Review

AMPK regulation of fatty acid metabolism and mitochondrial biogenesis:

Implications for obesity
Hayley M. ONeill
a,b,
, Graham P. Holloway
c
, Gregory R. Steinberg
b
a
University of Melbourne, Department of Medicine, St. Vincents Institute of Medical Research, Melbourne, Victoria 3065, Australia
b
McMaster University, Department of Medicine, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5
c
University of Guelph, Department of Human Health and Nutritional Science, Guelph, Ontario, Canada N1G 2W1
a r t i c l e i n f o
Article history:
Available online 28 June 2012
Keywords:
AMPK
Obesity
Insulin resistance
Exercise
Fatty acid
Mitochondria
a b s t r a c t
Skeletal muscle plays an important role in regulating whole-body energy expenditure given it is a major
site for glucose and lipid oxidation. Obesity and type 2 diabetes are causally linked through their associ-
ation with skeletal muscle insulin resistance, while conversely exercise is known to improve whole body
glucose homeostasis simultaneously with muscle insulin sensitivity. Exercise activates skeletal muscle
AMP-activated protein kinase (AMPK). AMPK plays a role in regulating exercise capacity, skeletal muscle
mitochondrial content and contraction-stimulated glucose uptake. Skeletal muscle AMPK is also thought
to be important for regulating fatty acid metabolism; however, direct genetic evidence in this area is
currently lacking. This review will discuss the current paradigms regarding the inuence of AMPK in
regulating skeletal muscle fatty acid metabolism and mitochondrial biogenesis at rest and during
exercise, and highlight the potential implications in the development of insulin resistance.
Crown Copyright 2012 Published by Elsevier Ireland Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
1.1. Insulin resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
1.2. AMPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
1.3. Regulation of AMPK activity: upstream kinases and allosteric regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
1.4. AMPK and exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
2. AMPK and fatty acid uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
3. Fatty acid handling (esterification and lipolysis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
3.1. GPAT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
3.2. Disturbances in lipid handling in obesity: HSL, ATGL and AMPK. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
3.3. Desnutrin/ATGL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
3.4. HSL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
4. AMPK and fatty acid oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
4.1. Regulation of acetyl-CoA carboxylase activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
4.2. Malonyl-CoA decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
5. AMPK-independent regulation of mitochondrial fatty acid oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6. AMPK and exercise-induced metabolic adaptations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6.1. Mitochondrial capacity and exercise tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
0303-7207/$ - see front matter Crown Copyright 2012 Published by Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mce.2012.06.019
Abbreviations: ACC2, acetyl-CoA carboxylase; AMPK, AMPK-activated protein kinase; AICAR, aminoimidazole-4-carboxamide-1-b-D-ribonucleoside; ATGL, adipose
triacylglycerol lipase; CaMKII, Ca
2+
/CaM-dependent protein kinase kinase; CPT-I, carnitine palmitoyl transferase; CREB, cAMP response element binding protein; DGAT,
diacylglyceride acyltransferase; GPA, guanadinopropionic acid; GPAT, glycerol-3-phosphate acyltransferase (GPAT); HDAC, class IIA histone diacetylases; HSL, hormone
sensitive lipase; MGCAT, monoglyceride acyltransferase; LCFA, long chain fatty acids; m.CoA, malonyl-CoA; MCD, malonyl-CoA decarboxylase; MEF, myocyte enhancer factor;
OXPHOS, oxidative phosphorylation; PGC-1
a
, peroxisome proliferator activated receptor c co-activator-1a; PKA, protein kinase A; SIRT1, situnin deacetylase; TZDs,
thiazoladinedione; ZMP, 5-aminoimidazoel4-carboxamide-1-D-riborfuronosil-5monophosphate.

Corresponding author at: McMaster University, Department of Medicine, 1280 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. Tel.: +1 905 521 2100x21689; fax:
+1 905 777 7856.
E-mail address: honeill@mcmaster.ca (H.M. ONeill).
Molecular and Cellular Endocrinology 366 (2013) 135151
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6.2. Fiber-type transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7. AMPK transcriptional regulation of mitochondrial oxidative genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7.1. p38 and p53. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7.2. PGC-1a. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7.3. AMPK and SIRT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
8. AMPK as a potential therapeutic target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
1. Introduction
1.1. Insulin resistance
Insulin resistance in skeletal muscle is a major factor in the
pathogenesis of type 2 diabetes (Yu et al., 2002). Chronic elevation
of plasma free fatty acids is commonly associated with impaired
insulin-mediated glucose uptake (Steiner et al., 1980; Frayn
et al., 1993), and often co-exists with obesity and type 2 diabetes
(Reaven and Chen, 1988). Skeletal muscle is the primary tissue
contributing to whole-body energy expenditure, and is the major
site for insulin-stimulated glucose disposal; therefore, responsive-
ness to insulin in this tissue greatly inuences whole-body glucose
homeostasis.
Insulin-mediated glucose uptake requires an intact signalling
cascade that involves a number of spatially distinct phosphoryla-
tion events, which result in moving glucose transporters (GLUT4)
to the plasma membrane, upregulating glucose transport into the
cell. Specically, in skeletal muscle, insulin mediates glucose up-
take by binding to its tyrosine kinase receptor on the outside of
the cell, causing further activation/phosphorylation of the insulin
receptor substrate (IRS) proteins-1 and 2 inside the cell. Activation
of IRS proteins trigger the activation of phosphatidlyinositol-3-ki-
nase (PI3), which promotes the interaction between phosphoinosi-
tide-dependent kinase (PDK) and Akt, and the subsequent
phosphorylation/activation of Akt/protein kinase B and inhibition
of AS160. The latter results in the recruitment of the glucose trans-
porter GLUT-4 to the plasma membrane and glucose uptake (Saltiel
and Kahn, 2001). There are a number of defects within this signal-
ling cascade that are associated with insulin resistance (Shulman,
2000; Steinberg, 2007); however, reduced IRS-phosphorylation
in response to insulin is the earliest and most pronounced defect
in the insulin signalling cascade (Steinberg, 2007). Therefore, an
important research question is to address the mechanistic causes
of insulin resistance.
A common observation in insulin resistant skeletal muscle is
that intramuscular lipid levels are elevated in obesity, raising the
possibility that alterations in lipid metabolism inuence insulin
signalling (Shulman, 2000; Bonen et al., 2004b; Steinberg, 2007).
Studies in humans (Pan et al., 1997; Krssak et al., 1999; Kraegen
et al., 2001) and mice (Kim et al., 2000, 2001) have demonstrated
a strong relationship between increased intramuscular triacylglyc-
erol (TAG) content and insulin resistance. In addition, acute eleva-
tions in plasma free fatty acid levels during lipid infusion reduce
insulin-mediated glucose uptake in humans (Boden and Jadali,
1991; Kelley et al., 1993; Boden et al., 1994; Roden et al., 1996)
and rats (Nolte et al., 1994; Jucker et al., 1997). In humans with
obesity and type 2 diabetes, TAG accumulation is associated with
increased rates of skeletal muscle fatty acid transport, and in-
creased translocation of fatty acid transporter fatty acid translo-
case (FAT/CD36) (FAT is the rodent homolog of human CD36
(Oquendo et al., 1989)) to the plasma membrane (Bonen et al.,
2004b; Aguer et al., 2010). Similarly, in obese and high-fat fed insu-
lin-resistant rats, increased fatty acid uptake is associated with
increased fatty acid transporter plasma membrane fatty acid bind-
ing protein (FABP
PM
) content and FAT/CD36 expression, respec-
tively (Turcotte et al., 2001; Hegarty et al., 2002).
