Physiol Rev 89: 1025–1078, 2009;
doi:10.1152/physrev.00011.2008.
AMPK in Health and Disease
GREGORY R. STEINBERG AND BRUCE E. KEMP
Protein Chemistry and Metabolism, St. Vincent’s Institute of Medical Research, University of Melbourne,
Fitzroy, Victoria, Australia
www.prv.org
0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society
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I. Introduction and Historical Background
A. Discovery of the “adenylate charge” regulatory kinase
B. Metabolic stress sensing in yeast
C. Emergence of AMPK subfamily
II. Structure and Regulation of AMPK
A. AMPK genes, transcripts, and variation
B. AMPK subunit composition: broad roles
C. Subunit structures
D. Nucleotide binding to the ␥ subunit
E. Mechanism of autoregulation
F. Myristoylation of the ␤ subunit
E. AMPK interaction with glycogen
F. AMPK substrate recognition
III. Regulation by Phosphorylation
A. Activating upstream kinases
B. Multisite subunit phosphorylation
IV. AMPK Regulation of Carbohydrate Metabolism
A. Glucose uptake
B. Glycolysis
C. Glycogen metabolism
D. Hepatic glucose production
E. Hypothalamic glucose sensing
V. AMPK Regulation of Lipid Metabolism
A. Fatty acid uptake
B. Fatty acid partitioning
C. Triacylglycerol turnover
D. Mitochondrial biogenesis
E. Cholesterol synthesis
VI. AMPK Regulation of Protein Metabolism, Cell Polarity, Growth, and Apoptosis
A. Protein synthesis
B. Cell growth and apoptosis
C. Regulation of cell polarity and ion flux
VII. Integrative Role of AMPK as a Regulator of Whole Body Energy Metabolism
A. Exercise
B. Regulation of appetite
C. Regulation by nutrients
D. Endocrine regulation
VIII. AMPK Dysregulation in Disease
A. Aging and longevity
B. Obesity and the metabolic syndrome
C. Cardiovascular disease and reperfusion injury
D. Cancer
E. Dementia, neurogenesis, and stroke
IX. Opportunities for Therapeutics
A. Metformin
B. Thiazolidinediones
C. Life-style interventions
D. Ciliary neurotrophic factor
E. Natural compounds
F. Small molecule activators
X. Conclusions and Future Directions
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GREGORY R. STEINBERG AND BRUCE E. KEMP
Steinberg GR, Kemp BE. AMPK in Health and Disease. Physiol Rev 89: 1025–1078, 2009; doi:10.1152/physrev.00011.2008.—
The function and survival of all organisms is dependent on the dynamic control of energy metabolism, when energy
demand is matched to energy supply. The AMP-activated protein kinase (AMPK) ␣␤␥ heterotrimer has emerged as
an important integrator of signals that control energy balance through the regulation of multiple biochemical
pathways in all eukaryotes. In this review, we begin with the discovery of the AMPK family and discuss the recent
structural studies that have revealed the molecular basis for AMP binding to the enzyme’s ␥ subunit. AMPK’s
regulation involves autoinhibitory features and phosphorylation of both the catalytic ␣ subunit and the ␤-targeting
subunit. We review the role of AMPK at the cellular level through examination of its many substrates and discuss
how it controls cellular energy balance. We look at how AMPK integrates stress responses such as exercise as well
as nutrient and hormonal signals to control food intake, energy expenditure, and substrate utilization at the whole
body level. Lastly, we review the possible role of AMPK in multiple common diseases and the role of the new age
of drugs targeting AMPK signaling.
A. Discovery of the “Adenylate Charge”
Regulatory Kinase
AMP-activated protein kinase (AMPK) is an ␣␤␥ heterotrimer comprising an ␣-catalytic subunit with ␤␥-regulatory subunits. Although mammalian AMPK was purified and sequenced in 1994 (76, 123, 341), it has a long
history dating back to independent studies in the laboratories of Ki Han Kim at Purdue University and David
Gibson at Indiana University, who reported a protein
kinase activity associated with acetyl-CoA carboxylase
(79) and HMG-CoA reductase, which are respectively (41)
the rate-limiting regulatory enzymes for fatty acid and
cholesterol synthesis. At this time, enzyme-catalyzed protein phosphorylation was emerging as a regulatory mechanism for enzymes in metabolic pathways such as glycogen
phosphorylase, glycogen synthase, pyruvate dehydrogenase, and pyruvate kinase (269). Both acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGR) were inactivated by their respective associated kinases present in
liver cytosol and could be reactivated by phosphatase
treatment (43, 224).
It was recognized early on that both the HMGR and
ACC kinase activities were stimulated by nucleotides. At
first, HMGR kinase was thought to be regulated by cAMP
(41), then ADP (61, 366), but it was subsequently shown
to be potently (micromolar range) activated by AMP (145)
with the apparent activation by ADP due to AMP contamination. Yeh et al. (565) were first to report that the ACC
kinase phosphorylation reaction was stimulated by AMP
and inhibited by high ATP, but they speculated that AMP
was acting via the ACC rather than the associated kinase.
Prophetically, Yeh et al. (565) referred to the AMP activation of ACC kinase-catalyzed reaction as regulation by
“adenylate energy charge,” a concept where enzyme activities are modulated allosterically by the ratio of [ATP]/
[ADP][AMP]. The “adenylate charge hypothesis” for the
metabolic coupling of anabolic and catabolic pathways
Physiol Rev • VOL
was proposed by Daniel Atkinson (25) and held that metabolic enzyme activities were regulated by the ratio of
adenine nucleotides (reviewed in Ref. 24).
The name AMP-activated protein kinase was first
proposed in 1988 when Munday et al. (351) demonstrated
that the enzyme formerly known as ACC kinase-3 was the
primary kinase responsible for the large reductions in
ACC Vmax. The name AMP-activated protein kinase was
adopted, in 1989 when HMGR kinase and ACC kinases
were recognized to be one and the same by Carling et al.
(77), who showed the respective kinases copurified
through six steps from rat liver. Because AMPK is activated under conditions of low energy charge and typically
inhibits anabolic reactions and promotes catabolism, it is
arguably the best example of the “adenylate charge hypothesis” in action. For example, in response to a reduction in the energy charge (decrease in ATP and increase in
AMP), AMPK switches off anabolic pathways such as
fatty acid, triglyceride, and cholesterol synthesis as well
as protein synthesis and transcription that consume ATP,
and switches on catabolic pathways that generate ATP,
such as fatty acid oxidation and glycolysis. While the
name AMPK has been universally adopted and was entirely appropriate when AMPK was considered a metabolic stress-sensing protein kinase under the primary influence of cellular AMP concentrations, it is now known
that Ca2⫹ pathways, mediated by calmodulin-dependent
kinase kinase ␤ can activate AMPK independent of cellular AMP levels. Moreover, AMPK influences energy metabolism at the organism level by promoting food intake.
For this reason, an alternate name (PKE energy kinase)
has been proposed to embrace AMPK’s broader mechanisms of regulation and physiological functions (544).
Nevertheless, the mammalian AMPK remains sensitive to
AMP (allosteric activation) whatever the upstream kinase
utilized; for this reason, AMPK remains appropriate. However, the yeast snf1 kinase (AMPK homolog), and possibly
other orthologs yet to be tested, is not activated by AMP.
Once the mechanism of control is understood in more
detail across the AMPK subfamily, the need for a possible
name change will become more apparent.
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I. INTRODUCTION AND HISTORICAL
BACKGROUND
AMPK IN HEALTH AND DISEASE
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C. Emergence of AMPK Subfamily
AMPK has homologs in all eukaryote organisms.
When mammalian AMPK was purified and sequenced
(339, 551), the catalytic subunit was found to correspond
to the Saccharomyces cerevisiae protein kinase Snf1
identified in 1986 (85). Genetic studies had revealed that
SNF1 mutants (sucrose nonfermenting) were unable to
grow on sucrose because of a defect in activation of
glucose-repressed genes when the carbon source was
switched from glucose to sucrose. One of these glucoserepressed genes is SUC2, which encodes invertase, the
enzyme secreted to hydrolyze sucrose into fructose and
glucose. SNF1 mutants also do not grow on nonfermentable carbon sources, such as glycerol, ethanol, and lactate, which also require derepression of glucose-repressed genes (reviewed in Refs. 80, 192, 236). It has been
estimated that Snf1 kinase controls the expression of over
400 genes out of several thousand, the transcription of
which changes with glucose deprivation (reviewed in Ref.
397). In the presence of glucose, the Mig1 repressor binds
the glucose-repressed gene promoters. But, in the absence of glucose, Snf1 kinase is activated and phosphorylates Mig1, altering its association with the transcriptional coactivators Cyc8 and Tup1 (383), as well as causing Mig1 to migrate to the cytoplasm and allow derepression
of glucose suppressed genes (reviewed in Refs. 184, 192).
Other substrates are involved as mutation of the Snf1
kinase site in Mig1 alone is not sufficient to block regulation. Recent studies from the laboratories of Thompson
and Berger and co-workers (238a) have shown that Snf1
kinase phosphorylates histone H2b on Thr-39, and mutation of this site alone is sufficient to give a ⌬Snf1 phenotype and block growth on nonfermentable carbon sources
(238). Although knowledge of Snf1 protein kinase’s role in
regulating transcription was well advanced by 1994, no
biochemical studies had been done and no metabolic
enzyme substrates were known. Following the discovery
that AMPK was a homolog of Snf1 kinase, it was quickly
found that ACC was a substrate for Snf1 kinase (339, 551),
and yeast ACC activity was inhibited by glucose deprivation provided Snf1 kinase was present (551). These observations provided compelling evidence that the AMPK/
Snf1 kinases were key protagonists in a highly conserved
metabolic stress-sensing pathway responsible for matching metabolic energy demand with supply. This conservation extends to plants, with the plant homolog RKIN1 able
to complement SNF1 in yeast (76). With glucose deprivation, it was expected that AMP would increase and trigger
Snf1 kinase activation. Indeed, Snf1p activity increases
dramatically with glucose withdrawal, and this correlated
with increases in AMP/ATP ratio (536); however, Snf1
kinase is not directly activated by AMP in vitro (339, 536).
The signaling mechanism for activation of Snf1 kinase in
response to glucose deprivation remains a conundrum.
Multiple mammalian AMPK subunit isoforms encoded by distinct genes were identified with two ␣ subunits (465), two ␤ subunits (91, 497), and three ␥ subunits
(95). The regulatory ␤ and ␥ subunits associated with the
mammalian ␣-catalytic subunit have yeast Snf1-interacting protein counterparts with the ␤ subunit related to the
S. cerevisiae GAL83-SIP1-SIP2 gene family and the ␥ subunit corresponding to Sn4 (158, 464, 549). The corresponding AMPK subunit genes are named PRKA followed
by the subunit identifier A1, A2, B1, B2, G1, G2, or G3 (e.g.,
PRKAG3). They are distributed across five chromosomes
[␣1(5p12), ␣2(1q31), ␤1(12q24.1), ␤2(1q21.1), ␥1(12q12-14),
␥2(7q35-36), ␥3(2q35)]. The AMPK ␣1 and ␣2 subunits are
similar (⬃550 residues), both having conserved NH2terminal catalytic domains and divergent COOH-terminal
tails. The AMPK catalytic domains are found in the
branches of the kinome belonging in the CAMK group
(calcium/calmodulin-dependent protein kinases) between
the MLCK and CaMK subfamilies (310). The 12 most
closely related kinases are MELK, BRSK2, BRSK1,
NUAK2, NUAK1, QIK, QSK, SIK, MARK4, MARK3,
MARK1, and MARK2. A feature of these AMPK-related
protein kinases is that they are activated by the AMPK
upstream kinase LKB1 (299). The AMPK ␤ subunits (⬃270
residues) vary in the first 65 residues but are otherwise
highly conserved. In contrast, the ␥ subunits vary in
length [␥1(331), ␥2(569), and ␥3(489)], but beyond their
diverse NH2 termini share a conserved COOH-terminal
⬃300 residues containing four CBS repeat sequences (see
below). Thus mammals differ from yeast in having multiple ␣ and ␥ subunits but only two ␤ subunits rather than
the three present in S. cerevisiae. There is even greater
diversity in plants and malaria, where ␤␥ subunit fusions
occur in addition to regular ␤ and ␥ subunits (see Ref.
397). The plant ␤␥ fusion subunits (␤3) consist of the
␤-carbohydrate-binding module (CBM) sequence fused to
an intact ␥.
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II. STRUCTURE AND REGULATION OF AMPK
A. AMPK Genes, Transcripts, and Variation
Since several of the AMPK subunits (␣1, ␥2, ␥3) can
occur in multiple forms generated by alternate initiation
or splicing, we have adopted nomenclature where the
longest transcript is denoted “.1” and shorter transcripts are
given higher values (e.g., ␥3.1, ␥3.2) (see Tables 1 and 2).
1. Human PRKAA1
The ␣1.3 isoform of 550 residues appears to be the
most commonly expressed alternate transcript. There is
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B. Metabolic Stress Sensing in Yeast
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TABLE
GREGORY R. STEINBERG AND BRUCE E. KEMP
1.
Human AMPK genes and transcripts
5. Human PRKAG1
Protein
Gene
Size, kb
Transcript, kb
Exons
␣1
␣2
␤1
␤2
␥1
␥2
␥3
PRKAA1
PRKAA2
PRKAB1
PRKAB2
PRKAG1
PRKAG2
PRKAG3
38.816
70.013
13.668
17.44
16.537
319.77
9.408
5
2.44
2.388
5.418
1.689
3.405
2.301
10
9
7
7
12
16
14
There is one SNPS reported within ␥1 exon 5
(Thr89Ser). While this is a conservative substitution, it is
nevertheless adjacent to Asp-90, which is the residue
responsible for binding the AMP ribose, and therefore
could conceivably have a subtle effect on AMPK regulation.
6. Human PRKAG2
2. Human PRKAA2
There are six SNPS reported within the exons with
two only in exons 3 (Ser84Ileu) and 7 (Val293Leu), respectively, giving rise to residue changes. In the case of
PRKAA2, intronic SNPs have been linked to cholesterol
and low-density lipoprotein (LDL)-cholesterol levels in a
cohort of 2,777 Caucasian females (459). One haplotype
was associated with lower total cholesterol and LDLcholesterol. Unfortunately, the underlying mechanisms of
intronic regulatory effects, which could explain such exciting associations, are not yet known. Several studies
have been undertaken to test for associations between
PRKAA2 SNPs and occurrence of type 2 diabetes, but
none has been confirmed (250).
3. Human PRKAB1
There are four SNPS reported within the ␤1 exons
but only two in exons 1 (Pro20Ala) and 3 (His135Leu),
respectively, giving rise to residue changes.
4. Human PRKAB2
There are five SNPS reported within the ␤2 exons
with only two in exons 4 (Val155Leu) and 5 (His211Tyr),
respectively, giving rise to residue changes.
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There are two additional ␥2 isoforms, with ␥2.2 (C)
transcribed from Met-44 in exon 2 (525 residues) and
isoform ␥2.3 (B) from Met-242 in exon 6 (328 residues).
There are two SNPs and one deletion [Met6Leu,
Pro198Leu, and (65– 67, SerProPhe)] reported for exons 1,
4, and 3, respectively. Rare mutations in ␥2 giving rise to
a cardiac glycogen storage phenotype are mentioned in
section IID.
7. Human PRKAG3
The ␥3 isoform 2 is initiated at Met-26 in exon 3 (␥3.2).
There are a total of nine reported SNPs, with six predicted
to alter the residue (Pro71Ala exon 3, Phe139Leu exon
4, Leu153Val exon 4, Gln260Arg exon 8, Arg340Trp
exon 10, and Ala484Val exon 13). The SNPs at 260, 340,
and 484 occur in the sequence containing the four
repeat CBS sequenes involved in nucleotide (AMP/ATP)
binding, but their effects if any on AMPK regulation
have not been reported. In a study of 761 obese subjects, Costford et al. (108) reported 87 variants in human PRKAG3 and its flanking regions. There were six
common variants, including three intronic polymorphisms, one synonymous variant, and two nonsynonymous variants (Pro71Ala and Arg340Trp). There were
81 rare variants of which 30 were coding variants.
Weyrick et al. (534) studied 1,061 subjects and found no
association between PRKAG3 SNPs and prediabetic
conditions (body fat distribution, insulin resistance, or
insulin secretion) but did detect a significant associa-
TABLE
2.
Alternate transcripts
Alternate Transcripts
Size, residues
Occurrence
␣1.1
␣1.2
␣1.3
␣2.1
␤1.1
␤2.1
␥1.1
␥2.1
␥2.2 (C)
␥2.3 (B)
␥3.1
␥3.2
574
559
550
552
270
272
331
569
525
328
489
444
Uncharacterized
Uncharacterized
Common
Common
Common
Common
Common
Common
Uncharacterized
Uncharacterized
Common
Uncharacterized
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also an alternate 10 exon transcript version encoding a
longer form of ␣, termed ␣L, that contains an in-frame
“cassette-type” exon 3A of 45 bp between exons 3 and 4
that encodes a 15-residue insert between the D- and E-helices of the kinase large lobe (see below). The transcript
is present in heart, liver, skeletal muscle, adipose tissue,
and peripheral blood lymphocytes but does not appear in
other mammals (B. J. W. van Denderen, unpublished
data). There are four SNPS reported with only two in
exons 1 (Met1Leu) and 9 (Val524Ala), respectively, giving rise
to residue changes. Alternate initiation or the Met1Leu SNP
could permit an upstream initiation at ⫺9 Met, which
provides for a 574 residue ␣1.1 isoform tranlated from 10
exons having the NH2-terminal sequence MRRLSSWRK-,
compared with MATAEKQKH- for the 550 residue isoform. The alternate possible 574 (␣1.1) and 559 (␣1.2)
residue isoforms have not been characterized biochemically.
AMPK IN HEALTH AND DISEASE
tion between LDL-cholesterol and apolipoprtein B-100
levels and two SNPs, Pro71Ala (rs692243) in exon 3 and
rs6436094 located in the 3’-untranslated region (UTR).
Both studies on PRKAG3 SNPs indicate that many more
SNPs will be discovered for the AMPK genes as more
sequencing is done on larger populations.
B. AMPK Subunit Composition: Broad Roles
Physiol Rev • VOL
C. Subunit Structures
The major structural elements of the AMPK subunit
sequences are illustrated in Figure 1. The ␣ subunit comprises an NH2-terminal kinase domain followed by an
autoinhibitory sequence (AIS) and a subunit interacting
domain (␤-SID) that binds ␤ (112, 227) (Fig. 1). The ␤
subunit contains two characterized elements, a mid molecule glycogen binding domain (GBD), now termed CBM
(215, 395), and a COOH-terminal subunit binding sequence responsible for binding ␣ and ␥ (␣␥-SBS) (227)
(Fig. 1). The ␥ subunit comprises four tandem repeat
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The AMPK subunits show differential tissue-specific
expression and activation. Early on, Northern blots revealed that AMPK ␣1 was relatively evenly distributed
across rat heart, liver, kidney, brain, spleen, lung, and
skeletal muscle (465), whereas AMPK ␣2 subunit was
highly abundant in rat skeletal muscle and, to a lesser
extent, in heart and liver followed by brain and kidney
and detectable in lung (465, 516). Quantitative real-time
PCR (qRT-PCR) of mouse tissues has shown that AMPK
␣1 is evenly distributed across liver, kidney, lung, heart,
red vastus, and brain, but with higher levels in adipose
tissue and lower levels in white vastus, spleen, and pancreas (N. Dzamko, unpublished data). The mouse tissue
qRT-PCR measures of AMPK ␣2 showed highest levels in
the red vastus, followed by white vastus then heart kidney
and liver, with very low levels seen in the lung, brain, and
adipose tissue and negligible amounts in the pancreas and
spleen (Dzamko, unpublished data).
The ␤1 subunit shows a widespread expression profile comparable to ␣1 based on Northern blots, qRT-PCR,
and immunoblotting. High expression of ␤2 occurs in
skeletal muscle and to a lesser extent heart (91, 497)
(Dzamko, unpublished data).
The tissue distribution of the ␥ subunits has been
studied reasonably comprehensively (95, 309). The ␥1
isoform is ubiquitously expressed, whereas the ␥3 isoform shows the most restricted expression, being confined to skeletal muscle (309). The ␥2 isoform is also
widely expressed but highest in the heart, followed by
brain, placenta, and skeletal muscle (95). Yu et al. (569)
found in mice the ␥3 is most highly expressed in fasttwitch glycolytic muscle (type IIb) with no detectable
expression in the slow-twitch oxidative soleus muscle
that contains type I fibers. Type IIa fast-twitch fibers and
red oxidative glycolytic-muscle have intermediate levels
of ␥3 expression. The expression profile of ␥3 reported
for the mouse is consistent with results from pigs carrying
the RN⫺ mutation (␥3 R200Q; Ref. 333), which have a
glycogen storage disease of the muscle with glycogen
accumulation in the white muscle (140). The longer isoform ␥3.1 (termed ␥L; Ref. 569) is the predominant form
in mouse muscle, whereas ␥3.2 (␥3S) is thought to be the
form expressed in human muscle (569).
