ArticleChronic Activation of g2 AMPK Induces Obesity and
Reduces b Cell FunctionGraphical AbstractHighlightsd An activating mutation of g2 AMPK in mice causes obesity
and impairs insulin secretion
d This occurs in part due to augmentation of ghrelin signaling-
dependent hyperphagia
d Humans with the homologous g2 mutation show key aspects
of the murine phenotype
d These findings have implications for therapeutic strategies
that aim to activate AMPKYavari et al., 2016, Cell Metabolism 23, 821–836
May 10, 2016 ª 2016 The Authors. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.cmet.2016.04.003Authors
Arash Yavari, Claire J. Stocker,
Sahar Ghaffari, ...,
Michael A. Cawthorne, Hugh Watkins,
Houman Ashrafian
Correspondence
arash.yavari@well.ox.ac.uk (A.Y.),
houman.ashrafian@cardiov.ox.ac.uk
(H.A.)
In Brief
AMPK is a promising therapeutic target
for obesity. Yavari et al. reveal the
potential consequences of chronic AMPK
activation in mice carrying an activating
g2 mutation, which results in obesity,
hyperphagia, and impaired insulin
secretion. Increased adiposity and
reduced b cell function are also observed
in humans bearing this mutation.Accession NumbersGSE73436
E-MTAB-3938
Cell Metabolism
ArticleChronic Activation of g2 AMPK Induces
Obesity and Reduces b Cell Function
Arash Yavari,1,2,3,21,* Claire J. Stocker,4,21 Sahar Ghaffari,2,3 Edward T. Wargent,4 Violetta Steeples,2,3 Gabor Czibik,2,3
Katalin Pinter,2,3 Mohamed Bellahcene,2,3 Angela Woods,5 Pablo B. Martı´nez de Morentin,6 Ce´line Cansell,6
Brian Y.H. Lam,7 Andre´ Chuster,8 Kasparas Petkevicius,7 Marie-Sophie Nguyen-Tu,9 Aida Martinez-Sanchez,9
Timothy J. Pullen,9 Peter L. Oliver,10 Alexander Stockenhuber,2,3 Chinh Nguyen,2,3 Merzaka Lazdam,2
Jacqueline F. O’Dowd,4 Parvathy Harikumar,4 Mo´nika To´th,11 Craig Beall,12 Theodosios Kyriakou,2,3 Julia Parnis,2,3
Dhruv Sarma,2,3 George Katritsis,2,3 Diana D.J. Wortmann,2,3 Andrew R. Harper,2,3 Laurence A. Brown,13 Robin Willows,5
Silvia Gandra,8 Victor Poncio,14 Ma´rcio J. de Oliveira Figueiredo,14 Nathan R. Qi,15 Stuart N. Peirson,13
Rory J. McCrimmon,12 Bala´zs Gereben,11 La´szlo´ Tretter,16,17 Csaba Fekete,11,18 Charles Redwood,2,3 Giles S.H. Yeo,7
Lora K. Heisler,6 Guy A. Rutter,9 Mark A. Smith,19 Dominic J. Withers,19 David Carling,5 Eduardo B. Sternick,8
Jonathan R.S. Arch,4 Michael A. Cawthorne,4 Hugh Watkins,2,3 and Houman Ashrafian1,2,3,20,*
1Experimental Therapeutics
2Division of Cardiovascular Medicine
Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DU, UK
3Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK
4The Buckingham Institute for Translational Medicine, University of Buckingham, Buckingham MK18 1EG, UK
5Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College London, London SW7 2AZ, UK
6Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen AB25 2ZD, UK
7University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge CB2 0QQ, UK
8Pos Graduac¸a˜o Cieˆncias Me´dicas, Faculdade Cieˆncias Me´dicas, Universidade Federal de Minas Gerais, Belo Horizonte-MG 31270-901,
Brazil
9Cell Biology and Functional Genomics, Division of Diabetes, Endocrinology, and Metabolism, Imperial College London,
London SW7 2AZ, UK
10MRC Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3PT, UK
11Department of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest 1083, Hungary
12Cardiovascular and Diabetes Medicine, Medical Research Institute, University of Dundee, Dundee DD1 9SY, UK
13Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, OX3 9DU, UK
14Universidade Estadual de Campinas, Campinas-SP 13083-970, Brazil
15Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor,
MI 48109, USA
16Department of Medical Biochemistry
17MTA-SE Laboratory for Neurobiochemistry
Semmelweis University, Budapest 1085, Hungary
18Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, Tupper Research Institute, Tufts Medical Center, Boston,
MA 02111, USA
19Metabolic Signalling Group, MRC Clinical Sciences Centre, Imperial College London, London W12 0NN, UK
20Experimental Therapeutics, Clinical Science Group, New Medicines, UCB Pharma S.A., Slough, Berkshire SL1 3WE, UK
21Co-first author
*Correspondence: arash.yavari@well.ox.ac.uk (A.Y.), houman.ashrafian@cardiov.ox.ac.uk (H.A.)
http://dx.doi.org/10.1016/j.cmet.2016.04.003SUMMARY
Despite significant advances in our understanding of
the biology determining systemic energy homeosta-
sis, the treatment of obesity remains a medical chal-
lenge. Activation of AMP-activated protein kinase
(AMPK) has been proposed as an attractive strategy
for the treatment of obesity and its complications.
AMPK is a conserved, ubiquitously expressed, heter-
otrimeric serine/threonine kinase whose short-term
activation has multiple beneficial metabolic effects.
Whether these translate into long-term benefits for
obesity and its complications is unknown. Here, we
observe that mice with chronic AMPK activation, re-
sulting frommutation of the AMPK g2 subunit, exhibitCell Metabolism 23, 821–836
This is an open access article undghrelin signaling-dependent hyperphagia, obesity,
and impaired pancreatic islet insulin secretion. Hu-
mans bearing the homologous mutation manifest a
congruent phenotype. Our studies highlight that
long-term AMPK activation throughout all tissues
can have adverse metabolic consequences, with im-
plications for pharmacological strategies seeking to
chronically activate AMPK systemically to treat meta-
bolic disease.
INTRODUCTION
Obesity affects an estimated 34.9% of adults in the United
States and is a major contributor to chronic diseases associated, May 10, 2016 ª 2016 The Authors. Published by Elsevier Inc. 821
er the CC BY license (http://creativecommons.org/licenses/by/4.0/).
with premature death or disability, including the metabolic syn-
drome type 2 diabetes mellitus (T2DM) and malignancy (Bauer
et al., 2014; Ogden et al., 2014). It develops in response to a
long-term imbalance between energy intake and expenditure.
While substantial progress has been made in understanding
the mammalian energy balance circuitry (Flier, 2004; Yeo and
Heisler, 2012), existing obesity medications exploiting these
pathways are few and of limited efficacy, complicating long-
term treatment strategies (Dietrich and Horvath, 2012).
An attractive target for obesity and related complications is
AMP-activated protein kinase (AMPK). AMPK is a phylogeneti-
cally conserved serine-threonine kinase that senses cellular en-
ergetic stress through binding of adenine nucleotides (Xiao
et al., 2011). AMPK exists in virtually all eukaryotes as a hetero-
trimeric complex consisting of a catalytic a subunit and regula-
tory b and g subunits, with multiple isoforms of each (two a, two
b, and three g) (Hardie, 2014). Once activated, AMPK triggers
catabolic ATP-generating processes while repressing anabolic
biosynthesis, to restore cellular energy homeostasis (Hardie,
2014).
In multicellular eukaryotes, the AMPK signaling system has
evolved to regulate feeding as well as cellular energy homeosta-
sis: its activation increases energy intake as well as conversion
to ATP. Thus, it integrates multiple nutritional, hormonal, and
cytokine inputs, co-ordinating whole-organism energy balance
(Kahn et al., 2005). In the hypothalamus, AMPK is subject to
physiologic regulation, with feeding repressing its activity and
fasting increasing it (Minokoshi et al., 2004). Hypothalamic
AMPK plays a key role in the orexigenic effect of ghrelin,
a gut-derived hormone signaling negative energy balance,
through effects on fatty-acid oxidation and mitochondrial respi-
ration, and by increasing presynaptic excitatory input firing rate
to orexigenic agouti-related protein (AGRP)-expressing neurons
(Andersson et al., 2004; Andrews et al., 2008; Lo´pez et al.,
2008; Minokoshi et al., 2004; Yang et al., 2011). Nontargeted re-
combinant adenoviral expression of constitutively active AMPK
in the mediobasal hypothalamus (MBH) is sufficient to acutely
increase food intake and body weight in mice, while expression
of dominant-negative AMPK has the opposite effects (Mino-
koshi et al., 2004). Acute central administration of activators
(AICAR) or inhibitors (compound C) of AMPK increases or re-
duces food intake, respectively (Kim et al., 2004). Targeted
loss-of-function experiments disrupting a2 AMPK in prototypi-
cal hypothalamic neurons regulating feeding behavior induce
divergent effects on body weight depending on the population
targeted (Claret et al., 2007). However, these diverse ap-
proaches provide limited and, occasionally, contradictory in-
sights into the systemic effects of long-term AMPK activation
(Viollet et al., 2010).
In the periphery, AMPK is modulated by, and contributes to,
the salutary effects of adipokines, including the effect of leptin
and adiponectin on fatty acid oxidation, and of adiponectin
on glucose utilization and insulin sensitivity (Minokoshi et al.,
2002; Yamauchi et al., 2002). The beneficial in vivo effects of
relatively short-term administration of AMPK agonists on overall
glucose and lipid metabolism have framed the hypothesis of
AMPK pathway activation as a therapeutic strategy for obesity
and T2DM (Cool et al., 2006; Zhang et al., 2009): for example,
metformin, the most widely prescribed oral drug for T2DM is822 Cell Metabolism 23, 821–836, May 10, 2016likely to act, at least in part, through AMPK activation (Foretz
et al., 2014). We sought to investigate this putatively beneficial
effect in a mouse model in which basal AMPK activity was
increased.
