 2001; Yeo et al. 2008) (Table 1).
Despite these metabolic improvements, however, the bene-
fits of fat adaptation with CHO restoration on exercise per-
formance were equivocal, with some studies (Lambert et
al. 2001; Rowlands and Hopkins 2002) reporting perform-
ance benefits during various endurance tests, and others
(Burke et al. 2000, 2002; Carey et al. 2001; Havemann et
al. 2006) showing no significant changes, or even a decre-
ment in performance of high-intensity cycling (1-km sprint
power output) (Havemann et al. 2006). It is possible that
many of these performance tasks have been undertaken at
exercise intensities at which CHO is the major fuel for
muscle metabolism: whereas rates of fat oxidation during
submaximal exercise (60%–70% maximal oxygen con-
sumption (
_
VO
2 max
)) are elevated after fat adaptation, there
is a reliance on CHO at intensities above 80%–85% of
_
VO
2 max
. Furthermore, it appears that there are ‘‘res-
ponders’’, who are able to benefit from such a diet-training
regime and ‘‘nonresponders’’, for whom performance is un-
changed or reduced. Regardless of the variability of per-
formance responses in the tests and situations that have
been investigated, we remain interested in the mechanisms
responsible for the dramatic alteration in substrate use dur-
ing exercise and the possibility that this protocol might
yield benefits for endurance athletes in applications that
have yet to be tested.
Fat adaptation followed by CHO restoration:
what are the mechanisms for the persistent
increase in fat oxidation?
There are several mechanisms that may explain the skele-
tal muscle adaptations induced by fat-adaptation strategies
(Fig. 2). These might reside with processes associated with
either, or both, fat and CHO oxidation and might be found
at the level of substrate transport at the sarcolemma, sub-
Fig. 1. Overview of the general experimental design for studies utilizing the model of fat adaptation and CHO restoration. HFAT, high-fat
diet; CHO, carbohydrate; HCHO, high-carbohydrate diet;
_
VO
2max
, maximal oxygen consumption.
16 Appl. Physiol. Nutr. Metab. Vol. 36, 2011
Published by NRC Research Press

Fig. 2. Endurance training and fat-adaptation-induced skeletal muscle adaptation with and without carbohydrate (CHO) restoration. (A) In-
tramyocellular and sarcolemmal environment in endurance-trained athletes before dietary manipulation. (B) Fat-adaptation-induced fluctua-
tions in skeletal muscle fuel availability, adaptations, and associated signaling pathways. (C) Fat adaptation followed by CHO-restoration-
induced fluctuations in skeletal muscle fuel availability, adaptations, and associated signaling pathways. Solid lines denote signaling that has
been verified experimentally after fat-adaptation studies, whereas dashed lines signify putative steps that are yet to be elucidated experi-
mentally. GLUT4, glucose transporter 4; FABPpm, plasma membrane fatty acid-binding protein; FAT/CD36, fatty acid translocase; Gly,
Glycogen; TG, intramuscular triglyceride; HSL, hormone-sensitive lipase; PDH, pyruvate dehydrogenase; AMPK, 5’AMP-activated protein
kinase; ACC, acetyl-CoA carboxylase; ;, decrease; :, increase; FFA, free fatty acids; P, phosphorylation; b-HAD, b-hydroxyacyl-CoA de-
hydrogenase.
Yeo et al. 17
Published by NRC Research Press

strate storage, breakdown of FA or glucose to acetyl-CoA,
or mitochondrial transport.
Membrane transport
The primary means of moving FA into contracting skele-
tal muscle cells is via the FA transporters, FA translocase
(FAT/CD36) and plasma membrane fatty acid-binding pro-
tein (FABPpm) (Nickerson et al. 2009; Glatz et al. 2010).
We have shown that 5 days of fat adaptation was associated
with increases in FAT/CD36 mRNA and protein content
(Fig. 2B) (Cameron-Smith et al. 2003), although in that
study, FABPpm mRNA and protein were unchanged; this
suggests that FAT/CD36 is more sensitive to changes in di-
etary fat content than FABPpm. The sensitivity of FAT/
CD36 to dietary changes was observed again when FAT/
CD36 protein content was found to return to pre-fat-
adaptation levels after CHO restoration (Yeo et al. 2008).