Collectively, these studies suggest an adaptation for enhanced
fatty acid uptake during times of lipid-oversupply, which may
contribute to accumulations of intramuscular lipids. However, re-
cent studies suggest that TAGs, per se, may not be the problem
since the accumulation of TAGs appears to be relatively innocuous,
but instead insulin resistance is caused by an accumulation of lipid
intermediates such as long-chain acyl CoA (LCACoA), diacylglycerol
(DAG) and ceramide (Steinberg, 2007). The accumulation of these
lipids within skeletal muscle, as a result of increased fatty acid up-
take, triggers the activation of a serine/threonine kinase cascade
involving the activation of protein kinase C (PKC) isoforms
(Yu et al., 2002; Moeschel et al., 2004; Yi et al., 2007), IkappaB
kinase-b (IKK-b) (Gao et al., 2002) and c-jun terminal amino kinase
(JNK) (Aguirre et al., 2000), which inhibit IRS signalling and Akt
phosphorylation. Suppressor of cytokine signalling 3 (SOCS3) also
directly interacts with insulin receptor and IRS proteins to inhibit
insulin signalling (Ueki et al., 2004; Steinberg et al., 2009). More
importantly, in obese skeletal muscle, PKC (Bell et al., 2000; Itani
et al., 2000; Kim et al., 2004a); IKK-b (Yuan et al., 2001; Arkan
et al., 2005), JNK (Hirosumi et al., 2002) and SOCS3 (Steinberg
et al., 2004b; Steinberg et al., 2006a; Watt et al., 2006) are elevated,
and the genetic ablation of these proteins have been shown to
be protective against obesity-induced insulin resistance. Taken
together, these studies indicate that strategies that limit the accu-
mulation of reactive intramuscular lipids may prevent the develop-
ment of obesity induced insulin resistance.
1.2. AMPK
AMPK is present in all tissues as a heterotrimeric complex con-
sisting of a catalytic a subunit and regulatory b and c subunits
(Xiao et al., 2007; Witczak et al., 2008). Both b and c subunits
are required for optimal activity of the a-catalytic subunit (Chen
et al., 1999). Multiple genes exist for each of the subunits (a1,
a2; b1, b2; c1, c2, c3), enabling the expression of 12 heterotrimer
combinations, which are expressed in a tissue-specic manner
(Mahlapuu et al., 2004). Alternative splice variants exist for a1
and c2, which further add to the potential diversity of the AMPK
abc heterotrimer.
In human skeletal muscle, the majority of AMPK complexes
contain both a2 and b2 subunits, and of these a2/b2 complexes,
20% associate with c3, whilst the remaining a1/b2 and a2/b2 asso-
ciate with c1 (Wojtaszewski et al., 2005). In agreement with these
ndings, AMPK b2 null mice have shown an essential role for this
subunit in regulating heterotrimer formation in skeletal muscle
(Steinberg et al. 2010). In mice, c3- and c2 AMPK is predominately
expressed in fast-twitch glycolytic extensor digitroum longus
(EDL) muscle compared to slow-twitch oxidative soleus muscle
(Barnes et al., 2004; Mahlapuu et al., 2004), whereas in gastrocne-
mius muscle, c1, c2 and c3 are evenly expressed (Mahlapuu et al.,
2004).
136 H.M. ONeill et al. / Molecular and Cellular Endocrinology 366 (2013) 135151
1.3. Regulation of AMPK activity: upstream kinases and allosteric
regulation
Reversible phosphorylation at Thr172 within the activation loop
of the a-subunit is the most potent activator of AMPK (>100-fold)
(reviewed in Oakhill et al. (2012)). Besides phosphorylation, AMPK
is also directly activated by both AMP and ADP, which bind to the
c-subunit (Sanders et al., 2007; Oakhill et al., 2011; Xiao et al.,
2011). Myristoylation of the b-subunit is required for AMP and
ADP to promote AMPK Thr172 phosphorylation and initiate AMPK
signalling; however, allosteric activation by AMP (3- to 5-fold)
does not require myristoylation (Oakhill et al., 2010). AMP and
ADP binding to the c-subunit is also thought to induce a conforma-
tional change in the kinase domain that protects AMPK Thr172
from dephosphorylation by the protein phosphatase 2 A and C
(PP2A and C); therefore, important for maintaining AMPK activity
(Sanders et al., 2007; Oakhill et al., 2011).
Two upstream kinases, LKB1 and Ca
2+
/CaM-dependent protein
kinase kinase (CaMKK), have been shown phosphorylate AMPK
Thr172 in mammalian cells. LKB1 is a heterotrimer complex with
regulatory proteins STRAD and MO25 (Hawley et al., 2003; Woods
et al., 2003). In skeletal muscle, studies in two independent models
lacking LKB1 have shown that LKB1 is the major AMPK kinase in
skeletal muscle. Skeletal muscle-specic deletion of LKB1 (LKB1-
MKO) from mice, results in greatly reduced a2 AMPK T172 and
ACC2 S221 phosphorylation following activation by aminoimidaz-
ole-4-carboxamide-1-b-D-ribonucleoside (AICAR- a cell-permeable
adenosine analog that can be phosphorylated to form 5-amin-
oimidazoel4-carboxamide-1-D-riborfuronosil-5
0
monophosphate
(ZMP)) or muscle contractions/exercise (Sakamoto et al., 2005;
Koh et al., 2006; Thomson et al., 2007b); however, AMPK a1 activity
does not appear to be substantially reduced. While CaMKK may also
activate AMPK in response to elevated intracellular Ca
2+
(Hawley
et al., 2005; Hurley et al., 2005; Woods et al., 2005) there is cur-
rently no genetic evidence supporting the importance of this kinase
in regulating skeletal muscle AMPK Thr172 phosphorylation.
1.4. AMPK and exercise
Skeletal muscle is a highly dynamic tissue that can increase the
rate of ATP turnover by >100-fold in response to exercise (Sahlin
et al., 1998). Under such conditions, AMP and ADP levels are
Fig. 1. AMPK regulation of lipid metabolism and mitochondrial content in skeletal muscle; role in exercise and obesity. Skeletal muscle AMPK activity is reduced in obesity
and may be an important therapeutic target for treating insulin resistance. AMPK is activated by low energy status ("AMP:ATP) e.g. exercise, hormones (adiponectin and
leptin, ; and " in obesity, respectively) and pharmacological agents (AICAR, GPA, metformin, resveratrol, TZDs, berberine, A-769662, PT1). AMPK is allosterically activated by
AMP binding, which promotes greater activation through phosphorylation by upstream kinases CaMKII and LKB1. ADP can also regulate AMPK activity by preventing
dephosphorylation by phosphatases. Once activated, AMPK regulates fatty acid uptake (FAT/CD36), esterication (inhibits GPAT), lipolysis (inhibits HSL, ATGL) and oxidation
(phosphorylation/inhibition of ACC2, which reduces m.CoA levels and relieves CPT-I inhibition allowing LCFA to enter mitochondria for beta-oxidation). AMPK has been
implicated in regulation of MCD activity, which converts m.CoA back to acetyl-CoA. CPT-I activity may also be regulated by m.CoA independent mechanisms (e.g. pH,
acetylation/phosphorylation, hormones, carnitine, LCFA, PKA). AMPK has been suggested to regulate mitochondrial biogenesis via phosphorylation of transcription factors
(CREB, HDAC, MEF) including PGC-1a, which is the master regulator of genes involved in oxidative metabolism. AMPK may regulate SIRT1 acetylation (via NAD+) and be
required for SIRT1- dependent deacetylation of PGC-1a. Pharmacological agents activate AMPK either directly or indirectly. AICAR is converted to the AMP analog ZMP,
whereas resveratrol, metformin, TZDs and berberine inhibit oxidative phosphorylation events (OXPHOS), which reduce AMP:ATP. AICAR can have non-specic effects as ZMP
can regulate alternative enzymes in addition to AMPK. A-76694 and PT1 directly activate AMPK by binding to AMPK subunits. Solid line () = AMPK known mechanism;
dotted lines () = proposed mechanism.
H.M. ONeill et al. / Molecular and Cellular Endocrinology 366 (2013) 135151 137
rapidly increased in an intensity-dependent manner and ATP levels
decline only slightly. Given the sensitivity of AMPK to changes in
nucleotides it is not surprising that AMPK is rapidly activated in
response to muscle contractions (electrical stimulation) or during
exercise (cycling exercise in humans and treadmill running in
mice) (Winder and Hardie, 1996; Fujii et al., 2000; Wojtaszewski
et al., 2000; Chen et al., 2003). The activation of skeletal muscle
AMPK a1 and a2 is dependent on exercise intensity, with a2 AMPK
activity increasing at moderate workloads starting at 40% of VO
2
Max and increasing progressively with higher intensity exercise.
