Acta Physiol 2011, 202, 323–335

REVIEW
Current understanding of increased insulin sensitivity after
exercise – emerging candidates

S. J. Maarbjerg, L. Sylow and E. A. Richter

Molecular Physiology Group, Department of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark

Received 31 August 2010, Abstract

revision requested 17 October
2010,
final revision received 16 February
2011,
accepted 22 February 2011
Correspondence: E. A. Richter,
Molecular Physiology Group,
Department of Exercise and Sport
Sciences, University of
Copenhagen, Copenhagen
DK-2100, Denmark.
E-mail: erichter@ifi.ku.dk
Exercise counteracts insulin resistance and improves glucose homeostasis in
many ways. Apart from increasing muscle glucose uptake quickly, exercise
also clearly increases muscle insulin sensitivity in the post-exercise period.
This review will focus on the mechanisms responsible for this increased insulin
sensitivity. It is believed that increased sarcolemmal content of the glucose
transporter GLUT4 can explain the phenomenon to some extent. Surprisingly
no improvement in the proximal insulin signalling pathway is observed at the
level of the insulin receptor, IRS1, PI3K or Akt. Recently more distal signalling
component in the insulin signalling pathway such as aPKC, Rac1, TBC1D4
and TBC1D1 have been described. These are all affected by both insulin and
exercise which means that they are likely converging points in promoting
GLUT4 translocation and therefore possible candidates for regulating insulin
sensitivity after exercise. Whereas TBC1D1 does not appear to regulate
insulin sensitivity after exercise, correlative evidence in contrast suggests
TBC1D4 to be a relevant candidate. Little is known about aPKC and Rac1 in
relation to insulin sensitivity after exercise. Besides mechanisms involved in
signalling to GLUT4 translocation, factors influencing the trans-sarcolemmal
glucose concentration gradient might also be important. With regard to the
interstitial glucose concentration microvascular perfusion is particular rele-
vant as correlative evidence supports a connection between insulin sensitivity
and microvascular perfusion. Thus, there are new candidates at several levels
which collectively might explain the phenomenon.
Keywords GLUT4, molecular signalling, muscle, TBC1D1, TBC1D4.

primarily caused by insulin resistance in liver, adipose

Introduction
The world is facing a huge challenge with the epidemic
prevalence of type 2 diabetes (T2D) throughout the
world (Day 2007). Epidemiological evidence strongly
associates the development of T2D with obesity and
physical inactivity (Wei et al. 1999). At the same time
longitudinal studies support that reduction in obesity
and increased physical activity are central in both
prevention and treatment of T2D (Eriksson & Lind-
garde 1991, Pan et al. 1997).
Central to the aetiology of T2D is impaired capacity
of insulin to regulate glucose homeostasis. This is
tissue and skeletal muscle. Skeletal muscle represents
around 40% of human body mass and might account
for up to 90% of insulin-stimulated glucose uptake
(DeFronzo et al. 1985). This highlights the potential of
factors that can increase insulin sensitivity in muscle.
Exercise training probably benefits from adaptations in
muscle such as increased capillary density, increased
expression of proteins regulating metabolism and
increased abundance of oxidative fibres. More impor-
tant might be that exercise training repeatedly induces a
post-exercise situation (Wojtaszewski & Richter 2006).
Increased insulin sensitivity in this period appears at

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Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x 323
Increased insulin sensitivity after exercise Æ S J Maarbjerg et al.
least partly to involve increased GLUT4 translocation
(Hansen et al. 1998, Geiger et al. 2006). However,
factors besides those involving GLUT4 translocation
might also contribute to the phenomenon.
The mechanisms involved in increased insulin sensi-
tivity after exercise attracts increasing attention because
of the continued growth in prevalence of T2D. Under-
standing these mechanisms can potentially be used in
drug development as well as in designing effective
lifestyle changes. In this review we will present our
current understanding of the possible mechanisms that
regulate increased insulin sensitivity after a single bout
of exercise.
Increased insulin sensitivity after exercise –
consequence of local changes in muscles
Improved insulin action after acute treadmill exercise
was first demonstrated in 1982 by Richter et al. in
perfused isolated rat hindquarters (Richter et al. 1982).
Shortly after, a study demonstrated that the phenome-
non could be ascribed to the previously contracted
muscles. This was done in a rat model where one leg
was stimulated electrically (via the sciatic nerve) to
contract while the opposite leg was resting. Subse-
quently both hindlimbs were perfused with a perfusate
containing insulin, and glucose uptake was observed to
be markedly increased in the previously contracted
muscles compared to the non-stimulated control mus-
cles (Richter et al. 1984). These early findings in rodents
have subsequently been supported by human studies
using the one-legged exercise model followed by an
euglycaemic hyperinsulinemic clamp (Richter et al.
1989, Wojtaszewski et al. 1997, 2000, Thong et al.
2003, Frosig et al. 2007). Dose–response studies have
demonstrated that increased insulin action occurs at
submaximal insulin concentrations and therefore
mainly is an outcome of decreased Km. This can be
visualized as a leftward shift in the dose–response curve
for insulin action on glucose uptake (Fig. 1)
(Wojtaszewski et al. 2002). This means that the phe-
nomenon is important at physiological insulin concen-
trations and therefore relevant for daily life. This
relevance is furthermore supported by studies demon-
strating increased insulin sensitivity after 30–60 min of
cycling in both healthy, insulin resistant and type 2
diabetic subjects (Bogardus et al. 1983, Devlin &
Horton 1985, Devlin et al. 1987, Mikines et al. 1988).
In addition to studies comparing corresponding
rested and exercised muscles within the same individual,
studies with other approaches furthermore indirectly
support that post-exercise increased insulin sensitivity is
a local phenomenon. Firstly it is shown that despite a
serum factor appears essential for developing increased
insulin sensitivity after exercise, intact systemic circula-
324 Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
Acta Physiol 2011, 202, 323–335
Figure 1 Exercise increases sensitivity of glucose uptake to
insulin in human muscle. Prior one-legged knee extensor
exercise improves insulin sensitivity of glucose uptake in the
exercised compared with the rested leg in healthy human
subjects as indicated by the reduction in insulin concentration
eliciting half maximal glucose uptake response (glucose uptake
is given as % of maximal increase). The graph is reproduced
from Wojtaszewski et al. (2002) and based on data extracted
from Richter et al. (1989). Reproduced with permission.
tion is not necessary (Cartee et al. 1989, Gulve et al.
1990, Gao et al. 1994a, Funai et al. 2010). Secondly, a
pivotal observation is that increased sensitivity of
glucose uptake at least partly can be explained by
increased insulin-stimulated sarcolemmal GLUT4 con-
tent (Hansen et al. 1998, Geiger et al. 2006). The issue
then is: What part of the GLUT4 translocation machin-
ery is amplified after a single bout of exercise?
Increased insulin sensitivity persist several
hours after exercise
It has been known for a long time that physical exercise
has acute beneficial effects on glucose homeostasis [For
review see (Goodyear & Kahn 1998)]. The overall
capacity of a single bout of exercise to improve
glycaemic control can be ascribed to several events in
relation to exercise. First, during muscle contractions
glucose uptake can increase up to 20-fold depending on
time and intensity (Rose & Richter 2005). However, in
the post-exercise state glucose uptake is also favoured
depending on the type and intensity of the preceding
exercise bout. The post-exercise state can be character-
ized by a period where the capacity for glucose uptake is
improved because of two phenomena: (1) residual effect
of the contraction-stimulated glucose uptake which is
independent of insulin (Fig. 2a) and (2) increased
sensitivity towards insulin (Fig. 2a,b) (Garetto et al.
1984, Richter et al. 1984). Contraction-stimulated glu-
cose uptake usually reverses completely within few
hours (Fig. 2a,b), in contrast, increased insulin sensitiv-
ity persists for longer time and has in some situations
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Acta Physiol 2011, 202, 323–335 S J Maarbjerg et al. Æ Increased insulin sensitivity after exercise
(a) Glucose utilization to the sarcolemma (Hansen et al. 1998, Geiger et al.
~ 0.5 h after exercise 2006). A straight forward explanation could therefore
14
Rest simply be that insulin signalling to GLUT4 transloca-
12
~ 0.5 h post exercise tion is generally enhanced. This is however not the case.
10 Human studies (Wojtaszewski et al. 1997, 2000, Frosig
et al. 2007) as well as rat studies (Goodyear et al. 1995,
μmol g–1 h–1

8 Funai et al. 2010) report increased insulin sensitivity

without simultaneous increase in proximal insulin
6
signalling. This includes measures such as insulin
4 receptor tyrosine kinase activity, insulin receptor sub-
strate 1 (IRS1) tyrosine phosphorylation, IRS1-asso-
2
ciated phosphatidylinositol-3 kinase (PI3K) activity,
0 Akt ser473/thr308 phosphorylation and GSK3 phos-
0 μU/mL 75 μU/mL phorylation/activity (Fig. 3) (Wojtaszewski et al. 1997,
Insulin concentration 2000). Actually IRS1-associated PI3K activity has by
some been reported to be blunted by prior exercise
(b) Glucose utilization (Goodyear et al. 1995, Wojtaszewski et al. 1997). On
14 ~ 2.5 h after exercise
the other hand a few studies do report increased Akt
Rest thr308 phosphorylation (Arias et al. 2007, Funai et al.
