Class IIa Histone Deacetylases Are
Hormone-Activated Regulators of FOXO
and Mammalian Glucose Homeostasis
Maria M. Mihaylova,1,5Debbie S. Vasquez,1,5Kim Ravnskjaer,2,5Pierre-Damien Denechaud,1,5Ruth T. Yu,3,5
Jacqueline G. Alvarez,3,5Michael Downes,3,5Ronald M. Evans,3,4,5Marc Montminy,2,5and Reuben J. Shaw1,4,5,*
1Molecular and Cell Biology Laboratory
2The Clayton Laboratories for Peptide Biology
3Gene Expression Laboratory
4Howard Hughes Medical Institute
5Leona and Harry Helmsley Center for Nutritional Genomics
The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
Class IIa histone deacetylases (HDACs) are signal-
dependent modulators of transcription with estab-
lished roles in muscle differentiation and neuronal
survival. We show here that in liver, class IIa HDACs
(HDAC4, 5, and 7) are phosphorylated and excluded
from the nucleus by AMPK family kinases. In
response to the fasting hormone glucagon, class
IIa HDACs arerapidly dephosphorylated and translo-
cated to the nucleus where they associate with the
promoters of gluconeogenic enzymes such as
G6Pase. In turn, HDAC4/5 recruit HDAC3, which
results in the acute transcriptional induction of these
genes via deacetylation and activation of FOXO
family transcription factors. Loss of class IIa HDACs
in murine liver results in inhibition of FOXO target
genes and lowers blood glucose, resulting in
increased glycogen storage. Finally, suppression of
class IIa HDACs in mouse models of type 2 diabetes
ameliorates hyperglycemia, suggesting that inhibi-
tors of class I/II HDACs may be potential therapeu-
tics for metabolic syndrome.
How multicellular organisms store and utilize nutrients in
response to changing environmental conditions is under the
control of hormones, as well as cell-autonomous nutrient and
energy sensors. Glucose homeostasis in mammals is primarily
maintained through a tight regulation of glucose uptake in
peripheral tissues in the fed state and production of glucose in
liver during fasting. After a meal, insulin signals the liver to atten-
glucose levels. Dysregulation of these processes contributes to
metabolic disorders such as type 2 diabetes (Biddinger and
Gluconeogenesis is largely regulated at the transcriptional
level of rate-limiting enzymes including glucose-6-phophatase
(G6pc; G6Pase) and phosphoenolpyruvate
(Pck1; PEPCK) via hormonal modulation of transcription factors
and coactivators including CREB, FOXO, HNF4a, GR, PGC-1a,
and C/EBPs (Viollet et al., 2009). Two major signaling pathways
signaling pathway and the LKB1/AMPK pathway. Insulin control
of gluconeogenesis is largely mediated through the serine/thre-
onine kinase Akt, which phosphorylates and inactivates
PGC-1a and the FOXO family of transcription factors, mainly
Foxo1 and Foxo3 in mammalian liver (Matsumoto et al., 2007;
Haeusler et al., 2010). Akt-dependent phosphorylation inacti-
cytoplasmic sequestration. In addition, FOXO is inhibited
through acetylation on up to six lysines, which reduces its
DNA-binding ability and alters its subcellular localization. The
Akt sites and acetylation sites are well conserved in metazoans
and across Foxo family members (Calnan and Brunet, 2008).
The LKB1/AMPK pathway is also a significant endogenous
inhibitor of gluconeogenesis (Shaw et al., 2005; Viollet et al.,
2009; Canto ´ and Auwerx, 2010). LKB1 is a master upstream
kinase that directly phosphorylates the activation loop of 14
kinases related to the AMP-activated protein kinase (AMPK). In
liver, AMPK activity is modulated by adipokines such as
adiponectin but is not thought to be regulated during physiolog-
ical fasting by blood glucose levels as they rarely fall low enough
to trigger ATP depletion (Kahn et al., 2005). However, a number
of pharmacological agents that trigger mild ATP depletion by
disrupting mitochondrial function can activate AMPK, including
the biguanide compound metformin, which is the most widely
used type 2 diabetes therapeutic worldwide. In addition to
AMPK, at least two other related LKB1-dependent kinases can
also suppress gluconeogenesis: Salt-Inducible Kinase 1 (SIK1)
and SIK2 (Koo et al., 2005). These LKB1-dependent kinases
Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc. 607
can all phosphorylate common downstream substrates to inhibit
gluconeogenesis, of which the CRTC2 coactivator is one
example, though it is likely that additional targets exist (Shackel-
ford and Shaw, 2009).
In addition to protein phosphorylation, acetylation of histones
feeding response in liver (Guarente, 2006). Three families of
deacetylases counteract the actions of the acetyltransferases
(HATs). Class I HDACs (HDAC1, 2, 3, and 8) are thought to be
classical histone deacetylases, though recently these have
been found to be associated with active transcriptional regions
(Wang et al., 2009) and nonhistone targets have been reported
(Gre ´goire et al., 2007; Canettieri et al., 2010). Class IIa HDACs
(HDAC4, 5, 7, and 9) are thought to be catalytically inactive
due to critical amino acid substitutions in their active sites
(Haberland et al., 2009) and are proposed to act as scaffolds
for catalytically active HDAC3-containing complexes in several
settings (Wen et al., 2000; Fischle et al., 2002). Similar to
FOXO, the localization of class IIa HDACs to the nucleus is
inhibited through phosphorylation on specific conserved resi-
dues (Ser259 and Ser498 in human HDAC5) and subsequent
14-3-3 binding resulting in cytoplasmic sequestration (reviewed
in Haberland et al., 2009). Based on their homology to Sir2 in
budding yeast, the class III family of HDACs are also known as
Sirtuins, and several mammalian Sirtuins are activated by
NAD+and thus serve as energy sensors (Houtkooper et al.,
2010; Haigis and Sinclair, 2010).