The identification of mutations in PRKAG2, which encodes the
ubiquitously expressed g2 subunit, characterized by increased
unstimulated AMPK activity and resulting in heart muscle dis-
ease, provides an opportunity to investigate the metabolic con-
sequences of AMPK activation in both mouse and man (Blair
et al., 2001; Folmes et al., 2009). We developed a gene-targeted
mouse model bearing the equivalent human R302Q PRKAG2
mutation, which causes a relatively benign cardiac phenotype
(Sternick et al., 2006). The goals of our study were (1) to generate
an experimental murine model of chronic AMPK activation, (2) to
delineate the physiological consequences of long-term AMPK
activation, and (3) to assess the metabolic impact of the same
mutation in man.
Here, we report that chronic AMPK activation in mice induces
hyperphagia and adult-onset obesity, with glucose intolerance
and impaired glucose-stimulated insulin secretion. We demon-
strate rescue of this phenotype through antagonism of ghrelin re-
ceptor signaling. Demonstrating the likely relevance of these
changes to energy metabolism in man, human g2 mutation car-
riers have increased adiposity, elevated fasting glucose, and
reduced estimates of islet b cell function, as in the mouse. Our
findings provide new insights into potentially adverse conse-
quences of long-term, tissue nonselective, pharmacological
AMPK activation and thereby inform strategies to treat metabolic
disease.
RESULTS
Generation and Analysis of R299Q g2 AMPK Knockin
Mice
To test the consequences of chronic AMPK activation in vivo, we
introduced an R299Qmutation (equivalent to humanR302Q) into
the murine Prkag2 gene. Knockin mice heterozygous (Het) for
the R299Q mutation were interbred to yield wild-type (WT) and
homozygous (Homo) mutant mice. Competitive multiplex PCR
from liver tissue, where g2 is significantly expressed (Cheung
et al., 2000), confirmed mutant transcript expression (Figure 1A).
We sought to determine the functional impact of R299Q g2 on
AMPK activity. Consonant with previous cellular studies (Folmes
et al., 2009), unstimulated g2-specific AMPK activity from iso-
lated equilibrated hepatocytes of homozygous R299Q g2 mice
was almost 3-fold elevated compared to WT (13.5 ± 0.7 versus
4.7 ± 0.4 pmol/min/mg, p < 0.0001; Figure 1B). Using a pan-b
AMPK subunit antibody for immunoprecipitation, we observed
a corresponding increase in total AMPK activity in hepatocytes
from homozygous R299Q g2 mice (Figure 1C); this increase
was also observed in white adipose tissue (WAT) and striated
muscle rapidly extracted under anesthesia to prevent changes
in AMPK activation during tissue harvesting (Figures S1A
and S1B, available online). Phosphorylation of the a subunit
residue Thr172, which is required for AMPK activation, was
also increased in homozygous R299Q g2 hepatocytes, confirm-
ing elevated AMPK activity (Figures 1D and 1E). In vivo cardiac
MRI revealed no evidence of significant cardiomyopathy in
mutant mice up to 40 weeks (data not shown).
Figure 1. R299Q g2 AMPK Mice Develop Obesity
(A) R299Q allelic discrimination plot from hepatic cDNA.
(B and C) Isolated hepatocyte basal g2-specific (B) and total (C) AMPK activity (n = 12).
(D and E) Representative immunoblot (D) and quantitation (E) of total a AMPKThr172 phosphorylation from isolated hepatocytes (n = 3).
(F) Male and female appearances aged 20 weeks.
(G) Growth curves on normal chow diet (n = 7).
(H) Total body fat mass at 4 and 40 weeks (n = 4–7).
(I) Hepatic H&E staining and steatosis quantification from male mice aged 40 weeks (n = 5); magnification 1003.
(J and K) Oral glucose tolerance and area (J) under the curve (AUC) for glucose (K) at 40weeks (n = 9). (J) *p < 0.05 versusWT. **p < 0.01Het versusWT. z p < 0.001
Homo versus WT.
(L and M) Insulin tolerance (L) and area above the curve (AAC) (M) for glucose at 40 weeks (n = 6). (L) *p < 0.05 Het versus WT. **p < 0.01 Homo versus WT. z
p < 0.01 Homo versus WT.
NTC, non-template control. Data are mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. See also Figures S1 and S2 and Table S1.
Cell Metabolism 23, 821–836, May 10, 2016 823
These results indicate that the R299Q g2 mutation induces a
basal gain of function in g2 AMPK and mild increase in total
AMPK activity.
Gain of Function in g2 AMPK Results in Age-Related
Obesity in Mice
We next examined the systemic consequences in mice of acti-
vating AMPK with the R299Q g2 mutation. Strikingly, R299Q
g2mice fed a normal chowdiet displayedmarked age-related in-
crease in body weight and size, most prominently in homozy-
gous males (Figures 1F, 1G, and S1C). While comparable in
weight and adiposity after weaning, we identified subtle alter-
ations in lean mass in R299Q g2 mice (Figures S1D and S1E).
Plasma and hepatic tissue levels of insulin-like growth factor 1
(IGF-1), a key effector of somatic growth, were comparable
across genotypes; however, we observed a trend (p = 0.05)
toward greater skeletal muscle IGF-1 levels in homozygous
R299Q g2 mice (Figures S1F–S1H). We found subtle changes
in expression of glycogen metabolism-related genes (Fig-
ure S2Q) but no differences in skeletal muscle glycogen content
(data not shown). At 40 weeks, R299Q g2 mice exhibited mark-
edly greater fat mass, consistent with obesity, and hepatic stea-
tosis (Figures 1H and 1I). Direct measurement of WAT depots
supported this, with evidence of white adipocyte hypertrophy
(Figures S1I and S1J). Obesity is associated with a chronic in-
flammatory state contributing to the development of insulin
resistance and T2DM (Hotamisligil, 2006). We identified in-
creases in plasma proinflammatory cytokines (Table S1) and up-
regulation of WAT expression of Tnf (encoding tumor necrosis
factor a) andAdgre1 (encodingmacrophage-restricted adhesion
G protein-coupled receptor E1, F4/80) (Figures S1K and S1L)
in 40-week-old R299Q g2 mice, consistent with systemic and
adipose inflammation.
Young pre-obese homozygous R299Q g2 mice exhibited
small reductions in plasma leptin compared to WT, with compa-
rable adiponectin (Table S1), but by 40 weeks displayed hyper-
leptinemia and hypoadiponectinemia (the latter with reduced
WAT expression; Figure S1M), consistent with obesity.
AMPK activation has been shown to improve insulin sensitivity
(Zhang et al., 2009). Evaluation of oral glucose and insulin toler-
ance (OGTT and ITT, respectively) in R299Q g2mice revealed no
differences toWT at 4weeks of age (Figures S1N, S1O, S1Q, and
S1R). To further explore insulin action in vivo, we used hyperin-
sulinemic-euglycemic clamps, coupled with isotopic [1-14C]-2-
deoxyglucose for assessment of tissue-specific glucose uptake
and [3-3H]-glucose tomeasure glucose turnover rate. Consistent
with the OGTT/ITT and the relatively minor contribution of g2
AMPK to total AMPK activity across most peripheral tissues
(80%–90% associated with the g1 isoform) (Cheung et al.,
2000), we found no significant differences inwhole-body glucose
turnover, basal hepatic glucose production (HGP), insulin-medi-
ated suppression of HGP, or glucose uptake of most tissues as-
sessed (Figures S2A–S2N). However, we observed a small but
significantly greater requirement for glucose in homozygous
R299Q g2 mice (p < 0.0001 for the effect of genotype on glucose
infusion rate, two-way ANOVA; Figures S2A and S2B), consis-
tent with a subtle increase in whole-body glucose utilization,
together with a trend (p = 0.05) toward increased glucose uptake
in gastrocnemius muscle (Figure S2I).824 Cell Metabolism 23, 821–836, May 10, 2016Hepatic steatosis reflects imbalance between triglyceride
acquisition and disposal (via fatty acid oxidation and triglyceride
export). The fatty acids required for triglyceride generation arise
from de novo lipogenesis (DNL) or extrinsic sources. AMPK has
been shown to exert beneficial effects on hepatic lipid meta-
bolism through its effects on fatty acid oxidation (via phosphor-
ylation of acetyl-CoA carboxylase; ACC) and lipogenesis (via
phosphorylation of sterol regulatory element binding protein
1c; SREBP-1c) (Li et al., 2011). We found no significant differ-
ence in hepatic SREBP-1c Ser372 phosphorylation between
genotypes (data not shown). However, assessment of hepatic
expression of lipogenesis-related genes revealed upregulation
of SREBP-1c target genes in heterozygous R299Q g2 mice,
including fatty acid synthase (Fasn; versus WT) and stearoyl-
CoA desaturase-1 (Scd1; versus homozygous R299Q g2) (Fig-
ure S2O). Examination of genes related to fatty acid oxidation
revealed upregulation of Cpt1a (catalyzing the rate-limiting
step of import of long-chain fatty acids into the mitochondrial
matrix) but downregulation of Acad1 (acyl-CoA dehydrogenase,
catalyzing the first step inmitochondrial beta oxidation) in R299Q
g2 mice (Figure S2O). As a functional correlate, quantification of
the rate of hepatic DNL in vivo—by measuring [3H]-glucose
incorporation into liver total lipids—revealed significantly greater
DNL in homozygous R299Q g2 mice (Figure S2P).
At 40 weeks, as expected with obesity, R299Q g2 mice dis-
played glucose intolerance (Figures 1J and 1K) and reduced in-
sulin sensitivity (Figures 1L and 1M). However, plasma insulin
levels before and after glucose challenge were lower in R299Q
g2 mice at 4 weeks and comparable to WT at 40 weeks (Figures
S1P and S1S), an observation we return to below.
Obesity in R299Q g2 AMPK Mice Is Driven by
Hyperphagia
We next evaluated energy balance in young adult mice when
genotypes were comparable in body weight, to avoid the con-
founding consequences of obesity per se (Tscho¨p et al., 2012).
R299Q g2 mice exhibited largely comparable levels of energy
expenditure (EE) and respiratory exchange ratio (RER) to WT
mice (Figures 2A–2F). Spontaneous locomotor activity did not
significantly differ (Figures S3A–S3D). We assessed adaptive
thermogenesis mediated by activated brown adipose tissue
(BAT): interscapular BAT (iBAT) weight, histology, and expres-
sion of key thermogenic genes were unchanged, as was the
thermic response to BRL 37344 (a b3-adrenoceptor-selective
agonist with lesser potency at the b2-adrenoceptor) (Figures
S3E–S3H). Re-evaluation at 40 weeks confirmed no reduction
in EE (data not shown).