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FIG. 1. AMPK subunit functional features. Mammalian AMPK subunits showing regions of shared known structure are shown (colored
cylinders). Numbers above ␣1 and ␤1 subunit cylinders denote NH2- and
COOH-terminal residues of crystal structures. Light blue cylinder in ␣
sequence denotes insert from S. cerevisiae structure and may not be
represented in mammalian AMPK. AIS, autoinhibitory sequence (312–
335); ␤-SID, ␤-subunit interacting domain. The ␤1 subunit CBM with
residues involved in sugar binding plus the phosphorylatable Ser-108 are
shown as side chains off the cylinder. ␣␥-SBS, ␣␥-subunit interacting
sequence. The ␥1 repeating CBS modules are shown with the residues
that form H-bonds with nucleotide 2⬘. 3⬘-Ribose hydroxyl groups are
shown above the ␥-subunit cylinder. Below the cylinder are shown the
basic residues that occupy the solvent accessible core of the ␥1 subunit
that make contacts with the nucleotide phosphates. The nucleotide
binding sites 1-4 are shown with the corresponding old nomenclature
used previously by Xiao et al. (556) and Jin et al. (235).
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GREGORY R. STEINBERG AND BRUCE E. KEMP
A
␤-sheet platform, the backbone extends over the ␣-subunit interacting domain. This segment of the ␤ sequence is
devoid of its own hydrophobic core but instead makes
hydrophobic contacts across the ␣-subunit interacting
domain (501). On the other hand, the ␤␥ interaction contains fewer hydrophobic interactions with the interface
stabilized by hydrogen bonding (501). Consistent with this
interpretation, alanine scanning mutagenesis of ␤ (260 –
270) has shown that Tyr-267 side chain is important and,
to a lesser extent, Thr-263 for stabilizing the ␤␥ interaction, whereas the remaining residues can be replaced by
Ala (226). The ␤ Tyr-267 is juxtaposed to ␥1 aromatic
residue, Phe-51 in the mammalian structure (556). The
␣-subunit COOH-terminal fragment forms a discreet ␣-helical/␤-strand domain that shares topology with components of the AMPK-related MARK kinases (499). The
Townley structure has provided a clear picture explaining
the earlier truncation mutagenesis results that showed
that the COOH-terminal 25 residues of ␤ (246 –270) were
essential for ␥ binding, whereas ␣ binding required 85
residues ␤ (186 –270) (227) (Fig. 2).
The ␥ subunit forms an elliptical disk ⬃60 Å in diameter and 30 Å in depth made up of the four CBS motifs
packed as pairs of Bateman domains (11, 501, 556). Each
CBS motif contributes a pair of antiparallel strands that
associate to form a Bateman domain with a pair of adenyl
binding sites (see below) (Figs. 1 and 3).
B
90°
CBM
αγ-SBS
Glycogen
binding
site
CBS2
CBS1
CBS4
CBS3
ADP
AMP
AMP
AMP
RS
FIG. 2. AMPK ␣␤␥ core structure. A: graphical representation of composite features from the mammalian AMPK heterotrimer and yeast
orthologs: in blue, the ␣-subunit structures comprising a mammalian ␤-subunit interacting domain (SID) and regulatory sequence possibly unique
to S. cerevisiae (RS); in green, the ␤-subunit structures [carbohydrate-binding molecule (CBM) from S. cerevisiae and ␣␥-subunit binding sequence
(SBS) from S. pombe]; in red, mammalian ␥-subunit structure. Three AMP molecules (yellow) evident in the mammalian AMPK structure and one
ADP molecule (orange) seen in the S. pombe structure are shown in stick representation. B: 90° rotation of A.
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sequences called CBS motifs, named after related sequences in cystathionine ␤ synthase first recognized by
Alex Bateman (35) (Fig. 1). The mammalian ␣␤␥ heterotrimer exists in solution as a single species (monomer)
based on light scattering (556) and analytical ultracentrifugation analyses (226).
Substantial progress has been made in the past 2
years towards the complete structure of the AMPK ␣␤␥
heterotrimer. The crystal structures of the isolated catalytic domain of the mammalian ␣2 (PDB ID: 2H6D) and S.
cerevisiae ␣ subunit have been solved (PDB ID: 2H6D)
(363, 420) together with the core ␣␤␥ interacting complex
comprising a COOH-terminal fragment of ␤ subunit, the
complete ␥1 subunit, and a C-terminal fragment of the ␣
subunit (11, 501, 556). Broadly speaking, the structures of
⬃73% of the ␣ subunit, 72% of the ␤ subunit, and 100% of
the ␥1 structure are now known notwithstanding some
regions of disorder in the ␣ and ␤ fragments (Figs. 1 and 2).
The major gap in our knowledge is the juxtaposition of
the ␣-subunit catalytic domain and its associated autoinhibitory sequence with the ␣␤␥ core complex. The first
structure of the ␣␤␥ core complex was reported for the
fission yeast Schizosaccharomyces pombe by Townley
et al. (501). The ␤ fragment forms a ␤-sheet structure that
serves as a subunit-binding platform for both ␣ and ␥, as
shown in Figure 2 [marked ␣␥-SBS green for both the ␣
fragment (blue) and ␥ (red)]. Beyond the ␤ subunit
AMPK IN HEALTH AND DISEASE
The S. cerevisiae structure shows additional features
to the mammalian and S. pombe structures, some of these
are species specific while others are likely to be common
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D. Nucleotide Binding to the ␥ Subunit
The structural studies have revealed that ␥ contains
four adenyl binding sites comprising a helix-loop-strand
motif with one contributed from each CBS unit (235, 501,
556). The mammalian ␥ structure was solved with 3 mol
of AMP bound, two of which were interchangeable with
ATP (see below) (556). In contrast, the S. pombe ␥ structures have been solved with either 1 mol of AMP bound
(501) or 2 mol of ADP bound, respectively (235). To
simplify the nomenclature for the binding sites, we have
proposed that they be named 1– 4 corresponding to their
CBS unit location (249). The adenyl binding sites are
surface pockets where the adenine moiety sits in a hydrophobic environment making hydrogen bonds to two
strands via backbone groups (556). There are no aromatic
residues in the AMP binding pockets, a feature of the
FIG. 3. The ␥1-subunit nucleotide binding sites. A: graphical representation of the composite nucleotide binding sites for mammalian ␥1
and S. pombe. Nucleotide binding sites 1-4 are shown with the corresponding old nomenclature used previously by Xiao et al. (556) and Jin
et al. (235) shown in parentheses. AMP molecules occupy sites 1, 3, and
4, with an ADP occupying site 2 from the S. pombe structure (see Fig. 1).
B: detailed view of AMP site 3 in mammalian ␥1, showing hydrogen
bonds between hydroxyl groups of the ribose moiety and Asp-244.
C: hydrogen bond interactions between phosphate groups of 3 AMP
molecules and basic residues lining the solvent accessible core of ␥1.
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to the architecture of mammalian AMPKs. In particular,
the ␤ subunit of the S. cerevisiae structure shows GBD
nestled alongside ␥, near the ␤␥ binding junction (11).
Previously, the GBD domain structures for ␤1 and ␤2 had
been solved in the presence of ␤-cyclodextrin (396). On
the basis of the ␤-cyclodextrin-GBD domain structure, it
was possible to identify the sugar-binding site (see below). The position of the ␤-cyclodextrin has been modeled into the S. cerevisiae structure (11) to provide a
guide to where glycogen is bound in the complex.
A larger fragment of the S. cerevisiae ␣ subunit was
crystallized: this now extends the known structure by
⬃30 residues (11). This makes it clear that the ␣-backbone path from the ␣-subunit interaction domain goes
down over the ␥ subunit to a helix-loop-helix structure
referred to as a regulatory sequence marked RS in the S.
cerevisiae structure (blue in Fig. 2). There is uncertainty
whether this feature will be found in the mammalian
␣-subunit structure, as S. cerevisiae and S. pombe both
have insert sequences in this region, depending on how
the alignments are done. Comparing the S. cerevisiae and
mammalian ␣-subunit alignments, there are ⬃110 common residues between the known COOH-terminal ␣ structure and the NH2-terminal kinase catalytic domain (Fig.
1). This segment of unknown structure contains the autoinhibitory sequence [␣ (313–335); see below].
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GREGORY R. STEINBERG AND BRUCE E. KEMP
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The ␥-subunit structures have provided an important
road map for understanding the structure-function relationships of naturally occurring glycogen storage disease
causing mutations. These mutations include the Hampshire pig
Rendement Napole (RN⫺) mutation in ␥3 (Arg302Gln) equivalent to Arg-70 in the ␥1 structure as well as 10 human
mutations in ␥2 that give rise to Wolf-Parkinson-White
cardiomyopathy (reviewed in Ref. 19). When mapped on
the human ␥1 structure, 8 of the 10 mutations occur in
amino acids whose side chains are in proximity to the
AMP binding sites (556). Notably, four mutations in ␥2
Arg302Gln ( ␥ 1 Arg-70), His383Arg ( ␥ 1 His-151),
Arg531Gly/Gln (␥1 Arg-299) correspond to the key phosphate interacting constellation of basic residues in the ␥1
solvent accessible core structure. While no diseasecausing mutations have been detected in ␥1, a transgenic
mouse with muscle specific expression of the ␥ 1
Arg70Gln mutation resulted in constitutive activation of
AMPK in the muscle and accumulation of glycogen (34).
In addition, in a screen of obese and lean individuals,
Costford et al. (108) have identified two families carrying
an Arg225Trp mutation in ␥3 who have double basal
glycogen levels and reduced intramuscular triglycerides.
This mutation occurs in CBS1 and corresponds to the
human ␥2 Arg302Gln and the pig ␥3 Arg200Gln mutation
mentioned above.
E. Mechanism of Autoregulation
Many protein kinases are autoregulated by a variety
of mechanisms (258). In the case of the CAMK branches
of the kinome to which AMPK belongs, smMLCK, skMLCK,
TTN, CaMK1, and CaMK2 have all been found to contain
autoregulatory sequences beyond the COOH terminus of
the catalytic domain which blocks catalytic activity (258).
In the case of TTN (213) and CaMK1 (168), structural
studies have shown that the autoregulatory sequence
binds to the protein substrate groove and extends into the
active site (257), although the extent of interaction is less
for CaMK2 (418). While both CaMK1 and TTN are members of the CAMK group in the kinome, they occur in
different branches/subfamilies. It is possible that the autoregulatory features may be more closely conserved
within each major branch of the CAMK group. In the case
of the calmodulin-activated protein kinases, the calmodulin binding sequence overlaps the autoregulatory sequence and provides a mechanism for relieving the autoinhibition when calmodulin binds (258). Typically truncation mutagenesis to remove the autoregulatory sequences
permits the kinase to become constitutively active (i.e.,
independent of calmodulin). Like many kinases (492),
AMPK has an absolute requirement for phosphorylation
of its activation loop Thr-172 for activity (186). In addition
to Thr-172 phosphorylation, it was found that the ␣1
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allosteric AMP binding site in phosphorylase. The adenyl
group is also anchored through its ribose 2⬘ and 3⬘ hydroxyl groups to Asp residues in three of the four CBS
units (CBS1, Asp90; CBS3, Asp245; CBS4, Asp317; see Fig.
1). These Asp residues are conserved across all species of
AMP ␥ subunits. In CBS2, the corresponding position for
the Asp in the structure is substituted with Arg-171, and in
the mammalian structure this site is unoccupied (556).
However, in the ADP-bound form of the S. pombe structure, both CBS2 and CBS4 are occupied with ADP showing that site 2 in CBS2 can bind nucleotide (235). In this
case, the equivalent ␥1 Arg-171 side chain is pointing
away and the ADP ribose 2⬘,3⬘ hydroxyls hydrogen bonds
to Asp-250, Gln-251, and Ser-252 (S. pombe) contributed
by the ␤-subunit 12-residue extended loop termed the ␤
flap (235). In the mammalian structure, the corresponding
␤-subunit loop carrying Asp-224 (h ␤1) is disordered
(556). Whether a nucleotide can bind to the mammalian
CBS2 with the aid of the ␤1 Asp-224 in the sequence
DPA252 remains to be determined.
A particularly striking feature of the ␥’s four AMP
binding sites is that the phosphate groups point inwards
in a solvent accessible core of the ␥ subunit that contains
a constellation of basic residues. Thus, rather than having
individual basic residues allocated to the phosphate
groups from each AMP, there is a high degree of connectivity (see Fig. 3). The key basic residues are Arg-70,
His-151, Arg-152, Lys-170, His-298, and Arg-299 (556). The
four CBS units make varying contribution of basic residues with Arg-70 from CBS1 and none from CBS3. The
CBS2 unit contributes His-151, R152, and Lys-170 and
CBS4 contributes His-298 and Arg-299 (Fig. 1).
As mentioned, the initial S. pombe structure contained a single AMP bound to CBS4 (501). This site was
also occupied in the mammalian structure but was found
not to exchange and was present during purification of
both the holoenzyme and the crystallized ␣␤␥ core complex (556). The CBS1 and CBS3 sites in the mammalian
structure bound AMP but would readily exchange ATP
(556). These results are consistent with the reported early
binding studies that found each Bateman domain (439)
bound a single mole of AMP or ATP.
Since no biochemical studies have been reported for
S. pombe, it is not clear whether nucleotide binding affects activity for either AMP or ADP. Mammalian AMPK
was initially thought to respond to ADP (61, 366), but it
was subsequently found that AMP was the genuine activator (145). What is clear is that mammalian AMPK is
activated allosterically by AMP and inhibited by ATP, and
the structure now identifies these as the exchangeable
sites CBS1 and CBS3. However, we are left with several
questions regarding the function of the nonexchangeable
AMP at the CBS4 site as well as the question of whether
the mammalian CBS2 site binds nucleotide at all.
AMPK IN HEALTH AND DISEASE
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Leu-137 in yeast ␥ (SNF4) gave a phenotype of constitutive AMPK activity (Snf1p) (441). Overall, the biochemical
data are strongly supportive of the ␥ pseudosubstrate
model, but the major uncertainty remains the structural
likelihood of the ␥ CBS2 domain adopting a different
structure to accommodate binding the ␣-subunit active
site.
The ␤ subunit also appears to contribute to the suppression of activity since either NH2-terminal truncation
of the first 65 residues up to the CBD (215) or the Gly2Ala
mutation, which blocks myristoylation (525), causes an
increase in activity. There is no structural information
available for the NH2 terminus of the ␤ subunit to provide
clues to the possible mechanism underlying its inhibitory
effects. The ␤1 NH2-terminal sequence contains adjacent
Ser-24 and Ser-25 that are autophosphorylated, indicating
that this sequence is accessible to the active site and
phosphorylation on Ser-24/25 may be involved in sustaining AMPK activation (340). The available evidence supports the concept that AMPK is autoregulated at multiple
levels involving all three subunits acting in concert. What
is missing at this point is structural data to evaluate the
proposed mechanisms.
F. Myristoylation of the ␤ Subunit
Myristoylation is a commonly used posttranslational
modification adding myristic acid (14 carbon lipophilic
group) typically to glycine-2 following processing on NH2terminal Met, but can also occur on Gly residues exposed
by endoproteinase activity (406). The myristoyl group
acts as a lipid anchor, tethering the host protein to cellular membranes and is widely used to secure signaling
proteins to membranes. The sequences of the mammalian
␤1 and ␤2 subunits (MGNTSSER) share NH2-terminal
consensus sequences (MGNXXS/T) for myristoylation
(Web resource: http://mendel.imp.ac.at/myristate/) (8).
With the use of ESI-mass spectrometry of the purified rat
liver AMPK (␣1␤1␥1), it was confirmed that the ␤1 subunit was myristoylated and phosphorylated (Mr 30,552,
30,635, and 30,722 of the myristoylated mono-, di-, and
tri-phosphorylated forms, respectively) (340). When AMPK is
expressed in insect cells, there is complete processing of
the ␤ NH2-terminal Met by Met-aminopeptidase, but incomplete myristoylation of Gly-2 giving rise to a mixture
of ␣␤␥ heterotrimers differing in their level of ␤-subunit
myristoylation (226). Supplementing the insect cell culture media with myristic acid does not overcome the
problem.
Myristolation of proteins is typically associated with
facilitating membrane binding, as is, for example, the case
with MARCKS protein ADP-ribosylation factor 1. However, in other instances, such as for calcineurin-B and the
PKA catalytic subunit, myristoylation may not affect
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(1-312) was active in the absence of ␥ or ␤, whereas a
longer version ␣1 (1–392) was inactive, consistent with
the presence of an autoregulatory sequence between 312
and 392 (112). Further detailed mapping of the autoinhibitory sequence has shown that it resides within a 23residue sequence 313–335 (380). Pang et al. (380) have
used homology modelling based on the crystal structure
of the AMPK-related kinase, MARK2 (382), to position the
autoinhibitory sequence of AMPK. The MARK kinases
occur on the same main branch of the CAMK group as
AMPK. The modeled AMPK autoinhibitory sequence is
present as a 3-helix unit (residues 290-335) juxtaposed to
the small lobe of the kinase. While there is low sequence
identity between MARK2 and AMPK (232) in this region,
the model is nevertheless attractive in that the positioning
of the autoregulatory sequence could modulate the orientation of the kinase small lobe to block catalytic activity.
The autoinhibitory sequence corresponds to the position
of the ubiquitin-associated domains (UBA) found within
other members of the AMPK-related protein kinase subfamily that includes MARK2 kinase. Furthermore, the
MARK2 kinase UBA domain plays an important role in
the regulation of phosphorylation by the upstream kinase
LKB1 (232). In support of this model, mutation of the
residues Leu328Gln and Val298Gly that are conserved
across the MARK family result in a constitutively active
AMPK (380). Formal proof of the model of AMPK autoregulation will depend on a crystal structure of the catalytic domain containing the autoregulatory sequence.
In addition to the autoregulatory sequences identified in the ␣ subunit, Hardie and colleagues (441) have
reported a pseudosubstrate sequence [human ␥ 1;
LFDAVSSLIRNKIH151R152LP(V155)IDPE159] within the
␥-subunit CBS2 sequence that closely resembles the consensus substrate recognition sequence for AMPK in ACC
(GISALQDGLAFHMRSSMS79GLHL) in terms of the juxtaposition of basic and hydrophobic residues (underlined)
with Val-55 occupying the site corresponding to the phosphorylatable Ser-79 in ACC1 (440, 441). This model requires the pseudosubstrate sequence in CBS2 to deform
and associate with the catalytic domain substrate-binding
site. It is envisaged that the binding of AMP to the ␥
subunit leads to association with His-151, Arg-152 and
dissociation of the pseudosubstrate sequence from the
active site substrate-binding groove. Since the orientation
of the catalytic domain to CBS2 is not yet known, there is
insufficient structural information to test whether this
model is plausible. Nevertheless, when the ␥2 pseudosubstrate peptide Val155Ser mutation was made, this residue
was autophosphorylated (441), implying that the autoregulatory sequence was accessible to the active site provided the phosphorylation was an intramolecular, rather
than an intermolecular, event. Further evidence in support of the model was obtained in yeast where mutation
of the corresponding residues of the p-3 Arg-146 and p-12
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GREGORY R. STEINBERG AND BRUCE E. KEMP
E. AMPK Interaction With Glycogen
FIG. 4. The ␤1-subunit carbohydrate binding module (CBM). The ␤1
CBM structure is shown in ribbon representation complexed with cyclodextrin. The key tryptophans (Trp-100, Trp-133) forming the sugar
binding cradle, and Lys-126 and Leu-146, are illustrated.
Initial bioinfromatics sequence/secondary structure
searches revealed a midmolecule sequence in the ␤ subunits related to the starch binding domains found in
plants (isoamylase domains) (215, 395). Most eukaryote ␤
subunits contain a carbohydrate-binding domain with the
exception of the plant AKINB3 subunits (164). The AMPK
␤ glycogen-binding domain has recently been reclassified
as a member of the CBM-containing family, CBM48
(http://afmb.cnrs-mrs.fr/CAZY). The mammalian AMPK
domain limits were established experimentally by partial
proteolysis (395), and a stable rat ␤1(68-163) fragment
was expressed and crystallized in the presence of ␤-cyclodextrin (an ␣ 1-4 linked heptaglucan), as had previously been performed for other starch binding domain
structural studies (396). The corresponding human ␤2
CBM structure has also been solved in the absence of
cyclodextrin (PDB ID: 2F15). The isolated ␤(68-163) domain was found to bind glycogen, which was blocked
with ␤-cyclodextrin Ka0.5 ⬃1.3 mM. Cell-based studies
with fluorescently tagged AMPK have also confirmed that
AMPK associates with glycogen and glycogen synthase
(215). However, it has recently been claimed that AMPK is
not found associated with isolated glycogen ␣-particles
(110 –290 nm) from rat liver (386). It is clear that more
detailed histochemical studies are required to establish
under what conditions and in which tissues AMPK is
associated with glycogen.
The ␤1 CBM structure revealed that cyclodextrin
bound in a sugar-binding pocket cradled between a pair of
aromatic residues, Trp-100, Trp-133 that accommodates
three sugars from the cyclodextrin (Figs. 1 and 4). When
other nearby contact residues including Asn-150 and Lys126 are mutated, there is a loss of glycogen binding (396).
Another feature of the structure is the extension of the
side chain of Leu-146 through the core of the bound
cyclodextrin. Solution studies using NMR have also been
reported, showing that the CBM binds maltohexose and
maltohepulose with KD values ⬍1 mM while shorter oligosaccharides bind substantially less strongly (256). The
NMR results also showed chemical shifts in all the key
sugar binding residues in the presence of maltoheptulose
with the exception of Trp-100. Because the CBM binds
most tightly to carbohydrates containing ␣ 1-4 glycosidic
linkages with a single glucose ␣ 1-6 branch, it has been
proposed that AMPK may bind to partially degraded glycogen (184, 256).
The physiological significance of AMPK’s association
with glycogen has been investigated in several studies.
