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One of the central regulators of cellular and organismal metabolism in eukaryotes is AMP-activated protein kinase (AMPK), which is activated when intracellular ATP production decreases. AMPK has critical roles in regulating growth and reprogramming metabolism, and has recently been connected to cellular processes such as autophagy and cell polarity. Here we review a number of recent breakthroughs in the mechanistic understanding of AMPK function, focusing on a number of newly identified downstream effectors of AMPK.
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The AMP-activated protein kinase (AMPK) signaling pathway
coordinates cell growth, autophagy, & metabolism
Maria M. Mihaylova and Reuben J. Shaw
Howard Hughes Medical Institute, Molecular & Cell Biology Laboratory, The Salk Institute, La
Jolla, CA 92037
One of the central regulators of cellular and organismal metabolism in eukaryotes is the AMP-
activated protein kinase (AMPK), which is activated when intracellular ATP levels lower. AMPK
plays critical roles in regulating growth and reprogramming metabolism, and recently has been
connected to cellular processes including autophagy and cell polarity. We review here a number of
recent breakthroughs in the mechanistic understanding of AMPK function, focusing on a number
of new identified downstream effectors of AMPK.
Core AMPK complex Components and Upstream Activators
One of the fundamental requirements of all cells is to balance ATP consumption and ATP
generation. AMPK is a highly conserved sensor of intracellular adenosine nucleotide levels
that is activated when even modest decreases in ATP production result in relative increases
in AMP or ADP. In response, AMPK promotes catabolic pathways to generate more ATP,
and inhibits anabolic pathways. Genetic analysis of AMPK orthologs in Arabidopsis1,
Saccharomyces cerevisiae2, Dictyostelium3, C. elegans4, Drosophila5, and even the moss
Physcomitrella patens6 has revealed a conserved function of AMPK as a metabolic sensor,
allowing for adaptive changes in growth, differentiation, and metabolism under conditions
of low energy. In higher eukaryotes like mammals, AMPK plays a general role in
coordinating growth and metabolism, and specialized roles in metabolic control in dedicated
tissues such as the liver, muscle and fat7.
In most species, AMPK exists as an obligate heterotrimer, containing a catalytic subunit (a),
and two regulatory subunits (β and γ). AMPK is hypothesized to be activated by a two-
pronged mechanism (for a full review, see8). Under lowered intracellular ATP levels, AMP
or ADP can directly bind to the γ regulatory subunits, leading to a conformational change
that protects the activating phosphorylation of AMPK9,10. Recent studies discovering that
ADP can also bind the nucleotide binding pockets in the AMPK γ suggest it may be the
physiological nucleotide for AMPK activation under a variety of cellular stresses18-11. In
addition to nucleotide binding, phosphorylation of Thr172 in the activation loop of AMPK is
required for its activation, and several groups have demonstrated that the serine/threonine
kinase LKB1 directly mediates this event12-14. Interestingly, LKB1 is a tumor suppressor
gene mutated in the inherited cancer disorder Peutz-Jeghers syndrome and in a significant
fraction of lung and cervical cancers, suggesting that AMPK could play a role in tumor
suppression15. Importantly, AMPK can also be phosphorylated on Thr172 in response to
calcium flux, independently of LKB1, via CAMKK2 (CAMKKβ) kinase, which is the
closest mammalian kinase to LKB1 by sequence homology16-19. Additional studies have
suggested the MAPKKK family member TAK1/MAP3K7 may also phosphorylate Thr172
The authors declare they have no competing financial interests.
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but the contexts in which TAK1 might regulate AMPK in vivo, and whether that involves
LKB1 still requires further investigation20, 21.
In mammals, there are two genes encoding the AMPK α catalytic subunit (α1 and α2), two β
genes (β1 and β2) and three γ subunit genes (γ1, γ2 and γ3)22. The expression of some of
these isoforms is tissue restricted, and functional distinctions are reported for the two
catalytic α subunits, particularly of AMP- and LKB1-responsiveness and nuclear
localization of AMPKα2 compared to the α123. However, the α1 subunit has been shown to
localize to the nucleus under some conditions24, and the myristoylation of the (β isoforms
has been shown to be required for proper activation of AMPK and its localization to
membranes25. Additional control via regulation of the localization of AMPK26-28 or
LKB129, 30 remains an critical underexplored area for future research.
Genetic studies of tissue-specific deletion of LKB1 have revealed that LKB1 mediates the
majority of AMPK activation in nearly every tissue type examined to date, though
CAMKK2 appears to be particularly involved in AMPK activation in neurons and T
cells31, 32. In addition to regulating AMPKα1 and AMPKα2 phosphorylation, LKB1
phosphorylates and activates another twelve kinases related to AMPK33. This family of
kinases includes the MARKs (1-4), SIKs (1-3), BRSK/SADs (1-2) and NUAKs (1-2) sub-
families of kinases34. Although only AMPKα1 and AMPKα2 are activated in response to
energy stress, there is a significant amount of crosstalk and shared substrates between
AMPK and the AMPK related kinases15.
Many types of cellular stresses can lead to AMPK activation. In addition to physiological
AMP/ADP elevation from stresses such as low nutrients or prolonged exercise, AMPK can
be activated in response to several pharmacological agents (see Figure 1). Metformin, the
most widely prescribed Type 2 diabetes drug, has been shown to activate AMPK35 and to do
so in an LKB1 dependent manner36. Metformin and other biguanides, such as the more
potent analog phenformin37, are thought to activate AMPK by acting as mild inhibitors of
Complex I of the respiratory chain, which leads to a drop of intracellular ATP levels38, 39.