However, FA transport proteins are present in the cyto-
plasm and on both the muscle and mitochondrial mem-
branes. Previous studies (Cameron-Smith et al. 2003; Yeo
et al. 2008) have been limited to total muscle measures
that provide no information regarding the compartment or
location where the protein changes occurred. Recent human
work has shown that while high-intensity training increased
total muscle FABPpm and FAT/CD36, the changes at the
sarcolemma were confined to FABPpm, as FAT/CD36 did
not change (the increase in FAT/CD36 occurred on the mi-
tochondrial membranes (discussed below)) (Talanian et al.
2010). In addition, it has also been shown that the FA
transport proteins can translocate to the membranes during
exercise. A recent study reported that FAT/CD36 content in-
creased in the sarcolemma during 2 h of cycling at *60%
_
VO
2 max
, whereas there was no movement of FABPpm
(N.S. Bradley, L.A. Snook, S.S. Jain, et al. 2010, personal
communication). Future work will be required to determine
if fat adaptation (and CHO restoration) alter the presence of
FA transporters on the sarcolemma at rest and whether exer-
cise translocates FA transport proteins to the muscle mem-
brane in these 2 situations (Figs. 2B and 2C).
To date, no study has investigated the effects of fat adap-
tation on the primary skeletal muscle glucose transport pro-
tein, GLUT4, and its locations in the cell. Although we
showed recently that total GLUT4 protein was unchanged
after fat adaptation with CHO restoration (Fig. 2C) (Yeo et
al. 2008), the effects of fat adaptation alone on both the loca-
tions of GLUT4 protein and the proteins associated with its
transport from subcellular compartments to the sarcolemma
need to be determined to assess whether fat-adaptation
strategies per se suppress glucose transport (Fig. 2B).
Trapping of glucose in the cell through phosphorylation
by hexokinase is an essential component of oxidation of
plasma-derived glucose and thus can be categorized with
mechanisms related to glucose transport. Invasive studies in
rodents show that phosphorylation of glucose is an impor-
tant step in muscle glucose uptake during exercise (Was-
serman and Ayala 2005). Although it is unknown whether
this regulatory process is important in humans, mechanisms
responsible for exercise-induced changes in hemodynamics
and glucose transporters that determine the importance of
hexokinase activity in glucose transport are similar between
rodents and humans. Therefore, it is assumed that glucose
transport during prolonged exercise in humans might be lim-
ited by a reduction in hexokinase activity. Fisher et al.
(1983) demonstrated that hexokinase activity was reduced
by 4-week fat adaptation, which might explain, in part, the
reduction in the capacity for glucose oxidation following fat
adaptation. In contrast, we have reported that plasma-
derived glucose disposal is unchanged after fat adaptation
and CHO restoration, suggesting that a reduction in glyco-
genolysis alone is responsible for reductions in CHO oxi-
dation (Burke et al. 2002). Thus, fat-adaptation protocols
of >1-week duration may be required to suppress plasma-
derived skeletal muscle glucose disposal, and further work
is required to examine whether hexokinase content and (or)
activity are altered after shorter protocols (<7 days), as
well as after CHO restoration.
Mitochondrial FA transport
Long-chain FA initially require transport into mitochon-
dria before they can be metabolized prior to oxidation (for
reviews see McGarry and Brown (1997); Kiens (2006); and
Holloway et al. (2008)). This process requires transport via
the mitochondrial carnitine palmitoyl transferase (CPT)
complex, with CPT1 believed to be the regulated enzyme;
the FA transport protein FAT/CD36 also appears to be in-
volved. FABPpm on the mitochondrial membrane does not
appear to play a role in FA transport but is structurally iden-
tical to aspartate aminotransferase, which is involved in
shuttling reducing equivalents into the mitochondria (Hol-
loway et al. 2007). Following training, the activity of CPTI
increased in the same proportion as the increase in mito-
chondrial content (Talanian et al. 2010). However, the
amount of FAT/CD36 on the mitochondrial membrane in-
creased to a greater extent than the increase in mitochondrial
volume following training (Talanian et al. 2010). Acute pro-
longed exercise (2 h of moderate-intensity cycling) also re-
sulted in the translocation of FAT/CD36 protein to the
mitochondrial membranes (Holloway et al. 2006). These re-
sults suggest that FAT/CD36 plays an important role as the
need for mitochondrial fat transport increases. Increases in
CPT1 activity have also been demonstrated after 15 (Goe-
decke et al. 1999) and 28 days (Fisher et al. 1983) of fat
adaptation, although a later study (Cameron-Smith et al.