High muscle glycogen levels correlated with reduced activation of AMPK in response to exercise (127, 415, 475,
531, 546) or 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR) (545), leading to the idea that AMPK
may be inhibited by glycogen. Intuitively, it seems reasonable that AMPK in mammals would be switched off as
energy stores such as glycogen become replete. However,
there has been conflicting data on whether glycogen is
inhibitory. We found that AMPK activity is not altered by
binding to glycogen, as measured by adding purified rat
liver AMPK to glycogen (395). However, this does not
exclude the possibility that glycogen can affect the AMPK
activation by an upstream kinase or inactivation by a
phosphatase. In the case of patients with an inherited
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membrane association (340). Proteins may undergo a
“myristoyl switch” where ligands (e.g., Ca2⫹-calmodulin,
GTP) or phosphorylation may trigger the myristoyl group
to flip inside the protein and dissociate from the membrane (406). Removal of the ␤-subunit myristoylation site
(Gly2Ala mutant) results in a fourfold activation of the
enzyme and relocalization of the ␤ subunit from a particulate extranuclear distribution to a more homogeneous
cell distribution (525). One interpretation of the increase
in AMPK activity in the absence of the myristoyl group is
that it may be tethering the NH2-terminal 65 residues,
which otherwise has an autoinhibitory effect. In the PKA
catalytic subunit structure, the myristoly group is positioned in a hydrophobic pocket at the tail of the large lobe
anchoring the PKA NH2-terminal A-helix. It is not known
whether something similar occurs with the AMPK ␣-subunit catalytic tail binding the ␤-myristoyl group. There is
also no information on whether AMPK could undergo a
“myristoyl switch” between membrane and soluble forms
in response to phosphorylation or ligand binding signals.
AMPK IN HEALTH AND DISEASE
F. AMPK Substrate Recognition
AMPK’s substrate recognition is determined by the
enzymes’s location, as well as residues present in the
local phosphorylation site sequence of its target substrates. Our knowledge of AMPK’s substrate specificity
has come from studies using synthetic peptides by Hardie
and others, as well as site-directed mutagenesis (96, 119,
332, 374, 440, 533). The general consensus motif for AMPK
includes hydrophobic residues in the P⫹4 and P-5 position relative to the Ser/Thr phosphorylation site at P0. The
P-5 position is anchored in a hydrophobic pocket positioned between the kinase large lobe helices ␣F and ␣G
around Leu-212. A basic residue, typically Arg, occurs in
the P-1 to P-4 positions. Snf1 exhibits a strong preference
for the P-3 basic residue (119, 533). There is also a positive influence of a second Arg at the P-6 position that
interacts with the kinase acidic residues Asp-215 and
Asp-217 slightly COOH-terminal to the ␣F helix (440). The
simple consensus motif can be written ␾X(B, X)XX(Ser/
Thr)XXX␾, where ␾ is a hydrophobic residue, B is basic,
and X is any residue (96, 119, 332, 440, 533). It is important
to recognize the consensus sequence requirements are a
guide only and that sites consistent with this arrangement
may not be phosphorylated in proteins for other overriding structural reasons while some naturally occurring
AMPK phosphorylation sites may not have all the consensus determinants. A good example is the AMPK phosphorylation site in endothelial nitric oxide synthase (eNOS) at
Ser-1177 (RIRTQS1177FSLQE), which does not have the ␾
residues at ⫺5 and ⫹4 (93). We found the synthetic
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peptide corresponding to the PKA phosphorylation
site (Ser-230) in the yeast transcription factor ADR1
(LTRRAS230FSAQ) is an excellent substrate for AMPK
without conforming to the ␾ at P⫹4 consensus requirement (332). Subsequent specificity studies employing mutagenesis of the recombinant ACoAC1 (60 –94) fragment
containing the Ser-79 phosphorylation site found that Gln
and Asn were well tolerated at the P⫹4 position (440).
This led Hardie and colleagues (440) to revise the consensus to emphasize that hydrophobic residue at P-5/P-4
and a basic residue at P-4/P-3 may be the more dominant determinants. In our view, the original consensus
sequence proposal for AMPK (119) has served the field
well with the recognition that there are multiple exceptions.
We have found that AMPK substrate recognition was
exquisitively sensitive to oxidation using the SAMS peptide rACoAC (73– 87) A77R86 – 87 (HMRSAMS79GLHLVK)
with Met oxidation at P-5 having the greatest impact
(332). Thus far, the possibility that oxidation of Met could
alter AMPK signaling pathways has not been investigated
in natural or pathological states. Several web-based programs can be used to analyze AMPK specificity. These
include Scansite (http://scansite.mit.edu/) (367), which
uses a specificity matrix of preferred residues within the
local phosphorylation site sequence to assign a probability for kinase sites. If one scans the mammalian data base
using the Scansite AMPK peptide based specificity motif,
the top hit is Ser-743 in the Tousled-like kinase sequence
PHMRRSNS743SGNLHMS (consensus motifs underlined).
However, we found recombinant Tousled-like kinase was
not a substrate for AMPK (L. Macaulay, L. A. Castelli, and
B. E. Kemp, unpublished data). Kobe and colleagues (59,
435) have developed an alternative approach called
Predikin which uses a prediction model of the kinase
substrate recognition groove to estimate the compatibility
of any putative substrate sequence being phosphorylated
(http://predikin.biosci.uq.edu.au). Predikin has been used
to predict all the possible Snf1 kinase substrates in the
yeast genome (60).
For other protein kinases such as the classic PKA
signaling system, there are multiple scaffold proteins
called AKAPs that serve to create signaling complexes
with substrates and signal terminators such as phosphatases and phosphodiesterases. For a recent review, see
Scott et al. (40).
While the AMPK ␤ subunit can localize the AMPK
to glycogen and membranes via its CBM and NH2terminal myristoyl group, respectively, no studies have
documented signaling complexes analogous to the PKA
AKAPs. We can anticipate this will change and AMPK
signaling complexes will be identified, especially where
multiple isoforms of the ␣␤␥ subunits are present in the
same cell.
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defect in glycogen phosphorylase (McArdle disease) and
associated excess skeletal muscle glycogen, AMPK is still
activated by exercise (365). In a recent comprehensive
study, McBride et al. (317) have reinvestigated the effects
of glycogen on AMPK. They show addition of glycogen to
rat liver AMPK is inhibitory, and when recombinant
AMPK is expressed in CCL13 cells, this inhibition is dependent on the presence of CBM. In agreement with Koay
et al. (256), they provide evidence that inhibition is mediated by the ␣136 linkages and require the key sugar
binding Trp residues in the CBM. Isomaltose, but not
maltose, inhibited rat liver AMPK with a half-maximal
effect at 16 mM (317). A series of synthetic branched
oligosaccharides with a single ␣ 1-6 linkages were found
to be more potent inhibitors of AMPK activity as well as
inhibiting AMPK phosphorylation by CaMKK␤ and LKB1
but not dephosphorylation by protein phosphatase 2C
(317). The inhibitory effect of the oligosaccharides is lost
when the CBM Trp residues are mutated, indicating that
the CBM is essential. These results have led Hardie and
colleagues (317) to propose that AMPK acts as a cellular
glycogen sensor through its CBM containing ␤ subunit.
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GREGORY R. STEINBERG AND BRUCE E. KEMP
III. REGULATION BY PHOSPHORYLATION
A. Activating Upstream Kinases
Like many protein kinases, AMPK has an obligate
requirement for phosphorylation by an upstream kinase
on Thr-172 in the activation loop of the ␣-subunit catalytic
domain (186). Three mammalian upstream kinases have
been identified for AMPK and are discussed below.
1. LKB1
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2. Calmodulin-dependent kinase kinase
Hawley et al. (189) were the first to report CaMKK
phosphorylated and activated AMPK, discovering when
they investigated the AMP dependence of partially purified fractions of liver AMPKK. However, CaMKK was
initially dismissed as a genuine AMPKK on the basis that
the partially purified liver kinase kinase was not calmodulin dependent (189). The discovery that AMPK could be
activated by Ca2⫹ ionophores in LKB1-deficient cells led
three independent groups to demonstrate CaMKK was
indeed an upstream kinase for AMPK. Using Hela cells
and LKB1-deficient MEFs, Hurley et al. (217) showed that
mannitol and ionomycin treatment activated AMPK Thr172 phosphorylation and that the response was inhibited
by STO-609, a relatively specific inhibitor for CaMKK.
AMPK signaling in these cells could also be suppressed
with siRNA for both CaMKK␣ and -␤ (217). Hawley et al.
(188) found that up to 3 ␮M concentrations on the ionophore A23187 activated AMPK in HeLa cells without significant changes in ATP/ADP ratios, and this could be
substantially blocked with 2.5 ␮M STO-609. Importantly,
these authors investigated the selectivity of STO-609 and
showed that other protein kinases, including AMPK itself,
were inhibited at 10 ␮M STO-609. CaMKK is highly expressed in brain, and K⫹ deplorization of brain slices
activates AMPK threefold, independent of any change in
cellular AMP/ATP ratio, indicating that CaMKK activation
of AMPK may be a major pathway in the brain (188).
Woods et al. (550) also showed that activation of
AMPK by ionomycin in HeLa cells was dependent on
CaMKK␤. These authors also investigated the relative
contribution of LKB1 and CaMKK in activating AMPK
using NIH3T3 fibroblasts (which contain both CaMKK␣
and -␤) and LKB1-deficient MEFs via a variety of treatments in conjunction with STO-609. Treatment of the
3T3NIH cells with STO-609 caused an ⬃20 and 50% inhibition of AMPK activation by ionomycin and AICAR, respectively. In contrast, in LKB1-deficient MEFs, there was
a dramatic reduction in the AICAR stimulation of AMPK,
but the residual was still inhibited by STO-609. For ionomycin-dependent activation of AMPK in the absence of
LKB1, the total activation was maintained and ⬃90% in
inhibited by STO-609; this is consistent with CaMKK being
responsible (550). Notwithstanding the question of selectivity of STO-609, the results indicate that in NIH3T3 cells
containing both LKB1 and CaMKK there is a contribution
from CaMKK with AICAR stimulation, which is otherwise
primarily mediated by LKB1. Woods et al. (550) also pro-
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The identification of AMPK upstream kinases proved
as difficult as that of AMPK, taking two decades. Mutation
screens in yeast for sucrose nonfermenting (Snf1) phenotypes did not reveal an upstream kinase. With the advent
of genome-wide studies and the systematic analysis of
protein complexes by mass spectrometry, vital clues
emerged concerning kinases that associated with members of the Snf1 kinase complex (Snf1, Snf4, Sip1, Sip2)
(161, 198). In particular, Pak1 (now termed Sak1) was
associated with snf1, and Tos3 was associated with Snf4.
Nath et al. (361) showed that Pak1 phosphorylated and
activated Snf1 kinase. A similar strategy was also employed independently by Hong et al. (205), who showed
that all three related kinases Tos3, Pak1, and Elm1 were
functional upstream kinases for Snf1 kinase. It required
the triple deletion mutant ⌬tos3, ⌬pak1, and ⌬elm1 to
exhibit the ⌬snf1 phenotype of failure to grow on raffinose (205). The presence of three complementry upstream
Snf1 kinase kinases explained why a single upstream
kinase had not been revealed by mutagenesis. Hong et al.
(205) also showed that the catalytic domain sequence of
mammalian LKB1 was the most closely related kinase to
the corresponding sequences in the three yeast kinases.
Furthermore, recombinant LKB1 phosphorylated and activated mammalian AMPK. Elm1 kinase was also shown
independently to activate Snf1 kinase through use of a
library of glutathione-S-transferase fusions containing all
119 yeast protein kinases (483). Again, it was found that
the triple mutant ⌬tos3, ⌬pak1, and ⌬elm1 had the ⌬snf1
phenotype (483). The possibility that LKB1 was the mammalian homolog to these three yeast Snf1 kinase kinases
was investigated, and rat liver AMPK kinase (AMPKK)
activity was shown to consist of a complex of LKB1 and
its two accessory subunits STRAD␣, ␤, and MO25␣/␤
(185).
LKB1 was also deduced as an upstream kinase for
AMPK by comparison of its peptide substrate specificity
requirements with the sequence around Thr-172 in the
AMPK ␣-subunit activation loop (445). LKB1 has a requirement for Thr at the phosphorylation acceptor site
and Arg at ⫺1 and Leu at ⫺2 features found in the
EFLRT172SCG sequence. With the use of LKB1-deficient
MEFs, it was found that there was negligible activation of
AMPK following treatment with AICAR or H2O2, but the
response could be restored by introducing wild-type (WT)
but not kinase-dead LKB1 alleles (445). Similar results
were also obtained with HeLa cells, which are LKB1
deficient on account of gene methylation (185, 445).
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AMPK IN HEALTH AND DISEASE
3. Tak1 kinase
The transforming growth factor (TGF)-␤-activated
kinase-1 was recently identified as an upstream kinase for
AMPK based on a genetic screen for mammalian kinases
that could rescue the triple deficient strain ⌬tos3, ⌬pak1,
and ⌬elm1 (342). Mice carrying a cardiac specific dominant negative mutation for Tak1 were reported to have a
Wolff-Parkinson-White syndrome reminiscent of the human AMPK ␥2 mutations (557). Tak1-deficient MEFs had
reduced AMPK activation to oligomycin, metformin, and
AICAR, leading the authors to propose a pathway where
Tak1 kinase is upstream of LKB1 and AMPK (557),
whereas the yeast study suggested Tak1 could directly
phosphorylate AMPK (342). Since the AMPK ␥2 mutations
giving rise to Wolff-Parkinson-White syndrome are gainof-function mutations with increased constitutive AMPK
activity, it is not clear how the dominant negative Tak1
gives the same phenotype if Tak1 is an upstream AMPK
kinase.
B. Multisite Subunit Phosphorylation
1. ␣- and ␤-Subunit phosphorylation
While the ␣ subunit Thr-172 is the major AMPK activating site phosphorylated by the upstream kinases
(LKB1, CaMKK␤, and Tak1), both ␣ and ␤ subunits have
multiple phosphorylation sites; however, the functional
role of these is relatively poorly understood. Initial studies by Mitchelhill et al. (340) revealed that native rat liver
␣1 was phosphorylated at Ser-485 and ␤1 at Ser-24/25,
Ser-108, and Ser-182. Further autophosphorylation sites
have been identified using recombinant protein (552)
[␣2Thr-258, ␣2Ser-491(␣1Ser-485) and ␤1Ser-96, Ser-101,
Ser-108]. With the use of immunoprecipitated AMPK from
rat liver and on-line capillary liquid chromatography ESIMS/MS, only ␣2Thr-258 and ␣2Ser-491 were confirmed
(552).
A) SECOND REGULATORY SITES. We have shown that ␣1Ser485 (␣2Ser-491) is involved in the negative regulation of
AMPK by PKA-mediated cAMP signaling (218). Other
studies in the heart also indicate that insulin activation of
Akt increases the phosphorylation of AMPK ␣1/␣2 at
Ser485/491 (209, 456) and inhibits AMPK signaling (37,
265). Ser-485 is in the ␣-subunit COOH-terminal ␤-binding
structure positioned close to ␥. It is not yet clear how the
phosphorylation signal is transmitted to ␣ or how this
inhibits AMPK Thr-172 phosphorylation and enzyme activity.
B) SITES OF UNKNOWN FUNCTION. We have identified a
number of additional phosphorylation sites (␣1 Thr-373,
Thr-379, Thr-481, Ser-485, Ser-499) by MS/MS sequencing
from tryptic digests of recombinant human ␣1 AMPK
phosphorylated in insect cells (Kemp et al., unpublished
data). A recent large-scale mouse liver phosphoproteomics study (517) reported these sites and others (␣1; Thr373, Thr-379, Ser-485, Ser-499, Ser-514, Ser-515, Thr-517),
but site identification was equivocal (summarized in Fig. 5)
and will need corroboration. The phosphoproteomics
analysis missed all known ␤ phosphopeptides and ␣ (Thr172). Even though the data are incomplete, it is striking
that there may be many phosphorylation sites in the
FIG. 5. The distribution of phosphorylation sites on AMPK␣1. The structures
of the ␣-subunit catalytic domain and the
COOH-terminal binding domain are
shown together with the recently reported
phosphorylation sites (see text for details).
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vided evidence to show that CaMKK␤ had a much stronger preference for AMPK as a substrate relative to
CaMKK␣, whereas CaMK-I was phosphorylated comparably by both kinase kinases (550). Using the yeast tripledeficient strain ⌬tos3, ⌬pak1 and ⌬elm1, these authors
showed that CaMKK␤ could rescue the ⌬snf1 phenotype
and allow growth on either glycerol-ethanol or raffinose
(550). This provides strong genetic evidence for the functional similarity of CaMKK␤ with Tos3, Sak1, and Elm1,
even though yeast are devoid of CaMKK␤.
Since CaMKK was established as an AMPK upstream
kinase using iononophores to promote Ca2⫹ signaling,
several studies have shown this pathway is operational
under normal physiological conditions. CaMKK mediates
both thrombin and bradykinin-dependent AMPK activation and stimulation of fatty acid oxidation in endothelial
cells (346, 463). Similarly, CaMKK␤ also mediates thyroid
hormone T3, stimulation of fatty acid oxidation in C2C12
myoblasts (559), and the effect of antigen receptor stimulation in T lymphocytes (486).
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GREGORY R. STEINBERG AND BRUCE E. KEMP
COOH terminus of the ␣ subunit (400 –550). Understanding the role of these sites will depend on identifying
physiological responses where their state of phosphorylation changes and interpreting this in relation to the
emerging structural information on AMPK and intersecting signaling pathways. For example, Ser-514 and Ser-515
are positioned at the ␣␤␥ junction and would seem poised
to affect signal coupling between the subunits.
IV. AMPK REGULATION OF CARBOHYDRATE
METABOLISM
A. Glucose Uptake
The uptake of glucose across the plasma membrane
is dependent on the glucose gradient as well as the expression of transmembrane proteins known as GLUTs,
which display Michaelis-Menten saturation kinetics. In
many tissues including skeletal muscle, GLUT1 and
GLUT4 predominate, with GLUT4 translocation to the
plasma membrane and t tubules considered rate-limiting
for the uptake of glucose in response to stimuli such as
muscle contraction and insulin; however, it should be
noted that under some situations, glucose phosphorylation by hexokinase also appears to be important (154,
155). Importantly, during exercise and/or skeletal muscle
contraction, glucose uptake is increased, an effect which
is independent of the proximal part of the insulin signaling pathway (for review, see Ref. 241). Therefore, the
finding that AICAR stimulated glucose uptake via a phosphatidylinositol 3-kinase (PI3K)-independent pathway
generated significant interest towards the therapeutic utility of AMPK to bypass insulin resistance (46, 190, 331). In
the heart, both AICAR and ischemia also activate AMPK,
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1. Rab GTPase-activating proteins: TBC1D1 and
TBC1D4
The signaling events, by which activation of AMPK
leads to the translocation of GLUT4, is an area of continuing excitement. Recent studies have revealed a critical role for the Rab GTPases TBC1D1 and TBC1D4
(AS160), a topic which has been discussed in detail in
several excellent reviews (82, 426, 572). AS160 contains
two phosphotyrosine-binding (PTB) domains at the NH2
terminus and a Rab-GAP (GTPase-activating protein) domain at the COOH terminus, which is proposed to promote hydrolysis of GTP to GDP by Rab protein(s) on the
GLUT4 storage vesicle (GSV) (246, 577). Subsequent studies demonstrated that AS160 was phosphorylated at five
Akt consensus sites (Ser-318, Ser-570, Ser-588, Thr-642,
and Thr-751) in response to insulin treatment in 3T3-L1
adipocytes (432), an effect also observed in skeletal muscle (67). Surprisingly, AS160 phosphorylation as assessed
using the PAS [the phospho (Ser/Thr)-Akt substrate] antibody was also detected following muscle contraction or
treatment with AICAR, conditions which do not increase
Akt phosphorylation, and the phosphorylation of AS160
correlated well with the increases in glucose uptake under these conditions. In cell-free assays, AMPK is shown
to directly phosphorylate AS160 predominately at Ser-588,
and to a lesser degree at Ser-341 and Thr-642, an effect
which directly enhances binding to 14-3-3 (163). Taken
together, these data suggest that the phosphorylation of
AS160 by AMPK directly regulates binding to 14-3-3,
which in turn controls GLUT4 vesicle recycling. In support of these findings, several groups using transgenic
mice with diminished AMPK signaling have demonstrated
that AS160 phosphorylation is markedly reduced in response to AICAR but not muscle contraction (266, 267,
502), consistent with changes in glucose uptake mediated
by these stimuli. However, recent studies which immunodepleted AS160 from muscle lysates have suggested
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Glycogen and starch provide rapidly accessible
stores of glucose for metabolism in eukaryotes. While we
will concentrate on AMPK’s role in mammalian carbohydrate metabolism, there is rapidly growing evidence that
the corresponding yeast and plant kinases play equally
important roles. In yeast, glycogen and trehalose serve as
carbohydrate reserves that accumulate late in the logarithmic growth phase in response to nutritional stress
(524). Snf1 is required for both the accumulation of glycogen as well as its maintenance by autophagy. Studies in
plants have revealed that SnRK1 is also important for
starch biosynthesis through transcriptional upregulation
of sucrose synthase and ADP-glucose pyrophosphorylase
(397). Futhermore, in transgenic plants overexpressing
SnRK1, there is starch accumulation (328) analogous to
mammals where increased AMPK activity either by naturally occurring mutations or transgenic models leads to
muscle glycogen accumulation (see below).
resulting in increased GLUT4 translocation and increased
glucose uptake (421).