In contrast, AMPK a1 activity only appears to be increased during
high intensity tetanic muscle contractions equivalent to >100% VO
2
Max (Chen and Hsieh, 2000; Fujii et al., 2000; Hayashi et al., 2000).
The activation of AMPK in skeletal muscle by AICAR promotes
glucose uptake and fatty acid oxidation in skeletal muscle, and this
has led to the suggestion that AMPK may be the primary mecha-
nism mediating the metabolic adaptations to exercise (Sabina
et al., 1985; Sullivan et al., 1994; Merrill et al., 1997). Since muscle
contractions bring about similar metabolic changes in skeletal
muscle to AICAR (increased AMPK activity, fatty acid metabolism
and glucose uptake), it is commonly believed that AMPK may
mediate some of the effects of exercise on metabolism. In agree-
ment with this idea, we have recently shown that skeletal muscle
AMPK b subunits are critical for controlling exercise tolerance and
glucose uptake during contractions (ONeill et al., 2011). The focus
of the current review is to discuss the role of AMPK in regulating
fatty acid metabolism and mitochondrial biogenesis, ndings
which are summarized in Fig. 1.
2. AMPK and fatty acid uptake
Increased rates of long-chain fatty acid (LCFA) uptake have been
observed in skeletal muscle of obese individuals (Bonen et al.,
2004b), as well obese (Luiken et al., 2001; Coort et al., 2004; Han
et al., 2007; Holloway et al., 2009a) and diabetic (Smith et al.,
2007; Bonen et al., 2009) Zucker rats. This provides a plausible
mechanism, in addition to the elevated levels of circulating plasma
free fatty acids (Boden, 2003), to account for the intramuscular
lipotoxic environment implicated in peripheral muscle insulin
resistance. Three types of transport proteins have been identied:
(1) a 40 kDa peripheral, FABP
PM
located on the outer leaet of the
plasma membrane (Stremmel et al., 1985; Schwieterman et al.,
1988; Isola et al., 1995); (2) a family of 63 kDa fatty acid trans-
port proteins (FATP1-6) that have at least six transmembrane
domains (Schaffer and Lodish, 1994; Hirsch et al., 1998; Gimeno
et al., 2003); and (3) a highly glycosolated 88 kDa FAT/CD36 that
has at least two transmembrane domains (Abumrad et al., 1993).
While research has shown a role for FATP1 and four in LCFA trans-
port (Kim et al., 2004b; DiRusso et al., 2005), less is known about
the regulation of the FATP family in response to physiological stim-
uli, obesity and insulin resistance.
Plasma membrane LCFA transport proteins appear to have
different efcacies in transporting LCFAs across the plasma mem-
brane, with FAT/CD36 displaying the greatest positive effect
(Nickerson et al., 2009). In addition, a number of studies utilizing
acute muscle contraction (Bonen et al., 2000), chronic low fre-
quency muscle stimulation (Koonen et al., 2004), and denervation
(Koonen et al., 2004) models suggest that FAT/CD36 moves be-
tween an intracellular depot and the plasma membrane, thereby
regulating LCFA import into muscle cells (reviewed in Bonen et
al. (2004a) and Holloway et al. (2008)). Collectively, these data
have implicated FAT/CD36 as a primary regulator of skeletal mus-
cle LCFA transport; however, FABPpm (Chabowski et al., 2005; Han
et al., 2007), FATP1 and 4 (Jain et al., 2009) also contribute. The
functional impact of the co-expression of these LCFA transporters
in skeletal muscle is unclear. However, speculations include the
belief that selected transporters channel fatty acids preferentially
into different metabolic fates or pools (e.g. oxidation or esterica-
tion). For example, in rats the overexpression of FATP1 in skeletal
muscle appears to upregulate lipid transport into the cell, and tar-
get these for oxidation (Holloway et al., 2011), while in human
myotubes over-expression of FAT/CD36 increases fatty acid uptake,
and targets these additional lipids for deposition, similar to that
seen in obese muscle (Garcia-Martinez et al., 2005).
Unlike the other fatty acid transporters, FAT/CD36 appears to
contribute to the development of insulin resistance, as in various
models of insulin resistance there is an increase in sarcolemmal
FAT/CD36, which occurs despite unaltered total cellular FAT/
CD36 protein expression (Luiken et al., 2001; Bonen et al., 2004b;
Coort et al., 2004; Han et al., 2007; Holloway et al., 2009a), suggest-
ing the induction of a permanent subcellular redistribution of FAT/
CD36 to the plasma membrane. In addition, myotubes established
from obese individuals and cultured in control media retain ele-
vated sarcolemmal FAT/CD36, suggesting an intrinsic factor within
the muscle contributes to the relocation of FAT/CD36 seen with
obesity (Aguer et al., 2010); however, the mechanism for this
remains unknown.
While the exact signalling mechanisms inducing FAT/CD36
translocation to the plasma membrane in muscle remain elusive,
AICAR can induce the translocation of all LCFA transport proteins
to the plasma membrane; therefore, AMPK may be involved
(Luiken et al., 2003; Chabowski et al., 2005). Other signalling cas-
cades have also been implicated, as inhibition of extra cellular
signalling receptor kinase (ERK1/2) can prevent contraction-in-
duced FAT/CD36 translocation (Turcotte et al., 2005) and activating
protein kinase C in cardiac myocytes induces FAT/CD36 transloca-
tion, suggesting a possible role for Ca
2+
(Luiken et al., 2004). This
has been further strengthened by recent work showing that fatty
acid transport is increased in response to caffeine, which increases
cytosolic Ca
2+
levels, a response attenuated with inhibitors for
CaMKII and CaMKK signalling (Abbott et al., 2009). Concomitantly,
inhibition also prevented caffeine-induced translocation of FAT/
CD36, but not FABPpm, to the plasma membrane. These data sug-
gest that Ca
2+
induces that translocation of FAT/CD36 to the plasma
membrane, increasing rates of LCFA transport into muscle. As dis-
cussed above, CaMKK activates AMPK; therefore, this may suggest
that while AMPK can regulate the translocation of fatty acid trans-
port proteins, CaMKK is required. In support of this, studies in
AMPKa2 KD have demonstrated that AMPK-independent path-
ways are important for regulating fatty acid uptake during contrac-
tions (Jeppesen et al., 2011). Similarly, AMPK is not required for
fatty acid uptake during low-intensity muscle contractions that
do not activate muscle AMPK (Raney et al., 2005). Therefore, whilst
AMPK may be important for regulation of fatty acid uptake in
response to pharmacological agents, AMPK-independent pathways
are required for regulating this process during exercise.
3. Fatty acid handling (esterication and lipolysis)
Esterication (TAG synthesis) and lipolysis (breakdown of
TAGs) are important processes involved in regulation of lipid levels
in skeletal muscle, and disturbances in the balance between the
two processes may contribute to insulin resistance due to a dispro-
portionate increase in reactive lipid species such as DAG.
3.1. GPAT
Glycerol-3-phosphate acyltransferase (GPAT) and diacylglyce-
ride acyltransferase (DGAT) catalyse the rst and nal rate-limiting
steps in TAG esterication and are commonly considered the most
138 H.M. ONeill et al. / Molecular and Cellular Endocrinology 366 (2013) 135151
important enzymes controlling the esterication pathway. It was
previously thought that GPAT existed as 2 isoforms, an outer mito-
chondrial (mtGPAT) and endoplasmic reticulum (microsomalG-
PAT) form, each being encoded by separate genes; however,
recent evidence suggests that at least 4 isoforms exist (mtGPAT1
and 2 and microsomalGPAT3 and 4) (Gimeno and Cao, 2008). mtG-
PAT contributes to 50% and 90% of total activity in liver (Nim-
mo, 1979) and skeletal muscle, respectively, whereas microsomal
GPAT represents the major isoform found in brown and white adi-
pose, contributing 85% of total activity (Gimeno and Cao, 2008).