12
~ 2.5 h post exercise
2009). However, the increased phosphorylation status
10 on thr308 is not reflected by increased Akt activity and
μmol g–1 h–1

therefore it probably has no functional relevance (Funai

8
6 Based on these negative findings it could be argued
4
2 additionally may control glucose uptake. If carry over
0
0 μU/mL 75 μU/mL
Insulin concentration
Figure 2 Glucose utilization and insulin-stimulated glucose
utilization at approximately 0.5 and 2.5 h after cessation of
exercise in rats. (a) Glucose utilization and insulin-stimulated
glucose utilization by perfused rat hindquarter approximately
0.5 h after high-intensity treadmill exercise. After cessation of
exercise the rat hindquarter was either perfused with a per-
fusate containing insulin at 0 lU ml)1 or 75 lU ml)1. (b)
Glucose utilization and insulin-stimulated glucose utilization
approximately 2.5 h after high-intensity treadmill exercise.
Data are extracted from Garetto et al. (1984). Reproduced
with permission.
been observed to last as long as 48 h (Mikines et al.
1988, Cartee et al. 1989). Increased insulin sensitivity
after a single bout of exercise is not dependent upon
elevated muscle GLUT4 content (Wojtaszewski et al.
1997). Supporting this contention are findings where
inhibition of protein synthesis does not affect the
increase in insulin sensitivity after exercise in rodents
(Fisher et al. 2002).
Proximal insulin signalling
As already mentioned increased insulin sensitivity after
exercise coincides with increased GLUT4 translocation
et al. 2009).
that it is more relevant to focus on steps that are
common for exercise and insulin stimulation and
effects from prior exercise exist in common signalling
steps, it may explain the additive response in glucose
uptake. Glucose uptake is classically regarded as regu-
lated by three main steps in vivo: delivery, transport and
intracellular metabolism. All three steps appear to be
regulated by exercise as well as by insulin and are
discussed below.
Intracellular metabolism
Intracellular metabolism covers phosphorylation of
glucose to glucose-6-phosphate and subsequent metab-
olism, which results in either incorporation into
glycogen or oxidation. In theory increased glucose
uptake could occur as a consequence of increased rate
of glucose phosphorylation by hexokinase II (HK II).
This could lower intracellular glucose concentration
and thereby drive glucose influx providing that the free
intracellular glucose concentration is not already neg-
ligible and that membrane permeability to glucose is
not negligible either. HK II activity is regulated by the
amount of HK II, the localization and the allosteric
inhibition mediated via glucose-6-phosphate (Wasser-
man & Halseth 1998). Whereas HK II expression does
not appear to regulate the increased insulin action after
exercise (Fisher et al. 2002), localization and allosteric
inhibition have (to our knowledge) not been investi-
gated. However, glucose metabolism downstream of

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Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x 325
Increased insulin sensitivity after exercise Æ S J Maarbjerg et al.
INS
IR
IRS1 PDK1
PI3K
Akt2
Rac1
Figure 3 Insulin signalling to GLUT4 translocation and stimulation of glucose uptake. During insulin stimulation after acute
exercise glucose uptake is enhanced as well as TBC1D4 phosphorylation and sarcolemmal GLUT4 content. The proximal insulin
signalling is not enhanced including insulin receptor (IR) tyrosine kinase activity, insulin receptor substrate 1 (IRS1) tyrosine
phosphorylation, IRS1-associated PI3K activity, Akt ser473/thr308 phosphorylation and GSK3 phosphorylation/activity.
glucose-6-phosphate appears to be increased during
insulin stimulation after exercise. This is exemplified
by increase in glycogen synthase activity as well as
increase in glucose incorporation into glycogen (Rich-
ter et al. 1982, Bogardus et al. 1983, Mikines et al.
1988, Wojtaszewski et al. 2000). Even though these
findings make it possible that intracellular metabolism
drives insulin-stimulated glucose uptake it is tradition-
ally regarded less likely. The traditionally view is based
on the premise that intracellular glucose concentration
is fairly low and does not accumulate during insulin
stimulation in either human (Katz et al. 1988) or rat
muscle (Richter et al. 1982, Cline et al. 1998). This
suggests no regulation by HK II or steps further
downstream in glucose handling. The traditional view
has, however, been challenged lately by Wasserman
and colleagues applying transgenic mouse models.
They showed that overexpression of HK II in mice
caused an approximately onefold increase in glucose
uptake during submaximal insulin stimulation com-
pared to wild type littermates. On the other hand a
reduction in GLUT4 expression by 50% in heterozy-
gous knockout mice (Fueger et al. 2004b) only signif-
icantly reduced insulin-stimulated glucose uptake in
mouse muscle when HK II was overexpressed (Fueger
et al. 2004a). Collectively these data suggest that the
phosphorylation step of glucose to glucose 6-phos-
phate might be an important barrier for insulin-
stimulated glucose uptake at least in mice in vivo.
However, whether these findings in transgenic mice
reflect human physiology remain unresolved. Further-
more, whereas HK II might be important for insulin-
326 Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
Acta Physiol 2011, 202, 323–335
GLUT
4
aPKC
TBC1D1
Rab-GTP
TBC1D4
GLUT GLUT GLUT
4 4 4
stimulated glucose uptake it should be remembered
that without GLUT4 insulin has negligible effects on
muscle glucose uptake (Ryder et al. 1999, Zisman
et al. 2000). Thus, based on these apparently conflict-
ing findings, especially the role of HK II in increased
insulin sensitivity after exercise needs to be further
investigated.
Glucose and insulin delivery to skeletal muscle
– potential role of microvascular perfusion
The interstitial glucose concentration is influenced by
plasma glucose concentration and delivery of blood to
the muscle. With respect to the delivery of blood,
several recent studies highlight the importance of
microvascular perfusion in relation to glucose metab-
olism (reviewed in Clark 2008). The hypothesis is that
increased microvascular perfusion elevates the average
interstitial concentration of glucose and thereby
favours glucose uptake via mass action. Microvascular
perfusion relates to the number of capillaries that is
perfused within a muscle and thereby gives a picture of
the perfusion conditions for nutrients and hormones
within the muscle. To highlight this matter microvas-
cular perfusion is also sometimes named nutritive flow
as oppose to bulk blood flow. Bulk blood flow simply
describes the total volume of blood passing through
the muscles per time. Changes in bulk blood flow do
not necessarily reflect changes in nutritive flow as the
two are regulated in different ways. Microvascular
perfusion is on one side mainly regulated by the pre-
capillary arterioles, whereas bulk blood flow is
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Acta Physiol 2011, 202, 323–335
governed by the larger resistance vessels. Vasodilation
of the pre-capillary arterioles will increase the portion
of blood perfusing the capillary bed and decrease the
portion of blood passing non-nutritive shunts in the
muscle.
Importantly, insulin as well as contractions/exercise
appear to increase microvascular perfusion; however it
should be remembered that the effect of insulin is small
compared to the effect of voluntary exercise (Kim A.
Sjøberg Stephen Rattigan, Erik A. Richter & Bente
Kiens, unpublished observations). Furthermore, the
two stimuli presumably rely on different mechanisms
to mediate this vascular effect (Vincent et al. 2003,
2004, Ross et al. 2007). This hypothesis is supported
by demonstration of an additive effect on microvascu-
lar perfusion when combining contractions and phys-
iological hyperinsulinemia. Importantly, Inyard et al.,
furthermore, showed that this additive effect on
microvascular perfusion is paralleled by the well-
established additive effect of contractions and insulin
on glucose uptake. This correlative evidence suggests
that microvascular perfusion takes part in regulating
glucose uptake. More correlative evidence exists in
insulin resistant models such as intralipid infusion
models (Clerk et al. 2002, Inyard et al. 2009, Liu et al.
2009), obese Zucker rats and people with T2D (Clark
2008). Whereas, microvascular perfusion was impaired
in these insulin resistant models, exercise training on
the other hand enhances insulin-stimulated glucose
uptake as well as insulin-stimulated microvascular
perfusion in rats (Rattigan et al. 2001). Time course
studies as well as mechanistic studies support the
hypothesis that microvascular perfusion to some extent
regulates glucose uptake. In rats insulin-stimulated
increase in microvascular perfusion precedes both
activation of insulin signalling and increase in glucose
uptake (Vincent et al. 2004) suggesting that microvas-
cular perfusion could regulate the early increase in
glucose uptake. Furthermore, blocking the synthesis of
the vasodilator NO by Nx-mono-methyl-l-arginine (l-
NMMA) abolishes the early increase in microvascular
perfusion as well as partially inhibits the early increase
in glucose disposal (Vincent et al. 2004). Collectively
this suggests rapid capillary recruitment as a contrib-
utor to the subsequent early increase in glucose uptake.