2003) reported that the mRNA and protein abundance of
CPT1 was unchanged after 5 days of fat adaptation. It is im-
portant to note that measures of mRNA and (or) protein are
not surrogates for CPT1 enzyme activity.
CPT1-mediated FA transport can be regulated by both its
abundance and allosteric inhibitors, the most prominent
being malonyl-CoA (M-CoA). In this regard, the allosteric
regulation of CPT1 activity is of great importance, at least
while at rest, in particular the regulation of M-CoA-mediated
inhibition of CPT1 induced by the 5’AMP-activated protein
kinase (AMPK) and the phosphorylation and suppression of
the rate-limiting enzyme in M-CoA synthesis, acetyl-CoA
carboxylase-b (ACC) (Winder and Hardie 1996). We re-
cently reported that the activation of AMPK and subse-
quent phosphorylation of ACC are increased at rest by
5 days of fat adaptation and 1 day of CHO restoration
(Fig. 2C) (Yeo et al. 2008). Given that the increase in
AMPK activity and ACC phosphorylation were observed
after CHO restoration, it seems reasonable to suggest that
18 Appl. Physiol. Nutr. Metab. Vol. 36, 2011
Published by NRC Research Press

these enzymes would be up-regulated after fat adaptation
alone (Fig. 2B). Thus, the regulation of CPT1 activity by
the AMPK–ACC–CPT1 axis might be a primary mediator
of fat adaptation induced up-regulation of FA oxidation
and might be involved in the sustained oxidation rate seen
after CHO restoration (Fig. 2C). Of note is that chronic
AMPK activation is known to increase muscle content of
FAT/CD36 in cardiac myocytes (Chabowski et al. 2006)
and therefore may be involved in the up-regulation of this
transporter, as described previously.
However, the problems with the above theory are that these
studies did not directly measure the M-CoA content or the ac-
tivity of the M-CoA degradation enzyme (M-CoA dehydro-
genase) in muscle. Although M-CoA appears to play a role in
regulating fat oxidation at rest, several studies in humans have
indicated that M-CoA levels do not decrease sufficiently dur-
ing moderate-intensity exercise to explain the increases in
LCFA oxidation (Odland et al. 1996, 1998; Roepstorff et al.
2005). It is not known how FA uptake into the mitochondria
is up-regulated in human skeletal muscle at the onset of exer-
cise, but it may involve increased substrate availability for
CPTI and decreased sensitivity to M-CoA, without changes
in the M-CoA concentration (Holloway et al. 2006).
Does priming the mitochondria with reduced
nicotinamide adenine dinucleotide (NADH) increase fat
oxidation?
Another possibility to explain the greater reliance of fat
oxidation following fat adaptation without concomitant in-
creases in mitochondrial volume arises from the results of
studies in which fat availability was acutely elevated during
exercise by increasing free FA (FFA) levels (Fig. 2B and
2C). In this scenario, there are increased rates of fat oxidation
and decreased glycogen breakdown and CHO oxidation
(Dyck et al. 1996; Romijn et al. 1995; Chesley et al. 1998).
Increased fat availability may have, in some way, increased
the availability of NADH and ATP from fat early in exercise,
thereby decreasing the mismatch between ATP utilization
and the ability to regenerate ATP, with the result that the ac-
cumulation of free ADP, AMP, and Pi is reduced and the key
CHO metabolizing enzymes, PHOS, PFK, and PDH are less
activated (Chesley et al. 1998; Spriet and Hargreaves 2006).
The presence of high blood FA availability at the onset of ex-
ercise (*1 h) may have elevated the oxidation of fat at rest,
thereby increasing the resting NADH level and (or) increas-
ing the concentrations of the intermediates in the b-oxidation
pathway, leading to a quicker onset of fat oxidation at the
start of exercise. The 5-day fat-adaptation protocol may sub-
ject athletes to prolonged periods during which FFA are
elevated both at rest and during intensive training sessions
(compared with the high-CHO diet) and the muscles are
primed toward using more fat without any increases in mi-
tochondrial volume. Increases in muscle membrane or mi-
tochondrial FA transport protein content would also help
in this regard. The fact that this effect is not totally re-
versed following CHO restoration suggests that the respon-
sible changes lie in the muscle and are retained. However,
no study to date has explored these possibilities in terms of
explaining the metabolic changes that have been reported
following fat adaptation and CHO restoration.