However, R299Q g2 mice were hyperphagic, most apparent
in male homozygotes (Figures 2G and 2H). Accordingly, we
focused on the male WT and homozygous R299Q g2 mice com-
parison for all subsequent experiments delineating the mecha-
nism(s) of hyperphagia. Pair-feeding experiments matching daily
food intake of homozygous R299Q g2mice to that ofWT normal-
ized their body weight (Figure 2I), confirming hyperphagia as the
principal driver of weight gain.
Taken together with the findings from the preceding section,
these results demonstrate that the effects of the R299Q g2 mu-
tation are spatially and temporally dynamic, with evidence of
some beneficial changes early on, consistent with the canonical
Figure 2. Energy Expenditure and Food
Intake of R299Q g2 AMPK Mice
(A–F) Energy expenditure and respiratory ex-
change ratio (RER) in males (A–C, n = 5) and
females (D–F, n = 7) at 6 weeks.
(G and H) Food intake in male (G) and female (H)
mice aged 8 weeks (male n = 11, female n = 4).
(I) Effect on body weight of pair-feeding homozy-
gous R299Q g2 mice to WT food intake (n = 6–12).
PF = pair fed. **p < 0.01 versus WT. ***p < 0.001
versus WT. ****p < 0.0001 versus WT. z p < 0.01
versus non-PF Homo. c p < 0.001 versus non-PF
Homo. ε p < 0.0001 versus non-PF Homo.
Data are mean ± SEM. *p < 0.05. **p < 0.01. See
also Figure S3.actions of AMPK activation in the periphery, but which are ulti-
mately likely to be overwhelmed by hyperphagia, leading to
obesity.
Chronic Activation of g2 AMPK Promotes AGRP Neuron
Excitability
To explore the hyperphagia driven by the R299Q g2mutation, we
examined central mechanisms regulating food intake in young
adult mice, focusing on the hypothalamus, a primary locus for
appetite regulation (Morton et al., 2006). We confirmed WT g2
expression in key nuclei implicated in energy homeostasis,
including the arcuate nucleus (ARC), by in situ hybridization
(ISH) (Figure 3A). Phosphorylation of ACC, a canonical AMPK
substrate, was increased in MBH lysates from R299Q g2 mice,
consistent with AMPK activation (Figures 3B and 3C).
The ARC integrates central and peripheral signals to regulate
food intake and contains two distinct populations of neurons,
distinguished by their expression of neuropeptides AGRP or
POMC (pro-opiomelanocortin), which promote and reduce
food intake, respectively (Flier, 2004). AGRP is expressed exclu-
sively in the ARC and is coexpressed with another potent orexi-
gen, neuropeptide Y (NPY). To assess whether the hyperphagiaCell Mof R299Q g2 mice was associated with
greater orexigenic neuropeptide expres-
sion, we undertook ARC laser-capture
microdissection followed by massive
parallel RNA sequencing (RNA-seq) and
observed an 
50% increase in both
Agrp and Npy (p < 0.001) but unaltered
Pomc expression in R299Q g2 mice
(Figures 3D–3F). Hypothalamic ISH
confirmed upregulated AGRP expression
(Figure 3G).
To determine whether changes in the
excitable properties of ARC NPY-ex-
pressing (i.e., AGRP) neurons contributed
to the R299Q g2 hyperphagic phenotype,
we crossed R299Q g2 mice with reporter
mice expressing hrGFP under the Npy
promoter (NPY-hrGFP); we made record-
ings from ARC NPY neurons from these
and control (WT/NPY-hrGFP) mice. We
identified a slightly more depolarizedresting membrane potential (Vm) of ARC AGRP neurons from
ad libitum-fed R299Q g2 mice (Figures 3H and 3I) and a nonsig-
nificant increase in spike frequency (Table S2). To investigate the
role of increased synaptic input, we bathed brain slices in GABAA
(g-aminobutyric acid) receptor ((+)-bicuculline) and glutamater-
gic receptor (NBQX and AP5) antagonists (‘‘synaptic inhibitors’’;
Figure 3J) and identified persistent differential changes in Vm,
suggesting an intrinsic difference in AGRP neuron excitability
(Figure 3K). No differences were observed in other biophysical
properties at baseline or in the presence of fast synaptic inhibi-
tors (Table S2).
These results implicate increased excitability of ARC AGRP
neurons and elevations of their cognate neuropeptides as rele-
vant electrical and molecular substrates for the hyperphagia of
R299Q g2 mice.
Hyperphagia Associated with Chronic g2 AMPK
Activation Is Dependent on Increased Ghrelin Receptor
Signaling
AGRP expression and neuronal firing rate increase with food
deprivation (Takahashi and Cone, 2005). We explored the effect
of fasting on subsequent feeding and weight gain in R299Q g2etabolism 23, 821–836, May 10, 2016 825
Figure 3. Hypothalamic Expression of g2 AMPK and
Consequences of Its Activation on ARC Neuropeptide
Expression and AGRP Neuron Electrophysiology
(A) Expression pattern of Prkag2 in normal murine hypothalamus
using digoxigenin ISH. Scale bar, 100 mm.
(B and C) Representative immunoblot (B) and quantitation (C) of
ACCSer79 phosphorylation in MBH (n = 6).
(D–F) ARC gene expression of orexigenic (Agrp, D and Npy, E)
and anorexigenic (Pomc, F) neuropeptides (n = 5). FPKM, frag-
ments per kilobase per million mapped reads.
(G) Hypothalamic Agrp expression by digoxigenin ISH and
quantification (n = 4). Scale bar, 100 mm.
(H–K) Current-clamp recordings from WT/NPY-hrGFP and ho-
mozygous R299Q g2/NPY-hrGFP ARC neurons at baseline (H)
and in the presence of fast synaptic inhibitors (J), together with
Vm scatterplots (I and K) (n = 14). Action potential spike ampli-
tudes truncated to demonstrate changes in Vm.
Data aremean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. See also
Table S2.
826 Cell Metabolism 23, 821–836, May 10, 2016
Figure 4. Influence of Physiological and Hormonal Modulation on Food Intake in R299Q g2 AMPK Mice
(A) Cumulative food intake following overnight fast (n = 11).
(B) Representative images and quantification of MBH FOS IR of WT/NPY-hrGFP and homozygous R299Q g2/NPY-hrGFPmice in fed and fasted states (n = 3–6).
Scale bar, 100 mm (top row) or 25 mm (lower rows).
(C) Acute feeding response of mice aged 6 weeks to peripheral ghrelin (30 mg, i.p.) (n = 5).
(D) Feeding response to 0.01 mg intracerebroventricular (i.c.v.) ghrelin (n = 7). x p < 0.0001 Homo ghrelin versus all other groups at 24 hr.
(E) Hypothalamic Bsx expression by ISH and quantification (n = 4). Scale bar, 100 mm.
(F) Effect of peripherally administered GHSR antagonist [D-Lys3]-GHRP-6 (200 nmol, i.p.) on food intake (n = 8).
(G) Effect of central [D-Lys3]-GHRP-6 (1 nmol, i.c.v.) on food intake (n = 8).
(H) Cumulative food intake after 4 weeks i.p. of [D-Lys3]-GHRP-6 (100 nmol twice daily) (n = 9–11).
(I) Cumulative food intake following MT-II (1 mg/kg, i.p.) as percent of vehicle-treated mice of the same genotype (n = 12–13).
Data are mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. See also Figure S4.mice, identifying exaggerated responses (Figures 4A and S4A).
Fasting-induced immunoreactivity (IR) of the immediate early
gene Fos, a marker of neuronal activation, was strikingly greaterin ARC NPY neurons of R299Q g2 mice, suggesting enhanced
fasting-induced neuronal activation (Figure 4B). During fasting,
circulating ghrelin conveys a negative energy balance signal toCell Metabolism 23, 821–836, May 10, 2016 827
Figure 5. ARC Transcriptome, Pathway Analysis, and Mediobasal Hypothalamic Mitochondrial Respiratory Activity in R299Q g2 AMPKMice
(A) Hierarchical clustering and heat map visualization of differentially expressed genes (1.5-fold change, FC; 361 genes) from the ARC of ad libitum-fed male mice
aged 8 weeks.
(B) Principle component analysis plot indicating segregation of genotypes.
(C) Top five canonical pathways in the ARC identified by pathway analysis.
(D) Venn diagram illustrating gene overlap in (C).
(E) Representative mitochondrial oxygen consumption trace from pooled mediobasal hypothalamic homogenates. Glutamate plus malate (GM), ADP, pyruvate
(Pyr), cytochrome c (Cyt c), carboxyatractylozide (CAT), uncoupler (FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone), and antimycin A (Anti) were
given as indicated.
(F) Effects of substrates on mediobasal hypothalamic mitochondrial oxygen consumption (n = 4–5 of 3 pooled mediobasal hypothalami).
(legend continued on next page)
828 Cell Metabolism 23, 821–836, May 10, 2016
the hypothalamus, exerting an orexigenic effect dependent upon
both NPY and AGRP expression (Chen et al., 2004). Given the
requirement for AMPK activation in ghrelin-evoked feeding (Lo´-
pez et al., 2008), we hypothesized that the heightened refeeding
of R299Q g2 mice reflected greater sensitivity to ghrelin’s orexi-
genic action. We tested the acute feeding response to a single
dose of ghrelin given peripherally (intraperitoneally, i.p.) or cen-
trally (intracerebroventricularly, i.c.v.) and found it significantly
greater in R299Q g2 mice (Figures 4C and 4D). Baseline plasma
active ghrelin levels were unaltered (Figure S4B). The brain-spe-
cific homeobox transcription factor (BSX) is expressed promi-
nently in the ARC where it is confined to virtually all adult
AGRP, but not POMC, neurons, playing a key role in post-fast
and ghrelin-induced feeding by directly regulating Npy and
Agrp transcription (Sakkou et al., 2007). Consistent with elevated
basal ARC Agrp and Npy expression, we found 2.5-fold greater
Bsx expression in freely fed R299Q g2 mice (Figure 4E).