Microsomal and mitochondrial GPAT can have different roles in
regulating lipid metabolism and have been reviewed previously
(Gimeno and Cao, 2008). In obese rodents, hepatic microsomal
and mtGPAT are elevated, and GPAT 1 and 4 null mice have re-
duced body weight, adiposity, hepatic and cardiac TAGs and in-
creased fatty acid oxidation (Hammond et al., 2002). In 3T3-Li
adipocytes, GPAT1 and 3 are increased during differentiation
(Hajra et al., 2000; Shan et al., 2010), which is a process that allows
increased lipid storage when maximum capacity for TAG accumu-
lation is reached. Deletion of both GPAT1 and 2 from adipocytes
abolishes TAG synthesis and lipid droplet formation (Harris et al.,
2011). In skeletal muscle from obese subjects, neither GPAT nor
DGAT activity nor expression is altered, which suggests that the
esterication pathway is not a major contributing factor to defects
in fatty acid metabolism (Li et al., 2011).
Pharmacological activation of AMPK with AICAR reduces GPAT
activity in multiple tissues in vivo (adipose, liver, skeletal muscle)
and in vitro (hepatocytes, C2C12 myotubes, 3T3L1 adipocytes)
(Park et al., 2002). Leptin also lowers GPAT activity, and genetic
deletion of GPAT1 in leptin-decient ob/ob mice reduces hepatic
lipids (Wendel et al., 2010). The activation of AMPK with fasting
or endurance exercise, also reduces GPAT1 activity in liver and adi-
pose (Ruderman et al., 2003; Assi et al., 2005), but not skeletal
muscle (Park et al., 2002; Ruderman et al., 2003; Watt et al.,
2004a). Thus, while there is substantial evidence suggesting that
reductions in GPAT activity mediated via AMPK are associated with
reduced TAG and DAG esterication in the liver, the importance of
GPAT for regulating fatty acid metabolism in skeletal muscle ap-
pears to be minimal.
3.2. Disturbances in lipid handling in obesity: HSL, ATGL and AMPK
Lipolysis is the process whereby TAGs are hydrolysed into free
fatty acids so they can be used for energy via b-oxidation. In obese
humans and rodents, it has been known for decades that basal
rates of adipose tissue lipolysis are enhanced, and this is in part
due an inability of insulin to suppress lipolysis. Lipolysis in adipose
tissue involves adipose triglyceride lipase (ATGL), hormone sensi-
tive lipase (HSL) and monoacylglycerol lipase (MAGL). Activation
of lipolysis is mediated via cAMP signalling induced by b-adrener-
gic stimulation (catecholamines) and subsequent activation of pro-
tein kinase A (PKA), and in skeletal muscle a similar pathway
exists. In obesity, whole-body catecholamine-induced lipolysis is
blunted, and this could be due to reduced expression of b2-adren-
ergic receptors (Reynisdottir et al., 1994), increased antilipolytic
properties of a2-adrenergic receptors (Mauriege et al., 1991) and
reduced HSL expression (Large et al., 1999; Langin et al., 2005) in
adipocytes; however, this is not observed in all studies (Langin
et al., 2005). Few studies have investigated the physiological role
of ATGL and HSL in regulating skeletal muscle lipolysis and
whether expression and activity of these proteins are altered in
obesity and insulin resistance; therefore, further studies are re-
quired to fully understand the contribution of each in regulating
this process.
3.3. Desnutrin/ATGL
ATGL expression appears to be reduced in obese and insulin-
resistant skeletal muscle, and correlates with increased TAG levels
in multiple tissues (Jocken et al., 2007); however, further studies
are required to conrm this. ATGL null mice have increased body
weight and adiposity, impaired exercise capacity and die prema-
turely due to cardiac failure (Haemmerle et al., 2006, 2011). Inter-
estingly, ATGL null mice have improved insulin sensitivity
(Haemmerle et al., 2006) and are protected against high-fat diet in-
duced insulin resistance despite increased TAG accumulation (Hoy
et al., 2011). This protection appears to be mediated via enhanced
hepatic insulin sensitivity as illustrated by greater glucose disposal
in liver, heart and adipose, which may be due to a tendency for
lower DAGs and ceramides (Hoy et al., 2011). There is no affect
of ATGL deciency on skeletal muscle insulin sensitivity (Hoy
et al., 2011). In humans, protein but not mRNA ATGL expression
correlates well with insulin sensitivity in adipose but not skeletal
muscle; rather, skeletal muscle ATGL correlates well with fatty acid
oxidation proteins (Yao-Borengasser et al., 2011).
Although it is not clear whether ATGL can also be phosphory-
lated by PKA, ATGL expression is upregulated by exercise training
(Alsted et al., 2009; Yao-Borengasser et al., 2011). AMPK activates
ATGL in both Caenorhabditis elegans (Narbonne and Roy, 2009)
and mammals (Ahmadian et al., 2011) through phosphorylation
of Ser308 and 406, respectively. In mice, AMPK is required for the
induction of PPARc target genes and brown adipose tissue (Vila-
Bedmar and Fernandez-Veledo, 2011). However, the physiological
importance of AMPK phosphorylation of ATGL in skeletal muscle
appears to be minimal as muscle fromATGL null mice do not fatigue
prematurely when contracted ex vivo (Huijsman et al., 2009). This is
likely a result of HSL having promiscuous lipolytic activity, inu-
encing the lipolysis of both TAG and DAG.
3.4. HSL
In obese adipose tissue and skeletal muscle, HSL activity is
reduced under basal resting conditions and in response to cate-
cholamines and exercise (Blaak et al., 1994, 2004; Blaak, 2004;
Watt et al., 2005; Jocken et al., 2007; Ryden et al., 2007; Jocken
et al., 2008a; Jocken et al., 2008b; Gaidhu et al., 2010). HSL expres-
sion in skeletal muscle is reduced in some (Large et al., 1999; Watt
et al., 2005, 2008; Jocken et al., 2008b) but not all obesity studies
(Jocken et al., 2008; Thrush et al., 2009; Gaidhu et al., 2010). Differ-
ences between studies may exist from sex differences, whereby
women have higher TAG levels and HSL expression but lower
HSL activity at rest and during exercise despite greater TAG utilisa-
tion during exercise compared to males (Roepstorff et al., 2006).
HSL null mice have reduced b-adrenergic lipolysis and are pro-
tected from high-fat diet induced (Harada et al., 2003) and genetic
(ob/ob) (Sekiya et al., 2004) insulin resistance, independent of
changes in body weight and adiposity, which increase similarly
compared to wildtype (Osuga et al., 2000; Wang et al., 2001;
Harada et al., 2003; Zimmermann et al., 2003; Sekiya et al.,
2004). Interestingly, despite lower TAG levels, HSL null mice accu-
mulate more DAG in adipose and skeletal muscle (Haemmerle
et al., 2002).
HSL can be phosphorylated on at least six sites (Ser 563, 566,
565, 600 and 660). Phosphorylation at Ser563, 659 and 660 are
considered the major PKA sites, which promote translocation to
lipid droplets and enhance lipolysis (Anthonsen et al., 1998). In hu-
man skeletal muscle, it has been suggested that the Ser563 site
may not be important for regulating HSL activity given phosphor-
ylation at this site is not increased during b-adrenergic stimulation
or exercise (Roepstorff et al., 2004; Roepstorff et al., 2006; Jocken
et al., 2008b). Rather, Ser659 appears to be more essential for
H.M. ONeill et al. / Molecular and Cellular Endocrinology 366 (2013) 135151 139
regulation of HSL activity during exercise (Roepstorff et al., 2006),
and importantly is not reduced in obese subjects (Jocken et al.,
2008b). AMPK directly phosphorylates HSL at Ser 565 and this
inhibits HSL activity (Garton et al., 1989; Roepstorff et al., 2004).