The loss of AMPK ␣2 but not AMPK ␣1 results in
abolished AICAR-stimulated glucose uptake (244), an effect also observed in AMPK ␥3 knockout mice (32) and
mice lacking AMPK ␤2 (Dzamko et al., unpublished data),
suggesting a key role for the AMPK ␣2, ␤2, ␥3 heterotrimer in the AMPK-dependent regulation of glucose uptake
in muscle. The expression of a constitutively active form
of AMPK␣1 is also sufficient to increase glucose uptake in
muscle cells (151). The stimulation of glucose uptake by
AICAR is mediated at least in part by increasing GLUT4
translocation to the plasma membrane (274, 421) but not
to t tubules (287). These effects may be dependent on
nitric oxide synthase (NOS) (152, 448), although these
data are equivocal (476).
AMPK IN HEALTH AND DISEASE
ing on the tissues examined. Interestingly, TBC1D1 has
been identified as a candidate for a severe obesity gene,
suggesting that defects in TBC1D1 signaling may contribute to obesity-related insulin resistance (284).
2. Insulin receptor substrate 1
It is well documented that a single bout of exercise
is capable of improving skeletal muscle insulin sensitivity in patients with type 2 diabetes, long after AMPK
signaling has returned to resting levels (128, 410). One
possible explanation for the enhanced insulin sensitivity postexercise relates to the finding that AMPK phosphorylates insulin receptor substrate 1 (IRS-1) at Ser789 in vitro and that AICAR treatment in C2C12 muscle
cells increases IRS-1 Ser-789 phosphorylation and insulin-stimulated IRS-1 associated PI3K activity (231)
(Fig. 6). However, future studies are required to deter-
FIG. 6. Glucose uptake in skeletal muscle. Schematic illustration of the interaction between AMPK, insulin signaling, and GLUT4 trafficking to
the plasma membrane. Both AMPK and Akt phosphorylate different sites of Rab GTPase activating protein (GAP) domains of TBC1D1 and AS160.
The phosphorylation of AS160 by Akt and AMPK results in 14-3-3 binding and increases GTP-bound Rab and GLUT4 exocytosis to the plasma
membrane. In contrast, while TBC1D1 is phosphorylated by both AMPK and Akt, only AMPK increases 14-3-3 binding and inactivation of TBC1D1,
suggesting that it is the predominant RabGAP regulating AMPK-stimulated GLUT4 exocytosis. The activation of AMPK also results in the
phosphorylation of the transcriptional coactivator PGC1␣, while also phosphorylating HDAC5, causing it to be sequestered out of the nucleus
resulting in enhanced GLUT4 promoter activity and mitochondrial biogenesis. Green arrow, stimulation/activation; red oval, inhibition/deactivation.
Dotted lines indicate proposed action, but direct phosphorylation site has not been identified.
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that in the experiments described above examining AS160
phosphorylation utilizing the PAS antibody were most
likely measuring a close relative to AS160, TBC1D1 (491).
In contrast to AS160, the phosphorylation of which by
both insulin and AMPK increases 14-3-3 binding/inhibition
recent studies (89) in L6 myotubes, demonstrate that
TBC1D1 is phosphorylated on Ser-237 and binds to 14-3-3
proteins in response to AMPK activation. Importantly,
while insulin promotes phosphorylation of Thr-596, it
does not result in 14-3-3 binding (89) or the inhibition of
TBC1D1 activity (87). TBC1D1 expression is high in glycolytic skeletal muscle compared with both soleus and
adipose tissue where AS160 appears to predominate.
Taken together, these data suggest there is a complementary system in which the phosphorylation of AS160
and TBC1D1 by Akt and AMPK is required for maximal
GLUT4 translocation. The hierarchical control/dependence of this pathway is differentially regulated depend-
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GREGORY R. STEINBERG AND BRUCE E. KEMP
mine whether this interaction occurs following exercise
or in response to hormonal stimuli in fully differentiated
skeletal muscle.
A second potential mechanism by which AMPK may
improve insulin sensitivity is through the suppression of
inhibitory serine phosphorylation of IRS1. The serine
phosphorylation of IRS by JNK, IKK, and S6 kinase is an
important factor contributing to the development of insulin resistance in obesity (210). Recent studies have shown
that the activation of AMPK by adiponectin results in
improvements in insulin sensitivity in C2C12 myotubes
(520). This improved insulin sensitivity is attributed to the
downregulation of mTOR/S6 kinase signaling (to be discussed in detail below) and is eliminated following the
overexpression of wild-type Rheb (Ras homology-enriched in brain) or a TSC2 mutant lacking the AMPK
phosphorylation site (TSC2 Ser1345Ala). It will be interesting to examine whether a similar phenomenon mediates improvements in insulin sensitivity postexercise in
obese insulin-resistant skeletal muscle.
B. Glycolysis
3. Transcriptional regulation of GLUT4
and hexokinase
Glycogen represents the most easily accessible largescale source of energy and is an important substrate in
muscle during exercise and in the liver during fasting. The
absolute level of glycogen is a function of both its production (glycogenesis) and its breakdown (glycogenolysis), which are mediated by the enzymes glycogen synthase (GS) and glycogen phosphorylase (GP), respectively. The role of AMPK in the regulation of glycogen
metabolism in muscle has been an area of great interest
for a number of laboratories and dates back to initial
studies by Carling and Hardie (78) demonstrating that
AMPK phosphorylates GS at Ser-7, a known inhibitory site
of the enzyme. Experiments several years later by Young
et al. (568) added further evidence for a role of AMPK in
glycogen metabolism by showing that activation of AMPK
by AICAR in rat muscle cell preparations caused an increase in GP activity. However, subsequent studies demonstrated that this was an artifact due to allosteric activation of
GP by the AICAR derivative 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranosyl-5⬘-monophosphate (ZMP) (302). Given
that AMPK causes an activation of GP and the suppression of GS, it was anticipated that AMPK would reduce
muscle glycogen levels; however, in vivo the chronic activation of AMPK using AICAR actually increased muscle
glycogen content and GS activities (20, 202). However,
subsequent studies demonstrated that the AICAR effect
on glycogen content and GS activities was due to an
increase in glucose uptake and cytosolic glucose and subsequently glucose-6-phosphate (G-6-P), which allosterically
activates GS independent of phosphorylation (20). These
findings are supported by studies in AMPK ␥3 mutant Hampshire pigs (333) and mice (32, 301), which have increased
AMPK signaling and muscle glycogen contents, while the
Physiol Rev • VOL
The cardiac isoform of 6-phosphofructokinase-2 (PFK2)
controls the synthesis and degradation of fructose 2,6bisphosphate, which in turn is a potent stimulator of
PFK1, a key enzyme in glycolysis (216). PFK2 activity is
regulated through phosphorylation within the regulatory
domain at the COOH terminus that contains phosphorylation sites at Ser-466 and Ser-483 (126). In the heart,
AMPK can increase the rate of glycolysis through phosphorylation of PFK2 at Ser-466 (313). There are four genes
for PFK-2 (PFK2FB1-4) in mammals with alternate splice
variants generating additional isoforms (411); in addition
to the cardiac isoform PKK2FB2, the ubiquitously expressed PKK2FB3 isoform is also phosphorylated (Ser461) and activated by AMPK and is responsible for stimulating glycolysis in leukocytes (314).
C. Glycogen Metabolism
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Increased insulin-stimulated glucose uptake following exercise may also be mediated through transcriptional
upregulation of rate-limiting enzymes for the uptake of
glucose, such as GLUT4 and hexokinase. In skeletal muscle, the chronic activation of AMPK by AICAR has been
shown to increase GLUT4 (581) and hexokinase II (HKII)
(480) transcription similar to exercise training (202, 242,
373, 541). Several years ago, studies by Salt et al. (429)
reported that AMPK␣2 was enriched in the nucleus in
cultured INS-1 and CCL13 cells but did not observe any
change in the cytoplasmic/nucleus ratio of ␣2 in response
to glucose deprivation (INS-1) or arsenite (CCL13) treatment that would be expected to activate AMPK in these
cells. More recently, AMPK␣2 has been shown to localize to the nucleus of human skeletal muscle following
exercise (323, 324, 475). The mechanism by which
AMPK increases GLUT4 expression (26, 391) involves
the transcription factors myocyte enhancer factor
(MEF) 2A and MEF 2D (295, 296). As illustrated in
Figure 6, the activity of these transcription factors may
be controlled through AMPK phosphorylation of both
peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1␣) (209) and histone deacetylase (HDAC) 5 (325). Taken together, these data support the concept that AMPK is a positive regulator of
GLUT4 and HKII gene transcription; however, it should
also be noted that studies using mice with mutated
AMPK signaling suggest that AMPK activation during
exercise is by no means obligatory (203, 245).
1. Phosphofructokinase 2
AMPK IN HEALTH AND DISEASE
1041
inverse is observed in AMPK ␣2 null mice (240). Therefore, while the phosphorylation of GS at Ser-7 by AMPK
does inhibit GS activity under basal conditions, stimuli
that increase the concentration of intracellular G-6-P
(such as AICAR-stimulated glucose uptake) are capable
of overcoming the inhibitory effects of Ser-7 phosphorylation by AMPK.
D. Hepatic Glucose Production
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FIG. 7. AMPK inhibits hepatic gluconeogenesis. The transcriptional
control of gluconeogenesis is mediated by CRTC2 and AMPK. During
fasting, CRTC2 is sequestered in the nucleus where it activates CREB by
enhancing interaction with the transcriptional coactivator PGC1␣ and
transcription factors HNF4␣ and Fox01 to control the expression of
PEPCK and G6Pase. The activation of AMPK by LKB1 (which also
phosphorylates the SIK1 kinase) results in the phosphorylation of
CRTC2 and results in its exocytosis from the nucleus by 14-3-3 proteins,
which in turn shuts down the gluconeogenic program. In addition,
AMPK directly reduces gluconeogenic gene expression by phosphorylating HNF4␣. Green arrow, stimulation/activation; red oval, inhibition/
deactivation. Dotted lines indicate proposed action, but direct phosphorylation site has not been identified.
regulated by AMPK and that this regulation may be important for the regulation of L-PK (279). AMPK has been
shown in vitro to phosphorylate HNF-4␣ on Ser-304, reducing the ability of the transcription factor to form the
homodimers required for stability and DNA binding (206).
This experiment provides a link between AMPK activation and the regulation of L-PK, but future studies are
required to determine whether this relationship is also
observed in vivo in genetic models of AMPK deficiency.
1. CREB-regulated transcription coactivator 2
Recently, CREB-regulated transcription coactivator 2
[CRTC2; formally known as transducer of regulated
CREB activity 2 (TORC2)] has emerged as a critical regulator of gluconeogenesis (264). Under fasting conditions,
glucagon triggers the transcription of gluconeogenic
genes via the cAMP-responsive factor CREB (CRE binding protein) and subsequent recruitment of the coactiva-
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The regulation of hepatic glucose production is critical for maintaining glucose homeostasis and is vital for
survival. As a result, this system has adapted a number of
diverse and redundant regulatory cues, which allow for
the storage of glucose as glycogen and triacylglycerols
following a meal and to conversely increase glucose output during a fast. Since AMPK is sensitive to changes in
hormones and nutrients, it is not surprising that it plays a
critical role in the regulation of this pathway by acting to
regulate a number of pathways in the liver and to sense
circulating glucose levels in the brain. The activation of
AMPK was first shown to inhibit hepatic glucose production by Bergeron et al. (44) using AICAR during a clamp in
rodents. However, studies by Wasserman and colleagues
(72) demonstrated the opposite effects in dogs even in the
presence of high levels of physiological insulin, an effect
believed to be mediated through indirect stimulation of
glycogenolysis by ZMP. The most convincing evidence
supporting a role for AMPK in inhibiting glucose production comes from AMPK ␣2 knockout mice which display
fasting hyperglycemia, glucose intolerance, and increased
hepatic glucose output (16, 519). Similarly, LKB1 floxed
mice injected with adenovirus Cre are also hyperglycemic
and exhibit increased mRNA expression of gluconeogenic
enzymes (446). In addition, studies in isolated hepatocytes treated with metformin (582) and adiponectin (16,
561) support an important role for AMPK in suppressing
hepatic glucose output effects mediated by reduced gluconeogenic gene expression as will be discussed below.
Hepatic glucose production is controlled through
phosphorylation of transcriptional coactivators and transcription factors, resulting in the inhibition or activation
of their cognate promoters, that in turn control the expression of enzymes critical for regulating lipid and carbohydrate metabolism (Fig. 7). Initial experiments demonstrated that the activation of AMPK negatively regulates the transcription of the gluconeogenic enzymes
L-type pyruvate kinase (L-PK) (117, 278), phosphoenolpyruvate carboxykinase (PEPCK) (300), and glucose-6phosphatase (G-6-Pase) (548) in response to elevated glucose. L-PK, along with GLUT-2 and other genes involved
in glucose and lipid metabolism, is regulated by the transcription factor hepatic nuclear factor 4␣ (HNF-4␣) (337).
It has recently been demonstrated that HNF-4␣ can be
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GREGORY R. STEINBERG AND BRUCE E. KEMP
E. Hypothalamic Glucose Sensing
In recent years, the role of central pathways in regulating hepatic glucose fluxes has gained considerable support
(393). Specifically, glucose-sensing neurons within the ventral medial hypothalamus have been shown to play a
critical role in counterregulatory responses to hypoglycemia, with several reports demonstrating that activation of
AMPK in the hypothalamus with AICAR (319, 320) or
2-deoxyglucose (10) increases hepatic glucose production. Glucose sensing also occurs in the portal vein with
the activation of AMPK being important for controlling
glucose uptake in skeletal muscle, although the signals
regulating this response are not understood (69).
A. Fatty Acid Uptake
The first step in the regulation of fatty acid (FA)
metabolism involves their movement across the plasma
membrane. In obesity, skeletal muscle displays an increased propensity towards the uptake of fatty acids,
suggesting this may be a principal defect contributing to
the accumulation of intramuscular lipids (56). While fatty
acids due to their hydrophobic nature may passively diffuse across plasma membranes via a flip-flop evidence
from genetic models of fatty acid translocase (FAT/
CD36), fatty acid binding protein (FABP) deficiency has
indicated that these key proteins also play an essential
role in mediating the process (for review, see Ref. 53).
The uptake of fatty acids is dependent on the metabolic
rate of the tissue, as both contraction (55) and exercise
(447) increase fatty acid uptake suggesting that AMPK
may be important. Supporting this idea are studies demonstrating that the pharmacological activation of AMPK
with AICAR increases both skeletal muscle (448, 473) and
heart (305, 447) fatty acid uptake. AMPK may also be
important in the regulation of FAT/CD36 transcription,
with endurance training and chronic electrical stimulation increasing protein expression levels (54), while fasting-induced increases in FAT/CD36 are suppressed in
AMPK ␥3 null mice (301). However, it should be cautioned that in muscle specific AMPK DN mice, total fatty
acid uptake is not altered in response to muscle contraction (137), a finding also supported by Turcotte et al. (506)
who have shown that an ERK inhibitor inhibits contraction-stimulated fatty acid uptake. If AMPK is involved to
some degree in fatty acid uptake, future studies will need
to identify whether AMPK phosphorylates FAT/CD36 directly or whether translocation is mediated indirectly as is
the case for the regulation of glucose uptake via TBC1D/
AS160.
V. AMPK REGULATION OF LIPID METABOLISM
During fasting, postabsorptive conditioned lipids are
the predominant substrate for the maintenance of whole
body energy metabolism (251). The ability to efficiently
store fuel in the form of energy-dense lipids and their
mobilization during times of low carbohydrate availability
was an essential development in evolution, allowing organisms to survive periods of famine and prolonged fasting. This coordinated release of free fatty acids from
adipose tissue combined with the ability to actively fine
tune the gradient between fat and carbohydrate metabolism in metabolically active tissues in response to a number of dynamic physiological stimuli requires an integrated metabolic system of control. This synchronized
regulation of metabolism occurs acutely, as well as
through transcriptional control by AMPK. A list of known
AMPK substrates and their role in regulating lipid metabolism is shown in Figure 8.
Physiol Rev • VOL
B. Fatty Acid Partitioning
Once fatty acids are taken up across the plasma
membrane and activated to fatty acyl-CoA, they can either
be directed towards oxidation or storage depending on
the metabolic demands of the tissue at the time. In addition, in lipogenic tissues such as adipose and liver, glucose can also be stored as lipid. AMPK plays a critical role
in determining the fate of glucose and fatty acids through
regulation of key substrates, as discussed below, and
summarized in Figure 8.
1. Acetyl-CoA carboxylase
Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl CoA to malonyl CoA, a metabolic intermediate, that is either incorporated into fatty acids during
their synthesis by fatty acid synthase or acts allosterically
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tor CBP and CRTC2 to the nucleus (Fig. 7). This recruitment leads to the expression of the coactivator PGC-1␣,
which in turn drives the transcription of PEPCK and
G-6-Pase. The phosphorylation of CRTC2 by AMPK and
AMPK-related kinases, salt-inducible-kinase1 (SIK1) and -2
promotes CRTC2 binding to 14-3-3 proteins in the cytoplasm and prevents the translocation of CRTC2 to the
nucleus, thereby reducing CREB-dependent mRNA expression of PEPCK and G-6-Pase (264). The mutation of
CRTC2 Ser171Ala blunts SIK1- and AICAR-mediated suppression of G-6-Pase, PEPCK, and PGC-1␣ expression in
primary hepatocytes, suggesting that CRTC2 might be a
critical downstream target of both AMPK and the AMPKrelated kinase SIK1 in the regulation of hepatic gluconeogenesis. Supporting this finding is the observation that
the hyperglycemia in liver-specific LKB1 null mice (446) is
much more severe than that seen in mice with liver deletion of both AMPK␣1 and -␣2 (16).
AMPK IN HEALTH AND DISEASE
1043
to inhibit carnitine palmitoyltransferase 1 (CPT1), which
controls transport of activated fatty acids into the mitochondria for oxidation (48, 350). ACC exists in two isoforms (3): ACC1 (also know as ACC␣) and ACC2 (also
known as ACC␤), with the major difference between the
isoforms being an NH2-terminal extension of 146 amino
acids in ACC2, localizing the enzyme to mitochondria (2).
This localization is thought to be important for ACC2
regulation of fatty acid oxidation, since malonyl-CoA will
be produced in close proximity to CPT1, which resides on
the outer mitochondrial membrane. Liver, brown adipose
tissue, and the brain contain both ACC1 and ACC2 isoforms, while skeletal muscle and cardiac muscle contain
predominantly ACC2, whilst white adipose tissue expresses predominantly the ACC1 isoform (3, 229).
Wakil and colleagues (4 – 6, 311) have provided evidence supporting a critical role of the two ACC isoforms
in the regulation of fatty acid metabolism using ACC1 and
ACC2 null mice. These studies have shown that whole
body ACC2 null mice have greatly reduced levels of skeletal muscle malonyl-CoA but normal levels of malonylCoA in adipose tissue and liver. This results in much
higher rates of fatty acid oxidation in skeletal muscle (4,
6) and surprisingly (given the low levels of ACC2 exPhysiol Rev • VOL
pressed) also adipose tissue (370). Importantly, these elevated rates of fatty acid oxidation result in a lean phenotype that protects mice against high-fat diet-induced
muscle and liver insulin resistance despite hyperphagia
(4, 6). An important note from these studies is that this
pronounced phenotype occurs despite normal ACC1 expression, which would be expected to compensate for the
reduced malonyl-CoA especially in tissues such as liver
and adipose tissue, thus highlighting the potential importance of the intracellular localization of malonyl-CoA for
the regulation of fatty acid oxidation. In the liver-specific
ACC1 null mice, there is a 70% reduction in malonyl-CoA
levels but no change in fatty acid oxidation (311). However, in contrast to these findings suggesting two autonomous pools of malonyl-CoA in the regulation of fatty
acid oxidation, studies by Savage et al. (436) using antisense oligonucleotide have found additive effects of
ACC1 and ACC2 inhibition on liver fatty acid oxidation,
indicating that in this model there is overlap and that
malonyl-CoA derived from ACC1 can suppress fatty acid
oxidation in liver tissue.
The whole body deletion of ACC1 is embryonic lethal, demonstrating the absolute requirement for the enzyme in fatty acid synthesis during development (5). In
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FIG. 8. Lipid metabolism and the role of AMPK. AMPK controls the fate of fatty acids in the cell by controlling rates of uptake by inducing the
translocation of CD36 to the plasma membrane. AMPK suppresses malonyl-CoA content by phosphorylating and inhibiting ACC1 and therefore
suppressing fatty acid synthesis and increasing mitochondrial ␤-oxidation, respectively. Futile cycling of fatty acids is suppressed by AMPK
inhibition of TG synthesis and TG hydrolysis through the phosphorylation of GPAT and HSL, respectively. AMPK also reduces FA synthesis by
inhibiting the transcription factor SREBP1c, which controls the entire synthesis pathway or by directly inhibiting the activity of FAS. Green arrow,
stimulation/activation; red oval, inhibition/deactivation. Dotted lines indicate proposed action, but direct phosphorylation has not been identified.
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GREGORY R. STEINBERG AND BRUCE E. KEMP
2. Malonyl-CoA decarboxylase
Malonyl-CoA levels are also regulated by decarboxylation by the enzyme malonyl-CoA decarboxylase (MCD)
(134). The overexpression of MCD in liver increases hePhysiol Rev • VOL
patic FA oxidation and rescues whole body insulin sensitivity in obese rats (12). While MCD activity is increased
in response to AICAR and contraction in skeletal muscle
(385, 423), this regulation appears to be indirect as AMPK
does not phosphorylate MCD in vitro (177). Increased
rates of fatty acid oxidation in the diabetic rat heart are
associated with elevated MCD activity (425), an effect
that is reversed in the presence of MCD inhibitors (135).
However, recent studies by Reszko et al. (407) have suggested that the primary determinant of malonyl-CoA production under physiological conditions and in the absence of inhibitors to MCD is the rate of acetyl-CoA
carboxylation.