Ghrelin’s orexigenic action is exclusively signaled via a single
receptor with unusually high ligand-independent constitutive ac-
tivity: the growth hormone secretagogue receptor (GHSR) (Holst
et al., 2003). GHSR is expressed in the ARC, where it colocalizes
with 
94% AGRP, but very few POMC neurons, and is respon-
sible for the majority of the acute feeding response to ghrelin
(Wang et al., 2014; Willesen et al., 1999). We examined whether
GHSR inhibition could ameliorate R299Q g2-associated hyper-
phagia and determined the effect of the selective GHSR antago-
nist, [D-Lys3]-GHRP-6, on post-fast refeeding. We observed a
markedly greater anorexigenic effect in R299Q g2 than WT
mice with peripheral or central [D-Lys3]-GHRP-6 (Figures 4F
and 4G). We next administered [D-Lys3]-GHRP-6 over 4 weeks
(i.p.) and found it to completely normalize R299Q g2 mice food
intake without effect in WT (Figure 4H).
In addition to ghrelin’s orexigenic action leading to sustained
positive energy balance, central ghrelin has been shown to pro-
mote adiposity independent of feeding by regulating WAT lipo-
genesis (Theander-Carrillo et al., 2006). However, we found no
significant differences in WAT expression of lipogenesis or fatty
acid oxidation-related genes assessed at 8 weeks (Figure S4C),
a finding that may reflect relative equipoise at this age between
the influence of central ghrelin signaling to promote lipogenesis
versus the direct antilipogenic effects of chronic AMPK activa-
tion in WAT to inhibit fatty acid uptake and promote lipolysis
(Gaidhu et al., 2009).
AGRP neurons inhibit anorexigenic POMC neurons and
antagonize the effects of POMC-derived a-melanocyte-stimula-
ting hormone (MSH) on melanocortin receptors (Cowley et al.,
2001). We considered whether a failure of central satiety net-
works further contributed to R299Q g2-induced hyperphagia.
To directly probe the functionality of the melanocortinergic
circuitry, we examined the response to melanotan-II (MT-II), a
melanocortin-3/4 receptor agonist. MT-II reduced food intake
in all genotypes, but with greater effect in WT (Figures 4I,
S4D, and S4E), suggesting reduced central melanocortinergic(G) In situ ROS generation detected by dihydroethidium (DHE) (red fluorescence) i
and homozygous R299Q g2/NPY-hrGFP mice (n = 5–7 mice). Scale bar, 25 mm.
(H and I) Quantification (H) and representative images (I) of MBH FOS and pS6 IR o
or 25 mm (lower rows).
Data are mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. See also Table S3.sensitivity in R299Q g2 mice that may reflect increased availabil-
ity of its endogenous competitive antagonist, AGRP (Ollmann
et al., 1997).
Thus, the R299Q g2 mutation lowers the threshold for feeding
by enhancing the gain on ghrelin-responsive orexigenic circuitry,
with GHSR inhibition sufficient to normalize hyperphagia.
Arcuate Nuclei from R299Q g2 AMPK Mice Display a
Gene Signature of Enhanced Oxidative Phosphorylation
Capacity and Ribosomal Biosynthesis
To delineate the signaling networks underlying the hyperphagia
of R299Q g2 mice, we analyzed ARC whole-transcriptome
profiles from freely fed mice, identifying 609 genes with signifi-
cant differential expression (Figures 5A and 5B). Ingenuity
pathway analysis identified highly significant overrepresentation
of several pathways, including oxidative phosphorylation
(OXPHOS; p = 8.1 3 1024) and mTOR signaling (p = 8.5 3
1010) (Figure 5C). We found significant overlap of genes within
these enriched pathways, with a substantial contribution from
mitochondrial respiratory chain components (including upregu-
lation of subunits of all four mitochondrial complexes and ATP
synthase) and ribosomal proteins, likely to promote enhanced
energetic capacity and macromolecular biosynthesis to support
sustained pro-orexigenic signaling (Figure 5D; Table S3).
To directly assess mediobasal hypothalamic mitochondrial
bioenergetic function, we utilized a modified substrate-uncou-
pler-inhibitor titration (SUIT) protocol (Pesta and Gnaiger, 2012)
to examine mitochondrial oxygen consumption (Figure 5E). We
identified a highly significant effect of genotype (p < 0.0001;
two-way ANOVA) and greater oxygen flux after glutamate plus
malate—complex I-linked substrates—followed by the addition
of pyruvate, consistent with upregulation of NADH-dependent
dehydrogenase activities and/or the overexpression of complex
I subunits (Figure 5F). In support of the latter, the ARC transcrip-
tome of R299Q g2 mice exhibited enrichment of many complex I
subunits (including mt-Nd3, Ndufb5, Ndufa5, Ndufv1, mt-Nd2,
Ndufb7, and others) (Table S3). The mitochondrial respiratory
chain is a major source of reactive oxygen species (ROS) in neu-
rons. Consistent with greater mitochondrial oxygen consump-
tion, assessment of in situ ROS suggested enhanced ROS pro-
duction in AGRP neurons from R299Q g2 mice (Figure 5G). In
AGRP neurons, ghrelin has been shown to enhance fatty acid
oxidation and mitochondrial respiration with consequent ROS
generation, the latter normally quenched by UCP2-associated
mitochondrial uncoupling (Andrews et al., 2008). We observed
no significant differences in ARC baseline Ucp2 expression,
however (data not shown), which may explain the discernible
signal for enhanced AGRP neuronal ROS in R299Q g2 mice.
Ribosomal protein S6, a structural component of the ribo-
some, is phosphorylated by ribosomal protein S6 kinase (S6K).
Phosphorylation of S6 is implicated in ghrelin’s orexigenic effect
(Hannan et al., 2003; Martins et al., 2012) and has been reported
to identify hypothalamic neurons regulated by food availabilityn arcuate NPY-hrGFP positive (green fluorescence) neurons of WT/NPY-hrGFP
f NPY-hrGFPmice in fed and fasted state (n = 3–6). Scale bar, 100 mm (top row)
Cell Metabolism 23, 821–836, May 10, 2016 829
Figure 6. Isolated Islet Insulin Secretion and Gene Expression Profile of R299Q g2 AMPK Mice
(A) Insulin secretion from isolated islets in response to variable glucose (n = 3).
(B andC) Representative perforated patch-clamp recordings of the electrical (B) andmembrane potential response (C) of isolated b cells to glucose level variation
(n = 6).
(D) Top 15 KEGG gene sets most significantly enriched for upregulated (red bar) and downregulated (blue bar) genes. Gene sets highly relevant to b cell function
highlighted in red.
(E) Plot of all measured genes ranked by log2 fold change in gene expression with those most upregulated in heterozygotes on the left.
(F and G) Enrichment plots of gene sets relevant to b cell function. Clustering of genes (black vertical lines) at the left or right side indicate enrichment for up-
regulated genes in the T2DM gene set (F) and for downregulated genes in the maturity onset diabetes of the young (MODY) (G) gene set.
(legend continued on next page)
830 Cell Metabolism 23, 821–836, May 10, 2016
(Knight et al., 2012). Fasting and ghrelin increase ARC pS6 IR in
activated (i.e., FOS positive) AGRP neurons (Villanueva et al.,
2009). Based on the hypothesis that pS6 induction corresponds
to significant AGRP neuronal activation, we predicted that fast-
ing would amplify the difference between R299Q g2 and WT
mice. Supporting this, we found greater induction of pS6 in acti-
vated AGRP cells from R299Q g2 following fasting compared to
WT mice (Figures 5H and 5I).
These data suggest that chronic g2 AMPK activation results
in adaptive changes in ARC gene expression profile, specifically
including critical OXPHOS components, with a corresponding
increase in mediobasal oxidative phosphorylation capacity
andactivity, adaptations likely tosustainenergetically costlyorexi-
genicAGRPneuronal activity,whichacts topromotehyperphagia.
The R299Q g2 AMPK Mutation Suppresses Islet Insulin
Release and Upregulates Genes Normally Repressed in
the b Cell
Returning to the observation of lower basal and glucose-stimu-
lated insulin levels in young pre-obese R299Q g2 mice (Fig-
ure S1P), we investigated whether this reflected an intrinsic
change in pancreatic insulin secretion. Evaluation of isolated islet
glucose-stimulated insulin secretion (GSIS) revealed a marked
reduction in R299Q g2 mice (Figure 6A). Insulin immunostaining
revealed comparable islet morphology across genotypes (Fig-
ures S5A–S5D). Pancreatic insulin content from aged mice was
comparable (Figure S5E).
To address the possibility that reduced GSIS reflected
impaired b cell glucose sensing, we next measured electrical re-
sponsivity of isolated b cells to glucose. Patch-clamp recordings
of b cells derived fromWT and R299Q g2 mice revealed indistin-
guishable electrical activity at high glucose and fully reversible
membrane hyperpolarization in response to low glucose, consis-
tent with normal regulation of membrane potential by KATP chan-
nels (Figures 6B and 6C). Whole-cell voltage-clamp analyses
revealed no difference in the current-voltage relationship or in
slope conductance before and after depletion of cellular ATP
to determine maximal KATP channel activity (Figures S5F–S5H),
suggesting the impaired GSIS of R299Q g2 mice to be KATP
channel independent.
To gain further insight into mechanisms potentially underlying
impaired GSIS, we evaluated the islet transcriptome with RNA-
seq. Assessment of differentially expressed functional gene
clusters revealed the clearest differences to be in the Het versus
WT islet transcriptome comparison, with T2DM as the 14th most
enriched gene set among upregulated genes (false discovery
rate; FDR 11.2%) and maturity onset diabetes of the young
(MODY) as the fifth most enriched gene set among the most
downregulated genes (FDR 5.9%) (Figures 6D–6G; Table S4).
Notable among the former included downregulation of the two
functional insulin genes (Ins1 and Ins2) andGck, encoding gluco-
kinase, critical for glucose sensing and whose loss of function
is associated with monogenic forms of diabetes (Ashcroft and
Rorsman, 2012). By contrast, high-affinity hexokinase isoforms(H) Enrichment plot of GSEA undertaken using a b cell disallowed gene set.