Another AMPK agonist, 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside (AICAR)
is a cell-permeable precursor to ZMP, which mimics AMP, and binds to the AMPKγ
subunits40. Interestingly, the chemotherapeutic pemetrexed, which is an inhibitor of
thymidylate synthase, also inhibits aminoimidazolecarboxamide ribonucleotide
formyltransferase (AICART), the second folate-dependent enzyme of purine biosynthesis,
resulting in increased intracellular ZMP and activation of AMPK, similar to AICAR
treatment41. Finally, a number of naturally occurring compounds including Resveratrol, a
polyphenol found in the skin of red grapes, have been shown to activate AMPK and yield
similar beneficial effects on metabolic disease as AICAR and metformin42, 43. Resveratrol
can rapidly activate AMPK via inhibition of the F1F0 mitochondrial ATPase38 and the
original studies suggesting that resveratrol directly binds and activates sirtuins have come
into question44, 45. Indeed, the activation of SIRT1 by resveratrol in cells and mice appears
to require increased NAD+ levels by AMPK activity46, 47.
The principle therapeutic mode of action of metformin in diabetes is via suppression of
hepatic gluconeogenesis7, 48, 49, though it remains controversial whether AMPK is
absolutely required for the glucose lowering effects of metformin50. Since metformin acts as
a mitochondrial inhibitor, it should be expected to activate a variety of stress sensing
pathways which could redundantly serve to inhibit hepatic gluconeogenesis, of which
currently AMPK is just one of the best appreciated. Critical for future studies will be
defining the relative contribution of AMPK and other stress-sensing pathways impacted by
metformin and the aforementioned energy stress agents in accurate in vivo models of
metabolic dysfunction and insulin resistance in which these agents show therapeutic benefit.
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Nonetheless, metformin, AICAR51, the direct small molecule AMPK activator A76966252,
and genetic expression of activated AMPK in liver53 all lower blood glucose levels, leaving
AMPK activation a primary goal for future diabetes therapeutics54. As a result of the diverse
beneficial effects of this endogenous metabolic checkpoint in other pathological conditions,
including several types of human cancer, there is an increasing interest in identifying novel
AMPK agonists to be exploited for therapeutic benefits.
AMPK coordinates Control of Cell Growth and Autophagy
In conditions where nutrients are scarce, AMPK acts as a metabolic checkpoint inhibiting
cellular growth. The most thoroughly described mechanism by which AMPK regulates cell
growth is via suppression of the mammalian target of rapamycin complex 1 (mTORC1)
pathway. One mechanism by which AMPK controls the mTORC1 is by direct
phosphorylation of the tumor suppressor TSC2 on serine 1387 (Ser1345 in rat TSC2).
However, in lower eukaryotes, which lack TSC2 and in TSC2-/- mouse embryonic
fibroblasts (MEFs) AMPK activation still suppresses mTORC1 55, 56. This led to the
discovery that AMPK also directly phosphorylates Raptor (regulatory associated protein of
mTOR), on two conserved serines, which blocks the ability of the mTORC1 kinase complex
to phosphorylate its substrates42.
In addition to regulating cell growth, mTORC1 also controls autophagy, a cellular process of
“self engulfment” in which the cell breaks down its own organelles (macroautophagy) and
cytosolic components (microautophagy) to ensure sufficient metabolites when nutrients run
low. The core components of the autophagy pathway were first defined in genetic screens in
budding yeast and the most upstream components of the pathway include the serine/
threonine kinase Atg1 and its associated regulatory subunits Atg13 and Atg1757, 58. In
budding yeast, the Atg1 complex is inhibited by the Tor-raptor (TORC1) complex59-61. The
recent cloning of the mammalian orthologs of the Atg1 complex revealed that its activity is
also suppressed by mTORC1 through a poorly defined mechanism likely to involve
phosphorylation of the Atg1 homologs ULK1 and ULK2, as well as their regulatory
subunits (reviewed in62). In contrast to inhibitory phosphorylations from mTORC1, studies
from a number of laboratories in the past year have revealed that the ULK1 complex is
activated via direct phosphorylation by AMPK, which is critical for its function in
autophagy and mitochondrial homeostasis (reviewed in63).
In addition to unbiased mass spectrometry studies discovering endogenous AMPK subunits
as ULK1 interactors64, 65, two recent studies reported AMPK can directly phosphorylate
several sites in ULK166, 67. Our laboratory found that hepatocytes and mouse embryonic
fibroblasts devoid of either AMPK or ULK1 had defective mitophagy and elevated levels of
p62 (Sequestrosome-1), a protein involved in aggregate turnover which itself is selectively
degraded by autophagy66. As observed for other core autophagy proteins, ULK1 was
required for cell survival following nutrient deprivation and this also requires the
phosphorylation of the AMPK sites in ULK1. Similarly, genetic studies in budding yeast68
and in C. elegans66 demonstrate that Atg1 is needed for the effect of AMPK on autophagy.
Interestingly, Kim and colleagues found distinct sites in ULK1 targeted by AMPK, though
they also found that AMPK regulation of ULK1 was needed for ULK1 function67. These
authors also mapped a direct mTOR phosphorylation site in ULK1 which appears to dictate
AMPK binding to ULK1, a finding corroborated by another recent study, though the details
differ69. Collectively, these studies show that AMPK can trigger autophagy in a double-
pronged mechanism of directly activating ULK1 and inhibiting the suppressive effect of
mTORC1 complex1 on ULK1 (see Fig. 2). Many of the temporal and spatial details of the
regulation of these three ancient interlocking nutrient-sensitive kinases (AMPK, ULK1,
mTOR) remains to be decoded.