Pharmacological AMPK activators AICAR (Corton et al., 1994;
Sullivan et al., 1994; Watt et al., 2004b), metformin and TZDs
(Ren et al., 2006; Bourron et al., 2010; Grisouard et al., 2011) and
hormones (adiponectin (Qiao et al., 2011; Wedellova et al.,
2011)) reduce b-adrenergic stimulated lipolysis whereas IL-6 en-
hances it (Mattacks and Pond, 1999; van Hall et al., 2003; Watt
et al., 2005). During exercise, AMPK appears to play a more domi-
nant role in regulating HSL activity at later stages of endurance
exercise (120 min, 6065% VO
2
Max) and under conditions of low
glycogen availability (and subsequently higher AMPK activity),
where HSL Ser565 phosphorylation in increased (Roepstorff et al.,
2004; Watt et al., 2004b). Interestingly, AMPK kinase dead mice
have suppressed HSL Ser565 phosphorylation in response to
AICAR, but normal increases in response to muscle contractions
(Dzamko et al., 2008).
4. AMPK and fatty acid oxidation
Whether or not skeletal muscle fatty acid oxidation is altered in
human obesity is of continuing debate with many studies demon-
strating increased (Steinberg et al., 2002; Bucci et al., 2011), no
change (Bonen et al., 2004b) or decreased (Kelley and Simoneau,
1994; Simoneau et al., 1999; Simoneau et al., 1995; Jong-Yeon
et al., 2002; Hulver et al., 2003; Gaster et al., 2004) rates of skeletal
muscle fatty acid oxidation in whole muscle. Interestingly, reduc-
tions in fatty acid oxidation may be due to reduced mitochondrial
content rather than impaired mitochondrial function/oxidation
(Boushel et al., 2007; Holloway et al., 2007). However, many stud-
ies over the past 15 years have provided important evidence that
activation of skeletal muscle AMPK by AICAR or hormones and
cytokines is associated with increases in fatty acid oxidation (for
review see Dzamko and Steinberg (2009)). Similarly, factors that
suppress AMPK activity in obesity such as TNFa (Steinberg et al.,
2006b) and resistin (Palanivel and Sweeney, 2005) are associated
with reductions in skeletal muscle fatty acid oxidation. Genetic
studies also support an important role for activation of skeletal
muscle AMPK as the over-expression of AMPK c3 increases fatty
acid oxidation and protects against high-fat diet induced accumu-
lation of TAGs in skeletal muscle (Barnes et al., 2004). Collectively,
these studies illustrate an important role for skeletal muscle AMPK
in regulating fatty acid oxidation under resting conditions.
AMPK is believed to regulate fatty acid oxidation in skeletal
muscle through phosphorylation of acetyl-CoA carboxylase (ACC)
2 on S221 (212 in mouse). ACC exists in 2 isoforms, ACC1 and
ACC2, each encoded by separate genes and differing in their tissue
distribution. ACC1 is ubiquitously expressed in many tissues, but is
highly expressed in lipogenic tissues such as liver and adipose
where it is believed to be present in the cytosol and regulate fatty
acid synthesis. ACC2 is the predominant isoform found in skeletal
muscle and is believed to be located in close proximity to the outer
mitochondrial membrane near carnitine palmitoyl transferase
(CPT)-I; the rate limiting enzyme that controls the transfer of cyto-
solic fatty acids into the mitochondria for beta oxidation. Whilst
both enzymes catalyse the carboxylation of acetyl-CoA to malo-
nyl-CoA, a critical role for ACC1 has been shown, as genetic
deletion of ACC1 frommice is embryonic lethal at E8.5 (Abu-Elheiga
et al., 2005). In regards to ACC2 and skeletal muscle, several studies
have shown that deletion of ACC2 results in reduced skeletal mus-
cle malonyl-CoA levels and enhanced fatty acid oxidation (Abu-
Elheiga et al., 2001, 2003; Oh et al., 2005; Choi et al., 2007; Hoehn
et al., 2010; Olson et al., 2010), similar to mice over-expressing
CPT-I (Bruce et al., 2009). Of these, the initial studies show protec-
tion from high-fat diet insulin resistance in mice lacking ACC2,
whereas later studies highlight a mere shift in substrate utilisation
(from oxidizing more carbohydrate and less fat) rather than an
increase in whole-body energy expenditure and weight loss, and
subsequent protection from diet-induced insulin resistance.
4.1. Regulation of acetyl-CoA carboxylase activity
ACC activity is regulated by both allosteric and covalent mech-
anisms. While ACC is allosterically activated by citrate, Munday
et al. showed that AMPK phosphorylation of ACC1 at Ser79 inhib-
ited ACC activity (Munday et al., 1988a). Later studies using AICAR
conrmed that Ser79 (equivalent Ser221 site on ACC2) was the
physiologically relevant phosphorylation site for ACC inhibition
by AMPK (Munday et al., 1988b). Studies in rats have demon-
strated that pharmacological activation of AMPK using AICAR, in-
creases fatty acid oxidation and ACC S79/S221 phosphorylation
while reducing malonyl-CoA (Merrill et al., 1997; Kaushik et al.,
2001; Smith et al., 2005). However, we have recently shown that
in mice over-expressing a muscle specic kinase dead form of a2
AMPK (AMPK KD) or in mice lacking AMPK b2, despite dramatic
reductions in ACC2 Ser221 phosphorylation skeletal muscle fatty
acid oxidation is not reduced and muscle TAG is not elevated
(Dzamko et al., 2008; Beck Jorgensen et al., 2009; Steinberg et al.,
2010). Similarly, LKB1-MKO mice also have normal basal levels of
fatty acid oxidation despite having undetectable AMPKa2 activity
and ACC phosphorylation (Thomson et al., 2007a,b).
In regards to exercise, there appears to be a mismatch between
AMPK activation, ACC2 Ser221 phosphorylation and fatty acid oxi-
dation. Fatty acid oxidation as a function of exercise intensity has
been reviewed elsewhere (Jeukendrup, 2002). During low-inten-
sity exercise, fat oxidation increases up until 65% (Jeukendrup,
2002); however, AMPK and ACC phosphorylation are only partially
increased at this workload. In contrast, during high-intensity exer-
cise where carbohydrates are preferentially utilized, AMPK is more
potently activated. Similarly, malonyl-CoA levels are not altered or
only modestly reduced despite substantial increases in fatty acid
oxidation in humans following low intensity exercise (Odland
et al., 1996; Roepstorff et al., 2005). Similarly, recent studies in
LKB1-MKO and AMPK KD mice also indicate a mismatch between
muscle contraction/exercise and rates of fatty acid oxidation
because despite substantial reductions in AMPK activity, ACC phos-
phorylation, malonyl-CoA levels and rates of fatty acid oxidation
are not different between mutant mice and wildtype littermates
(Thomson et al., 2007a; Dzamko et al., 2008). Taken altogether
these studies suggest three distinct possibilities (1) AMPK indepen-
dent kinases are responsible for phosphorylating Ser 221 of ACC in
response to AICAR and muscle contractions; (2) residual AMPK
activity in both the LKB1 and AMPK KD models are sufcient to
account for the ACC2 phosphorylation observed; and (3) ACC2
Ser221 phosphorylation is not critical for regulating malonyl-CoA
levels and fatty acid oxidation. Further studies in ACC Ser79/
221Ala knock-in mice will determine the contribution of AMPK
phosphorylation of ACC in regulating fatty acid oxidation at rest
and during exercise.
4.2. Malonyl-CoA decarboxylase
Malonyl-CoA levels are also regulated by the enzyme malonyl-
CoA decarboxylase (MCD), which converts malonyl-CoA back to
acetyl-CoA. Early studies in rat heart showed a role for MCD in reg-
ulating fatty acid oxidation (Dyck et al., 1998). Since then, it has
been shown that genetic knockdown of MCD results in elevated
malonyl-CoA levels and reduced rates of fatty acid oxidation in
both human and rodent skeletal muscle (Bouzakri et al., 2008;
140 H.M. ONeill et al. / Molecular and Cellular Endocrinology 366 (2013) 135151
Koves et al., 2008). MCD activity is enhanced by AICAR and con-
traction (Saha et al., 2000; Park et al., 2002; Kuhl et al., 2006) in
skeletal muscle, but it is not known whether AMPK plays a direct
role in regulating MCD activity. Incubation of MCD immunoprecip-
itates from muscle supernatant with recombinant AMPK increases
MCD activity (Park et al., 2002). However, an alternative study
showed that AMPK does not directly phosphorylate MCD
(Habinowski et al., 2001). Lysine acetylation of MCD is also impor-
tant for regulating enzyme activity (Rainwater and Kolattukudy,
1982) however, the importance of AMPK in regulating MCD acety-
lation has not been established.