(I and J) Baseline (30 min, I) and stimulated (+30 min, J) plasma insulin level follo
twice daily (n = 9).
Data are mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. See als(Hk1, Hk2, and Hk3) were upregulated. Gene set enrichment
analysis (GSEA) using a customized ‘‘b cell disallowed’’ set con-
structed from genes which we have shown to be highly selec-
tively repressed in mature b cells (Pullen et al., 2010) demon-
strated significant enrichment for upregulated genes (FDR
0.87%), including genes with potential to alter glucose meta-
bolism and thereby insulin secretion (Acot7 and Ldha), and
genes relevant to oxidative stress (Cat, Gsta4, and Mgst1), cell
proliferation (Cxcl12, Igfbp4, Nfib, and Pdgfra), and exocytosis
(Arhgdib and Mylk) (Figure 6H). Several of these disallowed
genes are also upregulated in humans with T2DM (Pullen and
Rutter, 2013). These data indicate that the R299Q g2 mutation
causes re-expression of b cell disallowed genes, with a profile
reminiscent of that of T2DM.
To determine whether, as in the hypothalamus, GHSR-based
signaling contributed to the g2-related islet phenotype, including
impaired GSIS, we evaluated glucose tolerance following GHSR
antagonism. [D-Lys3]-GHRP-6 normalized the insulin secretory
response of R299Q g2 mice 30 min post-glucose without
affecting glucose tolerance or basal insulin levels (Figures 6I,
6J, and S5I).
The Corresponding R302Q g2 AMPK Mutation in Man Is
Associated with Increased Adiposity, Reduced Basal b
Cell Function, and Elevated Plasma Glucose
Heterozygous human carriers of the R302Q g2 missense muta-
tion—orthologous to R299Q in mice—have a relatively mild car-
diac phenotype (Sternick et al., 2006). A systemic metabolic
phenotype has not been described for this or other pathogenic
PRKAG2 variants. To explore this possibility, we examined
26 adults heterozygous for the R302Q g2 mutation (R302Q ±)
and 44 genotype-negative siblings (mean age 41.2 ± 2.6 and
38.6 ± 2.3 years, respectively; mean ± SEM). None had cardiac
contractile dysfunction or a diagnosis of T2DM.
We observed small nonsignificant increases in body weight
(male 80.6 ± 2.9 versus 78.2 ± 4.6 kg; female 68.2 ± 2.1 versus
66.3 ± 3.0 kg), height, body mass index, and waist-to-hip ratio
in R302Q carriers versus controls (Table S5). Evaluation of
adiposity blind to genotype identified greater skinfold thickness
in R302Q carriers in the majority of sites assessed and, when
summated, was significantly increased in both sexes (Figures
7A–7F and S6A–S6D). Enhanced adiposity has been causatively
linked to elevation of hepatic biomarkers, a likely consequence of
hepatic steatosis (Fall et al., 2013; Jo et al., 2009). Consistentwith
their increased adiposity, R302Q carriers had significantly higher
plasma g-glutamyl transferase and bilirubin levels, but compara-
ble hepatic aminotransferases (Figures 7G, 7H, S6E, and S6F).
We found greater fasting glucose (5.0 ± 0.1 versus 4.6 ±
0.1 mmol/L, p < 0.05) and a trend to lower fasting insulin
(33.7 ± 2.9 versus 42.2 ± 4.3 pmol/L, p = 0.10) in R302Q carriers
(Figures 7I and 7J). To confirm the signal for elevated glucose,
we measured the percentage of glycated adult hemoglobin
(HbA1c), used clinically as a marker of long-term glycemic expo-
sure and diabetes risk (Zhang et al., 2010), observing higherwing glucose tolerance test in mice treated with 100 nmol [D-Lys3]-GHRP-6 i.p.
o Figure S5.
Cell Metabolism 23, 821–836, May 10, 2016 831
Figure 7. Adiposity and Glucose Homeosta-
sis of Human R302Q g2 AMPK Mutation
Carriers
(A–D) Individual skinfold thickness measures
of triceps (A), biceps (B), subscapular (C), and
suprailiac (D) sites in male heterozygous R302Q
carriers (R302Q ±, n = 13) and controls (n = 19).
(E and F) Summated skinfold thickness measures
for males (E) and females (F) (latter control n = 25,
R302Q ±, n = 13).
(G and H) Scatterplots of plasma bilirubin (G) and
g-glutamyl transferase (g-GT) (H).
(I–K) Scatterplots of fasting plasma glucose (I)
and insulin (J), together with haemoglobin A1c
(HbA1c) (K).
(L) Homeostatic model assessment (HOMA) of
basal b cell function (%B).
Data are mean ± SEM. *p < 0.05. **p < 0.01. See
also Figure S6 and Table S4.HbA1c in R302Qcarriers (5.38%±0.09%versus 5.13%±0.05%,
p < 0.01) (Figure 7K).
We applied the homeostatic model assessment (HOMA2), a
well-validated, nonlinear model used to assess basal b cell func-
tion (%B) and insulin sensitivity (%S) in man (Levy et al., 1998), to
infer the impact of the R302Q g2 mutation on basal b cell insulin
secretion and insulin sensitivity. We found lower HOMA %B in
R302Q carriers (62.2% ± 3.6% versus 82.7% ± 5.4%, p <
0.05), but comparable HOMA%S, consistent with reduced basal
b cell activity but preserved insulin sensitivity (Figures 7L and
S6G). Oral glucose tolerance was comparable between groups
(Figures S6H–S6J).
Our results indicate that chronic g2AMPK activation inman re-
capitulates key features of the murine phenotype, including
increased adiposity and reduced basal b cell function. The latter
is likely to contribute to chronically higher plasma glucose con-
centrations, as reflected in increased HbA1c.
DISCUSSION
In eukaryotes, AMPK has been co-opted from its role as a critical
cell-autonomous energy sensor to having a central function in
systemic energy accounting (Chantranupong et al., 2015).832 Cell Metabolism 23, 821–836, May 10, 2016Here, we use a gene-targeting approach
in mice to infer the integrated systemic
effects of chronic AMPK activation. We
identify striking metabolic sequelae of an
R299Q g2 mutation, including hyperpha-
gia leading to obesity and impaired insulin
secretion contributing to glucose intoler-
ance. We observe a gene dose-response
effect (with R299Q g2 heterozygotes
manifesting a largely intermediate pheno-
type); greater basal gene expression of
the prototypical hypothalamic orexigenic
peptide, AGRP; and corresponding in-
crease in activity of neurons character-
ized by this peptide, likely lowering the
threshold for eating. We infer an impor-tant role for ghrelin-based signaling in the hyperphagia of
R299Q g2 mice on the basis of the rescue resulting from
GHSR antagonism. We also identify derepression of a set of
genes normally absent in mature pancreatic islet b cells, a
feature of human T2DM, and an associated intrinsic impairment
of b cell function in R299Q g2 mice. Highlighting phylogenetic
conservation of this pathway in systemic caloric accounting,
members of families carrying an identical g2 mutation exhibit
key aspects reminiscent of the murine phenotype including
enhanced adiposity and reduced basal b cell function resulting
in elevated plasma glucose.
By increasing basal g2 AMPK activity, the R299Q mutation
may be conceptualized as signaling a tonic ‘‘starvation cue,’’
enhancing gain on central orexigenic signaling to restore a
perceived whole-body energy deficit. While a number of mecha-
nismsmay contribute to increased feeding in our model of global
AMPK activation, we demonstrate exaggerated food intake
post-fasting and marked sensitivity to exogenous ghrelin,
together with mitigation of hyperphagia by antagonism of the
only known ghrelin receptor. GHSR is expressed widely across
the CNS, including hypothalamic nuclei involved in dietary ho-
meostasis and sites mediating hedonic feeding such as the
ventral tegmental area, hippocampus, and amygdala (Mason
et al., 2014). However, GHSR-bearing AGRP neurons in the ARC
mediate a substantial proportion of ghrelin-evoked feeding
(Wang et al., 2014). Supporting this view, in our model, R299Q
g2 ARC AGRP neurons exhibited increased excitability and firing
frequency, albeit with a rate that falls short of statistical signifi-
cance, likely due to large intercell variability (spike frequency
6.2 ± 0.8 versus 4.8 ± 0.7 Hz, p = 0.21).
A specific role for AMPK activation within AGRP neurons has
been proposed, linking ghrelin-GHSR binding to enhancement
of fatty acid b-oxidation and mitochondrial respiration (Andrews
et al., 2008). Consistent with this and other (Dietrich et al., 2013)
data highlighting a role for mitochondrial function in central
feeding regulation, we found a striking upregulation of genes en-
coding mitochondrial respiratory chain complex and ribosomal
protein subunits in the ARC of R299Q g2 mice. These bioener-
getic and biosynthetic adaptations are anticipated to support
increased neurosecretory and synaptic function required by
orexigenic neurons to drive food intake (Liu et al., 2012). As a cor-
ollary, we observed greater mitochondrial respiration in theMBH
of R299Q g2mice, a finding consistent with enhancedmitochon-
drial activity that may reflect enhanced mitochondrial fatty acid
oxidation induced by tonic AMPK activation. Notably, modula-
tion of fatty acid metabolism has been demonstrated to be a
key mediator of ghrelin’s orexigenic action, with a particular
role for the VMH (Lo´pez et al., 2008). While the ubiquitous
expression of g2 AMPK and the systemic model used do not
localize g2 AMPK activation (or ARC gene expression signature)
to AGRP neurons alone, upregulation of Agrp and Npy expres-
sion, unaltered Pomc expression, intrinsic hyperexcitability,
and exaggerated FOS and pS6 induction in AGRP neurons to
fasting all support substantial colinearity between AMPK and
AGRP neuronal activation in the ARC.
AMPK activation in the hypothalamus and in the periphery
is likely to have pleiotropic effects on glucose metabolism. The
metabolic phenotype of R299Q g2 mice was therefore notable
for its consistent hypoinsulinemia. In line with our previous
in vitro findings (da Silva Xavier et al., 2003; Tsuboi et al.,
2003), isolated islet studies demonstrated a b cell-intrinsic
contribution to impairment in GSIS in R299Q g2 mice, together
with re-expression of b cell ‘‘disallowed’’ genes implicated in
loss of cell differentiation and altered metabolic configuration
(Kone et al., 2014). This pancreatic phenotype reflects an impor-
tant facet of AMPK’s complex integrated response to maintain
energy homeostasis.