Intramuscular triacylglycerol storage and breakdown
Levels of glycogen and intramuscular triacylglycerol
(IMTG) storage can affect the rates of CHO and fat oxida-
tion during exercise (Spriet and Watt 2003). The changes in
muscle glycogen content in relation to fat adaptation and
CHO restoration have already been discussed. However,
there are also likely to be many changes associated with the
storage of IMTG. Little current information exists, but
IMTG levels were increased by the dietary fat periodization
protocol (Fig. 2C) (Yeo et al. 2008) and there was a trend
toward increased maximal activity of hormone-sensitive li-
pase (HSL) (20%) after fat adaptation and CHO restoration
(Fig. 2C) (Stellingwerff et al. 2006). However, the overall
TG content in muscle at rest following a training session ul-
timately depends on the balance between the rates of FA up-
take, oxidation, and storage and the rate of TG hydrolysis.
The esterification of FFA to TG requires acylation by acyl-
CoA synthetase and the sequential addition of FFA to a
glycerol backbone via a series of 4 enzymes with the activ-
ities of glycerol-3-phosphate acyltransferase (GPAT) and di-
acylglycerol acyltransferase (DGAT), believed to be
regulatory. On the breakdown side, adipose triacylglycerol
lipase (ATGL) and HSL are believed to work hierarchically
to regulate complete TG hydrolysis. ATGL initiates lipolysis
by specifically removing the first FFA from TG to produce
diacylglycerol substrate, which is then hydrolyzed by HSL
to generate an additional FFA and monoglycerol (MG) sub-
strate. MGs are converted to FFA and glycerol by MG li-
pase in the final step of lipolysis (Watt and Spriet 2010).
An increase in IMTG levels following fat adaptation sug-
gests increased synthesis over degradation during the rest
periods between daily workouts. Although all the key en-
zymes may be up-regulated by the fat-adaptation paradigm,
the activities of DGAT and GPAT may be dominant at rest,
whereas ATGL and HSL are more active during exercise.
The likely signals for up-regulating these proteins are the
chronic decrease in insulin concentration and the increase in
plasma FFA that occur with the fat-adaptation paradigm.
FFA are known ligands for the family of peroxisome
proliferator-activated receptors, transcription factors that are
known to up-regulate fat-metabolizing proteins (Ehrenborg
and Krook 2009). Work in this area is mainly speculative
because little has been done to determine the changing dy-
namics of IMTG handling that may ultimately contribute to
a greater reliance on fat oxidation following fat adaptation.
High-fat diets and PDH activity
Fat adaptation also has profound effects on the regulation
of PDH, the key enzyme regulating the oxidation of CHO in
muscle. It has been shown that high-fat diets rapidly down-
regulate the amount of the PDH protein in the active form
(PDHa) at rest (Peters et al. 1998, 2001). This is accom-
plished by rapid up-regulation of the enzyme PDH kinase
(PDK), which moves PDH to the inactive form. This re-
sponse decreases the oxidation of CHO in the face of less
than optimal CHO intake. It is believed that the reduction
in circulating insulin concentration and the increased FFA
levels during the high-fat diet rapidly induce these changes
(Peters et al. 2001). It has been shown that, during exercise
following fat adaptation (Putman et al. 1993) and CHO re-
storation (Stellingwerff et al. 2006), PDH activation is re-
Yeo et al. 19
Published by NRC Research Press

duced at rest and over a range of exercise intensities
(Fig. 2C). Accordingly, CHO oxidation is reduced and,
along with other changes affecting muscle PHOS, glycoge-
nolysis is suppressed and glycogen is spared (Putman et al.
1993; Stellingwerff et al. 2006).