3. Fatty acid synthase
Fatty acid synthase (FAS) is a multifunctional enzyme found in lipogenic tissues such as adipose and liver,
which catalyzes the synthesis of long-chain fatty acids,
primarily palmitate, using acetyl-CoA and malonyl-CoA as
substrates (Fig. 8). A decade ago, Foretz et al. (147)
demonstrated that AMPK inhibited the glucose-stimulated
transcription of FAS. FAS expression is regulated through
the transcription factor sterol regulatory element binding
protein 1c (SREBP1c), which is a critical transcription
factor regulating many lipogenic genes (FAS, GPAT,
SCD1, etc.). In the liver, overexpression of a CA-AMPK
(146) or treatment with metformin (582) reduces SREBP1c
gene expression, but the exact mechanism by which
AMPK regulates this transcription factor is still unknown.
Recent studies have suggested that FAS may also be
regulated postranscriptionally by AMPK, as treatment
with AICAR or peroxynititrites which activate AMPK inhibits FAS activity within minutes in 3T3-L1 cells (13).
These effects were inhibited by the overexpression of an
AMPK dominant negative or following treatment with
Compound C (13). Further studies utilizing 32P labeling
confirmed that both AICAR and metformin dramatically
increased the incorporation of label into FAS, an effect
associated with increased Thr phosphorylation at an unidentified site (13). As obesity-associated hepatic steatosis is associated with increased SREBP1c and FAS, identifying the exact mechanism by which AMPK inhibits their
activity may help reveal novel treatments for this prevalent condition.
C. Triacylglycerol Turnover
The regulation of triglyceride (TG) turnover is a balance between biosynthetic pathways responsible for TG
synthesis and hydrolysis (Fig. 8). In recent years, the
control of this pathway has become an area of great
interest because even though total TG content in muscle
and liver does not appear to adversely affect insulin sensitivity, lipid intermediates in the form of DG, ceramides,
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terms of cross-talk between malonyl-CoA produced by
ACC1 and ACC2, the ACC1 liver-specific null mice have
reduced accumulation of liver triacylglycerol (TAG) when
fed a short-term high-sucrose/low-fat diet (311). Similarly,
studies by Savage et al. (436) using anti-sense oligonucleotides have also found that the suppression of ACC1
inhibits lipogenesis, whereas a reduction in ACC2 has no
effect on lipogenesis, supporting the theory of independent pools of malonyl-CoA for the regulation of lipogenesis. However, a recent report in ACC1 liver-specific null
mice questions this conclusion because their ACC1 liverspecific null mice had increased expression levels of
ACC2 and normal fatty acid synthesis and importantly
were not protected against fatty liver disease in response
to a high-sucrose diet (183). Overall, there is not a clear
consensus on the degree of malony-CoA overlap between
synthesis and oxidative pathways between ACC isoforms.
Future studies, in mice with targeted tissue-specific
knock ins, of dead ACC2 or ACC1 that preserve the respective enzyme architectures may be necessary to clarify
this important question.
The short-term regulation of ACC is achieved by
reversible phosphorylation and to a lesser extent, allosteric regulation by citrate (350). Early experiments using
liver purified ACC and AMPK concluded that AMPK phosphorylated ACC at three sites: Ser-79, Ser-1200 (351), and
subsequently Ser-1215 (124). A limited proteolysis approach suggested that Ser-79 was the major site responsible for the inhibition of ACC activity (124), and this was
later confirmed using site-directed mutagenesis (176). Experiments using AICAR confirmed that Ser-79 is the physiologically relevant phosphorylation site for inhibition of
ACC1 by AMPK (107), but due to the NH2-terminal extension, the equivalent site in ACC2 is Ser-221 (1). While
initial studies conducted in vitro showed that Ser-79 phosphorylation may be inhibited by PKA phosphorylation of
Ser-77 (352), this was not observed in hepatocytes treated
with glucagon, suggesting the Ser-77 phosphorylation is
not physiologically significant (453), a finding also confirmed in rodent skeletal muscle (542). Phosphorylation
of ACC1 (Ser-79) and ACC2 (Ser221) by AMPK is commonly used as an in vivo measure of AMPK signaling in
response to a variety of stimuli ranging from hormones to
exercise, but so far, genetic evidence supporting their
physiological importance at the whole animal level has
not been available. In this regard, we have recently generated mice carrying Ser/Ala knock ins for these sites that
may provide clues to their physiological importance in
regulation of fatty acid metabolism.
AMPK IN HEALTH AND DISEASE
and long-chain acyl-CoA activate serine threonine kinases
and protein phosphatases that directly inhibit insulin signaling (530). One hypothesis is that in obesity and insulin
resistance there is a mismatch between rates of esterification and hydrolysis, which results in futile cycling and
the build up of reactive lipid intermediates, which in turn
may impede insulin sensitivity.
1. Glycerol-3-phosphate acyl-transferase
Physiol Rev • VOL
also critical for regulating lipid esterification and in turn
insulin sensitivity, suggesting that AMPK regulation of
these enzymes may be a second point of regulation in this
important pathway.
2. Hormone-sensitive lipase
The breakdown of TG is accomplished through sequential hydrolysis of three FA residues.
While researchers have known for several decades
that adipose tissue lipolysis is sensitive to catabolic stimuli (49, 200, 412), it was several decades before the protein responsible for much of this lipolytic activity, justly
named hormone-sensitive lipase (HSL), was purified from
rat epidydimal fat pads. While HSL is known to be classically activated by PKA phosphorylation at serines 563,
659, and 660 (17), experiments with adipocytes and skeletal muscle suggested that AMPK inhibits this activation
through phosphorylation at Ser-565 (107, 159, 160, 415,
482, 528, 531). Indeed, recent studies in 3T3-L1 adipocytes
expressing DN and CA-AMPK mutants or adipocytes from
AMPK ␣1 knockout mice support the role of AMPK inhibiting ␤-adrenergic-stimulated lipolysis in adipose tissue
(122). Since AMPK is regulated by a variety of hormonal
stimuli (as discussed below), it will be important to test
whether these hormones control lipolysis in vivo via
AMPK. In addition, the recent finding that adipose tissue
triglyceride lipase (ATGL) is an important TG lipase suggests that there may be opportunities to regulate lipid
hydrolysis upstream of HSL.
AMPK also appears to negatively regulate HSL activity during muscle contraction. In both resting (354) and
contracting (455) muscle, TG hydrolysis is suppressed in
response to AMPK activation by AICAR. Consistent with
reductions in TG hydrolysis, PKA-stimulated HSL activity
is reduced in L6 myotubes treated with AICAR (531) or
following overexpression of a constitutively active AMPK
(532). In vivo evidence supporting a role of AMPK inhibition of HSL stems from findings in human skeletal muscle
that enhanced activation of AMPK, due to glycogen depletion prior to exercise, prevents exercise-induced increases in HSL activity (531). Similarly, prolonged endurance exercise (90 min) that results in significant activation of AMPK corresponds with reduced HSL activity and
increased Ser-565 phosphorylation (528). It should be
noted that this inhibitory effect of AMPK on HSL activity
has not been observed in all studies (415); however, the
majority of studies indicate that increased activation of
AMPK is capable of overriding elevations in epinephrine
and PKA stimulation of HSL.
D. Mitochondrial Biogenesis
A reduction in mitochondrial density is believed to be
a critical factor contributing to the accumulation of intra-
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The synthesis of TG is regulated in part by the supply
of the substrates glycerol-3-phosphate (from carbohydrate metabolism) and fatty acyl-coenzyme A supplied
through intracellular hydrolysis and/or transport from exogenous sources. The first committed step in phospholipid and TG synthesis is the formation of lysophosphatidic acid from glycerol-3-phosphate. GPAT is the ratelimiting enzyme in this reaction and is reported to exist as
both mitochondrial (mt) and microsomal isoforms based
on N-ethylmaleimide (NEM) inhibition. TG formation is
regulated by the mitochondrial 828 residue isoform (accession number Q8N1G6) that is an integral membrane
protein (221). The mtGPAT gene consists of 19 exons on
chromosome 10q25.2. Null mice have reductions in body
weight, total adipose tissue weight, and lower hepatic TG
content (181), while overexpression of GPAT reduces
fatty acid oxidation and increases TG esterification (296).
The activation of AMPK by AICAR reduces hepatic GPAT
activity and TG esterification (354). Similarly, the activation of AMPK in liver and adipose tissue by endurance
exercise also reduced mtGPAT activity (385) effects,
which were not observed in skeletal muscle, likely due to
its relatively low expression compared with lipogenic
tissues such as liver and adipose. In agreement, we have
also detected no significant reduction of mtGPAT activity
following endurance exercise in human skeletal muscle
(529). Despite the significant correlative data, perhaps the
best evidence supporting a role of AMPK in regulating
mtGPAT activity comes from Muoio et al. (354) who
reported that incubating mtGPAT with recombinant
AMPK inhibits the enzymes activity. In this study, it remains possible that an associated protein may be inhibiting mtGPAT activity as direct GPAT phosphorylation was
not shown and AMPK was added to a mitochondrial fraction of GPAT from rat liver that was from only partially
purified rat liver. While the inspection of the mtGPAT
sequence does reveal several likely AMPK sites in the
80-residue cytoplasmic sequence, future studies are required to determine whether the effects of AMPK on
GPAT activity are direct and the potential phophorylation
sites involved. Interestingly, fasting that activates liver
AMPK (543) inhibits both GPAT and DGAT activity; effects that are promptly reversed by refeeding, which terminates AMPK signaling (22). Several recent studies (83,
97, 297, 343, 571) have shown that DGAT1 and DGAT2 are
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GREGORY R. STEINBERG AND BRUCE E. KEMP
Physiol Rev • VOL
ings have also been confirmed with chronic AICAR treatment (245, 387, 480).
E. Cholesterol Synthesis
HMGR is the rate-limiting enzyme for isoprenoid
and cholesterol synthesis and a substrate for AMPK.
The geranylgeranyl and farnesyl groups derived from
the mevalonate pathway play an important role in providing lipid anchors for many signaling proteins including the small GTP binding proteins Rab, Rac, and Rho
which regulate vesicular transport and cytoskeletal
membrane interactions (329). Deletion of HMGR is embryonic lethal at the blastocyst stage, indicating that
the mevalonate pathway is indispensable for development (371). HMGR is highly regulated with its transcription and translation under negative-feedback control mediated by sterols and nonsterol metabolites derived from mevalonate. There is also diurnal and
dietary-induced changes in cholesterol synthesis in the
liver that follow changes in liver HMGR mRNA. The
catalytic activity of HMGR is inhibited by phosphorylation (42) on Ser-872 by AMPK (871 in mouse or 872 in
humans) (101). The crystal structure of the dephosphorylated catalytic portion of human HMGR (460 – 888)
has been solved (228), and inspection of the structure
indicates that phosphorylation may act by altering
NADPH affinity since the adjacent Arg-871 is directly
involved in binding NADPH. However, further work is
required to confirm this mechanism kinetically (374).
HMGR is inhibited by ATP depletion, and with the use
of transfected cells, it has been found that mutation of
HMGR Ser871Ala renders it insensitive to AMPK-mediated inhibition in response to ATP depletion. Phosphorylation of Ser-871 does not play a role in HMGR turnover (574) in vivo, and the posttranscriptional feedback
downregulation of the Ser871Ala mutant HMGR is normal when cells are incubated with mevalonate, 25hydroxycholesterol, or LDL (433). The finding that adiponectin (500, 561) activates AMPK and reduces cholesterol synthesis and atherosclerosis in ApoE-deficient
mice (376) suggests that the hormonal regulation of
HMGR via AMPK may be critical for regulating cholesterol metabolism in vivo.
VI. AMPK REGULATION OF PROTEIN
METABOLISM, CELL POLARITY, GROWTH,
AND APOPTOSIS
A. Protein Synthesis
Protein synthesis accounts for a large proportion of
cellular energy use, and inhibition of this pathway is
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muscular lipids and insulin resistance (389). While it has
been known for several years that one of the most rapid
and robust responses to exercise training is an increase in
mitochondrial content and capacity (201), Winder et al.
(541) were the first to demonstrate that chronic AICAR
treatment stimulated mitochondrial biogenesis. Similar
results were also observed following treatment with the
creatine analog ␤-guanidionapropriate (␤-GPA), which
competitively inhibits creatine uptake and lowers ATP
content (45). The critical role of AMPK in regulating
mitochondrial biogenesis basally and in response to
chronic AMPK activators such as ␤-GPA or AICAR was
proven to be causally related, as these effects were eliminated in mice with decreased AMPK signaling (242, 245,
496, 584).
Transcription factors and coactivators control mitochondrial biogenesis. Nuclear respiratory factor-1 and -2
(NRF-1, NRF-2) are critical transcriptional regulators of
nuclear genes encoding all five electron chain complexes
(322). Importantly, NRF expression is increased by the
chronic activation of AMPK using ␤-GPA (45). Another
important regulator of mitochondrial content is the inducible coactivator of nuclear receptors, PGC1␣, which is
increased in response to activation of AMPK and is reduced in AMPK␣2 null (222) and DN mice (531). Recent
studies by Jager et al. (230) demonstrate that PGC1␣
coimmunoprecipitates with AMPK ␣2 and that it can directly phosphorylate PGC1␣ on Thr-177 and Ser-538 (Fig.
6). Increased phosphorylation is proposed to cause an
increase in PGC-1␣ protein action on the PGC-1␣ promoter resulting in increased expression of PGC1␣, as well
as GLUT4 (as discussed previously) and mitochondrial
oxidative genes such as cytochrome c and uncoupling
protein (UCP) 1. Since PGC-1␣ activity and expression
are reduced in type 2 diabetes (344), activators of AMPK,
such as adiponectin, may reverse defects in mitochondrial
content and potentially the insulin resistance observed in
this population (98).
Despite the robust AMPK-dependent effects of AMPK
activators at rest in stimulating mitochondrial biogenesis,
Jorgensen and co-workers (243, 245) have shown that a
lack of AMPK ␣2 does not attenuate training-induced
mitochondrial biogenesis, suggesting that other pathways
during exercise may be more critical. Similar results are
also reported in LKB1 null mice (496). One possibility may
involve calcium signaling, since the treatment of muscle
with calcium ionophores rapidly induces mitochondrial
biogenesis (372, 553); however, it should be noted that in
both models ␣1 activation in response to endurance exercise is relatively normal. In addition to regulating mitochondrial biogenesis, AMPK has also been shown to specifically regulate the expression of UCP3, an effect which
may be independent of changes in mitochondrial density.
Zhou et al. (583) demonstrated that AICAR and hypoxia
increased UCP3, suggesting a role for AMPK. These find-
AMPK IN HEALTH AND DISEASE
Rheb GAP activity resulting in the inhibition of mTORC1
signaling (225). Subsequent studies have confirmed these
findings by demonstrating that the deletion of LKB1 and,
subsequently, AMPK signaling, results in a hyperactive
mTORC1 (106, 444). The second mechanism by which
AMPK impairs protein synthesis is by directly phosphorylating the mTOR binding partner raptor at Ser-722 and
Ser-792 (175). This phosphorylation by AMPK leads to
binding of raptor to 14-3-3 proteins and therefore causes
inactivation of the mTOR complex. Lastly, mTOR is also
inhibited through feedback inhibition by S6 kinase (7) in
skeletal muscle in response to endurance and resistance exercise training (23). The recent findings that the mTORC
pathway controls many aspects of metabolism in addition to
protein synthesis, including glucose homeostasis (511), fat
metabolism (7), and orexigenic peptide expression (109),
suggest that AMPK modulation of mTORC may mediate
many of the reported effects of AMPK in the regulation of
metabolism, but future studies are indeed warranted to fully
understand these interactions.
FIG. 9. AMPK inhibition of protein metabolism. Schematic illustrating the major pathways by which AMPK reduces protein synthesis and may
induce apoptosis. AMPK inhibits the TORC1 complex by phosphorylating the tumor suppressor complex TSC1/TSC2, which negatively regulates
TORC1 by inactivating the GTPase Rheb. Phosphorylation of TSC1/TSC2 may also mediate the effects of autophagy. TORC1 activity is also impaired
through phosphorylation of Raptor, which leads to 14-3-3 binding and physical separation of the TORC1 complex. Lastly, AMPK inhibits the initiation
of mRNA translation by inhibiting peptide elongation by activating the eEF-2 kinase. Secondary effects of TORC1 inhibition by AMPK may be to
reduce S6 kinase activity, which is a negative regulator of IRS signaling, which may explain the positive effects of AMPK on insulin sensitivity. Green
arrow, stimulation/activation of AMPK; red oval, inhibition/deactivation.
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therefore an important mechanism by which to maintain
cellular ATP under metabolic stress. AMPK inhibits protein synthesis at multiple points (Fig. 9). It phosphorylates
and activates eukaryote elongation factor 2 kinase on
Ser-398, which in turn phosphorylates eEF2 and inhibits
protein synthesis (62). It also inhibits the mammalian
target of rapacycin complex (mTORC) pathway (for review, see Ref. 275). This pathway is regulated through
phosphorylation of tuberous sclerosis 2 (TSC2), which
acts as a GTPase-activating protein (GAP) for the small
GTPase Rheb. Rheb in turn directly binds the kinase
domain of mTOR, activating the complex which in turn
activates the S6 kinase while inhibiting 4E-BP1 (Fig. 9).
While insulin and insulin-like growth factor I (IGF-I) signaling stimulate protein synthesis and the activation of
mTORC, AMPK counteracts these effects to inhibit protein synthesis in skeletal muscle (52, 535), liver (208, 405),
and cardiac muscle (86, 207). This effect is mediated by
AMPK via three distinct mechanisms. The first involves
the phosphorylation of TSC2 at Ser-1387, which enhances
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GREGORY R. STEINBERG AND BRUCE E. KEMP
der a number of conditions including cancer cell proliferation.
B. Cell Growth and Apoptosis
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C. Regulation of Cell Polarity and Ion Flux
Cells maintain an electrical potential difference (voltage) across their plasma membranes so that the cytoplasm is usually electrically negative relative to the extracellular fluid. This resting membrane potential is essential
for cell survival and is maintained through generation of
electrical potentials between ions mediated through active and inactive transport. In quiescent cells, the maintenance of this resting membrane potential is by far the
greatest consumer of cellular ATP, and the resting membrane potential plays a central role in the excitability of
nerve and muscle cells. Thus far, AMPK has not been
shown to play a role in maintaining membrane potential.
An important aspect of maintaining ionic equilibrium
involves the regulation of cell volume. Hallows and colleagues have identified that an important role for AMPK is
in the inhibition of the activity of the cystic fibrosis transmembrane conductance regulator Cl⫺ channel (CFTR)
(180) and epithelial Na⫹ channel (eNaC) (74). Although
AMPK directly phosphorylates CFTR to regulate its activity (180), the inhibition of eNaC involves upregulation of
the ubiquitin Ligase Nedd4 –2 which reduces EnaC expression in the plasma membrane (47). In the kidney,
the Na⫹-K⫹-2Cl⫺ cotransporter (NKCC1 and NKCC2) is
highly expressed in the distal nephron and is important
for regulating whole body sodium balance. An increase in
osmotic stress activates AMPK (148), increasing the activity of NKCC2 and thereby helping to maintain osmotic
balance (149). AMPK also regulates the activity and recruitment of sarcolemmal K (ATP) channels in cardiomyocytes (481).
In Drosophila, AMPK-null mutants are lethal with
severe abnormalities in cell polarity and mitosis, similar
to those of lkb1-null mutants (280). Constitutive activation of AMPK restored many of the phenotypes of lkb1null mutants, suggesting that AMPK mediates the polarityand mitosis-controlling functions of LKB1. Two studies
suggest that these effects may be mediated through
AMPK’s ability to promote transepithelial resistance and
tight junction assembly through activation of a calcium
switch which prevents tight junction disassembly (579,
580). The mechanisms and phosphorylation events implicated in this process are not known. Recently, it was
reported the calcium-activated potassium channel
KCa3.1, which is expressed at the basal lateral membrane
of many epithelial cells, is inhibited by AMPK (255). On
the basis of two-hybrid analysis, it is proposed that the
AMPK ␥1 subunit interacts with the COOH-terminal tail of
the channel. However, it is not yet clear that AMPK directly phosphorylates KCa3.1 or another associated protein.
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In addition to direct effects on protein synthesis,
AMPK may also inhibit cell growth and proliferation. One
mechanism by which this may occur appears to be
through AMPK induction of G1/S phase cell cycle arrest.
This effect is associated with the accumulation of the
tumor suppressor p53 and of the cyclin-dependent kinase
inhibitors p21 and p27, which act downstream of p53 and
may be dependent on phosphorylation of p53 on Ser-15
(223, 239, 404, 555). A recent study has demonstrated that
p27Kip1 may be the critical point of convergence in nutrient regulation of cell-cycle progression and the balance
between autophagy and apoptosis. While AMPK directly
phosphorylates p27Kip1 at Thr-198, this phosphorylation is
very weak and unlikely to occur in vivo, suggesting that
additional kinases downstream of LKB1-AMPK probably
regulate p27 stability in vivo, which would be expected to
inhibit cell cyle progression (293). In skeletal muscle,
AMPK increases the expression of the ubiquitin ligases
muscle atrophy F-box (MAFbx) and muscle RING finger 1
(MuRF1) protein, suggesting that AMPK may also be important for the atrophic transcriptional program executed
under various conditions of skeletal muscle wasting
(268). However, the physiological significance of these
findings is unclear, since exercise training is known to
prevent sarcopenia in the elderly. Taken together, these
data suggest that the effects of AMPK on cell growth may
be mediated by many different pathways, each with tissue
explicit specificity; this will make it challenging to understand which key pathways are involved.