The systemic phenotype of the R299Q g2 knockin model is
spatially and temporally dynamic, with evidence for early benefi-
cial effects of peripheral AMPK activation (e.g., mild improve-
ment in insulin sensitivity), which may account for their relatively
benign lipid, hormonal, adipocytokine, and transaminase profile,
consistent with AMPK’s anticipated canonical actions in the pe-
riphery. A notable exception to this concept of benefit from ‘‘pe-
ripheral’’ AMPK activation is the finding of intrinsic impairment in
GSIS in R299Q g2 mice. The subtle signal for metabolic benefit
arising from AMPK activation in this model is likely to reflect g2
AMPK’s small contribution to overall AMPK activity in most pe-
ripheral tissues (Cheung et al., 2000). In contrast, we identify
clear negative consequences of chronic central AMPK activa-
tion—principally, ghrelin-dependent hyperphagia and potentially
centrally mediated upregulation of hepatic de novo lipogen-esis—ultimately overwhelming the beneficial peripheral effects
and resulting in obesity and frank systemic insulin resistance,
the adverse glucoregulatory consequences of which are further
exacerbated by abnormal GSIS.
Unlike congenic mice, which are otherwise genetically sub-
stantially homogeneous, humans have genetic heterogeneity,
reducing the penetrance of any given allele. Notwithstanding
this and the fact that only human subjects with heterozygous
g2 AMPK mutations are available for study, the finding that hu-
man R302Q carriers have increased adiposity and abnormal
glucose homeostasis is instructive. Consonant with the mouse
model, HOMA-derived indices suggested that increased
glucose and HbA1c reflected primary changes in b cell secretory
function rather than systemic insulin sensitivity. Extrapolating
metabolic findings from mice to humans, we observed a subtle
increase in adiposity in human R302Q carriers compared to
marked obesity in R299Q g2 mice. Beyond fundamental biolog-
ical interspecies differences, the context of the mutation is likely
to be important. Human obesity is complex, with its development
and maintenance reflecting interaction between genetic, envi-
ronmental, psychological, and societal factors (Spiegelman
and Flier, 2001). These considerations are less germane to the
laboratory mouse with ad libitum access to food (Martin et al.,
2010). In contrast, the robustness of the altered b cell function
signal emerging from both mice and human experiments under-
lines the conserved importance of AMPK activation in mamma-
lian insulin secretion.
Strictly, our data pertain to the consequences of activation
of AMPK complexes containing only the g2 regulatory subunit.
However the ubiquity of the g2 subunit in the relevant metabolic
tissues and the low isoform specificity of AMPK activating
agents reinforce the likely generalizability of our observations
(Cheung et al., 2000; Jensen et al., 2015). Our findings suggest
important ramifications for long-term tissue-indiscriminate
pharmacological activation of AMPK and highlight the potential
for AMPK activators—depending on relative tissue activation,
blood-brain barrier permeability, and duration of use—to have
adverse metabolic sequelae. As a corollary, in parallel to
AMPK activators for the treatment of diabetes and obesity,
AMPK inhibitors have also been developed for the same indica-
tions (Scott et al., 2015). Our study sounds a note of caution for
those seeking to develop potent generalized AMPK activators,
and reinforces a rationale for a more nuanced pharmacological
strategy.
EXPERIMENTAL PROCEDURES
Mouse Care and Husbandry
Procedures were approved by the institutional ethical review committees of
the University of Oxford and the University of Buckingham and carried out
in accordance with the British Home Office Animals (Scientific Procedures)
Act 1986 incorporating European Directive 2010/63/EU. Mice were socially
housed with littermates under controlled conditions (20C–22C, humidity,
12 hr light-dark) andmaintained on a standard rodent chow diet (Teklad Global
Diet; Harlan Laboratories) with water provided ad libitum.
Generation of R299Q g2 Knockin Mice
The knockin mousemodel of the human R302Q PRKAG2mutation was gener-
ated by targeting the orthologous murine gene and introducing the mutation
into the equivalent position (R299Q) in exon 7 in conjunction with genOway
(see also Supplemental Experimental Procedures).Cell Metabolism 23, 821–836, May 10, 2016 833
Primary Hepatocyte Isolation, Culture, and AMPK Activity Assay
Primary hepatocyte isolation and SAMS assay determination of AMPK activity
were undertaken as described (Davies et al., 1989; Woods et al., 2011).
Hyperinsulinemic Euglycemic Clamps
Clamp studies were performed on unrestrained, conscious mice after a 5–6 hr
fast as described (Ayala et al., 2011).
Arcuate Nucleus Laser Capture Microdissection and RNA-Seq
Total RNA isolation was undertaken from microdissected ARC samples ob-
tained from 14 mm coronal sections using a QIAGEN RNeasy Plus Micro kit
as described (Jovanovic et al., 2010). RNA-seq was carried out on an Illumina
Hiseq 2500 systemwith pathway analysis performed using Ingenuity software.
OXPHOS Protocol
Mediobasal hypothalamic oxygen consumption was measured using a high-
resolution respirometry system (Oxygraph-2k) on pooled samples using amodi-
fied substrate-uncoupler-inhibitor titration protocol (Pesta and Gnaiger, 2012).
Hypothalamic Electrophysiology
Ex vivo slice electrophysiology from ad libitum-fed homozygous R299Q g2/
NPY-hrGFP and WT g2/NPY-hrGFP mice was performed as described (Claret
et al., 2007; Smith et al., 2015).
Food Intake Studies
Food intake and drug sensitivity studies were undertaken in 6-week-old mice
housed individually. MT-II (1 mg/kg i.p.) was administered after an overnight
fast, or for ghrelin (30 mg i.p.) and [D-Lys3]-GHRP-6 (200 nmol i.p.) in the freely
fed state.
Intracerebroventricular Injection
The lateral cerebral ventricle was cannulated under stereotaxic control. After
recovery, mice were fasted overnight, then injected with either artificial cere-
brospinal fluid, [D-Lys3]-GHRP-6 (1 nmol), or ghrelin (0.01 mg).
Islet Insulin Secretion and b Cell Electrophysiology
Glucose-stimulated insulin secretion measured from isolated islets after over-
night culture and whole b cell current-clamp recordings were performed as
previously described (Beall et al., 2010; Sun et al., 2010).
Islet RNA-Seq
RNA isolation, RNA deep sequencing, and analysis were conducted as previ-
ously described (Kone et al., 2014; Martinez-Sanchez et al., 2015).
Human Study
The protocol was approved by the local institutional Research Ethics Commit-
tee. All subjects provided full written informed consent prior to participation.
PCR amplification and fluorescent dideoxy sequencing was undertaken for
exon 7 of PRKAG2 in all individuals, using proband DNA as positive control.
Statistical Analysis
Results are shown as mean ± SEM. Data were analyzed by two-tailed Stu-
dent’s t test or ANOVA (parametric), or Mann-Whitney or Kruskal-Wallis test
(non-parametric), respectively, using GraphPad Prism Software (version 6.0).
ACCESSION NUMBERS
The accession number for the arcuate RNA-seq data reported in this paper
is GEO: GSE73436 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=
GSE73436). The accession number for the pancreatic islet RNA-seq data re-
ported in this paper is ArrayExpress: E-MTAB-3938.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
six figures, and five tables and can be found with this article online at http://
dx.doi.org/10.1016/j.cmet.2016.04.003.834 Cell Metabolism 23, 821–836, May 10, 2016AUTHOR CONTRIBUTIONS
A.Y. designed research, performed experiments, analyzed data, and wrote the
paper; C.J.S. and E.T.W. designed and performed experiments and analyzed
data; K. Pinter designed the targeting strategy and constructed the R299Q g2
gene-targeting vector; S. Ghaffari, V.S., G.C., M.B., A.W., P.B.M., C.C.,
B.Y.H.L., K. Petkevicius, M.-S.N.-T., A.M.-S., T.J.P., P.L.O., A.S., C.N., M.L.,
J.F.O., P.H., M.T., C.B., T.K., J.P., D.S., G.K., D.D.J.W., A.R.H., L.A.B.,
R.W., N.R.Q., B.G., L.T., C.F., and M.A.S. performed and analyzed experi-
ments; A.C., S. Gandra, V.P., M.J.O., and E.B.S. undertook human phenotyp-
ing; C.J.S., S.N.P., R.J.M., C.F., C.R., G.S.H.Y., L.K.H., G.A.R., M.A.S., D.J.W.,
D.C., E.B.S., J.R.S.A., M.A.C., and H.W. designed experiments and/or com-
mented on the paper; H.A. directed the study and cowrote the paper.
ACKNOWLEDGMENTS
We thank Sandra Stobrawa and colleagues (Genoway Lyon) for generating
R299Q g2 mice; families participating in the R302Q phenotyping study; Well-
come Trust Centre for Human Genetics High-Throughput Genomics Group
(grant 090532/Z/09/Z) for sequencing data; Hermes Pardini for human
biochemistry; Karen McGuire, Kate Thomson, and Jessica Woodley (Oxford
Medical Genetics Laboratories) for R302Q genotyping; Keith Burling (Core
Biochemical Assay Laboratory Cambridge) and Tertius Hough (MRC, Harwell
Oxford) for murine biochemistry; Paul Trayhurn for comments; and Parisa Ya-
vari for artwork support. This work utilized Core Services supported by grants
DK089503 (MNORC) and DK020572 (MDRC) of the NIH to the University of
Michigan. C.B. is supported by a Diabetes UK RD Lawrence Fellowship (13/
0004647). C.F. and B.G. are supported by the Hungarian National Brain
Research Program. L.K.H. is supported by the Wellcome Trust (WT098012)
and BBSRC (BB/K001418/1). G.A.R. was supported by a Wellcome Trust
Senior Investigator Award (WT098424AIA), MRC Programme Grant (MR/
J0003042/1), and a Royal Society Wolfson Research Merit Award. A.Y. was
funded by a Wellcome Trust Research Training Fellowship (086632/Z/08/Z)
and is supported by the UK National Institute for Health Research. A.Y. (RE/
08/004), H.W., and H.A. acknowledge support from the BHF Centre of
Research Excellence, Oxford. This work was supported by a grant from the
MRC to H.A. and H.W. (MR/K019023/1).