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Interestingly, AMPK both triggers the acute destruction of defective mitochondria through a
ULK1-dependent stimulation of mitophagy, as well as stimulating de novo mitochondrial
biogenesis through PGC-1α dependent transcription (see below). Thus AMPK controls
mitochondrial homeostasis in a situation resembling “Cash for Clunkers” in which existing
defective mitochondria are replaced by new fuel-efficient mitochondria (Fig. 3). One context
where AMPK control of mitochondrial homeostasis may be particularly critical is in the
context of adult stem cell populations. In a recent study on haematopoetic stem cells, genetic
deletion of LKB1 or both of the AMPK catalytic subunits phenocopied fibroblasts lacking
ULK1 or the AMPK sites in ULK1 in terms of the marked accumulation of defective
Beyond effects on mTOR and ULK1, two other reported targets of AMPK in growth control
are the tumor suppressor p5371 and the CDK inhibitor p2772, 73, though the reported sites of
phosphorylation do not conform well to the AMPK substrate sequence found in other
substrates. The recent discovery of AMPK family members controlling phosphatases74
presents another mechanism by which AMPK might control phosphorylation of proteins,
without being the kinase to directly phosphorylate the site.
Control of Metabolism via Transcription and Direct effects on Metabolic
AMPK was originally defined as the upstream kinase for the critical metabolic enzymes
Acetyl-CoA carboxylase (ACC1 & ACC2) and HMG-CoA reductase, which serve as the
rate-limiting steps for fatty-acid and sterol synthesis in wide-variety of eukaryotes75, 76. In
specialized tissues such as muscle and fat, AMPK regulates glucose uptake via effects on the
RabGAP TBC1D1, which along with its homolog TBC1D4 (AS160), play key roles in
GLUT4 trafficking following exercise and insulin77. In fat, AMPK also directly
phosphorylates lipases, including both hormone sensitive lipase (HSL)78 and adipocyte
triglyceride lipase (ATGL)79, an AMPK substrate also functionally conserved in C.
elegans4. Interestingly, mammalian ATGL and its liberation of fatty acids has recently been
shown to be important in rodent models of cancer-associated cachexia80. Whether AMPK is
important in this context remains to be seen.
In addition to acutely regulation of these metabolic enzymes, AMPK is also involved in a
adaptive reprogramming of metabolism through transcriptional changes. Breakthroughs in
this area have come through distinct lines of investigation. AMPK has been reported to
phosphorylate and regulate a number of transcription factors, coactivators, the
acetyltransferase p300, a subfamily of histone deacetylases, and even histones themselves.
In 2010, Bungard et al., reported that AMPK can target transcriptional regulation through
phosphorylation of histone H2B on Serine3681. Cells expressing a mutant H2B S36A
blunted the induction of stress genes upregulated by AMPK including p21 and cpt1c81, 82. In
addition, AMPK was chromatin immunoprecipitated at the promoters of these genes making
this one of the first studies to detect AMPK at specific chromatin loci in mammalian cells81.
Notably, Serine36 in H2B does not conform well to the AMPK consensus83; further studies
will reveal whether this substrate is an exception or whether this phosphorylation is
indirectly controlled.
AMPK activation has also recently been linked to circadian clock regulation, which couples
daily light and dark cycles to control of physiology in a wide variety of tissues through
tightly coordinated transcriptional programs84. Several master transcription factors are
involved in orchestrating this oscillating network. AMPK was shown to regulate the stability
of the core clock component Cry1 though phosphorylation of Cry1 Ser71, which stimulates
the direct binding of the Fbox protein Fbxl3 to Cry1, targeting it for ubiquitin-mediated
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degradation24. Importantly, this is the first example of AMPK-dependent phosphorylation
inducing protein turnover, although this is a common mechanism utilized by other kinases.
One would expect additional substrates in which AMPK-phosphorylation triggers
degradation will be discovered. Another study linked AMPK to the circadian clock via
effects on Casein kinase85, though the precise mechanism requires further investigation. A
recent genetic study in AMPK-deficient mice also indicates that AMPK modulates the
circadian clock to different extents in different tissues86.
As the role of transcriptional programs in the physiology of metabolic tissues is well-
studied, many connections between AMPK and transcriptional control have been found in
these systems. Importantly, many of the transcriptional regulators phosphorylated by AMPK
in metabolic tissues are expressed more ubiquitously than initially appreciated and may be
playing more central roles tying metabolism to growth. One example that was recently
discovered is the lipogenic transcriptional factor Srebp187. Srebp1 induces a gene program
including targets ACC1 and FASN that stimulate fatty acid synthesis in cells. In addition to
being a critical modulator of lipids in liver and other metabolic tissues, Srebp1 mediated
control of lipogenesis is needed in all dividing cells as illustrated in a recent study
identifying Srebp1 as a major cell growth regulator in Drosophila and mammalian cells88.
AMPK was recently found to phosphorylate a conserved serine near the cleavage site within
Srebp1, suppressing its activation87. This further illustrates the acute and prolonged nature
of AMPK control of biology. AMPK acutely controls lipid metabolism via phosphorylation
of ACC1 and ACC2, while mediating long-term adaptive effects via phosphorylation of
Srebp1 and loss of expression of lipogenic enzymes. AMPK has also been suggested to
phosphorylate the glucose-sensitive transcription factor ChREBP89 which dictates
expression of an overlapping lipogenic gene signature with Srebp190. Adding an extra
complexity here is the observation that phosphorylation of the histone acetyltransferase p300
by AMPK and its related kinases impacts the acetylation and activity of ChREBP as well91.
Interestingly, like Srebp1, ChREBP has also been shown to be broadly expressed and
involved in growth control in some tumor cell settings, at least in cell culture92.
Similarly, while best appreciated for roles in metabolic tissues, the CRTC family of
transcriptional co-activators for CREB and its related family members may also play roles in
epithelial cells and cancer93. Recent studies in C. elegans revealed that phosphorylation of
the CRTC ortholog by AMPK is needed for AMPK to promote lifespan extension94,
reinforcing the potentially broad biological functions of these coactivators. In addition to
these highly conserved targets of AMPK and its related kinases, AMPK has also been
reported to phosphorylate the nuclear receptors HNF4α (NR2A1)95 and TR4 (NR2C2)96, the
coactivator PGC-1α97 and the zinc-finger protein AREBP (ZNF692)98, though development
of phospho-specific antibodies and additional functional studies are needed to further define
the functional roles of these events.