This hypothesis is interesting with respect to the
increased insulin sensitivity after exercise as glucose
clearance increases faster in the exercised than in the
rested leg. Importantly time course studies reveal that
this faster response in glucose clearance occurs without
any temporal differences in insulin signalling between
the rested and exercised leg (Wojtaszewski et al. 1997,
2000). It could be hypothesized that the faster response
in glucose uptake during insulin stimulation after
exercise in vivo is the result of a potentiation of
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S J Maarbjerg et al. Æ Increased insulin sensitivity after exercise
microvascular perfusion which, however, does not lead
to increased insulin signalling. Still, increased micro-
vascular perfusion might elevate interstitial glucose
concentrations considerably faster than interstitial
insulin concentration because glucose is a much
smaller molecule than insulin. This could explain
why faster increase in glucose uptake can be observed
without significant changes in the timing of insulin
signalling. Thus, it seems possible that increased
microvascular perfusion is responsible for the faster
increase in glucose uptake during insulin stimulation
after exercise. However, this role of microvascular
perfusion needs to be investigated further.
Signalling to GLUT4 translocation
As mentioned previously increased insulin sensitivity
after exercise is not associated with increased proximal
insulin signalling. Therefore, other signalling pathways
have to be considered as discussed below.
Atypical protein kinase C
In the recent years, several novel insulin signalling
molecules have been characterized downstream of PI3K
(Fig. 3). One of them is atypical protein kinase C
(aPKC) which is activated in skeletal muscle both in
response to insulin stimulation (Farese et al. 2007,
Frosig et al. 2007) and exercise (Chen et al. 2002,
Richter et al. 2004, Sajan et al. 2009). During insulin
stimulation aPKC activation probably involves three
events: allosteric binding of PIP3, Thr410 phosphory-
lation by PDK1 and Thr 560 autophosphorylation
(Farese 2002). The downstream signalling of aPKC
to GLUT4 translocation is, however, still far from
clarified.
Whereas aPKC appears to be important for insulin-
stimulated glucose uptake (Bandyopadhyay et al. 1997,
2000, Etgen et al. 1999, Farese et al. 2007), the
increased aPKC activity during exercise does not seem
to be crucial for exercise induced glucose uptake (Sajan
et al. 2009). Considering the apparent importance of
aPKC in insulin signalling, amplification of aPKC
signalling after exercise could, however, potentially
increase insulin action. In a recent one-legged exercise
study we evaluated this 4 h after exercise. We observed
a more potent allosteric aPKC activation by PIP3 from
previously exercised muscles when compared to rested
muscles (Frosig et al. 2007). Whether this has any
functional role during insulin stimulation in vivo is hard
to judge since no knowledge about absolute PIP3
concentration or PIP3 location in relation to aPKC
exists during insulin stimulation. The role of aPKC in
increased insulin sensitivity post-exercise remains to be
further investigated.
327
Increased insulin sensitivity after exercise Æ S J Maarbjerg et al.
Rac1
Another protein involved in insulin signalling is Rac1
which is downstream of PI3K. Rac1 is a small GTPase
which is active when bound to GTP. Via GTPase
activating proteins (GAPs) Rac1 can hydrolyse the
bound GTP to GDP and become inactive. Correspond-
ingly Rac1 can be reactivated via guanine exchange
factors (GEFs) that mediate the exchange of the GDP to
GTP.
Mechanistic studies suggest that Rac1 regulates
insulin-stimulated glucose uptake as knock down of
Rac1 in cells (JeBailey et al. 2007, Ueda et al. 2008) as
well as muscle specific Rac1 knock out in mouse muscle
(Ueda et al. 2010) abolish insulin mediated GLUT4
translocation. On the other hand, ectopic expression of
a constitutively active Rac1 mutant as well as a
constitutively active Rac1 GEF induces GLUT4 trans-
location in the absence of insulin (JeBailey et al. 2007,
Ueda et al. 2008). These studies report that the reduc-
tion of insulin mediated GLUT4 translocation with
Rac1 knock down/knock out occurs without affecting
Akt or TBC1D4 signalling. This suggests that insulin
signalling downstream of PI3K may bifurcate into two
arms: an Akt/aPKC arm and a Rac1 arm (Fig. 3). The
crucial role of Rac1 is suggested to involve induction of
cortical actin remodelling (Patel et al. 2004, Randhawa
et al. 2008). Furthermore, it is proposed that the Rac1
arm regulates GLUT4 vesicle retention beneath the
membrane while the Akt arm regulates GLUT4 vesicle
docking/fusion (Ishikura et al. 2008, Randhawa et al.
2008). Apart from being involved in insulin-stimulated
glucose uptake preliminary data from our laboratory
show Rac1 activation during exercise and muscle
contractions (L. Sylow, T.E. Jensen, and E.A. Richter,
unpublished observations). We furthermore observed
that the actin depolymerizing agent, Latrunculin B,
decreases contraction-stimulated glucose uptake in
mouse muscles (L. Sylow, E.A. Richter and T.E. Jensen,
unpublished observations). This collectively suggests
that the actin cytoskeleton and its regulators, such as
Rac1, may be important in mediating contraction-
stimulated glucose uptake. However, the role of Rac1 in
relation to increased insulin sensitivity post-exercise is
still unexplored.
Role of TBC1D4 and TBC1D1 in insulin and exercise
induced glucose uptake
Whereas Rac1 is scarcely explored in mature muscle,
members of the Tre-2, BUB2, CDC16, 1 domain family
(TBC1) are currently being investigated intensively in
skeletal muscle. The first family member identified as
being related to glucose metabolisms was Akt substrate
of 160 kDa (AS160) which today is often referred to as
328 Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
Acta Physiol 2011, 202, 323–335
TBC1D4. This protein was initially identified as a
signalling molecule downstream of Akt linking insulin
signalling to GLUT4 trafficking in adipocytes (Kane
et al. 2002, Sano et al. 2003, Zeigerer et al. 2004).
Recently data from skeletal muscle support this concept
derived from studies in adipocytes (Kramer et al.
2006b, Chen et al. 2011) and furthermore suggest a
role of TBC1D4 in contraction-induced glucose uptake
(Kramer et al. 2006b).
The Rab-GAP domain of TBC1D4 seems to be
crucial for the effects on GLUT4 trafficking (Kane et al.
2002, Sano et al. 2003). The mechanism is probably
that the Rab-GAP function in the basal state promotes
target Rabs to exist on the inactive GDP-bound form
blocking GLUT4 translocation. Insulin stimulation on
the other hand deactivates the Rab-GAP function and
thereby favours the active GTP-bound form of target
Rabs, which ultimately results in GLUT4 translocation
(Kane et al. 2002, Sano et al. 2003).
Subsequent to the identification of TBC1D4, another
family member, TBC1D1, was discovered. Apart from
having a similar structure, TBC1D4 and TBC1D1 also
share several key features such as the Rab-GAP domain,
two phosphotyrosine-binding domains and a calmodu-
lin-binding domain (CBD). Similar to TBC1D4, the
TBC1D1 Rab-GAP function seems to regulate GLUT4
translocation and glucose uptake in response to insulin as
well as contractions (Kramer et al. 2006b, An et al.
2010, Vichaiwong et al. 2010). However, the proteins
also have several differences, first the expression pattern
in various species and tissues is diverse (Taylor et al.
2008, Treebak et al. 2009). Secondly, TBC1D4 and
TBC1D1 to a great extent have diverse phosphorylation
sites which are targeted by different kinases (Geraghty
et al. 2007, Chen et al. 2008, Pehmoller et al. 2009,
Treebak et al. 2010). These circumstances allow for
distinct regulation of the two paralog proteins. In this
context it is important to state that as the two proteins are
of similar size and both recognized by the Phospho Akt
Substrate (PAS) antibody, one should be careful when
interpreting PAS data ascribed to TBC1D4 if TBC1D4
has not been immunoprecipitated before western blot-
ting. This is particularly important in white mouse
muscle since the expression of TBC1D1 is much higher
than the expression of TBC1D4 (Taylor et al. 2008).
Regulation via phosphorylation
Phosphorylation of certain TBC1D1 and TBC1D4
residues seems to inhibit the Rab-GAP function, which
then leads to target Rab activation and eventually
promotes GLUT4 translocation (Cartee & Funai 2009).
The mechanistic link between TBC1D1/TBC1D4 and
Rab proteins seems to involve interaction between
TBC1D1/TBC1D4 phosphor motifs and 14-3-3 proteins
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Acta Physiol 2011, 202, 323–335
(Ramm et al. 2006, Geraghty et al. 2007, Chen et al.
2008, 2011).