Interestingly, refeeding with CHO following a high-fat
diet has been shown to quickly decrease PDK activity
(45 min to 3 h), but the suppression of PDHa activity and
impaired oxidation of CHO persisted for at least 3 h at rest
(Bigrigg et al. 2009). However, following 24 h of CHO re-
storation, the prediction would be that CHO oxidation would
return to normal at rest and once exercise begins. However,
CHO oxidation only returned part way to normal, and PDH
activation was still blunted during moderate and all-out ex-
ercise (Stellingwerff et al. 2006). While the subjects in this
latter study were highly trained and exercised throughout the
fat adaptation and the subjects in the Bigrigg et al. (2009)
study were recreationally active and did no activity outside
of daily living (detrained) during 6 days on the high-fat
diet, the results of both studies are consistent with a linger-
ing effect of fat adaptation on muscle fuel choice. Addi-
tional work is required to examine other key sites regulating
CHO and fat metabolism in skeletal muscle following fat
adaptation and 24 h of CHO restoration.
The suppression of PDH activity and CHO oxidation fol-
lowing fat adaptation and CHO restoration may have a neg-
ative effect on high-intensity exercise. Maximal PDH
activity increases following moderate- and high-intensity
aerobic training (LeBlanc et al. 2004; Perry et al. 2008) and
high CHO oxidation rates are essential for optimal perform-
ance at very high aerobic work rates (e.g., 90%–100% of
_
VO
2 peak
). Therefore, the persistence of down-regulated PDH
activity following fat-adaptation strategies, even with CHO
restoration, suggests that such paradigms may not be advis-
able in circumstances in which exercise of sustained higher
intensity is required. Indeed, it may explain the impaired
performance of 1-km cycling sprints interspersed within an
endurance cycling task following the fat adaptation and
CHO restoration protocol (Havemann et al. 2006).
Summary and directions for future research
The results from the studies reviewed here suggest that
the metabolic adaptations that favour a persistent increase
in fat oxidation following fat adaptation are likely to be ex-
plained by a number of different mechanisms. Clearly, there
is an increase in the ability of skeletal muscle to transport,
store, and oxidize FFA, with some, but not all, of these
adaptations evident in the face of restored muscle glycogen
or increased exogenous CHO availability (i.e., glucose feed-
ing before or during exercise after fat adaptation). Recent
work examining the regulation of fat metabolism in skeletal
muscle has demonstrated the importance of measuring the
compartmentalization–localization and translocation of FA
transport proteins, and these approaches are needed to exam-
ine how fat adaptation and exercise interactions may alter
this regulation. Strong experimental support for a reduction
in the ability to oxidize glucose is lacking. Clear mecha-
nisms linking fat adaptation to suppression of glucose me-
tabolism, such as glucose transport, oxidation, and enzymes
linked to glycogen synthesis and breakdown, are important
missing pieces of a puzzle that might partly explain some
of the observed adaptive responses. Because CHO oxidation
remains depressed even in the face of increased CHO avail-
ability (either in the form of restored muscle glycogen stores
and (or) in the provision of large amounts of exogenous
CHO), the data demonstrating suppression of both PDH ac-
tivity and glycogenolysis are important and imply a clear
impairment of the muscle’s ability to oxidize CHO. Such
impairment would be expected to hinder performance in
many endurance sports that require at least some portion of
work to be completed at or near
_
VO
2 peak
.
Practical issues surround the fine-tuning of fat-adaptation
strategies, primarily to determine positive responders vs.
nonresponders and then to optimize regimes for those ath-
letes for whom dietary periodization strategies are benefi-
cial. Whether a longer CHO restoration period would be
able to ‘‘rescue’’ the high-fat-diet-induced impairments to
PDH, such that rates of muscle glycogenolysis are not com-
promised during high-intensity work, remains to be tested
experimentally. Of course, any up-regulation of glycogen
metabolism is likely to cause a reciprocal down-regulation
of lipid metabolism and any diet-induced increases in fat
oxidation observed during submaximal exercise after fat
adaptation may be obliterated in such a scenario. In the fi-
nal analysis, the practical difficulties associated with the
preparation of, and compliance with, fat-adaptation proto-
cols mean that it is highly desirable to identify the mini-
mal time required to up-regulate FA oxidation and how
long this perturbation persists in the face of CHO restora-
tion. Finally, if scientists can provide the answers to some
of these questions, it will be left to coaches and athletes to
explore how best to fit dietary-periodization strategies into
macro- and microcycles of endurance-training programs.
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