5. AMPK-independent regulation of mitochondrial fatty acid
oxidation
Resting malonyl-CoA levels in both rat (Winder et al., 1989; Ma-
clean and Winder, 1995; Chien et al., 2000) and human (Odland
et al., 1996, 1998) skeletal muscles are substantially higher than
the measured IC
50
of CPT-I for malonyl-CoA (McGarry et al.,
1983; Starritt et al., 2000), suggesting CPT-I activity and rates of
fatty acid oxidation should be negligible in vivo. These data suggest
that the regulation of fatty acid oxidation at the level of the mito-
chondria is not exclusively restricted to the content of malonyl-
CoA (Odland et al., 1996; Kim et al., 2002). Indeed, it has previously
been shown that the ability of malonyl-CoA to inhibit CPT-I is
attenuated during prolonged exercise and with physiological
reductions in cytosolic pH (Bezaire et al., 2004; Holloway et al.,
2006). In addition, other groups have provided evidence that
CPT-I is post-translationally regulated, as various kinases alter
either CPT-I/malonly-CoA sensitivity and/or CPT-I activity (Kerner
et al., 2004). The N-terminal domain of CPT-I has been shown to
dictate the sensitivity of malonyl-CoA (Shi et al., 1999, 2000),
and it has been suggested that a chemical link between the N-
and the C-termini is essential in maintaining malonyl-CoA sensitiv-
ity within the liver isoform of CPT-I (Faye et al., 2005). Therefore,
future studies examining phosphorylation sites within this region
of CPT-I might prove insightful. In addition to this non-classical
regulation, the cytoskeletal network has been shown to inhibit
CPT-I activity in the liver (Velasco et al., 1997), an effect attenuated
by cytoskeletal phosphorylation via signalling pathways that are
activated during exercise (Velasco et al., 1997, 1998a,b). This raises
the spectra that these mechanisms exist in skeletal muscle.
Although this remains to be shown, a recent report has provided
evidence that CPT-I and the voltage-dependent anion channel
(VDAC) directly interact (Lee et al., 2011), and VDAC is known to
be regulated by the cytoskeletal network (Rostovtseva et al.,
2008). Therefore, the ability of the cytoskeletal network to regulate
mitochondrial fatty acid oxidation in muscle remains a possibility.
In addition to the classical positioning of CPT-I as required for
lipid transport into mitochondria, several proteins have been iden-
tied that can augment mitochondrial membrane lipid transport.
One example is the plasma membrane transporter FAT/CD36,
which has unexpectedly been found on mitochondrial membranes
(Campbell et al., 2004; Bezaire et al., 2006; Schenk and Horowitz,
2006; King et al., 2007; Aoi et al., 2008; Holloway et al., 2009b;
Sebastian et al., 2009; Smith et al., 2011), where it appears to reg-
ulate fatty acid oxidation in a CPT-I independent manner (Smith
et al., 2011). Other mechanisms have also been shown to inuence
mitochondrial fatty acid oxidation independent of CPT-I, including
the abundance of the FATP family on mitochondrial membranes
(Sebastian et al., 2009), the acetylation status of mitochondrial pro-
teins (Hirschey et al., 2010; Huang et al., 2010), complex I glutath-
ionylation (Hurd et al., 2008), and/or phosphorylation of electron
transport chain complexes (Acin-Perez et al., 2009). Clearly the
regulation of mitochondrial fatty acid metabolism extends beyond
the cellular content of malonyl-CoA, and additional work is
required to elucidate the complexities of this system.
6. AMPK and exercise-induced metabolic adaptations
It is well established that endurance exercise training is a major
stimulus for increasing substrate utilization and mitochondrial
content (Holloszy, 1967; Gollnick et al., 1973; Holloszy and Booth,
1976), and improving insulin sensitivity. Therefore, exercise is
important for people with obesity and type 2 diabetes given mito-
chondrial content and metabolic inexibility often accompanies
these diseases. AMPK has been proposed as a key molecule in elic-
iting metabolic adaptations to exercise. Initial studies using phar-
macological activators of AMPK have shown potent effects of
mitochondrial protein expression and enzyme activities. In
rodents, b-guanadinopropionic acid (b-GPA) feeding, which chron-
ically activates AMPK by depleting phosphocreatine stores, results
in increased mitochondrial content, enzyme activities and gene
expression; effects not observed in AMPK decient mice, and found
to be mediated through enhanced peroxisome proliferator acti-
vated receptor c co-activator-1 a (PGC-1a) binding to nuclear
respiratory factor (NRF)-1 (Zong et al., 2002). AICAR also increases
PGC-1a and expression and activity of a number of mitochondrial
oxidative proteins (citrate synthase, cytochrome c oxidase and
uncoupling protein 3) in rodent muscle and L6 myotubes but not
in AMPK a2KO or transgenic mice (Winder et al., 2000; Stoppani
et al., 2002; Suwa et al., 2003; Jorgensen et al., 2005, 2007; Reznick
and Shulman, 2006). Chronic pharmacological activation of AMPK
has been shown to induce metabolic adaptations similar to exer-
cise-training; however, LKB1-MKO and AMPK decient mouse
models where a single subunit has been mutated (AMPK a2KO,
a2iTg and c3KO) show normal increases in mitochondrial markers
in response to an acute bout of treadmill exercise and chronic
voluntary wheel running (Jorgensen et al., 2005, 2007; Rockl
et al., 2007; Thomson et al., 2007b). These ndings suggest that
AMPK-independent pathways are also involved or alternatively
residual AMPK activity in these models may be sufcient to allow
for normal mitochondrial adaptations in response to exercise.
6.1. Mitochondrial capacity and exercise tolerance
Mitochondrial oxidative capacity is important for endurance
exercise performance, and has been highlighted by recent ndings
in muscle specic PGC-1a/b null mice (Zechner et al., 2010). In
untrained mice, AICAR is a potent stimulus for improving exercise
capacity (Narkar et al., 2008) and recently, Leick and colleagues
have shown that PGC-1a is required for AICAR-induced expression
of mitochondrial proteins in mouse skeletal muscle (Leick et al.,
2010). AMPK decient mouse models where a single subunit has
been mutated have also shown AMPK is important for exercise
tolerance (Fujii et al., 2007; Lee-Young et al., 2009b; Maarbjerg
et al., 2009; Steinberg et al., 2010); however, under basal conditions
mitochondrial content is normal or modestly reduced (Zong et al.,
2002; Jorgensen et al., 2005, 2007; Rockl et al., 2007; Garcia-Roves
et al., 2008; Beck Jorgensen et al., 2009; Lee-Young et al., 2009b;
Steinberg et al., 2010). In contrast, mice lacking LKB1 or both AMPK
b subunits in skeletal muscle have drastically impaired exercise
tolerance as well as reduced mitochondrial content, protein expres-
sion and enzyme activities (Thomson et al., 2007b; ONeill et al.,
2011). It is difcult to assess the contribution of reduced mitochon-
drial content to impaired exercise performance in these models
given a number of pharmacological and genetic strategies have pro-
vided evidence for a functional role of AMPK in angiogenesis
(Zwetsloot et al., 2008; Stahmann et al., 2010), blood ow
(Lee-Young et al., 2009b), ber-type specication (Rockl et al.,
H.M. ONeill et al. / Molecular and Cellular Endocrinology 366 (2013) 135151 141
2007; Lee-Young et al., 2009a) and glucose uptake (Mu et al., 2001;
Zong et al., 2002; Jensen et al., 2007a,b; Jorgensen et al., 2007; Lefort
et al., 2008; ONeill et al., 2011), which may also contribute to
impaired exercise performance. However, a recent report has found
that muscle oxidative capacity is the best predictor of exercise
performance in humans (Jacobs et al., 2011).