In human skeletal muscle up to seven TBC1D4
residues can be phosphorylated in response to insulin
(Treebak et al. 2009, 2010). Even though several of the
sites are Akt motifs, the PAS antibody probably mainly
recognizes thr642 and only to a minor extent ser588
(Sano et al. 2003, Geraghty et al. 2007). Increased
TBC1D4 PAS/thr642 phosphorylation in response to
insulin is well-conserved as it is observed in both human
(Karlsson et al. 2005, Hojlund et al. 2008, Treebak
et al. 2009), rat (Arias et al. 2004, Bruss et al. 2005)
and mouse skeletal muscle (Taylor et al. 2008, Chen
et al. 2011). This could indicate that phosphorylation of
PAS sites, probably in combination with some of the
other sites, is important for insulin action. This idea is
supported by time course as well as dose–response
studies in isolated rat muscle where changes in TBC1D4
PAS phosphorylation are paralleled by changes in
glucose uptake in response to insulin (Arias et al.
2004, Funai et al. 2009, 2010). Furthermore, correla-
tive evidence between impaired insulin-stimulated
TBC1D4 PAS phosphorylation and impaired insulin-
stimulated glucose uptake in both people with T2D
(Karlsson et al. 2005, Vind et al. 2011) and women
with polycystic ovary syndrome (Hojlund et al. 2008)
supports this role in muscle glucose metabolism. This
correlative evidence in addition highlights a potential
clinical relevance of TBC1D4. This is further supported
by another study demonstrating an association between
acanthosis nigricans associated insulin resistance and a
premature stop codon mutation (R363X) in the
TCB1D4 gene (Dash et al. 2009). Even though the
mechanistic explanation for this finding remains unre-
solved it provides genetic evidence for a link between
TBC1D4 and insulin action.
Currently the strongest arguments favouring a role of
TBC1D4 derive from two mechanistic studies by
Kramer et al. (2006a,b) and Chen et al. (2011). Kramer
et al. (2006a,b) introduced two different TBC1D4
mutants into mouse muscle via electroporation. A
mutant unable to be phosphorylated on four critical
phosphorylation sites (the 4P mutant; first time used by
Sano et al. 2003), suggests that TBC1D4 phosphoryla-
tion mediates around 50% of glucose uptake in
response to insulin (Kramer et al. 2006b). Interestingly,
this negative effect on glucose uptake is reversed when
introducing a mutant that additionally has an inactive
Rab-GAP domain. Chen et al. 2011 observed whole
body glucose intolerance as well as decreased insulin-
stimulated glucose uptake and GLUT4 translocation in
muscle from mice with a TBC1D4 thr642Ala knockin
mutation (Chen et al. 2011). Collectively these studies
show that phosphorylation of certain phosphorylation
sites on TBC1D4 via14-3-3 binding and deactivation of
Ó 2011 The Authors
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
S J Maarbjerg et al. Æ Increased insulin sensitivity after exercise
the Rab-GAP function promotes glucose uptake in
response to insulin.
Similar to insulin, contraction is also able to increase
TBC1D4 PAS/thr 642 phosphorylation. However,
despite that mechanistic evidence also supports a role
of TBC1D4 during contractions (Kramer et al. 2006b) a
crucial role in contraction-induced glucose uptake is
questioned by other studies (Cartee & Funai 2009). One
of the main arguments against a crucial role of TBC1D4
PAS phosphorylation in contraction-induced glucose
uptake is that it does not increase until after 40–60 min
of bicycling (Sriwijitkamol et al. 2007, Treebak et al.
2007); whereas glucose uptake increases at the onset of
exercise. However, it remains possible that contraction
regulates TBC1D4 via phosphorylation of sites not
recognized by the PAS antibody.
TBC1D1 on the other hand appears to be a promising
candidate regarding contraction-induced glucose
uptake. Whereas TBC1D4 has several insulin responsive
sites, TBC1D1 in contrast has several contractions/
AICAR responsive sites (Taylor et al. 2008, Vichaiwong
et al. 2010). However, the TBC1D4 insulin responsive
site thr642 is conserved as thr596 on TBC1D1. Not
surprisingly this site on TBC1D1 is also recognized by
the PAS antibody (Taylor et al. 2008, Pehmoller et al.
2009). Other studies suggest that this homologue site on
TBC1D1 and TBC1D4 is an AMP-activated kinase
(AMPK) site as well as an Akt site (Pehmoller et al.
2009). However, in contrast to TBC1D4, TBC1D1 has
more predicted AMPK sites than the thr596 (Chen et al.
2008, Taylor et al. 2008, Treebak et al. 2010, Vichai-
wong et al. 2010). This presumption was confirmed
recently as phosphorylation of up to three different sites
increased with exercise/contraction and AICAR in an
AMPK dependent manner (Pehmoller et al. 2009, Frosig
et al. 2010, Vichaiwong et al. 2010). Several studies
support that AMPK is required for normal contraction-
induced glucose uptake at least during ex vivo condi-
tions (Jensen et al. 2009). Therefore, the dependency of
TBC1D1 phosphorylation on AMPK may suggest that
TBC1D1 is a link between increased AMPK activity and
GLUT4 translocation during muscle contraction.
The idea of TBC1D1 being that link is supported by
two very recent studies. These studies applied muscle
electroporation of TBC1D1 mutants (two different
4P mutants) that are unable to be phosphorylated at
particular sites. Whereas Vichaiwong et al. 2010
targeted four predicted AMPK sites (mouse ser231,
thr499, ser660 and ser700), An et al. 2010 targeted
thr596 in addition to three predicted AMPK sites (mouse
ser231, thr499 and ser621). Both studies observed a 20–
35% reduction in contraction-induced glucose uptake.
Furthermore, An et al. showed that this effect is reversed
in a double mutant additionally containing a mutation
that inactivates the Rab-GAP function. This collectively
329
Increased insulin sensitivity after exercise Æ S J Maarbjerg et al.
suggests that phosphorylation of four particular sites
deactivates the Rab-GAP function of TBC1D1 during
contraction and thereby eventually induces glucose
uptake. Consequently, TBC1D1 appears to regulate
contraction-induced glucose uptake similar to the way
TBC1D4 regulates insulin induced and maybe also
contraction-induced glucose uptake. However, it is
interesting that insulin induced glucose uptake was
normal when expressing the 4P mutant of TBC1D1
generated by An et al. while it was reduced when the
obesity-associated TBC1D1 R125W mutant was ex-
pressed. This suggests that even though TBC1D1 regu-
lates both contraction and insulin induced glucose
uptake it occurs via distinct mechanisms.
Regulation via CBD
Whereas the Rab-GAP function of TBC1D4 and
TBC1D1 appears to be regulated by certain phosphor-
ylation sites (Kramer et al. 2007, Sakamoto & Holman
2008, An et al. 2010, Vichaiwong et al. 2010), the role
of the common CBD is far less examined. As muscle
contractions are associated with increased calcium
availability allowing for calcium calmodulin interac-
tion, the CBD could be hypothesized to influence the
function of TBC1D4 and TBC1D1 during contractions
and maybe also into recovery. It is therefore not
surprising that mutations that block calmodulin binding
of the TBC1D4 CBD reduce contraction (40%) but not
insulin induced glucose uptake in muscle. A TBC1D4
mutant additionally containing a deactivating point
mutation in the Rab-GAP domain restored contraction-
induced glucose uptake. This demonstrates that the
CBD via deactivation of the Rab-GAP function can
induce glucose uptake. (Kramer et al. 2007). It is
noteworthy that expressing a double mutant (CBD &
4P) did not have greater effect on glucose uptake than
either mutation alone. Whether the CBD plays any role
in post-exercise insulin sensitivity remains to be inves-
tigated. Furthermore, it needs to be investigated
whether the CBD on TBC1D1 has a similar function.
In conclusion TBD1D4 regulates muscle glucose
uptake in response to insulin and likely also in response
to contraction. Similarly TBC1D1 appears to regulate
insulin as well as contraction-induced glucose uptake
yet presumably via distinct mechanisms. However, also
other mechanisms, that are not dependent on TBC1D1
and/or TBC1D4, appear responsible for a substantial
part of contraction and insulin induced glucose uptake.
Possible role of TBC1D4 and TBC1D1 in
increased insulin sensitivity after exercise
As already mentioned, distal signalling steps common for
insulin and contraction to GLUT4 translocation are of
330 Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
Acta Physiol 2011, 202, 323–335
special interest with respect to increased insulin sensitivity
after exercise. The reason is that an increased GLUT4
membrane content at least to some extent appears to be
responsible for the increased insulin sensitivity. In that
respect it is notable that both TBC1D1 and TBC1D4 are
phosphorylated in response to insulin as well as contrac-
tion and exercise (Bruss et al. 2005, Taylor et al. 2008).
What is even more important is that combining insulin
and contractions in isolated EDL muscle mediates an
additive response on TBC1D4 PAS/thr642 phosphoryla-
tion (Kramer et al. 2006a). This additive effect is inter-
esting because it parallels the additive effect of insulin and
contraction on GLUT4 translocation (Gao et al. 1994b)
and thereby suggests TBC1D4 as a convergent point prior
to GLUT4 translocation.