Another recently described set of transcriptional regulators targeted by AMPK and its
related family members across a range of eukaryotes are the class IIa family of histone
deacetylases (HDACs)99-105. In mammals the class IIa HDACs comprise a family of four
functionally overlapping members: HDAC4, HDAC5, HDAC7, and HDAC9106 Like
CRTCs, class IIa HDACs are inhibited by phosphorylation by AMPK and its family
members, resulting in 14-3-3 binding and cytoplasmic sequestration. Recently, we
discovered that similar to CRTCs, in liver the class IIa HDACs are dephosphorylated in
response to the fasting hormone glucagon, resulting in transcriptional increases that are
normally opposed by AMPK. Once nuclear, class IIa HDACs bind FOXO family
transcription factors, stimulating their de-acetylation and activation,104 increasing
expression of gluconeogenesis genes including G6Pase and PEPCK. Collectively, these
findings suggest AMPK suppresses glucose production through two transcriptional effects:
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reduced expression of CREB targets via CRTC inactivation and reduced expression of
FOXO target genes via class IIa HDAC inactivation (Figure 4). It is worth noting that while
AMPK activation inhibits expression of FOXO gluconeogenic targets in the liver, in other
cell types AMPK is reported to stimulate a set of FOXO-dependent target genes in stress
resistance via direct phosphorylation of novel sites in FOXO3 and FOXO4 (though not
FOXO1)107, an effect which appears conserved in C. elegans108. Ultimately, defining the
tissues, isoforms, and conditions where the AMPK pathway controls FOXO via
phosphorylation or acetylation is an important goal for understanding how these two ancient
metabolic regulators are coordinated.
In addition to phosphorylating transcription regulators, AMPK has also been shown to
regulate the activity of the deacetylase SIRT1 in some tissues via effects on NAD+
levels109, 110. As SIRT1 targets a number of transcriptional regulators for deacetylation, this
adds yet another layer of temporal and tissue specific control of metabolic transcription by
AMPK. This has been studied best in the context of exercise and skeletal muscle
physiology, where depletion of ATP activates AMPK and through SIRT1 promotes fatty
acid oxidation and mitochondrial gene expression. Interestingly, AMPK was also implicated
in skeletal muscle reprogramming in a study where sedentary mice were treated with
AICAR for 4 weeks and able to perform 44% better than control vehicle receiving
counterparts111. This metabolic reprogramming was shown to require PPARβ/δ111 and
likely involves PGC-1α as well97, though the AMPK substrates critical in this process have
not yet been rigorously defined. Interestingly, the only other single agent ever reported to
have such endurance reprogramming properties besides AICAR is Resveratrol112, whose
action in regulating metabolism is now known to be critical dependent on AMPK47.
Control of Cell Polarity, Migration, & Cytoskeletal Dynamics
In addition to the ample data for AMPK in cell growth and metabolism, recent studies
suggest that AMPK may control cell polarity and cytoskeletal dynamics in some settings113.
It has been known for some time that LKB1 plays a critical role in cell polarity from simpler
to complex eukaryotes. In C. elegans114 and Drosophila115, LKB1 orthologs establish
cellular polarity during critical asymmetric cell divisions and in mammalian cell culture,
activation of LKB1 was sufficient to promote polarization of certain epithelial cell lines116.
Initially, it was assumed that the AMPK-related MARKs (Microtubule Affinity Regulating
Kinases), which are homologs of C. elegans par-1 and play well-established roles in
polarity, were the principal targets of LKB1 in polarity117. However, recent studies also
support a role for AMPK in cell polarity.
In Drosophila, loss of AMPK results in altered polarity118, 119 and in mammalian MDCK
cells, AMPK was activated and needed for proper re-polarization and tight junction
formation following calcium switch120, 121. Moreover, LKB1 was shown to localize to
adherens junctions in MDCK cells and E-cadherin RNAi led to specific loss of this
localization and AMPK activation at these sites30. The adherens junctions protein Afadin122
and a Golgi-specific nucleotide exchange factor for Arf5 (GBF1)123 have been reported to
be regulated by AMPK and may be involved in this polarity122, though more studies are
needed to define these events and their functional consequences. In Drosophila AMPK
deficiency altered multiple polarity markers, including loss of myosin light chain (MLC)
phosphorylation118. While it was suggested in this paper that MLC may be a direct substrate
of AMPK, this seems unlikely as the sites do not conform to the optimal AMPK substrate
motif. However, AMPK and its related family members have been reported to modulate the
activity of kinases and phosphatases that regulate MLC (MLCK, MYPT1), so MLC
phosphorylation may be indirectly controlled via one of these potential mechanisms.
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Another recent study discovered the microtubule plus end protein CLIP-170 (CLIP1) as a
direct AMPK substrate124. Mutation of the AMPK site in CLIP-170 caused slower
microtubule assembly, suggesting a role in the dynamic of CLIP-170 dissociation from the
growing end of microtubules. It is noteworthy that mTORC1 was also previously suggested
as a kinase for CLIP-170125, introducing the possibility that like ULK1, CLIP-170 may be a
convergence point in the cell for AMPK and mTOR signaling. Consistent with this, besides
effects on cell growth, LKB1/AMPK control of mTOR was recently reported to control
cilia126 and neuronal polarization under conditions of energy stress127. In addition, the
regulation of CLIP-170 by AMPK is reminiscent of the regulation of MAPs (microtubule
associated proteins) by the AMPK related MARK kinases, which are critical in Tau
hyperphosphorylation in Alzheimer's models128, 129. Indeed AMPK itself has been shown to
target the same sites in Tau under some conditions as well130.