6.2. Fiber-type transformation
Aerobic exercise training also induces a transformation in
myosin heavy chain isoform expression, inducing a fast to slow
transition associated with an increase in oxidative potential. Mito-
chondrial biogenesis and ber-type transitions can occur by dis-
tinct mechanisms; however, both are important for enhancing
skeletal muscle mitochondrial oxidative capacity. Under basal
conditions, forced expression of PGC-1a promotes glycolytic-to-
oxidative bre-type transformation and mitochondrial biogenesis
in skeletal muscle (Lin et al., 2002) with enhanced endurance exer-
cise capacity (Calvo et al., 2008). The role of AMPK in mediating
ber-type shifts is equivocal. AMPK b1b2M-KO, a2iTg, a2KO and
c3KO mice have normal ber distribution (Jorgensen et al., 2004;
Rockl et al., 2007), whereas AMPK c1 gain-of-function mice have
increased type IIa/x bers and higher basal mitochondrial enzyme
activities (Rockl et al., 2007). However, AMPK c3 gain-of-function
(R225Q) mice also display increased mitochondrial biogenesis
but not a ber-type shift (Garcia-Roves et al., 2008).
In response to exercise training, AMPK but not PGC-1a is impor-
tant for IIb to IIa/x (glycolytic to oxidative) ber-type transforma-
tion in response in response to endurance exercise training (Rockl
et al., 2007; Geng et al., 2010). The clinical signicance of this is
that humans with insulin resistance or type 2 diabetes tend to have
more glycolytic type IIx (glycolytic) skeletal muscle bers than
healthy individuals, and a number of studies have shown that
ber-type distribution correlates with insulin resistance (reviewed
in Zierath and Hawley (2004)). Therefore, AMPK may be important
for the insulin sensitizing effects of endurance exercise training. In
summary, AMPK signalling appears to be important for skeletal
muscle mitochondrial content; therefore, understanding the
pathways involved in regulating mitochondrial biogenesis are
important for the develop of new therapeutics that target obes-
ity-induced insulin resistance.
7. AMPK transcriptional regulation of mitochondrial oxidative
genes
Early studies in puried rat liver showing AMPK a2 localises to
the nucleus suggested that AMPK may play a direct role in regulat-
ing gene transcription (Salt et al., 1998). Since then, additional
studies have shown similar ndings in human skeletal muscle in
response to exercise (McGee et al., 2003) and low glycogen avail-
ability (Steinberg et al., 2006c). Recently, Ju et al. (2011) have
shown that skeletal muscle AMPK a1 can also translocate to the
nucleas to potentiate neurogeneration in Huntingtons disease.
AMPK a2 can phosphorylate a number of transcriptional substrates
to enhance mitochondrial transcription in skeletal muscle and
these include PGC-1a, p53, cAMP response element binding pro-
tein (CREB), class IIA histone diacetylases (HDAC), forkhead box
proteins (FOXO) and sirtuin deacetylase (SIRT) 1 (reviewed in
McGee and Hargreaves (2010)).
7.1. p38 and p53
AMPK has also been proposed to activate the p38 MAPK path-
way, which can regulate PGC-1a expression and mitochondrial
biogenesis at rest and following exercise training (Puigserver
et al., 2001; Akimoto et al., 2005; Wright et al., 2007; Pogozelski
et al., 2009). However, evidence from AMPK a2iTg mice show that
p38 and AMPK are distinct pathways, additionally; Ca
2+
is required
for p38 MAPK induction of mitochondrial biogenesis (Wright et al.,
2007). p53 is also important for mitochondrial biogenesis
(Ho et al., 2007). Treatment with AICAR (Imamura et al., 2001)
and constitutively active AMPK (Jones et al., 2005) induces p53
phosphorylation at Ser15. Exercise and muscle contraction have
also been shown to increase phosphorylation at this residue and
may be responsible for metabolic adaptations in response to exer-
cise training. p53 KO mice have reduced mitochondrial content and
PGC-1a expression in skeletal muscle; however, exercise-induced
adaptations are intact in these mice, suggesting alternative path-
ways (p38 or AMPK) are also important (Saleem et al., 2009).
7.2. PGC-1a
PGC-1a is considered a master transcriptional regulator of genes
involved in oxidative metabolism. In addition to autoregulation
of PGC-1a expression through interaction with myocyte
enhancer factor (MEF) 2 family of transcription factors, PGC-1a
activity is also regulated by posttranslational modications (PTM)
through phosphorylation (Jager et al., 2007), deacetylation (Canto
and Auwerx, 2009) and sumoylation (Rytinki and Palvimo, 2008).
Post-translational activationof PGC-1aleads to increased transcrip-
tionof target genes as well as PGC-1aitself. AMPKcandirectly phos-
phorylate PGC-1a on Thr1722 and Ser538, and mutation of these
sites to Ala abolishes AICAR-induced increase in PGC-1a phosphor-
ylation in skeletal muscle (Jager et al., 2007). Recent evidence in
human skeletal muscle shows AICAR can induce the expression of
both isoforms of PGC-1 (a and b) similar to exercise via an alterna-
tive splice variant (Norrbomet al., 2011). Similar ndings have been
observedinrodents andincombinationwithcatecholamines (Miura
et al., 2007; Chinsomboon et al., 2009; Tadaishi et al., 2011); further
supporting a role for AMPKin the regulation of PGC-1. As previously
mentioned, AMPK can also phosphorylate other transcription
factors such as CREB (Thomson et al., 2008) and HDAC (McGee
et al., 2008), which have also been implicated in PGC-1a-mediated
transcription; therefore, dissecting the exact mechanism by which
AMPK regulates PGC-1a is difcult. Interestingly, both PGC-1a KO
(Adhihetty et al., 2009) and AMPK b1b2M-KO (ONeill et al., 2011)
mice have reduced mitochondrial content, despite either normal
AMPKactivity (Leick et al., 2009, 2010) or increased PGC-1a expres-
sion (ONeill et al., 2011), respectively. In contrast, PGC-1a null mice
have normal increases in mitochondrial adaptations to exercise
training (Leick et al., 2008; Adhihetty et al., 2009) similar to AMPK
a2 null mice (Jorgensen et al., 2005, 2007), suggesting that residual
PGC-1a and/or AMPK activity is sufcient for normal exercise-in-
duced increases in mitochondrial capacity. Findings of a compensa-
toryincrease inPGC-1aexpressioninAMPKb1b2M-KOmaysuggest
that post translational regulation of PGC-1a by AMPK may also be
important for the regulation of mitochondrial content under basal
conditions; however, whether this is the case during exercise
remains to be determined. Further studies in AMPK b1b2M-KO
and PGC-1a/b null mice will reveal this.
7.3. AMPK and SIRT1
Recently, the NAD+-dependent protein deacetylase SIRT1 has
been proposed as a regulator of exercise-induced mitochondrial
biogenesis in skeletal muscle due to its ability to increase PGC-
1a activity through deacetylation. Increased PGC-1a deacetylation
occurs in response to pharmacological activation (resveratrol and
small molecule activators) (Howitz et al., 2003; Lagouge et al.,
2006; Timmers et al., 2011) and energy deprivation (fasting, calorie
restriction and exercise) (Rodgers et al., 2005; Chen et al., 2008;
142 H.M. ONeill et al. / Molecular and Cellular Endocrinology 366 (2013) 135151
Canto et al., 2009; Canto and Auwerx, 2010), which elevate
NAD + levels (see reviews Alcain and Villalba (2009) and Canto
and Auwerx (2011)). SIRT1 has numerous protective metabolic ef-
fects against those associated with a high-fat diet, and this has
been highlighted in SIRT1 decient and gain of function mouse
models (Chen et al., 2008; Cohen et al., 2009; Purushotham et al.,
2009; Ramadori et al., 2010; Schug et al., 2010; Xu et al., 2010)
(see review Herranz and Serrano (2010)). However, recent reports
in muscle-specic SIRT1 KO mice reveal a non-essential role for
SIRT1 in regulating PGC-1a deacetylation and mitochondrial bio-
genesis following an acute bout of treadmill exercise and chronic
voluntary wheel running (Philp et al., 2011), suggesting that
PGC-1a deacetylation occurs via SIRT-1 independent pathways.