As previously mentioned, TBC1D4 PAS phosphory-
lation increases after prolonged exercise in both humans
(Deshmukh et al. 2006, Sriwijitkamol et al. 2007,
Treebak et al. 2007) and rats (Funai et al. 2009,
2010). Interestingly this increase is sustained at least
3–4 h into recovery after exercise in rats (Arias et al.
2007, Funai et al. 2009, 2010) and humans (Sriwijitka-
mol et al. 2007). Funai et al. (2009, 2010) furthermore
observed the same pattern for TBC1D4 thr642 phos-
phorylation. A human study using one-legged exercise,
however, failed to see increased TBC1D4 PAS/thr642
phosphorylation 4 h into recovery. Nevertheless, small
but significant increases were observed on four other
TBC1D4 sites in that study (Treebak et al. 2009). Thus,
it is generally agreed that TBC1D4 phosphorylation is
sustained some time into recovery, however, there are
some differences between studies about which sites are
affected.
In both humans and rats the increased basal phos-
phorylation of TBC1D4 3–4 h into recovery is additive
to the effect of insulin stimulation. In addition, Funai
et al. 2009 observed that 3 h of chow feeding instead of
fasting abolished increased insulin action as well as
increased TBC1D4 PAS/thr642 phosphorylation. More-
over, 27 h fasting post-exercise concomitantly increased
insulin action and TBC1D4 PAS/thr642 phosphoryla-
tion. Collectively this suggests that TBC1D4 may take
part in regulating increased insulin sensitivity post-
exercise; (Fig. 3) however studies with a mechanistic
approach remain to clarify this.
Regarding the TBC1D4 paralog TBC1D1, it is less
clear whether it is involved in increased insulin sensi-
tivity after exercise. For instance, even though TBC1D1
PAS phosphorylation increased around 50% during an
exercise bout in rats, the phosphorylation level had
returned to basal levels after 3 h (Funai et al. 2009).
Furthermore, the insulin effect on TBC1D1 PAS phos-
phorylation was identical in the rested and previously
exercised condition. Thus, a role for TBC1D1 in post-
exercise insulin sensitivity is less certain at this point.
Ó 2011 The Authors
Acta Physiol 2011, 202, 323–335
Other stimuli influencing insulin sensitivity
Correlative evidence suggests that decreased glycogen
levels after exercise regulate insulin sensitivity as well as
GLUT4 translocation (Wojtaszewski et al. 2002). In
addition glycogen depleted rat muscles are more insulin
sensitive and activate Akt more than muscles with high
glycogen levels (Derave et al. 2000). However, several
studies speak against a central role of glycogen, first of all
increased insulin sensitivity has been shown to persist
beyond the point of full glycogen re-synthesis (Cartee
et al. 1989). Secondly AICAR treatment in isolated
muscle, which activates AMPK but causes no reduction
in glycogen, has also been observed to increase insulin
sensitivity (Fisher et al. 2002). Recently, some studies
question whether the phenomenon of increased insulin
sensitivity after exercise describes a unique situation or
whether it relies on a general phenomenon regarding
GLUT4 translocation. The reason for this is that
increased insulin sensitivity has not only been observed
to occur after AICAR stimulation and muscle contrac-
tions, but also after hypoxia and maximal insulin
stimulation in rodents (Fisher et al. 2002, Geiger et al.
2006). This suggests that stimulation of glucose trans-
port by any agent is accompanied by subsequent
increased insulin sensitivity. This phenomenon has been
hypothesized to occur as the result of movement of
GLUT4 into regions where they are more susceptible to
translocation to the surface membrane upon subsequent
insulin signalling. Whether this is in fact the case and
what the molecular mechanism could be remains to be
investigated further. However, recent findings in humans
have shown that insulin sensitivity measured by the
clamp technique is not increased when performed 4 h
after a preceding clamp. This suggests that the findings in
isolated rat muscle may not apply to human whole body
physiology (Lucidi et al. 2010).
Finally, it is apparent that exercise does not always
increase insulin sensitivity and may even decrease it. For
instance, eccentric contractions (lengthening contractions)
which cause muscle damage induce insulin resistance (Asp
& Richter 1996, Asp et al. 1996). Muscle damage
decreases GLUT4 protein content (Asp et al. 1995a,b)
because of decreased transcription of the GLUT4 gene
(Kristiansen et al. 1997). Furthermore, muscle damaging
exercise has been shown to impair insulin signalling in
muscle (del Aguila et al. 2000). Together, decreased
GLUT4 content and decreased insulin signalling likely
explains decreased insulin sensitivity of glucose uptake in
muscle damaged by eccentric contractions.
Conclusion
The current understanding of the mechanisms respon-
sible for increased insulin sensitivity after exercise is far
Ó 2011 The Authors
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
S J Maarbjerg et al. Æ Increased insulin sensitivity after exercise
from complete; however new promising players have
entered the field recently. The current consensus is that
increased insulin action is restricted to the active
muscles and mainly caused by increased translocation
of GLUT4 to the surface membrane. Thorough inves-
tigations of the proximal insulin signalling pathway
have shown that the increased GLUT4 translocation is
not paralleled by amplification of proximal insulin
signalling in general. However, recent investigations
have highlighted the importance of TBC1D1 as well as
TBC1D4 in insulin and contraction-induced glucose
uptake. Whereas both proteins are phosphorylated in
response to insulin and contraction, findings until now
primarily support TBC1D4 to be involved in increased
insulin sensitivity after exercise. Perhaps this phosphor-
signature of TBC1D4 combined with potentiation of
other signalling steps at the level of aPKC and/or Rac1
could account for the increased GLUT4 membrane
content. A further scenario could be that increased
membrane permeability is accompanied by elevated
insulin-stimulated microvascular perfusion in the post-
exercise state which could favour glucose uptake
further. Thus, perhaps the combined effect of several
small amplifications in insulin signalling and in micro-
vascular perfusion can explain the relative great
enhancement in insulin action on glucose uptake after
a single bout of exercise.
Conflicts of interest
No conflicts of interest exist.
The work of the authors is supported by the Danish Medical
Research Council, The NovoNordisk Foundation, The Lund-
beck Foundation and the UNIK research program Food, Fitness
and Pharma. Jonas Thue Treebak is thanked for offering
valuable comments during preparation of this manuscript.
References
del Aguila, L.F., Krishnan, R.K., Ulbrecht, J.S., Farrell, P.A.,
Correll, P.H., Lang, C.H., Zierath, J.R. & Kirwan, J.P.
2000. Muscle damage impairs insulin stimulation of IRS-1,
PI 3-kinase, and Akt-kinase in human skeletal muscle. Am J
Physiol Endocrinol Metab 279, E206–E212.
An, D., Toyoda, T., Taylor, E.B., Yu, H., Fujii, N., Hirshman,
M.F. & Goodyear, L.J. 2010. TBC1D1 regulates insulin-
and contraction-induced glucose transport in mouse skeletal
muscle. Diabetes 59, 1358–1365.
Arias, E.B., Kim, J. & Cartee, G.D. 2004. Prolonged incuba-
tion in PUGNAc results in increased protein O-Linked
glycosylation and insulin resistance in rat skeletal muscle.
Diabetes 53, 921–930.
Arias, E.B., Kim, J., Funai, K. & Cartee, G.D. 2007. Prior
exercise increases phosphorylation of Akt substrate of
160 kDa (AS160) in rat skeletal muscle. Am J Physiol
Endocrinol Metab 292, E1191–E1200.
331
Increased insulin sensitivity after exercise Æ S J Maarbjerg et al.
Asp, S. & Richter, E.A. 1996. Decreased insulin action on
muscle glucose transport after eccentric contractions in rats.
J Appl Physiol 81, 1924–1928.
Asp, S., Daugaard, J.R. & Richter, E.A. 1995a. Eccentric
exercise decreases glucose transporter GLUT4 protein in
human skeletal muscle. J Physiol 482(Pt 3), 705–712.
Asp, S., Kristiansen, S. & Richter, E.A. 1995b. Eccentric
muscle damage transiently decreases rat skeletal muscle
GLUT-4 protein. J Appl Physiol 79, 1338–1345.
Asp, S., Daugaard, J.R., Kristiansen, S., Kiens, B. & Richter,
E.A. 1996. Eccentric exercise decreases maximal insulin ac-
tion in humans: muscle and systemic effects. J Physiol 494
(Pt 3), 891–898.
Bandyopadhyay, G., Standaert, M.L., Zhao, L., Yu, B., Avi-
gnon, A., Galloway, L., Karnam, P., Moscat, J. & Farese,
R.V. 1997. Activation of protein kinase C (alpha, beta, and
zeta) by insulin in 3T3/L1 cells. Transfection studies suggest
a role for PKC-zeta in glucose transport. J Biol Chem 272,
2551–2558.