Finally, an independent study suggested a role for AMPK in polarizing neurons via control
of PI3K localization131. Here, AMPK was shown to directly phosphorylate Kinesin Light
Chain 2 (KLC2) and inhibit axonal growth via preventing PI3K localization to the axonal
tip. Interestingly, a previous study examined the related protein KLC1 as a target of AMPK
and determined it was not a real substrate in vivo132. Further experiments are needed to
clarify whether AMPK is a bona fide kinase for KLC1 or KLC2 in vivo and in which
Emerging themes and future directions
An explosion of studies in the past 5 years has begun decoding substrates of AMPK playing
roles in a variety of growth, metabolism, autophagy, and cell polarity processes. An
emergent theme in the field is that AMPK and its related family members often redundantly
phosphorylate a common set of substrates on the same residues, though the tissue expression
and condition under which AMPK or its related family members are active vary. For
example, CRTCs, Class IIa HDACs, p300, Srebp1, IRS1, and tau are reported to be
regulated by AMPK and/or its SIK and MARK family members depending on the cell type
or conditions. As a example of the complexity to be expected, SIK1 itself is transcriptionally
regulated and its kinase activity is modulated by Akt and PKA so the conditions under
which it is expressed and active will be a narrow range in specific cell types only, and
usually distinct from conditions where AMPK is active. Delineating the tissues and
conditions in which the 12 AMPK related kinases are active remains a critical goal for
dissecting the growth and metabolic roles of their shared downstream substrates. A much
more comprehensive analysis of AMPK and its family members using genetic loss of
function and RNAi is needed to decode the relative importance of each AMPK family
kinase on a given substrate for each cell type.
Now with a more complete list of AMPK substrates, it is also becoming clear that there is a
convergence of AMPK signaling with PI3K and Erk signaling in growth control pathways,
and with insulin and cAMP-dependent pathways in metabolic control. The convergence of
these pathways reinforces the concept that there is a small core of rate-limiting regulators
that control distinct aspects of biology and act as master coordinators of cell growth,
metabolism, and ultimately cell fate. As more targets of AMPK are decoded, the challenge
will be in defining more precisely which targets are essential and relevant for the beneficial
effects of AMPK activation seen in pathological states ranging from diabetes to cancer to
neurological disorders. The identification of these downstream effectors will provide new
targets for therapeutically treating these diseases by unlocking this endogenous mechanism
that evolution has developed to restore cellular and organismal homeostasis.
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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
The authors want to apologize for the many primary studies in the AMPK field that could be not covered due to
space limitations. Work in the authors' laboratory is, or has been, funded by the NIH grants R01 DK080425 and
1P01CA120964, an American Cancer Society Research Scholar Award, the American Diabetes Association Junior
Faculty Award, and a Howard Hughes Medical Institute Early Career Scientist Award. We also thank the Adler
Family Foundation and the Leona M. and Harry B. Helmsley Charitable Trust for their generous support.
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Figure 1. The AMPK signaling pathway
AMPK is activated when AMP and ADP levels in the cells rise due to variety of
physiological stresses, as well as pharmacological inducers. LKB1 is the upstream kinase
activating it in response to AMP increase, whereas CAMKK2 activates AMPK in response
to calcium increase. Activated AMPK directly phosphorylates a number of subtrates to
acutely impact metabolism and growth, as well as phosphorylating a number of
transcriptional regulators that mediate long term metabolic reprogramming. Shown are all
the best-established substrates to date-those needing further in vivo examination are
italicized. Question marks denote candidate substrates whose identified phosphorylation
sites diverge from the established optimal substrate motif (which all the others conform to).
A full lineup of the identified AMPK phosphorylation sites in these substrates in
Supplemental Table 1. Substrates in red have been reported to serve as substrates of other
AMPK family members (SIK1, SIK2, MARKs, SADs) in vivo in addition to being
substrates of AMPK.
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Figure 2. The Ras/ PI3K/ mTOR pathways intersect the LKB1/AMPK pathway at multiple
LKB1, the upstream kinase for AMPK, is the tumor suppressor gene mutated in Peutz–
Jeghers syndrome (PJS), as well a significant fraction of sporadic lung cancers and cervical
cancers. PJS patients share a number of clinical features with patients inheriting defective
PTEN or TSC tumor suppressors, perhaps due to their control of common biochemical
pathways, best understood currently being the mammalian target of rapamycin complex 1
(mTORC1) pathway. Extensive cross-regulation of the LKB1/AMPK pathway by the
oncogenic Ras and PI3K pathways has been discovered, which may explain how these
commonly mutated oncogenes also try to circumvent this endogenous tumor suppressor
pathway. The ULK1/hATG1 kinase complex has emerged recently as a central node
receiving inputs from both AMPK and mTORC1. A number of kinases that can
phosphorylate specific residues in LKB1 or AMPK have been identified (upper inset),
though the contexts in which most of these regulatory events occur is poorly defined at
present, as is the functional impact of these phosphorylation events on AMPK signaling. The
BHD tumor suppressor and its partner FNIP1, as well as the sestrin family of proteins, have
also been implicated as being upstream or downstream of AMPK and mTOR depending on
the context.
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Figure 3. AMPK acts as a mitochondrial “Cash for Clunkers”
Activated AMPK acutely triggers the destruction of existing defective mitochondria via
ULK1-dependent mitophagy and simultaneously triggers the biogenesis of new
mitochondria via effects on PGC-1a dependent transcription. These dual processes
controlled by AMPK have the net effect of replacing existing defective mitochondria with
new functional mitochondria. This two-pronged control of mitochondria homeostasis by
AMPK will have a number of physiological and pathological conditions where it plays a
critical role, and a few are illustrated here.
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Figure 4. AMPK control of transcription
AMPK regulates several physiological processes through phosphorylation of transcription
factors and co-activators. It shares substrates with its AMPK family related kinases to
negatively regulate gluconeogenesis in the liver by phosphorylation and inhibition of the
CRCT2 and Class IIa HDACs. These phosphorylation events induce binding to 14-3-3
scaffold proteins and sequestration of these transcription regulators into the cytoplasm.