Another possible mechanism by which AMPK may
regulate cell growth involves AMPK reducing the cytoplasmic-to-nuclear ratio of the RNA-binding protein HuR,
which in turn reduces mRNA stability of critical cell cycle
regulators such as cyclins A and B1 (523). Additional
studies also implicate AMPK in the regulation of transcription factors and coactivators, which might regulate
cell growth and proliferation, especially in cancer. In the
prostate cancer cell line, DU145, activation of AMPK with
hypoxia and blockade of AMPK activity with a dominant
negative mutant demonstrated that AMPK was essential
for the transcriptional activity of the hypoxia inducible
factor 1 (HIF1) (281). The protein products of the HIF1
target genes function to increase oxygen delivery and to
enhance metabolic adaptation to anaerobic conditions,
requisites for tumorgenesis (281). AMPK has also been
shown in vitro to phosphorylate the transcriptional coactivator p300 on Ser-89, reducing its affinity for nuclear
receptors (564). The physiological significance of p300
phosphorylation by AMPK is still unknown; however, this
event has the ability to influence transcription levels of a
large set of genes, which may be critical for regulating cell
growth and survival and may therefore be important un-
AMPK IN HEALTH AND DISEASE
VII. INTEGRATIVE ROLE OF AMPK A S A
REGULATOR OF WHOLE BODY
ENERGY METABOLISM
1. Glucose uptake
A. Exercise
During exercise and/or skeletal muscle contraction,
glucose uptake is increased, an effect which is independent of the proximal part of the insulin signaling pathway
(for review, see Ref. 241). The role of AMPK in regulating
exercise-stimulated glucose uptake has been an area of
considerable interest in recent years due to the therapeutic potential of stimulating glucose uptake via alternative
pathways in insulin-resistant skeletal muscle. As discussed, many studies have examined the role of AMPK in
mediating glucose uptake utilizing the pharmacological
AMPK activator AICAR in resting (noncontracting) skeletal muscle. Based on the above studies that demonstrated positive associations between contraction, AMPK
activity, and glucose uptake, it was somewhat surprising
that in transgenic AMPK dominant negative mice (157,
348) and AMPK ␣2 null animals (244) that ex vivo contraction-stimulated glucose uptake was either normal
(157, 244) or only modestly reduced (157, 233, 348) during
tetanic muscle contractions. Even though all of the above
models have some degree of residual AMPK signaling,
e.g., some remaining ␣1 activity, these findings collectively point towards a system dependent on more than
one signaling pathway.
2. Fatty acid oxidation
During exercise/muscle contractions, the activation
of AMPK is associated with increased phosphorylation
and deactivation of ACC2 and MCD (219, 247, 331, 413,
496a, 514, 540), which results in reductions in malonyl
CoA levels (538). Despite the strong correlations between
the activation of AMPK and the stimulation of fatty acid
oxidation under many conditions, especially in rodents,
there is a mismatch between rates of fatty acid oxidation
and the activation of AMPK/ACC signaling. One example
of this dichotomy is observed in LKB1-deficient mice,
which despite markedly blunted resting and contraction
stimulated phosphorylation of ACC (259, 427, 493, 496),
malonyl-CoA levels and rates of resting palmitate oxidation are not altered and the ability of contraction to
suppress malonyl-CoA is only marginally impaired (493).
We have also demonstrated that although contractionstimulated AMPK activity is reduced in AMPK DN mice,
ACC phosphorylation and fatty acid oxidation are maintained (137). In addition, it has also been reported that
low-intensity contraction can increase fatty acid oxidation independent of increased AMPK activity (402, 403)
and that AICAR and contraction may have additive effects
on fatty acid oxidation (455).
In exercising humans, there are also a number of
specific examples in which there is a mismatch between
AMPK signaling and fatty acid oxidation. One example
involves observations following endurance exercise training, which increases lipid oxidation during exercise but
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It is well documented that exercise or lack thereof is
a modifiable risk factor for a multitude of common diseases ranging from diabetes and cardiovascular disease to
certain cancers and even depression (129, 505). Despite
the tremendous clinical importance of exercise in the
prevention and treatment of these common diseases, the
molecular mechanisms mediating the positive effects of
exercise remained elusive for many years. Therefore, one
of the most influential findings in AMPK research was the
discovery that the enzyme was activated by muscle contractions (219, 514) and exercise in both rodents (540)
and humans (90, 156, 547) in an intensity-dependent manner (90, 92, 125, 477, 546, 547). Studies in LKB1 null mice
have shown that the activation of AMPK␣2 by exercise is
entirely dependent on phosphorylation by LKB1 (259, 427,
496), while rather surprisingly maintaining AMPK ␣1
phosphorylation (259).
During intense exercise, increased demands by contracting sarcomeres and Ca2⫹ pumps in the sarcoplasmic
reticulum can increase ATP turnover by in excess of
100-fold. In humans, this dramatic increase in ATP demand is nearly precisely matched by rapidly increasing
oxidative and nonoxidative phosphorylation of substrates
derived from intra- and extracellular lipid and carbohydrate stores (460). Recent studies in AMPK ␣2 (245) and
LKB1 (427) null mice have highlighted the importance of
AMPK in the maintenance of energy charge during contraction, since these mice have an elevated AMP/ATP
ratio in isolated muscles contracted ex vivo, independent
of complications related to substrate delivery and perfusion. This reduction in the capacity to maintain ATP in
response to muscle contraction may contribute to the
reduced exercise capacity of LKB1 KOs (496) as well as
muscle-specific AMPK dominant negative mice (347).
Since AMPK␣1 and ␣2 are also increased in the heart with
exercise intensity (110), it is unknown whether a reduced
exercise capacity is due principally to effects in skeletal
muscle or is the result of secondary effects that limit
blood flow and perfusion (347). Rather surprisingly,
AMPK ␣2 DN mice do not have an elevated cardiac AMP/
ATP ratio during treadmill running, suggesting this may
not be the case (356). Taken together, these studies highlight the critical importance of AMPK for the maintenance
of skeletal muscle energy balance during exercise; however, as discussed below, this may not be due to impaired
metabolism of substrates.
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3. Blood flow
During exercise there is an increased demand for
exogenous fatty acids and glucose by contracting muscle,
which is met by increases in blood flow and capillary
recruitment, ultimately resulting in improvements in muscle perfusion. Using L-NAME (a pharmacological NOS
inhibitor) and AICAR, Shearer et al. (448) have shown that
AMPK stimulation of skeletal muscle glucose but not fatty
acid uptake is dependent on nitric oxide. This effect may
be mediated by AMPK phosphorylation of eNOS which
phosphorylates the eNOS at Ser-1177 to activate NOS
both in vitro and during ischemia in rat hearts (93) and
contracting human skeletal muscle (90). These data suggest that the phosphorylation of NOS by AMPK in endothelial cells and myocytes is an important link between
metabolic stress and substrate delivery and muscle perfusion. However, direct studies measuring muscle perfusion in genetic models of AMPK deficiency in response to
exercise and hormonal activators of AMPK are needed to
fully address the physiological significance of this relationship.
B. Regulation of Appetite
The hypothalamus receives input from circulating
nutrients such as glucose and fatty acids (438) as well as
hormonal signals derived from the pancreas, adipose tissue, and the gut (438). The integration of these signals
occurs within the arcuate nucleus (ARC)-containing neurons resulting in alterations in the expression of key
orexigenic (neuropeptide Y, agouti related peptide) and
anorexigenic (proopiomelanocortin) neuropeptides, which reciprocally regulate food intake, peripheral energy expenditure, and insulin sensitivity (438). Studies have demonPhysiol Rev • VOL
strated that AMPK is an important link in integrating these
nutrient signals. AMPK catalytic subunits colocalize with
neuropeptide Y (NPY)-expressing neurons and play a role
in feeding control. Hypothalamic AMPK is activated by
fasting and inhibited by refeeding (335). In elegant experiments where adenovirus encoding dominant negative
(DN) and constitutively active (CA) mutations of AMPK
were injected into the ventral medial hypothalamus, Minokoshi et al. (335) showed that a CA-AMPK increased
body weight and food intake while a DN-AMPK had the
opposite effect. The hyperphagia observed with CAAMPK administration was related to increased expression
of arcuate NPY and agouti-related peptide (AgRP) when
in the fasted state while the reciprocal relationship was
observed in the fed state with the DN-AMPK. The injection of AICAR intracerebroventricularly also increases
AMPK and food intake (15). In a neuroblastoma cell line,
the modulation of cellular ATP and AMPK by glucose,
2-deoxyglucose, pyruvate, or ATP synthesis inhibitors alters the expression of AgRP. Similarly, the fatty acid
synthase inhibitor C75 was also found to suppress AMPK
activity and NPY expression in arcuate-containing neurons, an effect that is reversed in the presence of AICAR
(252). Additional anorexigenic signals, namely, insulin,
glucose, and refeeding, also suppress AMPK activity in the
brain, whilst the appetite-stimulating agouti-related protein activates hypothalamic AMPK (335).
The one caveat of all of the studies described above
is that they have examined AMPK signaling in the context
of hypothalamic sections, which contain multiple populations of neuronal (AgRP, POMC, NPY, etc.) and nonneuronal cell types, which may alter AMPK activity and appetite regulation in unknown ways. Surprisingly, mice
with a whole body deletion of AMPK␣1 or ␣2 have normal
food intake and energy expenditure, suggesting that in the
hypothalamus the ␣ isoforms may substitute for one another in response to appetite control (519). However,
recent studies by Claret et al. (99) utilizing a genetic
approach to delete AMPK␣2 from AgRp-containing hypothalamic neurons have found that the deletion of AMPK
results in a lean phenotype, while the effects of deleting
AMPK from POMC neurons has the opposite effect, as
would be expected and surprisingly resulted in obesity.
Importantly, these studies illustrate that AMPK is not
required for leptin and insulin’s effects on KATP channel
activation and appetite regulation, but is essential for
glucose sensing. An important consideration of the current data in AMPK null mice is that compensatory pathways during development may be upregulated to compensate for the lack of AMPK signaling in specific neurons
that were not present using transient infections using
adenovirus. In addition, the experiments described by
Claret et al. (99) were completed in mice with tissuespecific AMPK␣2 deletion but on a whole body AMPK␣1
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results in the suppression of AMPK signaling (318). The
inverse is also observed during sprint exercise, which
maximally activates AMPK signaling (90, 92) but reduces
fatty acid oxidation (416). Similarly, in exercising women
who have higher rates of fatty acid oxidation than men at
the same relative intensity, AMPK activity is markedly
lower, although ACC phosphorylation is only marginally
suppressed (414, 489). Taken together, these studies suggest that either 1) AMPK is not required and an alternative
ACC kinase(s) exists which assists in the regulation of
fatty acid oxidation during muscle contraction or 2) ACC2
is not required for the regulation of fatty acid oxidation
and alternative enzymes are more critical for controlling
malonyl-CoA production. Future studies in muscle-specific double-KO (␣1␣2 or ␤1␤2) mice or those lacking the
ability of AMPK to phosphorylate ACCS221 should be
revealing as to which of these possibilities is most important and may lead to the identification of alternative regulators of fatty acid oxidation.
AMPK IN HEALTH AND DISEASE
1. Circadian clock
An emerging role of AMPK is in the regulation of
circadian rhythm, which is set by a core of clock genes.
These genes are regulated in a negative-feedback loop
by casein kinase I␧ (CKI␧), which phosphorylates Per
and Cry proteins, suppressing their own transcription.
Since food intake and energy metabolism are synchronized to a circadian rhythm, it was hypothesized that
AMPK may play a role in regulating this pathway (510).
Treatment of cells with metformin shortened the circadian clock by reducing Per expression, suggesting that
the activation of AMPK may be playing a role. In vitro
phosphorylation confirmed that AMPK phosphorylated
CKI␧ but not Per directly. Further analysis confirmed
that AMPK phosphorylation of CKI␧ at Ser-389 dramatically increased CKI␧ activity inducing the degradation
of mPer2, effects that were confirmed both in fibroblast
cells and in vivo in mice treated with metformin. These
data suggest that the circadian regulation of AMPK may
be critical to understand the normal biological rhythms
of metabolic enzymes and perhaps appetite warranting
future studies in this area.
Physiol Rev • VOL
C. Regulation by Nutrients
1. Free fatty acids
Increasing fatty acid availability results in the upregulation of fatty acid oxidation while concomitantly
reducing endogenous rates of fatty acid oxidation and
glucose metabolism (133) consistent with the Randle hypothesis (401). However, an unresolved question has been
how increased extracellular fatty acid availability leads to
this “feed-forward” activation of fatty acid metabolism.
Recent studies in heart (100) and skeletal muscle (144,
307, 532) have revealed that AMPK may be sensitive to the
“lipid status” of the cell and that activation may be influenced by intracellular fatty acid availability independent
of the cellular AMP levels. The activation of AMPK by
fatty acids is, alike to AMP, resulting in allosteric activation leading to an increased ability of LKB1 to phosphorylate the AMPK holoenzyme in vitro (532). Importantly,
this effect is not observed when only AMPK ␣ 1-312 is
expressed, demonstrating that the effect of fatty acids
requires interaction with the ␤ or ␥ subunits of AMPK
(532). It appears that at least in muscle cells the feedforward activation of fatty acid oxidation by fatty acids is
completely dependent on AMPK signaling, suggesting this
is an important mechanism by which muscle responds to
alterations in substrate availability (532). In support of
these findings, genetic models that alter fatty acid handling as observed in mice null for steroyl-CoA desaturase
1 (130) or the fatty acid binding protein AP2/MAL (73,
307) have increased AMPK in liver and skeletal muscle,
respectively, and are protected from diet-induced obesity,
suggesting a potentially important role for fatty acid sensing in vivo. During fasting, AMPK activity and fatty acid
oxidation are increased, suggesting that an elevation of
circulating fatty acids may mediate this effect.
2. High-density lipoprotein cholesterol
Circulating high-density lipoprotein (HDL) is often
low in patients with the metabolic syndrome, and it is well
documented that high levels of HDL are protective against
atherosclerosis. The major apolipoprotein of HDL, apoAI,
activates AMPK in endothelial cells and increases endothelial NOS phosphorylation and activity (132). Recent
studies have shown APOA-I stimulation of AMPK signaling in C2C12 myotubes and that in skeletal muscle and
liver of ApoAI null mice, AMPK phosphorylation is reduced (182). The authors suggest this as a potentially
important role of HDL in regulating glucose and lipid
metabolism in skeletal muscle and liver and that HDL may
be important for protecting against diabetes. Future studies are required to determine whether the insulin-sensitizing effects of HDL are dependent on AMPK and to
understand the mechanisms by which HDL may regulate
AMPK signaling.
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background that has previously been reported to have
hematopoietic defects; this may have influenced the findings (518, 519). Future studies using inducible models of
AMPK deficiency or constitutive activation directly in
AgRP and POMC-expressing neurons will be important to
validate the role of AMPK in nutrient sensing and in
response to hormonal stimuli in the hypothalamus.
It is currently unclear whether AMPK effects on neuropeptide expression are mediated directly or through
inhibition of its downstream substrates ACC and mTOR.
Surprisingly, whole body AMPK␣1 and ␣2 null mice appear to have normal food intake, suggesting that the
effects of AMPK on appetite may be indirect. The activation of AMPK would be expected to inhibit ACC, increasing rates of fatty acid oxidation and reducing long-chain
fatty acyl-CoAs. Since the infusion of long-chain acylCoAs directly into the hypothalamus (369) or the chemical (368) and genetic (191) inhibition of hypothalamic
CPT-1 leads to reduced food intake, this is consistent with
the role of AMPK’s effects on orexigenic peptide expression. Another possibility is that AMPK regulation of
mTOR may be critical seeing that the inhibition of mTOR
using leucine, leptin, or rapamycin reduces food intake
(109). While a recent proteomics screen has identified 12
in vitro substrates of AMPK in the brain, many of which
were related to glycolysis (504), future studies are required to determine whether AMPK directly affects neuropeptide gene expression or whether this is via indirect
mechanisms possibly involving ACC and mTOR.
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3. Amino acids
D. Endocrine Regulation
Adipocytes and resident macrophages secrete a wide
variety of hormones and cytokines commonly called “adipokines.” These signal changes in adipose tissue mass and
energy status to other organs that control fuel usage, such
as the brain, skeletal muscle, and liver. The secretion of
adipokines such as leptin, adiponectin, interleukin-6, visfatin, TNF-␣, retinol binding protein-4, and resistin are
altered in obesity and are linked with many aspects of the
metabolic syndrome. The discovery that AMPK is a central node utilized by hormones to regulate metabolism has
been an exciting development in the field of AMPK biology. This is summarized in Figure 10 and is discussed
below.
1. Leptin
Leptin signaling in the hypothalamus is essential for
the regulation of body mass and neuroendocrine homeostasis (124a). However, leptin’s effects on metabolism are not limited to the hypothalamus as almost all
tissues examined express leptin receptors (490). Leptin
acts directly in isolated skeletal muscle to increase fatty
acid oxidation (353) in an AMPK-dependent manner
(336), and this process appears to involve an increase in
the AMP/ATP ratio (336, 469, 526). The acute activation of
AMPK is limited to ␣2-containing heterotrimers (484) in
oxidative muscle fibers (336), but the reason for this
tissue-specific activation is still not clear.
In addition to the direct activation of leptin in skeletal muscle, there is also a delayed action of leptin that
requires inputs from the central nervous system (CNS).
Physiol Rev • VOL
FIG. 10. Hormonal regulation of AMPK signaling. In the brain,
increased AMPK promotes food intake. In skeletal muscle, AMPK activation promotes energy expenditure through upregulation of fatty acid
oxidation, glucose uptake, and mitochondrial biogenesis. In the liver,
AMPK suppresses hepatic glucose production and lipogenesis. Green
arrow, stimulation/activation of AMPK; red oval, inhibition/deactivation.
The CNS-mediated activation of AMPK involves the stimulation of ␣-adrenergic signaling (336) and is dependent
on the melanocortin (MC) system, since intracerebroventricular delivery of an MC4 receptor antagonist inhibits
leptin activation of AMPK in skeletal muscle while a
melanocortin agonist increases AMPK (487). Contrary to
the situation in muscle, leptin administration results in
suppression of AMPK activity in the paraventricular and
arcuate sections of the hypothalamus (15, 335). The effects of leptin on AMPK signaling in the brain appear to be
downstream of the MC receptor as intracerebroventricular administration of the MC4 receptor agonist MT-II inhibits AMPK activity in the paraventricular nucleus
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As discussed above, AMPK regulates protein translation via at least two mechanisms: phosphorylation and
activation of the eukaryotic elongation factor 2 kinase
(eEF2K), and inhibition of the mTOR-signaling pathway.
Since the regulation of protein synthesis by mTOR is
highly dependent on amino acid availability, it would
seem logical that AMPK may also be regulated by amino
acids. Recent studies by Gleason et al. (165) using MIN-6
cells have demonstrated that AMPK activity is suppressed
by amino acids. This effect was found to be independent
of mTOR, since treatment with rapamycin did not alter
the ability of amino acids to inhibit AMPK phosphorylation. Subsequent studies using nonmetabolizable amino
acids suggested that the inhibitory effect of amino acids
on AMPK was indirect and was likely due to the welldocumented stimulatory effects of amino acids on mitochondrial glutamate dehydrogenase, which results in enhanced tricarboxylic acid cycle flux, which would be
anticipated to reduce cellular AMP content (165).
AMPK IN HEALTH AND DISEASE
activation of AMPK by IL-6 is essential for increases in
fatty acid oxidation and glucose uptake. The mechanisms
by which IL-6 increases AMPK are presently not understood.
3. TNF-␣
TNF-␣ is directly implicated in the pathogenesis of
insulin resistance in both humans and rodents, effects
which have been linked to inflammatory signaling and
serine phosphorylation of IRS1 (392, 512). TNF-␣ treatment downregulates fatty acid oxidation and inhibits
AMPK signaling, an effect which is reversed following the
siRNA of protein phosphatase 2C (PP2C) (470). TNF-␣
infusion into lean animals increases PP2C expression,
resulting in a reduction in AMPK signaling, fatty acid
oxidation, and increased intramuscular diacylglycerol accumulation, protein kinase C (PKC)-␧ and -␪ activation,
and the development of skeletal muscle insulin resistance. Importantly, when TNF-␣ was neutralized in ob/ob
mice, or when TNF-␣ signaling was genetically ablated
(ob/ob TNFR ⫺/⫺ mice), the reduced AMPK activation
observed in obese animals was reversed. These studies
provide mechanistic evidence supporting a common link
between inflammation and defective fatty acid metabolism in obesity while also highlighting an important role
by which PP2C regulates AMPK signaling. Similar studies
have also recently been reported in vascular smooth muscle (457). Future studies are required to determine
whether TNF neutralization, which has been shown to
improve insulin sensitivity in large-scale trials in patients
with rheumatoid arthritis (170, 254), is associated with
increased AMPK signaling and reductions in skeletal muscle lipid.
4. Resistin
2. Interleukin-6
The interleukin-6 (IL-6) family, also known as “gp130
cytokines,” consists of IL-6, IL-11, leukemia inhibitory
factor (LIF), oncostatin M (OsM), cardiotrophin 1 (CT-1),
ciliary neurotrophic factor (CNTF), and cardiotrophinlike cytokine (CLC) (57). While IL-6 is elevated with obesity, the discovery that IL-6 can be produced and released
from skeletal muscle during exercise (143) suggested a
possible alternative role of IL-6 in regulating metabolism.