Bandyopadhyay, G., Kanoh, Y., Sajan, M.P., Standaert, M.L.
& Farese, R.V. 2000. Effects of adenoviral gene transfer of
wild-type, constitutively active, and kinase-defective protein
kinase C-lambda on insulin-stimulated glucose transport in
L6 myotubes. Endocrinology 141, 4120–4127.
Bogardus, C., Thuillez, P., Ravussin, E., Vasquez, B., Nari-
miga, M. & Azhar, S. 1983. Effect of muscle glycogen
depletion on in vivo insulin action in man. J Clin Invest 72,
1605–1610.
Bruss, M.D., Arias, E.B., Lienhard, G.E. & Cartee, G.D. 2005.
Increased phosphorylation of Akt Substrate of 160 kDa
(AS160) in rat skeletal muscle in response to insulin or
contractile activity. Diabetes 54, 41–50.
Cartee, G.D. & Funai, K. 2009. Exercise and insulin: conver-
gence or divergence at AS160 and TBC1D1? Exerc Sport Sci
Rev 37, 188–195.
Cartee, G.D., Young, D.A., Sleeper, M.D., Zierath, J., Wall-
berg-Henriksson, H. & Holloszy, J.O. 1989. Prolonged in-
crease in insulin-stimulated glucose transport in muscle after
exercise. Am J Physiol 256, E494–E499.
Chen, H.C., Bandyopadhyay, G., Sajan, M.P., Kanoh, Y.,
Standaert, M., Farese, R.V. Jr & Farese, R.V. 2002. Acti-
vation of the ERK pathway and atypical protein kinase C
isoforms in exercise- and aminoimidazole-4-carboxamide-1-
beta-d-riboside (AICAR)-stimulated glucose transport.
J Biol Chem 277, 23554–23562.
Chen, S., Murphy, J., Toth, R., Campbell, D.G., Morrice, N.A.
& MacKintosh, C. 2008. Complementary regulation of
TBC1D1 and AS160 by growth factors, insulin and AMPK
activators. Biochem J 409, 449–459.
Chen, S., Wasserman, D.H., MacKintosh, C. & Sakamoto, K.
2011. Mice with AS160/TBC1D4-Thr649Ala knockin
mutation are glucose intolerant with reduced insulin sensi-
tivity and altered GLUT4 trafficking. Cell Metab 13, 68–79.
Clark, M.G. 2008. Impaired microvascular perfusion: a con-
sequence of vascular dysfunction and a potential cause of
insulin resistance in muscle. Am J Physiol Endocrinol Metab
295, E732–E750.
Clerk, L.H., Rattigan, S. & Clark, M.G. 2002. Lipid infusion
impairs physiologic insulin-mediated capillary recruitment
332 Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
Acta Physiol 2011, 202, 323–335
and muscle glucose uptake in vivo. Diabetes 51, 1138–
1145.
Cline, G.W., Jucker, B.M., Trajanoski, Z., Rennings, A.J. &
Shulman, G.I. 1998. A novel 13C NMR method to assess
intracellular glucose concentration in muscle, in vivo. Am J
Physiol 274, E381–E389.
Dash, S., Sano, H., Rochford, J.J., Semple, R.K., Yeo, G.,
Hyden, C.S., Soos, M.A., Clark, J., Rodin, A., Langenberg,
C. et al. 2009. A truncation mutation in TBC1D4 in a family
with acanthosis nigricans and postprandial hyperinsulin-
emia. Proc Natl Acad Sci USA 106, 9350–9355.
Day, C. 2007. Metabolic syndrome, or what you will: defini-
tions and epidemiology. Diab Vasc Dis Res 4, 32–38.
DeFronzo, R.A., Gunnarsson, R., Bjorkman, O., Olsson, M. &
Wahren, J. 1985. Effects of insulin on peripheral and
splanchnic glucose metabolism in noninsulin-dependent
(type II) diabetes mellitus. J Clin Invest 76, 149–155.
Derave, W., Hansen, B.F., Lund, S., Kristiansen, S. & Richter,
E.A. 2000. Muscle glycogen content affects insulin-stimu-
lated glucose transport and protein kinase B activity. Am J
Physiol Endocrinol Metab 279, E947–E955.
Deshmukh, A., Coffey, V.G., Zhong, Z., Chibalin, A.V.,
Hawley, J.A. & Zierath, J.R. 2006. Exercise-induced phos-
phorylation of the novel Akt substrates AS160 and filamin A
in human skeletal muscle. Diabetes 55, 1776–1782.
Devlin, J.T. & Horton, E.S. 1985. Effects of prior high-
intensity exercise on glucose metabolism in normal and
insulin-resistant men. Diabetes 34, 973–979.
Devlin, J.T., Hirshman, M., Horton, E.D. & Horton, E.S.
1987. Enhanced peripheral and splanchnic insulin sensitivity
in NIDDM men after single bout of exercise. Diabetes 36,
434–439.
Eriksson, K.F. & Lindgarde, F. 1991. Prevention of type 2
(non-insulin-dependent) diabetes mellitus by diet and phys-
ical exercise. The 6-year Malmo feasibility study. Diabeto-
logia 34, 891–898.
Etgen, G.J., Valasek, K.M., Broderick, C.L. & Miller, A.R.
1999. In vivo adenoviral delivery of recombinant human
protein kinase C-zeta stimulates glucose transport activity in
rat skeletal muscle. J Biol Chem 274, 22139–22142.
Farese, R.V. 2002. Function and dysfunction of aPKC isoforms
for glucose transport in insulin-sensitive and insulin-resistant
states. Am J Physiol Endocrinol Metab 283, E1–E11.
Farese, R.V., Sajan, M.P., Yang, H., Li, P., Mastorides, S.,
Gower, W.R. Jr, Nimal, S., Choi, C.S., Kim, S., Shulman,
G.I., Kahn, C.R., Braun, U. & Leitges, M. 2007. Muscle-
specific knockout of PKC-lambda impairs glucose transport
and induces metabolic and diabetic syndromes. J Clin Invest
117, 2289–2301.
Fisher, J.S., Gao, J., Han, D.H., Holloszy, J.O. & Nolte, L.A.
2002. Activation of AMP kinase enhances sensitivity of
muscle glucose transport to insulin. Am J Physiol Endocrinol
Metab 282, E18–E23.
Frosig, C., Sajan, M.P., Maarbjerg, S.J., Brandt, N., Roe-
pstorff, C., Wojtaszewski, J.F., Kiens, B., Farese, R.V. &
Richter, E.A. 2007. Exercise improves phosphatidylinositol-
3,4,5-trisphosphate responsiveness of atypical protein kinase
C and interacts with insulin signalling to peptide elongation
in human skeletal muscle. J Physiol 582, 1289–1301.
Ó 2011 The Authors
Acta Physiol 2011, 202, 323–335
Frosig, C., Pehmoller, C., Birk, J.B., Richter, E.A. &
Wojtaszewski, J.F. 2010. Exercise-induced TBC1D1 Ser237
phosphorylation and 14-3-3 protein binding capacity in
human skeletal muscle. J Physiol 588, 4539–4548.
Fueger, P.T., Hess, H.S., Bracy, D.P., Pencek, R.R., Posey,
K.A., Charron, M.J. & Wasserman, D.H. 2004a. Regula-
tion of insulin-stimulated muscle glucose uptake in the
conscious mouse: role of glucose transport is dependent on
glucose phosphorylation capacity. Endocrinology 145,
4912–4916.
Fueger, P.T., Hess, H.S., Posey, K.A., Bracy, D.P., Pencek,
R.R., Charron, M.J. & Wasserman, D.H. 2004b. Control of
exercise-stimulated muscle glucose uptake by GLUT4 is
dependent on glucose phosphorylation capacity in the con-
scious mouse. J Biol Chem 279, 50956–50961.
Funai, K., Schweitzer, G.G., Sharma, N., Kanzaki, M. &
Cartee, G.D. 2009. Increased AS160 phosphorylation, but
not TBC1D1 phosphorylation, with increased postexercise
insulin sensitivity in rat skeletal muscle. Am J Physiol
Endocrinol Metab 297, E242–E251.
Funai, K., Schweitzer, G.G., Castorena, C.M., Kanzaki, M. &
Cartee, G.D. 2010. In vivo exercise followed by in vitro
contraction additively elevates subsequent insulin-stimulated
glucose transport by rat skeletal muscle. Am J Physiol
Endocrinol Metab 298, E999–E1010.
Gao, J., Gulve, E.A. & Holloszy, J.O. 1994a. Contraction-
induced increase in muscle insulin sensitivity: requirement
for a serum factor. Am J Physiol 266, E186–E192.
Gao, J., Ren, J., Gulve, E.A. & Holloszy, J.O. 1994b. Additive
effect of contractions and insulin on GLUT-4 translocation
into the sarcolemma. J Appl Physiol 77, 1597–1601.