AMPK also regulates transcription factors via inducing their degradation (Cry1), preventing
their proteolytic activation and translocation to nucleus (Srebp1), and by disrupting protein-
protein (p300) or protein-DNA interactions (Arebp, HNF4a). AMPK has also been shown to
directly control phosphorylation of Histone 2B on Serine 36 as well as indirectly controlling
SIRT1 activity via increasing NAD+ levels.
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... AMPK is a serine/threonine kinase that is activated by AMP and plays a major role in regulating metabolic homeostasis [12,13]. By being sensitive to AMP concentrations, agonist); and (3) Compound C (30 nM; AMPK inhibitor). ...
... AMPK plays a large role in a number of metabolic diseases and has therefore become an important therapeutic target [13,23,55,56]. Impairments associated with metabolic factors such as IR and neuroinflammation seen with aging and in AD are known to exacerbate A and Tau accumulation, leading to impaired brain function [57][58][59]. ...
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Neuronal growth and synaptic function are dependent on precise protein production and turnover at the synapse. AMPK-activated protein kinase (AMPK) represents a metabolic node involved in energy sensing and in regulating synaptic protein homeostasis. However, there is ambiguity surrounding the role of AMPK in regulating neuronal growth and health. This study examined the effect of chronic AMPK activation on markers of synaptic function and growth. Retinoic-acid-differentiated SH-SY5Y human neuroblastoma cells were treated with A-769662 (100 nM) or Compound C (30 nM) for 1, 3, or 5 days before AMPK, mTORC1, and markers for synapse function were examined. Cell morphology, neuronal marker content, and location were quantified after 5 days of treatment. AMPK phosphorylation was maintained throughout all 5 days of treatment with A-769662 and resulted in chronic mTORC1 inhibition. Lower total, soma, and neuritic neuronal marker contents were observed following 5 d of AMPK activation. Neurite protein abundance and distribution was lower following 5 days of A-769662 treatment. Our data suggest that chronic AMPK activation impacts synaptic protein content and reduces neurite protein abundance and distribution. These results highlight a distinct role that metabolism plays on markers of synapse health and function.
... The master metabolic regulator, AMP-activated protein kinase (AMPK), is also traditionally considered to be an enzyme that mediates metabolic adjustment during starvation. Indeed, caloric restriction activates the AMPK pathways to maintain energy homeostasis by stimulating ATGL [66,67]. Interestingly, AMPK is also activated by certain drugs and xenobiotics, most of which act by inhibiting mitochondrial function [66]. ...
In recent years, observations of distinct organisms have linked the quality of the environment experienced by a given individual and the sex it will develop. In most described cases, facing relatively harsh conditions resulted in masculinization, while thriving in favorable conditions promoted the development of an ovary. This was shown indistinctively in some species presenting a genetic sex determination (GSD), which were able to sex-reverse, and in species with an environmental sex determination (ESD) system. However, this pattern strongly depends on evolutionary constrains and is detected only when females need more energy for reproduction. Here, I describe the mechanisms involved in this environmentally driven sex allocation (EDSA), which involves two main energy pathways, lipid and carbohydrate metabolism. These pathways act through various enzymes and are not necessarily independent of the previously known transducers of environmental signals in species with ESD: calcium–redox, epigenetic, and stress regulation pathways. Overall, there is evidence of a link between energy level and the sexual fate of individuals of various species, including reptiles, fish, amphibians, insects, and nematodes. As energy path- ways are evolutionarily conserved, this knowledge opens new avenues to advance our understanding of the mechanisms that allow animals to adapt their sex according to the local environment.
... AMP-dependent protein kinase (AMPK) also plays a key role in autophagy [117]. Studies have shown that Fyn gene deletion increases AMPK activity depending on LKB1 regulation [118,119]. ...
Src family kinases (SFKs) are non-receptor tyrosine kinases and play a key role in regulating signal transduction. The mechanism of SFKs in various tumors has been widely studied, and there are more and more studies on its role in the kidney. Acute kidney injury (AKI) is a disease with complex pathogenesis, including oxidative stress (OS), inflammation, endoplasmic reticulum (ER) stress, autophagy, and apoptosis. In addition, fibrosis has a significant impact on the progression of AKI to developing chronic kidney disease (CKD). The mortality rate of this disease is very high, and there is no effective treatment drug at present. In recent years, some studies have found that SFKs, especially Src, Fyn, and Lyn, are involved in the pathogenesis of AKI. In this paper, the structure, function, and role of SFKs in AKI are discussed. SFKs play a crucial role in the occurrence and development of AKI, making them promising molecular targets for the treatment of AKI.
... Given amino acids (Suzuki et al., 2021), fatty acids (Turner et al., 2014), and glucose (Sylow et al., 2017) are important energy sources, AMPK signaling is proposed to regulate intestinal nutrient absorption. Furthermore, AMPK has been shown to regulate intestinal barrier function (Mihaylova & Shaw, 2011;Sun et al., 2017). In addition to the functions mentioned above, AMPK also has great potential to be a therapeutic target of intestinal diseases, which cause life-threatening damage to humans around the world. ...
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As one of the most important organs in animals, the intestine is responsible for nutrient absorption and acts as a barrier between the body and the environment. Intestinal physiology and function require the participation of energy. 5′-adenosine monophosphate-activated protein kinase (AMPK), a classical and highly expressed energy regulator in intestinal cells, regulates the process of nutrient absorption and barrier function and is also involved in the therapy of intestinal diseases. Studies have yielded findings that AMPK regulates the absorption of glucose, amino acids, and fatty acids in the intestine primarily by regulating transportation systems, as we detailed here. Moreover, AMPK is involved in the regulation of the intestinal mechanical barrier and immune barrier through manipulating the expression of tight junctions, antimicrobial peptides, and secretory immunoglobulins. In addition, AMPK also participates in the regulation of intestinal diseases, which indicates that AMPK is a promising therapeutic target for intestinal diseases and cancer. In this review, we summarized the current understanding regarding how AMPK regulates intestinal nutrient absorption, barrier function, and intestinal diseases.