This paper is dedicated to the memory of the late Professor Michael A.
Cawthorne.
Received: September 21, 2015
Revised: March 1, 2016
Accepted: April 1, 2016
Published: April 28, 2016
REFERENCES
Andersson, U., Filipsson, K., Abbott, C.R., Woods, A., Smith, K., Bloom, S.R.,
Carling, D., and Small, C.J. (2004). AMP-activated protein kinase plays a role in
the control of food intake. J. Biol. Chem. 279, 12005–12008.
Andrews, Z.B., Liu, Z.W., Walllingford, N., Erion, D.M., Borok, E., Friedman,
J.M., Tscho¨p, M.H., Shanabrough, M., Cline, G., Shulman, G.I., et al. (2008).
UCP2 mediates ghrelin’s action on NPY/AgRP neurons by lowering free radi-
cals. Nature 454, 846–851.
Ashcroft, F.M., and Rorsman, P. (2012). Diabetes mellitus and the b cell: the
last ten years. Cell 148, 1160–1171.
Ayala, J.E., Bracy, D.P., Malabanan, C., James, F.D., Ansari, T., Fueger, P.T.,
McGuinness, O.P., and Wasserman, D.H. (2011). Hyperinsulinemic-euglyce-
mic clamps in conscious, unrestrained mice. J. Vis. Exp. http://dx.doi.org/
10.3791/3188.
Bauer, U.E., Briss, P.A., Goodman, R.A., and Bowman, B.A. (2014). Prevention
of chronic disease in the 21st century: elimination of the leading preventable
causes of premature death and disability in the USA. Lancet 384, 45–52.
Beall, C., Piipari, K., Al-Qassab, H., Smith, M.A., Parker, N., Carling, D., Viollet,
B., Withers, D.J., and Ashford, M.L. (2010). Loss of AMP-activated protein ki-
nase alpha2 subunit in mouse beta-cells impairs glucose-stimulated insulin
secretion and inhibits their sensitivity to hypoglycaemia. Biochem. J. 429,
323–333.
Blair, E., Redwood, C., Ashrafian, H., Oliveira, M., Broxholme, J., Kerr, B.,
Salmon, A., Ostman-Smith, I., and Watkins, H. (2001). Mutations in the
gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic
cardiomyopathy: evidence for the central role of energy compromise in dis-
ease pathogenesis. Hum. Mol. Genet. 10, 1215–1220.
Chantranupong, L., Wolfson, R.L., and Sabatini, D.M. (2015). Nutrient-sensing
mechanisms across evolution. Cell 161, 67–83.
Chen, H.Y., Trumbauer, M.E., Chen, A.S., Weingarth, D.T., Adams, J.R.,
Frazier, E.G., Shen, Z., Marsh, D.J., Feighner, S.D., Guan, X.M., et al. (2004).
Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and
agouti-related protein. Endocrinology 145, 2607–2612.
Cheung, P.C., Salt, I.P., Davies, S.P., Hardie, D.G., and Carling, D. (2000).
Characterization of AMP-activated protein kinase gamma-subunit isoforms
and their role in AMP binding. Biochem. J. 346, 659–669.
Claret, M., Smith, M.A., Batterham, R.L., Selman, C., Choudhury, A.I., Fryer,
L.G., Clements, M., Al-Qassab, H., Heffron, H., Xu, A.W., et al. (2007). AMPK
is essential for energy homeostasis regulation and glucose sensing by
POMC and AgRP neurons. J. Clin. Invest. 117, 2325–2336.
Cool, B., Zinker, B., Chiou, W., Kifle, L., Cao, N., Perham, M., Dickinson, R.,
Adler, A., Gagne, G., Iyengar, R., et al. (2006). Identification and characteriza-
tion of a small molecule AMPK activator that treats key components of type 2
diabetes and the metabolic syndrome. Cell Metab. 3, 403–416.
Cowley, M.A., Smart, J.L., Rubinstein, M., Cerda´n, M.G., Diano, S., Horvath,
T.L., Cone, R.D., and Low, M.J. (2001). Leptin activates anorexigenic POMC
neurons through a neural network in the arcuate nucleus. Nature 411, 480–484.
da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S.K., and Rutter,
G.A. (2003). Role for AMP-activated protein kinase in glucose-stimulated insu-
lin secretion and preproinsulin gene expression. Biochem. J. 371, 761–774.
Davies, S.P., Carling, D., and Hardie, D.G. (1989). Tissue distribution of the
AMP-activated protein kinase, and lack of activation by cyclic-AMP-depen-
dent protein kinase, studied using a specific and sensitive peptide assay.
Eur. J. Biochem. 186, 123–128.
Dietrich, M.O., and Horvath, T.L. (2012). Limitations in anti-obesity drug devel-
opment: the critical role of hunger-promoting neurons. Nat. Rev. Drug Discov.
11, 675–691.
Dietrich, M.O., Liu, Z.W., and Horvath, T.L. (2013). Mitochondrial dynamics
controlled by mitofusins regulate Agrp neuronal activity and diet-induced
obesity. Cell 155, 188–199.
Fall, T., Ha¨gg, S., Ma¨gi, R., Ploner, A., Fischer, K., Horikoshi, M., Sarin, A.P.,
Thorleifsson, G., Ladenvall, C., Kals, M., et al.; European Network for
Genetic and Genomic Epidemiology (ENGAGE) consortium (2013). The role
of adiposity in cardiometabolic traits: a Mendelian randomization analysis.
PLoS Med. 10, e1001474.
Flier, J.S. (2004). Obesity wars: molecular progress confronts an expanding
epidemic. Cell 116, 337–350.
Folmes, K.D., Chan, A.Y., Koonen, D.P., Pulinilkunnil, T.C., Baczko´, I., Hunter,
B.E., Thorn, S., Allard, M.F., Roberts, R., Gollob, M.H., et al. (2009). Distinct
early signaling events resulting from the expression of the PRKAG2
R302Q mutant of AMPK contribute to increased myocardial glycogen. Circ
Cardiovasc Genet 2, 457–466.
Foretz, M., Guigas, B., Bertrand, L., Pollak, M., and Viollet, B. (2014).
Metformin: from mechanisms of action to therapies. Cell Metab. 20, 953–966.
Gaidhu, M.P., Fediuc, S., Anthony, N.M., So, M., Mirpourian, M., Perry, R.L.,
and Ceddia, R.B. (2009). Prolonged AICAR-induced AMP-kinase activation
promotes energy dissipation in white adipocytes: novel mechanisms inte-
grating HSL and ATGL. J. Lipid Res. 50, 704–715.
Hannan, K.M., Brandenburger, Y., Jenkins, A., Sharkey, K., Cavanaugh, A.,
Rothblum, L., Moss, T., Poortinga, G., McArthur, G.A., Pearson, R.B., and
Hannan, R.D. (2003). mTOR-dependent regulation of ribosomal gene tran-
scription requires S6K1 and is mediated by phosphorylation of the carboxy-
terminal activation domain of the nucleolar transcription factor UBF. Mol.
Cell. Biol. 23, 8862–8877.Hardie, D.G. (2014). AMPK—sensing energy while talking to other signaling
pathways. Cell Metab. 20, 939–952.
Holst, B., Cygankiewicz, A., Jensen, T.H., Ankersen, M., and Schwartz, T.W.
(2003). High constitutive signaling of the ghrelin receptor—identification of a
potent inverse agonist. Mol. Endocrinol. 17, 2201–2210.
Hotamisligil, G.S. (2006). Inflammation and metabolic disorders. Nature 444,
860–867.
Jensen, T.E., Ross, F.A., Kleinert, M., Sylow, L., Knudsen, J.R., Gowans, G.J.,
Hardie, D.G., and Richter, E.A. (2015). PT-1 selectively activates AMPK-g1
complexes in mouse skeletal muscle, but activates all three g subunit com-
plexes in cultured human cells by inhibiting the respiratory chain. Biochem.
J. 467, 461–472.
Jo, S.K., Lee, W.Y., Rhee, E.J., Won, J.C., Jung, C.H., Park, C.Y., Oh, K.W.,
Park, S.W., and Kim, S.W. (2009). Serum gamma-glutamyl transferase activity
predicts future development of metabolic syndrome defined by 2 different
criteria. Clin. Chim. Acta 403, 234–240.
Jovanovic, Z., Tung, Y.C., Lam, B.Y., O’Rahilly, S., and Yeo, G.S. (2010).
Identification of the global transcriptomic response of the hypothalamic
arcuate nucleus to fasting and leptin. J. Neuroendocrinol. 22, 915–925.
Kahn, B.B., Alquier, T., Carling, D., and Hardie, D.G. (2005). AMP-activated
protein kinase: ancient energy gauge provides clues to modern understanding
of metabolism. Cell Metab. 1, 15–25.
Kim, M.S., Park, J.Y., Namkoong, C., Jang, P.G., Ryu, J.W., Song, H.S., Yun,
J.Y., Namgoong, I.S., Ha, J., Park, I.S., et al. (2004). Anti-obesity effects of
alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated
protein kinase. Nat. Med. 10, 727–733.
Knight, Z.A., Tan, K., Birsoy, K., Schmidt, S., Garrison, J.L., Wysocki, R.W.,
Emiliano, A., Ekstrand, M.I., and Friedman, J.M. (2012). Molecular profiling
of activated neurons by phosphorylated ribosome capture. Cell 151, 1126–
1137.
Kone,M., Pullen, T.J., Sun, G., Ibberson, M., Martinez-Sanchez, A., Sayers, S.,
Nguyen-Tu, M.S., Kantor, C., Swisa, A., Dor, Y., et al. (2014). LKB1 and AMPK
differentially regulate pancreatic b-cell identity. FASEB J. 28, 4972–4985.
Levy, J.C., Matthews, D.R., and Hermans, M.P. (1998). Correct homeostasis
model assessment (HOMA) evaluation uses the computer program.
Diabetes Care 21, 2191–2192.