Garetto, L.P., Richter, E.A., Goodman, M.N. & Ruderman,
N.B. 1984. Enhanced muscle glucose metabolism after
exercise in the rat: the two phases. Am J Physiol 246, E471–
E475.
Geiger, P.C., Han, D.H., Wright, D.C. & Holloszy, J.O. 2006.
How muscle insulin sensitivity is regulated: testing of a
hypothesis. Am J Physiol Endocrinol Metab 291, E1258–
E1263.
Geraghty, K.M., Chen, S., Harthill, J.E., Ibrahim, A.F., Toth,
R., Morrice, N.A., Vandermoere, F., Moorhead, G.B.,
Hardie, D.G. & MacKintosh, C. 2007. Regulation of mul-
tisite phosphorylation and 14-3-3 binding of AS160 in
response to IGF-1, EGF, PMA and AICAR. Biochem J 407,
231–241.
Goodyear, L.J. & Kahn, B.B. 1998. Exercise, glucose trans-
port, and insulin sensitivity. Annu Rev Med 49, 235–261.
Goodyear, L.J., Giorgino, F., Balon, T.W., Condorelli, G. &
Smith, R.J. 1995. Effects of contractile activity on tyrosine
phosphoproteins and PI 3-kinase activity in rat skeletal
muscle. Am J Physiol 268, E987–E995.
Gulve, E.A., Cartee, G.D., Zierath, J.R., Corpus, V.M. &
Holloszy, J.O. 1990. Reversal of enhanced muscle glucose
transport after exercise: roles of insulin and glucose. Am
J Physiol 259, E685–E691.
Hansen, P.A., Nolte, L.A., Chen, M.M. & Holloszy, J.O.
1998. Increased GLUT-4 translocation mediates enhanced
insulin sensitivity of muscle glucose transport after exercise.
J Appl Physiol 85, 1218–1222.
Ó 2011 The Authors
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
S J Maarbjerg et al. Æ Increased insulin sensitivity after exercise
Hojlund, K., Glintborg, D., Andersen, N.R., Birk, J.B., Tree-
bak, J.T., Frosig, C., Beck-Nielsen, H. & Wojtaszewski, J.F.
2008. Impaired insulin-stimulated phosphorylation of Akt
and AS160 in skeletal muscle of women with polycystic
ovary syndrome is reversed by pioglitazone treatment. Dia-
betes 57, 357–366.
Inyard, A.C., Chong, D.G., Klibanov, A.L. & Barrett, E.J.
2009. Muscle contraction, but not insulin, increases micro-
vascular blood volume in the presence of free fatty acid-
induced insulin resistance. Diabetes 58, 2457–2463.
Ishikura, S., Koshkina, A. & Klip, A. 2008. Small G proteins in
insulin action: Rab and Rho families at the crossroads of
signal transduction and GLUT4 vesicle traffic. Acta Physiol
(Oxf) 192, 61–74.
JeBailey, L., Wanono, O., Niu, W., Roessler, J., Rudich, A. &
Klip, A. 2007. Ceramide- and oxidant-induced insulin
resistance involve loss of insulin-dependent Rac-activation
and actin remodeling in muscle cells. Diabetes 56, 394–403.
Jensen, T.E., Wojtaszewski, J.F. & Richter, E.A. 2009. AMP-
activated protein kinase in contraction regulation of skeletal
muscle metabolism: necessary and/or sufficient? Acta Physiol
(Oxf) 196, 155–174.
Kane, S., Sano, H., Liu, S.C., Asara, J.M., Lane, W.S., Garner,
C.C. & Lienhard, G.E. 2002. A method to identify serine
kinase substrates. Akt phosphorylates a novel adipocyte
protein with a Rab GTPase-activating protein (GAP)
domain. J Biol Chem 277, 22115–22118.
Karlsson, H.K., Zierath, J.R., Kane, S., Krook, A., Lienhard,
G.E. & Wallberg-Henriksson, H. 2005. Insulin-stimulated
phosphorylation of the Akt substrate AS160 is impaired in
skeletal muscle of type 2 diabetic subjects. Diabetes 54,
1692–1697.
Katz, A., Nyomba, B.L. & Bogardus, C. 1988. No accumula-
tion of glucose in human skeletal muscle during euglycemic
hyperinsulinemia. Am J Physiol 255, E942–E945.
Kramer, H.F., Witczak, C.A., Fujii, N., Jessen, N., Taylor,
E.B., Arnolds, D.E., Sakamoto, K., Hirshman, M.F. &
Goodyear, L.J. 2006a. Distinct signals regulate AS160
phosphorylation in response to insulin, AICAR, and con-
traction in mouse skeletal muscle. Diabetes 55, 2067–2076.
Kramer, H.F., Witczak, C.A., Taylor, E.B., Fujii, N., Hirsh-
man, M.F. & Goodyear, L.J. 2006b. AS160 regulates insu-
lin- and contraction-stimulated glucose uptake in mouse
skeletal muscle. J Biol Chem 281, 31478–31485.
Kramer, H.F., Taylor, E.B., Witczak, C.A., Fujii, N., Hirsh-
man, M.F. & Goodyear, L.J. 2007. Calmodulin-binding
domain of AS160 regulates contraction- but not insulin-
stimulated glucose uptake in skeletal muscle. Diabetes 56,
2854–2862.
Kristiansen, S., Jones, J., Handberg, A., Dohm, G.L. & Richter,
E.A. 1997. Eccentric contractions decrease glucose trans-
porter transcription rate, mRNA, and protein in skeletal
muscle. Am J Physiol 272, C1734–C1738.
Liu, Z., Liu, J., Jahn, L.A., Fowler, D.E. & Barrett, E.J. 2009.
Infusing lipid raises plasma free fatty acids and induces
insulin resistance in muscle microvasculature. J Clin Endo-
crinol Metab 94, 3543–3549.
Lucidi, P., Rossetti, P., Porcellati, F., Pampanelli, S., Candel-
oro, P., Andreoli, A.M., Perriello, G., Bolli, G.B. & Fanelli,
333
Increased insulin sensitivity after exercise Æ S J Maarbjerg et al.
C.G. 2010. Mechanisms of insulin resistance after insulin-
induced hypoglycemia in humans: the role of lipolysis.
Diabetes 59, 1349–1357.
Mikines, K.J., Sonne, B., Farrell, P.A., Tronier, B. & Galbo, H.
1988. Effect of physical exercise on sensitivity and respon-
siveness to insulin in humans. Am J Physiol 254, E248–
E259.
Pan, X.R., Li, G.W., Hu, Y.H., Wang, J.X., Yang, W.Y., An,
Z.X., Hu, Z.X., Lin, J., Xiao, J.Z., Cao, H.B. et al. 1997.
Effects of diet and exercise in preventing NIDDM in people
with impaired glucose tolerance. The Da Qing IGT and
Diabetes Study. Diabetes Care 20, 537–544.
Patel, N.A., Apostolatos, H.S., Mebert, K., Chalfant, C.E.,
Watson, J.E., Pillay, T.S., Sparks, J. & Cooper, D.R. 2004.
Insulin regulates protein kinase CbetaII alternative splicing
in multiple target tissues: development of a hormonally
responsive heterologous minigene. Mol Endocrinol 18, 899–
911.
Pehmoller, C., Treebak, J.T., Birk, J.B., Chen, S., MacKintosh,
C., Hardie, D.G., Richter, E.A. & Wojtaszewski, J.F. 2009.
Genetic disruption of AMPK signaling abolishes both con-
traction- and insulin-stimulated TBC1D1 phosphorylation
and 14-3-3 binding in mouse skeletal muscle. Am J Physiol
Endocrinol Metab 297, E665–E675.
Ramm, G., Larance, M., Guilhaus, M. & James, D.E. 2006. A
role for 14-3-3 in insulin-stimulated GLUT4 translocation
through its interaction with the RabGAP AS160. J Biol
Chem 281, 29174–29180.
Randhawa, V.K., Ishikura, S., Talior-Volodarsky, I., Cheng,
A.W., Patel, N., Hartwig, J.H. & Klip, A. 2008. GLUT4
vesicle recruitment and fusion are differentially regulated by
Rac, AS160, and Rab8A in muscle cells. J Biol Chem 283,
27208–27219.
Rattigan, S., Wallis, M.G., Youd, J.M. & Clark, M.G. 2001.
Exercise training improves insulin-mediated capillary
recruitment in association with glucose uptake in rat hind-
limb. Diabetes 50, 2659–2665.
Richter, E.A., Garetto, L.P., Goodman, M.N. & Ruderman,
N.B. 1982. Muscle glucose metabolism following exercise in
the rat: increased sensitivity to insulin. J Clin Invest 69, 785–
793.
Richter, E.A., Garetto, L.P., Goodman, M.N. & Ruderman,
N.B. 1984. Enhanced muscle glucose metabolism after
exercise: modulation by local factors. Am J Physiol 246,
E476–E482.
Richter, E.A., Mikines, K.J., Galbo, H. & Kiens, B. 1989.