... AMPK is an essential regulator of cellular energy status, and can maintain energy homeostasis via autophagy 33 . The phosphorylation of AMPK is caused by increasing AMP:ATP ratios and metabolic stress, which in turns upregulates SIRT1 expression and ultimately induces autophagy 34,35 . ...
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Isosteviol sodium (STVNa) is a beyerane diterpene synthesized via acid hydrolysis of stevioside, which can improve glucose and lipid metabolism in animals with diabetes. However, it remains unknown whether STVNa can exhibit a therapeutic effect on nonalcoholic fatty liver disease (NAFLD) and its underlying mechanism. We hypothesize that autophagic initiation may play a key role in mediating the development of NAFLD. Herein, we assessed the effects of STVNa on NAFLD and its underlying mechanisms. The results demonstrated that STVNa treatment effectively ameliorated NAFLD in rats fed high-fat diet (HFD). Moreover, STVNa decreased the expression of inflammation-related genes and maintained a balance of pro-inflammatory cytokines in NAFLD rats. STVNa also reduced lipid accumulation in free fatty acid (FFA)-exposed LO2 cells. In addition, STVNa attenuated hepatic oxidative stress and fibrosis in NAFLD rats. Furthermore, STVNa enhanced autophagy and activated Sirtuin 1/adenosine monophosphate-activated protein kinase (Sirt1/AMPK) pathway both in vivo and in vitro, thus attenuating intracellular lipid accumulation. In summary, STVNa could improve lipid metabolism in NAFLD by initiating autophagy via Sirt1/AMPK pathway. Therefore, STVNa may be an alternative therapeutic agent for treatment of NAFLD.
With the prevalence of obesity and associated comorbidities, studies aimed at revealing mechanisms that regulate energy homeostasis have gained increasing interest. In 1994, the cloning of leptin was a milestone in metabolic research. As an adipocytokine, leptin governs food intake and energy homeostasis through leptin receptors (LepR) in the brain. The failure of increased leptin levels to suppress feeding and elevate energy expenditure is referred to as leptin resistance, which encompasses complex pathophysiological processes. Within the brain, LepR-expressing neurons are distributed in hypothalamus and other brain areas, and each population of the LepR-expressing neurons may mediate particular aspects of leptin effects. In LepR-expressing neurons, the binding of leptin to LepR initiates multiple signaling cascades including janus kinase (JAK)–signal transducers and activators of transcription (STAT) phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT), extracellular regulated protein kinase (ERK), and AMP-activated protein kinase (AMPK) signaling, etc., mediating leptin actions. These findings place leptin at the intersection of metabolic and neuroendocrine regulations, and render leptin a key target for treating obesity and associated comorbidities. This review highlights the main discoveries that shaped the field of leptin for better understanding of the mechanism governing metabolic homeostasis, and guides the development of safe and effective interventions to treat obesity and associated diseases.
The 5′-adenosine monophosphate-activated protein kinase (AMPK) is an important metabolic regulator. Its allosteric drug and metabolite binding (ADaM) site was identified as an attractive target for direct AMPK activation and holds promise as a novel mechanism for the treatment of metabolic diseases. With the exception of lusianthridin and salicylic acid, no natural product (NP) is reported so far to directly target the ADaM site. For the streamlined assessment of direct AMPK activators from the pool of NPs, an integrated workflow using in silico and in vitro methods was applied. Virtual screening combining a 3D shape-based approach and docking identified 21 NPs and NP-like molecules that could potentially activate AMPK. The compounds were purchased and tested in an in vitro AMPK α 1 β 1 γ 1 kinase assay. Two NP-like virtual hits were identified, which, at 30 µM concentration, caused a 1.65-fold (± 0.24) and a 1.58-fold (± 0.17) activation of AMPK, respectively. Intriguingly, using two different evaluation methods, we could not confirm the bioactivity of the supposed AMPK activator lusianthridin, which rebuts earlier reports.
Using an in-cell AMPK activation assay, we have developed structure-activity relationships around a hit pyridine dicarboxamide 5 that resulted in 40 (R419). A particular focus was to retain the on-target potency while also improving microsomal stability and reducing off-target activities, including hERG inhibition. We were able to show that removing a tertiary amino group from the piperazine unit of hit compound 5 improved microsomal stability while hERG inhibition was improved by modifying the substitution of the central core pyridine ring. The SAR resulted in 40, which continues to maintain on-target potency. Compound 40 was able to activate AMPK in vivo after oral administration and showed efficacy in animal models investigating activation of AMPK as a therapy for glucose control (both db/db and DIO mouse models).