Indeed, studies highlighting this possibility were findings
that IL-6 can increase skeletal muscle fatty acid oxidation
(63, 388, 513) and glucose uptake (75), effects observed
both in vitro and in vivo in rodents and humans. Interestingly, basal AMPK signaling is markedly suppressed in
liver, adipose tissue, and skeletal muscle of IL-6 KO mice,
an effect which persists following swimming (248). Recent studies in myotubes (9, 75) have found that the
Physiol Rev • VOL
Resistin (or FIZZ3) is an adipocyte-derived secretory
factor first identified as a novel transcript produced exclusively by adipocytes (478). Despite the significant interest generated by the discovery of resistin in 2001, very
little is known about the intracellular signaling pathways
by which resistin induces its metabolic effects. A consistent finding in vivo is that resistin suppresses liver and
muscle AMPK signaling (30, 398, 434), an effect also observed in L6 muscle cells (378). The mechanisms mediating this inhibition of AMPK signaling are still unclear, and
it is unknown whether the observed in vivo effects are
due to direct effects on AMPK signaling or may be mediated through indirect pathways. One possibility may be
that resistin-induced increases in SOCS3 (479) may inhibit
leptin signaling through to AMPK, but future studies are
required to establish the mechanisms involved.
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(PVN), while both refeeding and leptin fail to inhibit
hypothalamic AMPK activity in MC4 receptor knockout
mice (335). Future studies are required to delineate the
upstream pathways mediating the acute and chronic effects of leptin on AMPK signaling and to understand how
leptin mediates opposite effects on AMPK, activating in
muscle but suppressing in hypothalamus.
Chronic leptin treatment is believed to help reverse
insulin resistance independent of caloric restriction by
reducing lipid storage in skeletal muscle and liver (451).
Chronic leptin administration increases skeletal muscle
AMPK ␣ and ␤ expression (472) and fatty acid oxidation
(466). There is also evidence in cultured myotubes that
AMPK activation by leptin increases AMPK␣2 nuclear
translocation and results in PPAR␣ transcription (484).
While leptin dramatically reduces lipid storage in the
liver, this effect is independent of AMPK and involves the
inhibition of PPAR␣ (285) and SCD1 (102). Chronic adenovirus-induced hyperleptinemia rapidly reduces adipocyte size and mass, induces mitochondrial biogenesis, and
concomitantly increases AMPK Thr-172 phosphorylation
in adipocytes (375, 522), but whether AMPK is required
for this process is currently unknown. Skeletal muscle
leptin resistance develops following high-fat feeding in
rodents (467) and is also prevalent in obese humans (471,
473). The development of leptin resistance in obese skeletal muscle is characterized by suppressed rates of leptinstimulated AMPK signaling (315, 469, 473, 526). Similarly,
high-fat feeding inhibits the ability of leptin to suppress
hypothalamic AMPK signaling (315, 474), an effect which
may be mediated by the suppressor of cytokine signaling-3 (SOCS3) (469); however, this requires further study.
In addition, while it is commonly believed that the insulinsensitizing effects of leptin are mediated through activation of AMPK, further evidence in genetic models of
AMPK deficiency is required to validate this hypothesis.
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GREGORY R. STEINBERG AND BRUCE E. KEMP
5. Ghrelin
Ghrelin is a gut-derived peptide hormone that promotes food intake (358) and has been shown to increase
AMPK activity in the hypothalamus (15). Ghrelin has differential effects on AMPK activity depending on the tissue
studied, with reports of ghrelin treatment suppressing
AMPK activity in liver (31, 262) whilst activating AMPK in
the heart (262). Ghrelin appears to have no effect on
skeletal muscle AMPK. This may be related to the fact
that ghrelin signaling is dependent on the G protein-coupled receptor (211) and therefore most likely on CAMKK
signaling.
Adiponectin is an abundant plasma protein that circulates as a range of multimers from low-molecularweight trimers to high-molecular-weight dodecamers
(409). Adiponectin levels are reduced in obesity (212),
and overexpression has been shown to reverse skeletal
muscle insulin resistance in models of genetic and dietinduced obesity (150, 316, 376, 560, 562). Adiponectin
activates AMPK and stimulates fatty acid oxidation and
glucose uptake in skeletal muscle (500, 561) and adipose
tissue (554). Purification and characterization of adiponectin multimers from human plasma suggest that highmolecular-weight adiponectin most potently stimulates
AMPK activity, at least in C2C12 cells (178). The activation of AMPK signaling is dependent on signaling through
the adiponectin receptor 1 (AdipoR1) (563) and requires
the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPL1); however, it is still unknown how
AAPL1 regulates AMPK signaling (312). The metabolic
effects of adiponectin also involve the suppression of
hepatic glucose output (103), an effect which is dependent on the activation of AMPK (16, 561). In agreement
with these findings, the deletion of the AdipoR1 receptor
in liver using siRNA results in reduced AMPK and increased gluconeogenesis and glucose intolerance (563).
In the hypothalamus, a recent study has found that
adiponectin trimers and hexamers, but not high-molecular-weight forms, increase appetite (271) via AdipoR1 activation of AMPK. Importantly, Kubota et al. (271) demonstrate that the activation of hypothalamic AMPK by
adiponectin reduces leptin sensitivity, leading to the proposal that through their central action leptin and adiponectin have opposite roles but when acting in concert
these two adipokines work to maintain fat levels/energy
stores through modulation of hypothalamic AMPK signaling (468).
7. Sex hormones: estradiol and testosterone
Estrogen and androgens play a critical role in regulating metabolism as highlighted by studies in mice bearPhysiol Rev • VOL
VIII. AMPK DYSREGULATION IN DISEASE
A. Aging and Longevity
Aging affects multiple aspects of metabolism, but it is
generally well accepted, no matter what pathways are
studied, that all organisms have a decreased ability to
tolerate metabolic stress upon aging. Studies in lower
organisms such as yeast and worms have indicated a
potentially important role of AMPK homologs in regulating these age-related changes. Lin et al. (295) have used
Snf1p, a yeast homolog of AMPK, to explore the genetic
and biochemical link between glucose metabolism and
aging. Snf1p is incorporated into a complex that contains
Snf4p (relative to AMPK ␥1), Sip1p, Sip2p, and Gal83
(relative to AMPK ␤1). Loss of the Snf1p activator Snf4p
produces a 20% increase in the life span of yeast cells,
whereas loss of Sip2p, a presumed repressor of the kinase, causes a rapid aging phenotype that appears to be
related to increased Snf1p activity (21). The authors
found that a prominent feature of the metabolic evolution
of aging in yeast cells was activation of the Snf1p kinase
pathway and increased gluconeogenesis and glucose storage. In Caenorhabditis elegans, the overexpression of
AMPK␣, designated aak-2, increases life span and appears to be required for increased longevity associated
with signaling via daf-2/insulin and the deacetylase sir-2.1
(114). AMPK is also required for the long-term survival of
the daur larva stage of C. elegans (360). In a followup
study, it was found that AMPK signaling was required to
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6. Adiponectin
ing inactivating mutations in either aromatase, the enzyme responsible for estrogen biosynthesis (ArKO), or in
the estrogen receptor ␣ (ERKO). In both models mice
develop age-onset obesity, hepatic steatosis, and insulin
resistance (193, 197, 237, 338). Estrogen directly activates
AMPK signaling and stimulates fatty acid oxidation in
C2C12 myotubes (116), suggesting that in these models
reduced AMPK signaling in muscle may contribute to the
observed phenotype. However, a complicating factor in
both the ArKO and ERKO mice is the finding of elevated
circulating androgens. Recent studies by McInnes et al.
(326) have shown that dihydrotestosterone infusion inhibits AMPK signaling in adipocytes, suggesting that increased adiposity in ArKO mice is due at least in part to
the increase in androgen-to-estrogen ratio resulting in the
inhibition of AMPK signaling rather than solely a lack of
estrogen. Interestingly, AMPK expression levels and activity are not altered in human skeletal muscle (414).
However, future studies are required to determine the
mechanisms by which estrogen and androgens modulate
AMPK activity and to determine the effects of these sex
hormones in other tissues critical for regulating metabolism.
AMPK IN HEALTH AND DISEASE
B. Obesity and the Metabolic Syndrome
The World Health Organization estimates that of the
more than 1 billion adults worldwide who are overweight,
300 million are clinically obese, defined as having a body
mass index equal to or greater than 30 kg/m2. Obesity is
associated with a number of health problems that are
often summarized together as the metabolic syndrome.
These include insulin resistance, type 2 diabetes, cardiovascular disease, and fatty liver disease. There are currently in excess of 170 million patients with type 2 diabetes, and this figure is expected to double by 2030 in line
with trends in obesity. Much of the interest in AMPK has
been generated because of the associations drawn between the metabolic syndrome and the pathways which
AMPK regulates at the cellular level leading to the suggestion that AMPK signaling may be suppressed with
obesity and that therapeutic activation of AMPK may
therefore be beneficial (419, 539). Evidence supporting or
refuting this conclusion will be discussed below.
In peripheral tissues such as heart, skeletal muscle,
and liver, AMPK activity is reduced in most genetic modPhysiol Rev • VOL
els of rodent obesity (33, 298, 462, 470, 521, 570). However, it appears that AMPK activity is not altered in skeletal muscle (315, 526) or the hypothalamus (315, 474) of
animals made obese by a high-fat diet, which is believed
to more directly mimic the development of human obesity
and insulin resistance. In obese human muscle, AMPK
protein expression and activity are unaltered (473), although others have reported modest reductions (29). In
human type 2 diabetic skeletal muscle, AMPK expression
and activity are also unaltered relative to subjects with a
similar body mass (199, 355). The sensitivity of AMPK to
allosteric activation by AMP (473) and LKB1 activity/
expression (88) is also unchanged in obesity and in obese
type 2 diabetic skeletal muscle, suggesting that the AMPK
system is indeed intact at least in moderate obesity. Similarly, AICAR has been demonstrated to increase AMPK
activity to a similar degree in obese skeletal muscle from
rodents (44, 68, 222, 394, 570) and humans (260, 327, 469,
473) relative to lean controls, although a recent study
suggests that there may be reduced sensitivity of AMPK to
aerobic exercise (461). Importantly, this activation of
AMPK by AICAR increases glucose uptake and fatty acid
oxidation in obese diabetic rodents (32, 44) and humans
(88, 115, 260, 469, 473), validating the therapeutic potential of an AMPK activator for bypassing skeletal muscle
insulin resistance. Taken together, these data suggest that
the downregulation of AMPK signaling may not be a primary defect preceding metabolic aberrations associated
with the development of obesity-related insulin resistance. However, it does appear that suppressed AMPK
signaling in severe obesity, as seen in obese genetic models, is likely to exacerbate aspects of the metabolic syndrome. Therefore, therapies that prevent or reverse this
reduction in AMPK signaling or artificially elevate it may
have therapeutic value as will be discussed in the following sections.
1. Hypertension, diabetic myopathy, and lipotoxicity
Regular physical exercise has advantageous effects
on blood pressure level in humans and also beneficially
influences blood lipid profile and glucose homeostasis in
individuals displaying features of the metabolic syndrome. Chronic AICAR treatment for 7 wk reduces blood
pressure in obese Zucker rats, but it is currently unknown
whether this effect is direct or is secondary to improvements in glucose and lipid homeostasis (68). Lipotoxic
cardiomyopathy is another prominent component of the
metabolic syndrome and is characterized by ectopic fat
accumulation in the heart. Wang and Unger (521) have
demonstrated that AMPK phosphorylation in cardiac
muscle of ob/ob mice and obese Zucker rats is reduced
relative to lean controls, an effect associated with increases in PP2C expression and cardiac lipid deposition,
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suppress the lipase ATGL and suppress the depletion of
lipid stores in the daur larva (359). This seems likely to be
a species-specific adaption as C. elegans contains a nonconserved insert around Ser-303, the AMPK phosphorylation site. Glucose reduction or 2-deoxy-D-glucose-induced
caloric restriction increases mitochondrial respiration,
and Schults et al. (437) have suggested reactive oxygen/
oxidative stress may be important for the longevity effects, but this view has been challenged (400). Greer et al.
(173) also showed that dietary restriction-induced longevity required AMPK, and this involved multisite phosphorylation of the transcription factor FOXO/DAF-16 previously known to mediate longevity in C. elegans.
In higher organisms, the role of AMPK in the regulation of aging is not clear, with some reports demonstrating a significant reduction in AMPK signaling in muscle
(399, 408) while others report no change (169, 379) or
even increased AMPK activity (494, 495). The effects of
metabolic stress such as hypoxia, caloric restriction, or
muscle contraction on AMPK signaling in aging are
equally equivocal with some studies reporting increased
activation (169, 379), while others have reported a reduction (349, 408). Interestingly, resveratrol (3,5,4⬘-trihydroxystilbene), which extends the life span of organisms
through Sir2, a conserved deacetylase, proposed to underlie the beneficial effects of caloric restriction, activates
AMPK (121, 220, 384, 575). While recent studies suggest
that the effect of Sir2 on longevity may have more to do
with altered insulin and IGF-I signaling than AMPK (292),
future studies examining longevity in AMPK null mice will
be important.
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GREGORY R. STEINBERG AND BRUCE E. KEMP
suggesting that reduced AMPK signaling may contribute
to this condition in obesity.
C. Cardiovascular Disease and Reperfusion Injury
1. Cardiac hypertrophy
Cardiac hypertrophy can occur due to a chronically
increased cardiac work load. Since the activation of
AMPK inhibits protein synthesis, it seems logical that
overt activation may protect against hypertrophy, although evidence supporting this conclusion is equivocal
with some studies presenting positive associations between AMPK activation and the development of cardiac
hypertrophy (18, 498) while others suggest that AMPK
activation can inhibit hypertrophic growth (86, 449).
Since Akt plays a critical role in regulating cardiac hypertrophy, Dyck and colleagues (86, 265) have demonstrated
that overt activation of this pathway and subsequently
enhanced rates of protein synthesis are mediated at least
in part through Akt inhibition of AMPK mediated through
␣1 Ser-485 phosphorylation. Consistent with this finding
are studies in adiponectin null mice that have diminished
AMPK signaling and are more susceptible to pressureoverload-induced cardiac hypertrophy (449). Based on
these findings, it would be expected that mouse models of
AMPK deficiency would have increased heart size, but in
most cases heart size is normal or even slightly reduced
(428), suggesting that compensatory pathways may have
developed or that these models must be studied under
conditions of increased cardiac work load.
2. Myocardial ischemia-reperfusion injury
Both AMPK ␣1 and ␣2 activities are elevated during
ischemia (272, 273), although only ␣2 phosphorylation
appears to be dependent on LKB1 signaling (428), possibly downstream of TAK1 (557). Increasing AMPK signaling results in GLUT4 translocation and the stimulation of
glucose uptake (421, 567) and fatty acid oxidation (272,
273), but whether or not this is beneficial for the recovery
of the heart during reperfusion is unknown with reports
utilizing a variety of different genetic models reporting
either positive (422, 450) or detrimental effects (558). As
discussed in detail in two recent reviews (131, 136), these
Physiol Rev • VOL
D. Cancer
The AMPK upstream kinase LKB1 is a tumor suppressor that is mutated in Peutz-Jegher syndrome, a rare
genetic disease leading to a predisposition towards intestinal polyps (hamartomas) and increased incidence of
epithelial cancers in many other tissues including breast
(234). While LKB1 mutations are uncommon in most cancers, somatic LKB1 inactivation is estimated to occur in
30 –50% of sporadically occurring lung adenocarcinomas,
⬃20% of squamous cell carcinomas, and ⬃10% of large
cell carcinomas of the lung, illustrating its tumor suppressor properties (234). LKB1 nonsense or frame shift mutations resulting in abnormal or truncated protein can occur
in both primary lung adenocarcinomas or in lung andenocarcinoma cell lines (81, 430). In the case of human
endometrial cancer, downregulation of LKB1 expression
has been detected by immunohistochemistry and was
inversely correlated with both tumor grade and stage
(104).
LKB1 (⫺/⫹) mice have been reported to have enhanced sensitivity to chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA)-induced squamous cell carcinoma (SCC) of the skin and lung (174). In this study,
restoration of wild-type LKB1 caused senescence in tumor-derived cell lines that was dependent on the Rb
pathway, but not on p53 or AMPK, indicating that AMPK
is not an obligate mediator of LKB1’s SCC suppressor
activity.
The role of LKB1 in activating AMPK has highlighted
the possibility that AMPK may be important in mediating
LKB1 tumor suppression pathways. Accordingly, activation of AMPK by pharmacological or other means might
be associated with reduced cancer incidence. Activation
of AMPK by AICAR treatment of the breast cancer cell
line MDA-MB-231 with AICAR blocks proliferation and
colony formation in culture as well as reducing tumor
growth in nude mice (485). Subsequently, these investigators have reported that the antiproliferative effects of
AICAR can be further enhanced with methotrexate treatment to reduce AICAR metabolism (39). AICAR has also
been found to inhibit cell proliferation and promote apoptosis in multiple acute lymphoblastic leukemia cells,
CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL,
TEL/AML1), and SupB15 (Bp-ALL, BCR/ABL) (443). The
adenosine kinase inhibitor 5⬘-iodotubericidin abolished
the effect of AICAR (443), indicating nucleoside phos-
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In the normal, healthy heart, ⬃10 – 40% of ATP utilization is produced via pyruvate oxidation, while the remaining 60 –90% of the ATP is derived from the oxidation
of fatty acids. The regulation of cardiac energy metabolism is altered under many pathological conditions such
as cardiac hypertrophy and myocardial ischemia-reperfusion injury, and since AMPK plays a critical role in regulating substrate use in the heart, its role in these pathologies has been examined.
equivocal findings may be due to the availability of fatty
acids as fatty acids may directly inhibit recovery following
ischemia. Therefore, it may be possible that AMPK may
protect against ischemia-reperfusion injury by increasing
glucose uptake if the potential negative consequences of
increased fatty acid oxidation are not present.
AMPK IN HEALTH AND DISEASE
E. Dementia, Neurogenesis, and Stroke
Both AMPK␣1 and -␣2 are highly expressed in the
brain (465), exhibiting a predominant neuronal localizaPhysiol Rev • VOL
tion (113) with some expression in glial cells such as
activated astrocytes and oligodendrocytes (509). AMPK in
astrocytes plays a role in ketogenesis and in the prevention of apoptosis (50, 51). Brain vessel remodeling is
dysregulated in neurodegenerative diseases such as Alzheimer’s disease (172), and a recent report suggests that
increased AMPK activity may be a contributing factor
(303). Early on it was found that AICAR protected hippocampal neurons against death induced by multiple
stresses including glucose deprivation, chemical hypoxia,
exposure to glutamate, and amyloid ␤-peptide (113).
Similar findings with regard to ischemia and reperfusion in the heart and activation of AMPK in neurons has
been shown to be both protective (113) or inhibitory (321)
depending on the conditions and cell types studied. However, a recent study has clarified these discrepancies using a focal model of stroke in AMPK ␣2 null mice, which
demonstrated that a lack of AMPK results in significantly
smaller infarct size compared with wild-type littermates
(291). How AMPK␣2 increases neuronal damage is still
unclear, but it has been speculated that it involves the
regulation of neuronal NO, which appears to be detrimental to recovery following ischemia (321). AMPK has recently been shown to be important in preventing retinal
degeneration in the Drosophila visual system using deletion of the ␤ subunit (458). Mutation of the Drosophila
AMPK ␥ subunit also results in progressive neurodegeneration phenotype (442, 503). Dasgupta and Milbrant
(120) have proposed that AMPK also has a development
role in brain in addition to its neuroprotective role. Mice
in which the ␤1 gene was interrupted by an insertion of
␤geo resulting in a fusion protein ␤1(exons 1-5)-␤geo had
profound brain abnormalities. The abnormality was associated with reduced phosphorylation of the retinoblastoma protein at Ser-804. However, the phenotype is certainly a consequence of aberrant fusion protein expression since the conventional ␤1 KO mice are otherwise
normal (442).
IX. OPPORTUNITIES FOR THERAPEUTICS
A. Metformin
Metformin is an oral biguanidine that reduces plasma
glucose and lipids and improves insulin sensitivity in patients with type 2 diabetes (129). Although biguanidines
became available for diabetes therapy in 1957, and have
since become the most widely used treatment for the
disease, the mechanisms by which they improve insulin
sensitivity is incompletely understood. Interest in AMPK
as a therapeutic target for the treatment of metabolic
syndrome gained rapid momentum after experiments
showed that AMPK was activated by metformin (357,
582). Metformin activation of AMPK is dependent on up-
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phorylation to ZMP was required. Thus studies showing
different activators of AMPK, namely, AICAR and metformin (discussed in detail below), can block growth of
breast cancer cells in culture are in line with the epidemiology data showing a reduced risk of cancer in diabetic
patients treated with metformin (142).
It is recognized that tumor development and progression are accompanied by marked changes in the expression and activity of key enzymes in lipid metabolism.
These include FAS whose transcription is regulated by
AMPK (as discussed previously) and is thought to play a
critical role in lipogenesis for cancer cell energy storage.
FAS is highly expressed in breast cancer and many common human carcinomas. Indeed, FAS is now regarded as
an important anticancer drug target (330, 515). Inhibition
of earlier steps in fatty acid synthesis such the AMPK
substrate ACC by the macrocyclic polyketide inhibitor
soraphen A has been shown to inhibit proliferation of
prostate cancer cells (38). Thus from this perspective any
loss of AMPK signaling to ACC in cancer cells would be
expected to promote survival. There is a further important link in breast cancer metabolism because BRCA1
associates with phosphorylated ACC1 (308, 345) and suppresses its dephosphorylation and activation (i.e., equivalent to augmenting the AMPK signal). Deletion mutations
in BRCA1 fail to block ACC1 dephosphorylation, thereby
contributing to elevated fatty acid synthesis in tumor cells
with BRCA1 mutations (65). AMPK readily associates
with ACC1, and this raises the possibility that a larger
signaling complex with BRCA1 may be present with other
yet unrecognized tumor suppressor functions.