Effect of exercise on insulin action in human skeletal muscle.
J Appl Physiol 66, 876–885.
Richter, E.A., Vistisen, B., Maarbjerg, S.J., Sajan, M., Farese,
R.V. & Kiens, B. 2004. Differential effect of bicycling
exercise intensity on activity and phosphorylation of atypical
protein kinase C and extracellular signal-regulated protein
kinase in skeletal muscle. J Physiol 560, 909–918.
Rose, A.J. & Richter, E.A. 2005. Skeletal muscle glucose up-
take during exercise: how is it regulated? Physiology (Beth-
esda) 20, 260–270.
Ross, R.M., Wadley, G.D., Clark, M.G., Rattigan, S. &
McConell, G.K. 2007. Local nitric oxide synthase inhibition
reduces skeletal muscle glucose uptake but not capillary
334 Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
Acta Physiol 2011, 202, 323–335
blood flow during in situ muscle contraction in rats. Diabetes
56, 2885–2892.
Ryder, J.W., Kawano, Y., Galuska, D., Fahlman, R.,
Wallberg-Henriksson, H., Charron, M.J. & Zierath, J.R.
1999. Postexercise glucose uptake and glycogen synthesis in
skeletal muscle from GLUT4-deficient mice. FASEB J 13,
2246–2256.
Sajan, M.P., Bandyopadhyay, G., Miura, A., Standaert, M.,
Nimal, S., Longnus, S.L., Van, O.E., Hainault, I., Foufelle,
F., Kahn, C.R., Braun, U., Leitges, M. & Farese, R.V. 2009.
AICAR and metformin, but not exercise, increase muscle
glucose transport through. Am J Physiol Endocrinol Metab
298, E179–E192.
Sakamoto, K. & Holman, G.D. 2008. Emerging role for
AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4
traffic. Am J Physiol Endocrinol Metab 295, E29–E37.
Sano, H., Kane, S., Sano, E., Miinea, C.P., Asara, J.M., Lane,
W.S., Garner, C.W. & Lienhard, G.E. 2003. Insulin-stimu-
lated phosphorylation of a Rab GTPase-activating protein
regulates GLUT4 translocation. J Biol Chem 278, 14599–
14602.
Sriwijitkamol, A., Coletta, D.K., Wajcberg, E., Balbontin,
G.B., Reyna, S.M., Barrientes, J., Eagan, P.A., Jenkinson,
C.P., Cersosimo, E., DeFronzo, R.A., Sakamoto, K. &
Musi, N. 2007. Effect of acute exercise on AMPK signaling
in skeletal muscle of subjects with type 2 diabetes: a
time-course and dose–response study. Diabetes 56, 836–
848.
Taylor, E.B., An, D., Kramer, H.F., Yu, H., Fujii, N.L., Roeckl,
K.S., Bowles, N., Hirshman, M.F., Xie, J., Feener, E.P. &
Goodyear, L.J. 2008. Discovery of TBC1D1 as an insulin-,
AICAR-, and contraction-stimulated signaling nexus in
mouse skeletal muscle. J Biol Chem 283, 9787–9796.
Thong, F.S., Derave, W., Urso, B., Kiens, B. & Richter, E.A.
2003. Prior exercise increases basal and insulin-induced p38
mitogen-activated protein kinase phosphorylation in human
skeletal muscle. J Appl Physiol 94, 2337–2341.
Treebak, J.T., Birk, J.B., Rose, A.J., Kiens, B., Richter, E.A. &
Wojtaszewski, J.F. 2007. AS160 phosphorylation is associ-
ated with activation of alpha2beta2gamma1- but not
alpha2beta2gamma3-AMPK trimeric complex in skeletal
muscle during exercise in humans. Am J Physiol Endocrinol
Metab 292, E715–E722.
Treebak, J.T., Frosig, C., Pehmoller, C., Chen, S., Maarbjerg,
S.J., Brandt, N., MacKintosh, C., Zierath, J.R., Hardie,
D.G., Kiens, B., Richter, E.A., Pilegaard, H. & Wojtaszew-
ski, J.F. 2009. Potential role of TBC1D4 in enhanced post-
exercise insulin action in human skeletal muscle. Diabeto-
logia 52, 891–900.
Treebak, J.T., Taylor, E.B., Witczak, C.A., An, D., Toyoda, T.,
Koh, H.J., Xie, J., Feener, E.P., Wojtaszewski, J.F., Hirsh-
man, M.F. & Goodyear, L.J. 2010. Identification of a novel
phosphorylation site on TBC1D4 regulated by AMP-acti-
vated protein kinase in skeletal muscle. Am J Physiol Cell
Physiol 298, C377–C385.
Ueda, S., Kataoka, T. & Satoh, T. 2008. Activation of the
small GTPase Rac1 by a specific guanine-nucleotide-ex-
change factor suffices to induce glucose uptake into skeletal-
muscle cells. Biol Cell 100, 645–657.
Ó 2011 The Authors
Acta Physiol 2011, 202, 323–335
Ueda, S., Kitazawa, S., Ishida, K., Nishikawa, Y., Matsui, M.,
Matsumoto, H., Aoki, T., Nozaki, S., Takeda, T., Tamori,
Y., Aiba, A., Kahn, C.R., Kataoka, T. & Satoh, T. 2010.
Crucial role of the small GTPase Rac1 in insulin-stimulated
translocation of glucose transporter 4 to the mouse skeletal
muscle sarcolemma. FASEB J 24, 2254–2261.
Vichaiwong, K., Purohit, S., An, D., Toyoda, T., Jessen, N.,
Hirshman, M.F. & Goodyear, L.J. 2010. Contraction regu-
lates site-specific phosphorylation of TBC1D1 in skeletal
muscle. Biochem J 431, 311–320.
Vincent, M.A., Barrett, E.J., Lindner, J.R., Clark, M.G. &
Rattigan, S. 2003. Inhibiting NOS blocks microvascular
recruitment and blunts muscle glucose uptake in response to
insulin. Am J Physiol Endocrinol Metab 285, E123–E129.
Vincent, M.A., Clerk, L.H., Lindner, J.R., Klibanov, A.L.,
Clark, M.G., Rattigan, S. & Barrett, E.J. 2004. Microvascular
recruitment is an early insulin effect that regulates skeletal
muscle glucose uptake in vivo. Diabetes 53, 1418–1423.
Vind, B.F., Pehmoller, C., Treebak, J.T., Birk, J.B.,
Hey-Mogensen, M., Beck-Nielsen, H., Zierath, J.R.,
Wojtaszewski, J.F. & Hojlund, K. 2011. Impaired insulin-
induced site-specific phosphorylation of TBC1 domain
family, member 4 (TBC1D4) in skeletal muscle of type 2
diabetes patients is restored by endurance exercise-training.
Diabetologia 54, 157–167.
Wasserman, D.H. & Halseth, A.E. 1998. An overview of
muscle glucose uptake during exercise. Sites of regulation.
Adv Exp Med Biol 441, 1–16.
Ó 2011 The Authors
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02267.x
S J Maarbjerg et al. Æ Increased insulin sensitivity after exercise
Wei, M., Gibbons, L.W., Mitchell, T.L., Kampert, J.B., Lee,
C.D. & Blair, S.N. 1999. The association between cardio-
respiratory fitness and impaired fasting glucose and type 2
diabetes mellitus in men. Ann Intern Med 130, 89–96.
Wojtaszewski, J.F. & Richter, E.A. 2006. Effects of acute
exercise and training on insulin action and sensitivity: focus
on molecular mechanisms in muscle. Essays Biochem 42,
31–46.
Wojtaszewski, J.F., Hansen, B.F., Kiens, B. & Richter, E.A.
1997. Insulin signaling in human skeletal muscle: time
course and effect of exercise. Diabetes 46, 1775–1781.
Wojtaszewski, J.F., Hansen, B.F., Gade, K.B., Markuns, J.F.,
Goodyear, L.J. & Richter, E.A. 2000. Insulin signaling and
insulin sensitivity after exercise in human skeletal muscle.
Diabetes 49, 325–331.
Wojtaszewski, J.F., Nielsen, J.N. & Richter, E.A. 2002. Invited
review: effect of acute exercise on insulin signaling and
action in humans. J Appl Physiol 93, 384–392.
Zeigerer, A., McBrayer, M.K. & McGraw, T.E. 2004. Insulin
stimulation of GLUT4 exocytosis, but not its inhibition of
endocytosis, is dependent on RabGAP AS160. Mol Biol Cell
15, 4406–4415.
Zisman, A., Peroni, O.D., Abel, E.D., Michael, M.D., Mauv-
ais-Jarvis, F., Lowell, B.B., Wojtaszewski, J.F., Hirshman,
M.F., Virkamaki, A., Goodyear, L.J., Kahn, C.R. & Kahn,
B.B. 2000. Targeted disruption of the glucose transporter 4
selectively in muscle causes insulin resistance and glucose
intolerance. Nat Med 6, 924–928.
335