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Casein phosphopeptides (CPPs) are good at calcium-binding and intestinal calcium absorption, but there are few studies on the osteogenic activity of CPPs. In this study, the preparation of casein phosphopeptide calcium chelate (CPP-Ca) was optimized on the basis of previous studies, and its peptide-calcium chelating activity was characterized. Subsequently, the effects of CPP-Ca on the proliferation, differentiation, and mineralization of MC3T3-E1 cells were studied, and the differentiation mechanism of CPP-Ca on MC3T3-E1 cells was further elucidated by RNA sequencing (RNA-seq). The results showed that the calcium chelation rate of CPPs was 23.37%, and the calcium content of CPP-Ca reached 2.64 × 105 mg/kg. The test results of Ultraviolet–Visible absorption spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) indicated that carboxyl oxygen and amino nitrogen atoms of CPPs might be chelated with calcium during the chelation. Compared with the control group, the proliferation of MC3T3-E1 cells treated with 250 μg/mL of CPP-Ca increased by 21.65%, 26.43%, and 28.43% at 24, 48, and 72 h, respectively, and the alkaline phosphatase (ALP) activity and mineralized calcium nodules of MC3T3-E1 cells were notably increased by 55% and 72%. RNA-seq results showed that 321 differentially expressed genes (DEGs) were found in MC3T3-E1 cells treated with CPP-Ca, including 121 upregulated and 200 downregulated genes. Gene ontology (GO) revealed that the DEGs mainly played important roles in the regulation of cellular components. The enrichment of the Kyoto Encyclopedia of Genes and Genomes Database (KEGG) pathway indicated that the AMPK, PI3K-Akt, MAPK, and Wnt signaling pathways were involved in the differentiation of MC3T3-E1 cells. The results of a quantitative real-time PCR (qRT-PCR) showed that compared with the blank control group, the mRNA expressions of Apolipoprotein D (APOD), Osteoglycin (OGN), and Insulin-like growth factor (IGF1) were significantly increased by 2.6, 2.0 and 3.0 times, respectively, while the mRNA levels of NOTUM, WIF1, and LRP4 notably decreased to 2.3, 2.1, and 4.2 times, respectively, which were consistent both in GO functional and KEGG enrichment pathway analysis. This study provided a theoretical basis for CPP-Ca as a nutritional additive in the treatment and prevention of osteoporosis.
Locus coeruleus (LC) is among the first brain areas to degenerate in Alzheimer's disease and. Parkinson's disease; however, the underlying causes for the vulnerability of LC neurons are not well defined. Here we report a novel mechanism of degeneration of LC neurons caused by loss of the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2). GPT2 Deficiency is a newly-recognized childhood neurometabolic disorder. The GPT2 enzyme regulates cell growth through replenishment of tricarboxylic acid (TCA) cycle intermediates and modulation of amino acid metabolism. In Gpt2-null mice, we observe an early loss of tyrosine hydroxylase (TH) positive neurons in LC and reduced soma size at postnatal day 18. Gpt2-null LC shows selective positive Fluoro-Jade C staining. Neuron loss is accompanied by selective, prominent microgliosis and astrogliosis in LC. We observe reduced noradrenergic projections to and norepinephrine levels in hippocampus and spinal cord. Whole cell recordings in Gpt2-null LC slices show reduced soma size and abnormal action potentials with altered firing kinetics. Strikingly, we observe early decreases in phosphorylated S6 in Gpt2-null LC, preceding prominent p62 aggregation, increased LC3B-II to LC3B-I ratio, and neuronal loss. These data are consistent with a possible mechanism involving deficiency in protein synthesis and cell growth, associated subsequently with abnormal autophagy and neurodegeneration. As compared to the few genetic animal models with LC degeneration, loss of LC neurons in Gpt2-null mice is developmentally the earliest. Early neuron loss in LC in a model of human neurometabolic disease provides important clues regarding the metabolic vulnerability of LC and may lead to new therapeutic targets.
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Axon-dendrite polarization is crucial for neural network wiring and information processing in the brain. Polarization begins with the transformation of a single neurite into an axon and its subsequent rapid extension, which requires coordination of cellular energy status to allow for transport of building materials to support axon growth. We found that activation of the energy-sensing adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK) pathway suppressed axon initiation and neuronal polarization. Phosphorylation of the kinesin light chain of the Kif5 motor protein by AMPK disrupted the association of the motor with phosphatidylinositol 3-kinase (PI3K), preventing PI3K targeting to the axonal tip and inhibiting polarization and axon growth.
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Resveratrol (3,5,4'-trihydroxystilbene) extends the lifespan of diverse species including Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. In these organisms, lifespan extension is dependent on Sir2, a conserved deacetylase proposed to underlie the beneficial effects of caloric restriction. Here we show that resveratrol shifts the physiology of middle-aged mice on a high-calorie diet towards that of mice on a standard diet and significantly increases their survival. Resveratrol produces changes associated with longer lifespan, including increased insulin sensitivity, reduced insulin-like growth factor-1 (IGF-I) levels, increased AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor- coactivator 1 (PGC-1) activity, increased mitochondrial number, and improved motor function. Parametric analysis of gene set enrichment revealed that resveratrol opposed the effects of the high-calorie diet in 144 out of 153 significantly altered pathways. These data show that improving general health in mammals using small molecules is an attainable goal, and point to new approaches for treating obesity-related disorders and diseases of ageing.
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Extracellular Ca2+ is essential for the development of stable epithelial tight junctions. We find that in the absence of extracellular Ca2+, AMP-activated protein kinase (AMPK) activation and glycogen synthase kinase (GSK)-3β inhibition independently induce the localization of epithelial tight junction components to the plasma membrane. The Ca2+-independent deposition of junctional proteins induced by AMPK activation and GSK-3β inhibition is independent of E-cadherin. Furthermore, the nectin-afadin system is required for the deposition of tight junction components induced by AMPK activation, but is not required for that induced by GSK-3β inhibition. Phosphorylation studies demonstrate that afadin is a substrate for AMPK. These data demonstrate that two kinases involved in regulating cell growth and metabolism act through distinct pathways to influence the deposition of the components of epithelial tight junctions.
As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.
AMPK is a ubiquitous sensor of cellular energy status in eukaryotic cells. It is activated by stresses causing ATP depletion and, once activated, maintains energy homeostasis by phosphorylating targets that activate catabolism and inhibit energy-consuming processes. Evidence derived from non-mammalian orthologs suggests that its ancestral role was in the response to starvation for a carbon source. We review recent findings showing that AMPK is activated by ADP as well as AMP, and discuss the mechanism by which binding of these nucleotides prevent its dephosphorylation and inactivation. We also discuss the role of the carbohydrate-binding module on the β subunit and the mechanisms by which it is activated by drugs and xenobiotics such as metformin and resveratrol.