Statins have been reported to have antitumor activity, and the relationship between statin use and breast
cancer risk has been investigated. One large study detected an 18% reduction in breast cancer incidence with
statin therapy (hydrophobic statins only) targeting HMGR
(84). AMPK phosphorylates and inactivates HMGR, thereby
inhibiting flux through the mevalonate pathway and production of cholesterol and isoprenoid groups for the posttranslational modification of many signaling proteins
(e.g., GTP-binding proteins that are either farnesylated or
geranylgeranylated). While there may be a possible link
between tumor suppression and AMPK regulation of
HMGR, there is a lack of evidence to support a mechanism at this point. Transgenic mice carrying Ser/Ala mutations in the AMPK phosphorylation sites of ACC1 and
HMGR have now become available, making it possible to
test whether blocking AMPK signaling to these enzyme
targets has an impact on tumor development or progression.
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inhibits respiratory chain complex 1 resulting in the suppression of ␤-oxidation, cancer cells treated with metformin require increased rates of glycolysis to compensate. Cells deficient in either AMPK or p53 are unable to
upregulate glycolysis, resulting in increased tumor cell
apoptosis in vivo. These studies suggest a potential benefit of metformin use in patients harboring p53-deficient
tumors, which are often resistant to existing forms of
chemotherapy or radiotherapy (70).
Metformin has also been shown to act as a growth
inhibitor of MCF7 breast cancer cells, an effect dependent
on the presence of AMPK (573). The growth inhibition
was associated with decreased mTOR and S6 kinase activation and was reversed by siRNA against AMPK. The
growth suppressor effects were specific, as shown by
the fact that HeLa cells, which are deficient in LKB1,
were unaffected by the metformin treatment. Metformintreated cells have suppressed oxidative phosphorylation
and compensate for this by increasing their rate of glycolysis, which is p53-dependent and raises the possibility
that p53-deficient tumor cells are unable to make this
metabolic switch in the presence of metformin (70). On
the other hand, a recent study has identified reductions in
cyclin D1 and Rb phosphorylation in response to metformin treatment as important for this drug’s antitumor
action on prostate cancer cells (424). Importantly, inhibition of AMPK using siRNAs to the ␣ subunits did not
prevent the antiproliferative effects of metformin,
strongly indicating that AMPK is not essential in this case
(424).
B. Thiazolidinediones
PPAR-␥ is a nuclear hormone receptor that is predominantly expressed in adipose tissue but also has important roles in skeletal muscle (194), liver (162), and
macrophages (195). Despite intensive research into the
role of PPAR␥, the mechanisms by which their synthetic
ligands, the thiazolidinediones (TZDs), cause insulin sensitization are incompletely understood. TZDs improve hepatic insulin sensitivity (196), but interestingly, they do
not consistently lower lipid content (288). TZDs activate
AMPK in skeletal muscle cells by increasing cellular adenine nucleotide levels (153, 277). In vivo, chronic treatment with TZDs increases skeletal muscle and cardiac
AMPK, but it is difficult to discern whether this effect is
direct or due to the potent effects of TZDs in stimulating
adiponectin production (521). Supporting the latter conclusion is the finding that rosiglitazone is unable to improve glucose tolerance or activate AMPK in ob/ob adiponectin null mice (270, 362). In light of recent studies
demonstrating an increased incidence of myocardial ischemic events with rosiglitazone treatment (204, 417), it is
interesting to speculate that this may be due to chronic
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take by the organic cation transporter (OCT1) (452), and
although original studies suggested that metformin activated AMPK independent of changes in nucleotides (153,
187), more recent studies utilizing ␥ mutants which are
insensitive to AMP have demonstrated that nucleotide
binding is essential for metformin activation of AMPK
(441). Both phenformin and metformin treatment of isolated rat hearts results in increased calculated cytosolic
AMP based on 31P-NMR measurements of phosphocreatine, ATP, and intracellular pH (578). In this study,
HPLC-based measurement of the total AMP/ATP ratio
showed no change with metformin treatment (578), perhaps indicating that a local pool of AMP associated with
the mitochondria may be responsible for AMPK activation. These findings are consistent with the well-documented effects of metformin, which is known to inhibit
respiratory chain complex I, leading to an inhibition of
mitochondrial respiration and the inhibition of ␤-oxidation of fatty acids which requires NAD⫹ as a cofactor
(138, 377, 585). A secondary mechanism by which metformin may activate AMPK involves the accumulation of
reactive nitrogen species that in turn stimulate the c-Src/
PI3K pathway generating a metabolite or other molecule
inside the cell to promote AMPK activation by the LKB1
complex (585). However, identification of this factor remains elusive.
The main site of metformin action appears to be the
liver, where AMPK is activated and is required to suppress
gluconeogenic and lipogenic gene expression (582). Studies in human HepG2 cells using a constitutively active or
dominant negative AMPK␣ have shown that AMPK is also
required for the lipid-lowering effects of metformin (576).
Metformin is ineffective in lowering blood glucose in
high-fat diet fed mice lacking LKB1 in the liver (446),
suggesting that the LKB1-AMPK pathway is required for
metformin action. Interestingly, metformin also reduces
inflammatory markers such as TNF-␣ (294) and C-reactive
protein (179), effects that are likely mediated by AMPK
inhibition of NF␬B transcription (71), suggesting a secondary mechanism by which AMPK may improve insulin
sensitivity.
Lastly, the possible involvement of AMPK in tumor
suppressor pathways prompted several groups to investigate whether there was a reduced incidence of cancer in
diabetic patients on metformin therapy. Diabetic patients
treated with metformin show ⬃30% reduction in deaths
from cancers versus those on either sulfonylurea or insulin therapy (142). However, in the Bowker et al. (58)
study, the authors point out that the differential risk could
be related either to deleterious effects of sulfonylurea and
insulin or a direct protective effect of metformin. A recent
study (70) suggests that one of the mechanisms by which
metformin may inhibit tumor growth is by preventing
p53-induced authophagy, an effect that is dependent on
the upregulation of fatty acid oxidation. Since metformin
AMPK IN HEALTH AND DISEASE
activation of AMPK in the heart, which may lead to glycogen storage disease, cardiac hypertrophy, and potentially greater cardiac ischemia (306).
C. Life-style Interventions
D. Ciliary Neurotrophic Factor
CNTF is a member of the IL-6 family of cytokines and
was initially identified as a neurotrophic factor to potentially treat severe neurodegenerative disorders such as
amyotrophic lateral sclerosis (ALS). CNTF rapidly induces weight loss in diet-induced (276) and genetic (ob/
ob, MC4R⫺/⫺) obesity (167, 454) at low doses without
causing the typical deleterious effects of other related
cytokines. This effect has been confirmed in recent clinical trials (141). CNTF increases AMPK activity in skeletal
muscle by acutely decreasing the ATP/AMP ratio, resulting in increased fatty acid oxidation (526). The administration of CNTF, at doses demonstrated to induce weight
loss, stimulated the expression of mitochondrial oxidative
genes in skeletal muscle, an effect which may involve the
activation of AMPK. Importantly, the stimulatory effect of
CNTF on AMPK activation and fatty acid oxidation reversed insulin resistance induced by acute exposure to
high fatty acid levels in rodents in vivo (527) and in vitro
(526); this effect was not maintained when cells were
infected with a dominant negative AMPK.
In the brain, CNTF and leptin display similar expression patterns and signaling homology within hypothalamic regions involved in food intake (14, 276). Therefore,
in many ways, it was not surprising that like leptin CNTF
reduces AMPK signaling in hypothalamic sections containing ARC neurons. Importantly, and unlike leptin, the
Physiol Rev • VOL
ability of CNTF to suppress AMPK in the hypothalamus
(474) and activate AMPK in skeletal muscle (526) persists
in leptin resistant mice fed a high-fat diet highlighting its
potential role in the therapeutic treatment of human obesity. CNTF also induces hypothalamic remodeling/neurogenesis, which may contribute to the resistance to rebound weight gain (261), although whether this involves
AMPK as has been shown for leptin (118) is unknown. A
recent study (139) has found that like skeletal muscle
AMPK is also activated by CNTF in neurons/astrocytes
leading to increases in fatty acid metabolism and mitochondrial gene expression. This effect may serve to protect neurons in time of metabolic stress. CNTF also activates AMPK in hepatocytes (214) and adipocytes (111).
Future studies are required to delineate the mechanisms
by which CNTF can have diverse effects in different aspects of the brain and to determine whether the effects of
CNTF on feeding are dependent on AMPK signaling.
E. Natural Compounds
1. ␣-Lipoic acid
The naturally occurring short-chain fatty acid ␣-lipoic acid (␣-LA) is an essential cofactor of mitochondrial
respiratory enzymes and is best known for its powerful
effects as an antioxidant. However, recent studies have
demonstrated impressive effects of ␣-LA on many aspects
of metabolic regulation; these effects appear to be largely
dependent on AMPK signaling. For example, in the arcuate region of the hypothalamus, ␣-LA reduces AMPK signaling and food intake (253), while in skeletal muscle it
activates AMPK enhancing glucose transport and fatty
acid oxidation (283); these effects are maintained in leptin- and insulin-resistant obese rodents. AMPK activation
by ␣-LA has also been observed in the heart (284) and the
endothelium (282) where it has been shown to prevent
and improve endothelial dysfunction by normalizing lipid
metabolism. Consistent with its role in enhancing insulin
sensitivity in vivo, it appears to concomitantly reduce
insulin secretion and content, as well as cell growth in
␤-cells (488).
2. Polyphenols and resveratrol
Polyphenols have been suggested to be the primary
active ingredients found in green tea or red wine, which
help mediate their reported beneficial effects on dyslipidemia, cardiovascular disease, and longevity. However,
the mechanism(s) of action of these compounds has remained largely a mystery, limiting their therapeutic potential. Several recent studies have demonstrated that
polyphenols such as resveratrol stimulate AMPK activity
in a variety of cell types including liver (575), skeletal
muscle (36, 384), neurons (121), and cancer (220). The
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Cross-sectional and retrospective epidemiological
studies have provided direct evidence that a lack of physical activity is strongly associated with impaired glucose
tolerance in obesity (390). Physical inactivity is considered an independent risk factor for insulin resistance and
type 2 diabetes and exercise (150 min/wk), with moderate
weight loss (⫺7%) reducing the incidence of diabetes by
58% in individuals with elevated fasting plasma glucose
(129). Importantly, this effect was greater than that
achieved by metformin treatment alone (⫺31%). Petersen
et al. (390) have demonstrated that improved glucose
tolerance with exercise training is related primarily to the
reversal of hepatic steatosis and a reduction in fasting
hepatic glucose production, while other studies also report a reduction in skeletal muscle lipid metabolites (64).
While exercise activates AMPK signaling in muscle and
liver, it remains to be tested in genetic models of AMPK
deficiency whether AMPK is sufficient and/or essential for
this effect.
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3. The traditional Chinese medicine berberine
Berberine is a naturally occurring bright yellow botanical isoquinoline alkaloid that has been commonly
used in traditional Chinese and Korean medicines as an
antimicrobial/fungal agent and as a treatment for type 2
diabetes. It has also been used historically to treat a
plethora of ailments as well as a fluorescent indicator for
heparin in mast cells. The clinical efficacy of this compound has been demonstrated in clinical trials where it
has been shown to restore euglycemia in type 2 diabetic
patients (364) and to reduce hypercholesterolemia (263);
however, the mechanisms mediating these effects are
only now being explored. Large oral doses of the drug are
required, and derivatives, including dihydroxyberberine,
with approximately fourfold greater potency are being
explored (508). Recent studies have demonstrated that
berberine potently activates AMPK in hepatocytes (66) as
well as skeletal muscle, L6 cells (94), and adipose tissue
(286). The activation of AMPK in these tissues was associated with the suppression of lipid synthesis and increases in both fatty acid oxidation and glucose uptake,
effects which may have contributed to the significant
improvements in insulin sensitivity in obese mouse models treated chronically with the extract (286). The activation of AMPK, analogous to other natural compounds
such as metformin, is thought to be due to berberine
inhibiting respiratory-complex I of the mitochondria, resulting in an elevated AMP/ATP ratio (508, 566). Berberine
activation of AMPK is not upstream kinase selective,
since activation in L6 cells is independent of CaMKK␤
inhibition by STO-609, presumably because LKB1 is acting
as the upstream kinase whereas in LKB1 ⫺/⫺ MEFS the
berberine-dependent activation is blocked by STO-609
pretreatment (508). In these experiments AICAR behaved
in the same way as berberine with respect to STO-609
inhibition, leading the authors to make the point that
inhibition of phosphatase activity by AMP or ZMP binding
to the ␥ subunit was driving AMPK activation (508).
Physiol Rev • VOL
4. Gut flora and AMPK
Despite the accepted role of both genetics and environmental factors in predicting the development of obesity, recent studies have demonstrated that rather surprisingly the type and number of bacteria colonizing the gut
and intestines are also critical (27, 289, 290, 507). A recent
study from the Gordon group (28) has shown that germfree mice, which are protected from diet-induced obesity,
have elevated AMPK signaling in skeletal muscle and
liver, an effect most likely attributable to a 50% increase in
muscle AMP content. Importantly, these effects are reversed following recolonization into the germ-free animals. Future studies are required to identify whether
alterations in AMPK signaling are required for the lean
phenotype and to determine whether changes in the hormonal milieu, related to a reduced caloric balance due to
incomplete digestion or the bacteria themselves may be
secreting a factor, which directly modulates cellular AMP
levels. This raises the exciting prospect of identifying
bacterial flora derived AMPK signaling suppressor molecule. In this event, antagonists would be expected to
block the bacterial flora impact and promote weight loss.
Alternatively, irrespective of the mechanisms involved,
these findings suggest that it may be possible to manipulate AMPK signaling and therefore weight gain through
the manipulation of gut flora by ingesting food enriched
with selected probiotics.
F. Small Molecule Activators
While metformin and to a lesser degree TZDs appear
to mediate many of their effects on lipid profiles and
insulin sensitivity through activation of AMPK, these
agents activate AMPK indirectly by altering nucleotide
levels and thus are nonspecific for AMPK. In addition,
these current treatments may also activate AMPK in tissues such as heart and brain where the activation of
AMPK is not desired. The great breadth of AMPK’s roles
linking numerous physiological events to energy metabolism makes a “drug target siren.” Nevertheless, there are
compelling arguments favoring the development of agonists that target AMPK specifically in skeletal muscle and
liver. Abbott Laboratories has pioneered this area and
identified a thienopyridone AMPK activator. This compound resulted from a chemical library screen of 700,000
compounds resulting in a single hit, nonnucleoside thienopyridone (A-592107, EC50 38 ␮M) AMPK activator. Optimization of this compound resulted in A-769662 with
substantially improved potency (EC50 0.8 ␮M) (105).
A-769662 inhibits fatty acid synthesis in isolated hepatocytes and reduces liver malonyl-CoA levels in rats. It also
decreases PEPCK, G-6-Pase, and FAS expression in ob/ob
mice, lowers plasma glucose, body weight, and plasma
and liver triglyceride levels. Although these properties
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activation of AMPK by resveratrol may be mediated by
AMP as resveratrol inhibits the mitochondrial F1 ATPase
(166). In muscle cells, the activation of AMPK by resveratrol has been linked to the stimulation of glucose transport (384) and mitochondrial biogenesis (36), while in
hepatocytes AMPK activation reduces lipid accumulation
due to high glucose. This effect is inhibited by a DNAMPK (575). Future studies in models of AMPK deficiency are needed to test in vivo whether the beneficial
effects of polyphenols do in fact require AMPK. At
present, this seems unlikely given the potent effects of a
SIRT1 activator, which improves insulin sensitivity in
obese Zucker rats without altering AMPK activity (334).
AMPK IN HEALTH AND DISEASE
Physiol Rev • VOL
activity of which is regulated by autoinhibitory mechanisms (258).
X. CONCLUSIONS AND FUTURE DIRECTIONS
AMPK is present in all tissues as an ␣␤␥ heterotrimer, and its expression is regulated by multiple genes
encoding each of the subunits (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, ␥3)
with differential tissue-specific expression and activity.
AMPK regulates cellular energy metabolism through direct effects on gene transcription and key metabolic enzymes. This typically occurs at multiple points in metabolic pathways as is illustrated by AMPK’s regulation of
lipid metabolism. At the whole body level, AMPK helps
control appetite, energy expenditure, and substrate utilization in response to exercise, nutrients, and cytokines
and may be linked to many physiological processes.
AMPK has now fully emerged from its historical orphan
status as a cellular metabolic stress sensor to a highly
integrated signaling junction for many biochemical pathways. Nevertheless, despite AMPK’s ascent to “super metabolic regulator” status, it is becoming increasingly clear
AMPK is just one of a number of players. Indeed, AMPK
can suffer from the “Heisenberg uncertainty” phenomenon where the harder you look the less it seems to be
essential, as appears to be the case for AMPK’s role in
exercise-induced glucose uptake. AMPK signaling may be
altered in a number of disease conditions; however, despite the convincing associations between AMPK signaling under a number of different conditions and treatments, there is nevertheless an urgent need for studies
using genetic models of AMPK deficiency/functional titration to fully evaluate the quantitative role of AMPK in
regulating physiological responses in vivo. We can look
forward to an exciting vista of future discoveries that
answer the following pressing questions: how significant
is AMPK to the etiology of type 2 diabetes? Will future
drug development lead to a repetoire of ␤1 and ␤2 specific
antagonists and agonists? Will AMPK targeted therapy
have a role in obesity treatment? Is AMPK a practical
target for cancer therapy? Can AMPK-activating drugs
mimic the beneficial metabolic effects of diet and exercise? Can AMPK therapy improve the quality of life in the
elderly?
ACKNOWLEDGMENTS
We are indebted to Drs. Jonathan S. Oakhill and Sebastian
B. Jorgensen for assistance in preparing the figures and to Anne
Johnston for editing the manuscript.
G. R. Steinberg is a Canadian Research Chair in Metabolism
Obesity and Type 2 Diabetes. Current address: G. R. Steinberg,
Dept. of Medicine, McMaster University, 1200 main St. W, Hamilton, Ontario, Canada L8N 375 (e-mail: [email protected]).
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were encouraging, A-769662 did not activate AMPK in
skeletal muscle and had poor bioavailability. The
A-769662 activation mechanism has turned out to be novel
and does not compete with AMP. However, A-769662 does
mimic the effects of AMP in allosterically activating
AMPK and preventing dephosphorylation (171). It does
not involve the AMP binding sites on the ␥ subunit, since
mutations that abolish AMP activation do not block
A-769662 activation (431). Instead, A-769662 activation
requires the ␤ subunit (431). Truncation mutagenesis of
the carbohydrate-binding domain (CBD) abolishes
A-769662 activation but not AMP activation. While the
CBD is important for A-769662 activation, it does not
appear to involve the glycogen/sugar-binding site, since it
does not inhibit CBD glycogen binding (171, 442). There is
a puzzling requirement for ␤-subunit Ser-108 since mutation to Ala abolishes drug activation (431, 442). A-769662
does not activate the isolated catalytic domain (␣ 1-312)
or the autoinhibited fragment (␣ 1-333) (171), suggesting
that it is primarily reversing the ␤-subunit autoinhibitory
actions rather than those of the ␣ subunit. A screen of 76
kinases indicates that A-769662 is neither a promiscuous
activator nor inhibitor of other kinases (171). In terms of
upstream kinase requirements, A-769662 activates AMPK
in HeLa cells, lacking LKB1 as well as in TAK1 null MEFs
(171). Like AMP, A-769662 inhibits dephosphorylation of
AMPK by phosphatase PP2C, which is interesting from a
mechanistic aspect because both ligands appear to have
distinct mechanisms of activation. We have recently
found that A-769662 is selective for the ␤1 isoform and
does not activate AMPK heterotrimers containing ␤2
(442) nor AMPK in tissues from ␤1 null mice.
Pang et al. (381) have recently reported a second
class of small molecule AMPK activator, termed PT1. This
compound was identified by a more targeted chemical
library screening of 3,600 diverse compounds with the
autoinhibited ␣-subunit fragment [␣1 (1-394)] and has an
EC50 8 ␮M for ␣1 and 12 ␮M for the corresponding ␣2
(1-398) fragment. Since the constitutively active AMPK
fragment ␣1 (1-312) is not activated by the PT1 compound, it is proposed that PT1 acts by reversing the
autoinhibition of the AIS (313-335). This is supported by
molecular modeling and mutagenesis studies showing
that PT1 activation depends on Glu-96 in the small lobe
and Lys-156 in the large lobe near the catalytic DFG motif
(381). However, these mutations also dramatically reduce
basal activity so are not necessarily fully informative. PT1
appears to activate the native ␣␤␥ heterotrimer since it
activates AMPK in L6 and HeLa cells (381). Both cholesterol and triglyceride synthesis are inhibited by PT1 in
HepG2 cells (381). Considerable further work is required
to determine the crystal structure of PT1 complexed with
AMPK as well as chemical optimization, target specificity,
and in vivo evaluation of the drug. This work sets the
stage for exploiting drug screening of other proteins, the
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GRANTS
We are supported by grants from the Australian Research
Council (ARC; to B. E. Kemp), National Health and Medical
Research Council (NHMRC; to G. R. Steinberg and B. E. Kemp),
National Heart Foundation (to B. E. Kemp), Diabetes Australia
Research Trust (to G. R. Steinberg), and Lord Mayor Charitable
Trust (to G. R. Steinberg). G. R. Steinberg and B. E. Kemp are
NHMRC Fellows, and B. E. Kemp is a Federation Fellow (ARC).
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