Li, Y., Xu, S., Mihaylova, M.M., Zheng, B., Hou, X., Jiang, B., Park, O., Luo, Z.,
Lefai, E., Shyy, J.Y., et al. (2011). AMPK phosphorylates and inhibits SREBP
activity to attenuate hepatic steatosis and atherosclerosis in diet-induced in-
sulin-resistant mice. Cell Metab. 13, 376–388.
Liu, T., Kong, D., Shah, B.P., Ye, C., Koda, S., Saunders, A., Ding, J.B., Yang,
Z., Sabatini, B.L., and Lowell, B.B. (2012). Fasting activation of AgRP neurons
requires NMDA receptors and involves spinogenesis and increased excitatory
tone. Neuron 73, 511–522.
Lo´pez, M., Lage, R., Saha, A.K., Pe´rez-Tilve, D., Va´zquez, M.J., Varela, L.,
Sangiao-Alvarellos, S., Tovar, S., Raghay, K., Rodrı´guez-Cuenca, S., et al.
(2008). Hypothalamic fatty acid metabolism mediates the orexigenic action
of ghrelin. Cell Metab. 7, 389–399.
Martin, B., Ji, S., Maudsley, S., and Mattson, M.P. (2010). ‘‘Control’’ laboratory
rodents are metabolically morbid: why it matters. Proc. Natl. Acad. Sci. USA
107, 6127–6133.
Martinez-Sanchez, A., Nguyen-Tu, M.S., and Rutter, G.A. (2015). DICER
inactivation identifies pancreatic b-cell ‘‘disallowed’’ genes targeted by
microRNAs. Mol. Endocrinol. 29, 1067–1079.
Martins, L., Ferna´ndez-Mallo, D., Novelle, M.G., Va´zquez,M.J., Tena-Sempere,
M., Nogueiras, R., Lo´pez, M., and Die´guez, C. (2012). Hypothalamic mTOR
signaling mediates the orexigenic action of ghrelin. PLoS ONE 7, e46923.
Mason, B.L., Wang, Q., and Zigman, J.M. (2014). The central nervous system
sites mediating the orexigenic actions of ghrelin. Annu. Rev. Physiol. 76,
519–533.
Minokoshi, Y., Kim, Y.B., Peroni, O.D., Fryer, L.G., Mu¨ller, C., Carling, D., and
Kahn, B.B. (2002). Leptin stimulates fatty-acid oxidation by activating AMP-
activated protein kinase. Nature 415, 339–343.Cell Metabolism 23, 821–836, May 10, 2016 835
Minokoshi, Y., Alquier, T., Furukawa, N., Kim, Y.B., Lee, A., Xue, B., Mu, J.,
Foufelle, F., Ferre´, P., Birnbaum, M.J., et al. (2004). AMP-kinase regulates
food intake by responding to hormonal and nutrient signals in the hypothala-
mus. Nature 428, 569–574.
Morton, G.J., Cummings, D.E., Baskin, D.G., Barsh, G.S., and Schwartz, M.W.
(2006). Central nervous system control of food intake and body weight. Nature
443, 289–295.
Ogden, C.L., Carroll, M.D., Kit, B.K., and Flegal, K.M. (2014). Prevalence of
childhood and adult obesity in the United States, 2011-2012. JAMA 311,
806–814.
Ollmann, M.M., Wilson, B.D., Yang, Y.K., Kerns, J.A., Chen, Y., Gantz, I., and
Barsh, G.S. (1997). Antagonism of central melanocortin receptors in vitro and
in vivo by agouti-related protein. Science 278, 135–138.
Pesta, D., and Gnaiger, E. (2012). High-resolution respirometry: OXPHOS pro-
tocols for human cells and permeabilized fibers from small biopsies of human
muscle. Methods Mol. Biol. 810, 25–58.
Pullen, T.J., and Rutter, G.A. (2013). When less is more: the forbidden fruits of
gene repression in the adult b-cell. Diabetes Obes. Metab. 15, 503–512.
Pullen, T.J., Khan, A.M., Barton, G., Butcher, S.A., Sun, G., and Rutter, G.A.
(2010). Identification of genes selectively disallowed in the pancreatic islet.
Islets 2, 89–95.
Sakkou, M., Wiedmer, P., Anlag, K., Hamm, A., Seuntjens, E., Ettwiller, L.,
Tscho¨p, M.H., and Treier, M. (2007). A role for brain-specific homeobox factor
Bsx in the control of hyperphagia and locomotory behavior. Cell Metab. 5,
450–463.
Scott, J.W., Galic, S., Graham, K.L., Foitzik, R., Ling, N.X., Dite, T.A., Issa,
S.M., Langendorf, C.G., Weng, Q.P., Thomas, H.E., et al. (2015). Inhibition of
AMP-activated protein kinase at the allosteric drug-binding site promotes islet
insulin release. Chem. Biol. 22, 705–711.
Smith, M.A., Katsouri, L., Irvine, E.E., Hankir, M.K., Pedroni, S.M., Voshol, P.J.,
Gordon, M.W., Choudhury, A.I., Woods, A., Vidal-Puig, A., et al. (2015).
Ribosomal S6K1 in POMC and AgRP neurons regulates glucose homeostasis
but not feeding behavior in mice. Cell Rep. 11, 335–343.
Spiegelman, B.M., and Flier, J.S. (2001). Obesity and the regulation of energy
balance. Cell 104, 531–543.
Sternick, E.B., Oliva, A., Magalha˜es, L.P., Gerken, L.M., Hong, K., Santana, O.,
Brugada, P., Brugada, J., and Brugada, R. (2006). Familial pseudo-Wolff-
Parkinson-White syndrome. J. Cardiovasc. Electrophysiol. 17, 724–732.
Sun, G., Tarasov, A.I., McGinty, J.A., French, P.M., McDonald, A., Leclerc, I.,
and Rutter, G.A. (2010). LKB1 deletion with the RIP2.Cre transgene modifies
pancreatic beta-cell morphology and enhances insulin secretion in vivo. Am.
J. Physiol. Endocrinol. Metab. 298, E1261–E1273.
Takahashi, K.A., and Cone, R.D. (2005). Fasting induces a large, leptin-depen-
dent increase in the intrinsic action potential frequency of orexigenic arcuate
nucleus neuropeptide Y/Agouti-related protein neurons. Endocrinology 146,
1043–1047.836 Cell Metabolism 23, 821–836, May 10, 2016Theander-Carrillo, C., Wiedmer, P., Cettour-Rose, P., Nogueiras, R., Perez-
Tilve, D., Pfluger, P., Castaneda, T.R., Muzzin, P., Schu¨rmann, A., Szanto, I.,
et al. (2006). Ghrelin action in the brain controls adipocyte metabolism.
J. Clin. Invest. 116, 1983–1993.
Tscho¨p, M.H., Speakman, J.R., Arch, J.R., Auwerx, J., Bru¨ning, J.C., Chan, L.,
Eckel, R.H., Farese, R.V., Jr., Galgani, J.E., Hambly, C., et al. (2012). A guide to
analysis of mouse energy metabolism. Nat. Methods 9, 57–63.
Tsuboi, T., da Silva Xavier, G., Leclerc, I., and Rutter, G.A. (2003). 50-AMP-acti-
vated protein kinase controls insulin-containing secretory vesicle dynamics.
J. Biol. Chem. 278, 52042–52051.
Villanueva, E.C., Mu¨nzberg, H., Cota, D., Leshan, R.L., Kopp, K., Ishida-
Takahashi, R., Jones, J.C., Fingar, D.C., Seeley, R.J., and Myers, M.G., Jr.
(2009). Complex regulation of mammalian target of rapamycin complex 1 in
the basomedial hypothalamus by leptin and nutritional status. Endocrinology
150, 4541–4551.
Viollet, B., Horman, S., Leclerc, J., Lantier, L., Foretz, M., Billaud, M., Giri, S.,
and Andreelli, F. (2010). AMPK inhibition in health and disease. Crit. Rev.
Biochem. Mol. Biol. 45, 276–295.
Wang, Q., Liu, C., Uchida, A., Chuang, J.C., Walker, A., Liu, T., Osborne-
Lawrence, S., Mason, B.L., Mosher, C., Berglund, E.D., et al. (2014). Arcuate
AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin.
Mol. Metab. 3, 64–72.
Willesen, M.G., Kristensen, P., and Rømer, J. (1999). Co-localization of growth
hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the
rat. Neuroendocrinology 70, 306–316.
Woods, A., Heslegrave, A.J., Muckett, P.J., Levene, A.P., Clements, M.,
Mobberley, M., Ryder, T.A., Abu-Hayyeh, S., Williamson, C., Goldin, R.D.,
et al. (2011). LKB1 is required for hepatic bile acid transport and canalicular
membrane integrity in mice. Biochem. J. 434, 49–60.
Xiao, B., Sanders, M.J., Underwood, E., Heath, R., Mayer, F.V., Carmena, D.,
Jing, C., Walker, P.A., Eccleston, J.F., Haire, L.F., et al. (2011). Structure of
mammalian AMPK and its regulation by ADP. Nature 472, 230–233.
Yamauchi, T., Kamon, J., Minokoshi, Y., Ito, Y., Waki, H., Uchida, S.,
Yamashita, S., Noda, M., Kita, S., Ueki, K., et al. (2002). Adiponectin stimulates
glucose utilization and fatty-acid oxidation by activating AMP-activated pro-
tein kinase. Nat. Med. 8, 1288–1295.
Yang, Y., Atasoy, D., Su, H.H., and Sternson, S.M. (2011). Hunger states
switch a flip-flop memory circuit via a synaptic AMPK-dependent positive
feedback loop. Cell 146, 992–1003.
Yeo, G.S., and Heisler, L.K. (2012). Unraveling the brain regulation of appetite:
lessons from genetics. Nat. Neurosci. 15, 1343–1349.
Zhang, B.B., Zhou, G., and Li, C. (2009). AMPK: an emerging drug target for
diabetes and the metabolic syndrome. Cell Metab. 9, 407–416.
Zhang, X., Gregg, E.W., Williamson, D.F., Barker, L.E., Thomas, W., Bullard,
K.M., Imperatore, G., Williams, D.E., and Albright, A.L. (2010). A1C level and
future risk of diabetes: a systematic review. Diabetes Care 33, 1665–1673.