Indirect pharmacological activation of SIRT1 by resveratrol, a com-
ponent found in the skin of grapes, has shown to improve insulin
sensitivity, mitochondrial biogenesis and endurance capacity in
wildtype but not AMPK a-decient mice, suggesting a link be-
tween AMPK and SIRT1 in mediating these effects (Um et al.,
2010). AMPK phosphorylation at Ser177 and Ser538 on PGC-1a is
important for PGC1-a deacetylation by SIRT1 in response to AICAR
(Canto et al., 2009); however, AMPK may also regulate SIRT1 activ-
ity by increasing NAD + levels; thereby inuencing PGC1-a deacet-
ylation through this mechanism also (Canto et al., 2009). Recent
evidence in AMPK c3 KO mice have shown that exercise-induced
increases in PGC-1a deacetylation are blunted in these mice (Canto
and Auwerx, 2010; Canto et al., 2010). Interestingly, but consistent
with normal mitochondrial biogenesis, these mice do not have re-
duced PGC-1a deacetylation under basal conditions suggesting
that residual AMPK activity may be sufcient. Further studies in
AMPK b1b2M-KO are required to fully elucidate the functional role
of AMPK and SIRT1 in regulating PGC-1a activity in response to
exercise.
Emerging evidence suggests that regulation of PGC1-a by its
associated acetyltransferase may also be important for the regula-
tion of PGC-1a transcriptional activity (Philp et al., 2011). PGC-1a
interacts with a number of acetyl-transferases including steroid
receptor coactivator receptor coactivator (SRC) 1, CREB binding
protein/p300 and the histone deacetylase general control non-
repressible (GCN) 5; however, only GCN5 possess a strong acetylat-
ing effect (Puigserver et al., 1999), which suppresses PGC-1a
mediated gene transcription in many cell lines (Lerin et al., 2006;
Gerhart-Hines et al., 2007). Therefore, it has been proposed that
GCN5 may also have a role in regulating mitochondrial content
and biogenesis as well as fatty acid oxidation in skeletal muscle
in response to energy deprivation (exercise and fasting); however,
little information in the literature currently exists.
In addition, receptor-interacting protein 140 (RIP140) is a tran-
scriptional coregulator highly expressed in adipose tissue and
skeletal muscle (Leonardsson et al., 2004), which has been shown
to bind and repress a wide array of transcription factors, including
directly binding to and antagonize PGC-1a (Hallberg et al. (2008)
and reviewed in Francis et al. (2003)). While little is known about
the regulation of RIP140, GPA feeding reduces RIP140 expression in
triceps but not soleus (Williams et al., 2009), suggesting a possible
link between AMPK regulation of PGC-1a through RIP140. In spite
of this, a recent study showed that reduced RIP140 is not required
for AICAR or exercise induced increases in mitochondrial content
(Frier et al., 2011). Clearly the role of AMPK in regulating both
RIP140 and GCN5 is an important area for future research and
understanding of the regulation of PGC-1a transcriptional activity.
8. AMPK as a potential therapeutic target
AMPK regulation of fatty acid metabolism and mitochondrial
content in response to pharmacological activation as well as
exercise has made AMPK a potential therapeutic target for treat-
ment of obesity and type 2 diabetes. Although initial small trials
found that skeletal muscle AMPK activity was normal with obesity
(Steinberg et al., 2004a) and type 2 diabetes (Hojlund et al., 2004),
a recent large scale genetic twin study found that both obesity and
type 2 diabetes cause reductions in AMPK activity without altering
AMPK expression (Mortensen et al., 2009). AICAR (Bergeron et al.,
2001; Buhl et al., 2001; Iglesias et al., 2002, 2004; Song et al.,
2002; Pold et al., 2005), metformin (Zhou et al., 2001; Hawley
et al., 2002; Musi et al., 2002) and TZDs (Lessard et al.,2006; Ye
et al., 2006) all activate skeletal muscle AMPK and improve insulin
sensitivity. However, it is currently not understood whether skele-
tal muscle AMPK is essential for their insulin sensitizing effects
and/or whether changes in fatty acid metabolismare required. Res-
veratrol, increases mitochondrial capacity and insulin sensitivity in
both obese humans (Timmers et al., 2011) and mice (Baur et al.,
2006), and these effects have been shown to require AMPK (Canto
et al., 2010; Um et al., 2010). Similarly, berberine, a known AMPK
activator that improves insulin sensitivity (Cheng et al., 2006;
Lee et al., 2006; Turner et al., 2008), promotes skeletal muscle
mitochondrial biogenesis though AMPK/SIRT1 activation/deacety-
lation, and reverses effects associated with a high-fat diet in rats
(Gomes et al., 2011). Hawley et al. have recently shown that both
resveratrol and berberine increase AMPK activity due to disrup-
tions in the cellular AMP/ATP ratio (Hawley et al., 2010). Alterna-
tively, AICAR can have non-specic effects and act as a potent
stimulus for other enzymes including stimulation of glycogen
phosphorylase (Young et al., 1996; Longnus et al., 2003) and inhi-
bition of fructose-1,6-bisphosphatase (Vincent et al., 1991) and s-
adenosylhomocysteine hydrolase (Fabianowska-Majewska et al.,
1994), which can also inuence metabolism. Therefore, it is
unclear whether the insulin sensitising effects of the current phar-
macological agents are mediated directly via AMPK.
In contrast, the direct AMPK-specic activators A-769662 and
PT1 appear to have protective effects against diet and genetic
induced obesity, (Cool et al., 2006; Pang et al., 2008; Zhou et al.,
2009). However, since A-769662 only activates AMPK b1-contain-
ing complexes, its effects on skeletal muscle (which consists of
almost entirely b2-containg complexes) is likely of negligible
importance (Scott et al., 2008), and instead, improvements in insu-
lin sensitivity are likely related to the activation of AMPK in liver
(Cool et al., 2006) and macrophages (Galic et al., 2011). In the
future it will be important to see whether direct activators of
AMPK b2 can be developed to increase AMPK activity specically
in skeletal muscle.
9. Conclusion
The importance of AMPK in regulating fatty acid metabolism
and mitochondrial biogenesis has been highlighted in this review.
Given that disturbances in these pathways contribute to insulin
resistance in people with obesity, it is critical to understand the
underlying mechanisms eliciting these effects, in order to develop
new strategies (exercise and pharmacological) that will combat the
growing problem of insulin resistance and type 2 diabetes. AMPK
has been implicated in regulation of fatty acid uptake (CD36, FABP,
FAPT), handling (lipolysis via HSL and esterication via GPAT), and
oxidation (via ACC2/malonyl-CoA), as well as mitochondrial bio-
genesis (via PGC-1a/SIRT1), all of which are either enhanced or
reduced in obesity and lead to accumulation of lipids within skel-
etal muscle that have been associated with impairing insulin sig-
nalling. Whilst much of the evidence on AMPK in regulating
these processes has been provided from studies using pharmaco-
logical agents, few studies have explored the role of AMPK in reg-
ulating these processes during acute and chronic exercise training,
H.M. ONeill et al. / Molecular and Cellular Endocrinology 366 (2013) 135151 143
which is also a potent stimulus for AMPK activation. In addition,
there is a lack of direct genetic evidence supporting a role for AMPK
in regulating these processes during exercise and in obesity.
Because the insulin sensitising effects of exercise are well known,
further investigation into the underlying mechanism are critical
to increase our knowledge about AMPK as a potential therapeutic
target for the treatment of obesity-induced insulin resistance.
Recent evidence showing AMPK-specic activators are protective
against and can rescue metabolic defects associated with obesity,
further support this idea and the need for further investigation.
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