We report here that phosphorylation of class IIa HDACs is
controlled in liver by LKB1-dependent kinases, but in response
to glucagon, class IIa HDACs are rapidly dephosphorylated
and translocate to the nucleus where they associate with the
G6pc and Pck1 promoters. Importantly, glucagon is known to
stimulate expression of these genes in hepatocytes through
PKA-mediated effects on CREB (Montminy et al., 2004) and
through effects on FOXO of an unknown mechanism (Matsu-
moto et al., 2007). We demonstrate that class IIa HDACs recruit
HDAC3 to gluconeogenic loci and regulate FOXO acetylation in
genes, and reduction of hyperglycemia in several mouse models
of type 2 diabetes, indicating that these proteins play key roles in
mammalian glucose homeostasis.
Class IIa HDAC Phosphorylation in Liver Is Controlled
by LKB1-Dependent Kinases
We sought to identify novel substrates of AMPK and its related
family members that mediate control of glucose and lipid metab-
olism in liver. In a previously described bioinformatics and
proteomic screen for substrates of AMPK family kinases (Gwinn
et al., 2008; Egan et al., 2011), we identified multiple candidate
phosphorylation sites in the class IIa HDAC family that are highly
conserved (Figure 1A) and represent well-established phosphor-
ylation sites governing their subcellular localization (Haberland
et al., 2009). Of the four class IIa family members in mammals,
we examined the protein expression of HDAC4, HDAC5, and
ificity of antibodies used for detecting endogenous proteins.
HDAC4, HDAC5, and HDAC7 were widely expressed and
present in C2C12 myoblasts, mouse embryonic fibroblasts,
and hepa1–6 liver-derived cells (Figure S1A available online). In
order to explore the function and regulation of the class IIa
HDACs in liver, we generated adenoviruses bearing hairpin
shRNAs against murine HDAC4, HDAC5, and HDAC7, which
efficiently knocked down each family member (Figure 1B). As
each family member was upregulated when another was
depleted (Figure 1B), to study loss of class IIa HDAC function it
was necessary to combine shRNAs of all three.
Phosphospecific antibodies were validated for detecting
endogenously phosphorylated HDAC4, HDAC5, and HDAC7
on their Ser259 and Ser498 sites (Figure 1B, Figure S1B,
Extended Results) and used to examine whether these sites in
each family member were regulated by LKB1-dependent
kinases in liver or hepatoma cell lines. Consistent with previous
reports suggesting that AMPK family members can target class
IIa HDACs in other cell types (Berdeaux et al., 2007; Dequiedt
et al., 2006; McGee et al., 2008; van der Linden et al., 2007),
RNAi depletion of LKB1 resulted in loss of basal Phospho-
Ser259 and Phospho-Ser498 of HDAC4 and HDAC5 in HepG2
and Huh7 hepatoma cells (Figure 1C). Moreover, treatment
with phenformin, a metformin analog that activates AMPK in an
LKB1-dependent manner, also led to an LKB1-dependent
increase in phosphorylation on Ser498 of HDAC4/5 (Figure 1C,
Figures S1C–S1E, Extended Results).
To examine the physiological conditions during which class IIa
HDACs are regulated by the LKB1 pathway, we utilized a condi-
tional deletion of the LKB1 gene in mouse liver (Shaw et al.,
2005). LKB1 deletion led to loss of basal Phospho-Ser259 and
Phospho-Ser498 in HDAC4/5/7, and acute treatment of mice
with the AMPK agonist metformin led to an increase in Phos-
pho-Ser498 in HDAC4/5/7 (Figure 1D), consistent with results
from hepatoma cell lines. Paralleling the effects seen with
metformin and phenformin, A769662, a direct AMPK-activating
small molecule (Cool et al., 2006), increased HDAC4/5/7 phos-
phorylation, particularly on the Ser498 sites (Figure S1F). Collec-
tively, these data indicate that class IIa HDACs are bona fide
in vivo targets suppressed by the LKB1 signaling pathway in liver
and can be further inhibited in response to the antidiabetic
The Fasting Hormone Glucagon Induces
Dephosphorylation and Nuclear Shuttling
of Class IIa HDACs
Considering the prominent basal phosphorylation of the HDACs
in primary hepatocytes and in livers of ad libitum fed mice
(Figures 1B and 1D), we sought to examine whether their phos-
phorylation may be controlled by physiological stimuli such as
fasting and refeeding. We discovered that HDAC4/5/7 phos-
phorylation in the liver was reduced under fasting conditions
and increased upon refeeding (Figure 2A). To examine whether
this was an adaptive response to fasting, or whether hormones
induced upon fasting could acutely mimic this effect, mice
were injected with the fasting hormone glucagon, which resulted
in reduced HDAC4/5/7 phosphorylation (Figure S2A). The
observed decrease of HDAC4/5/7 phosphorylation by glucagon
608 Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc.
paralleled decreased phosphorylation of CRTC2, another
protein whose localization is controlled by LKB1-dependent
kinases and 14-3-3 binding (Screaton et al., 2004). To further
and localization of the HDACs in primary hepatocyte cultures.
Consistent with the high basal levels of endogenous HDAC4/5/
7 phosphorylation observed in primary hepatocytes, GFP-
tagged HDAC5 was basally excluded from the nuclei of these
cells (Figure 2D). Treatment with glucagon induced rapid loss
of endogenous HDAC4/5/7 phosphorylation (Figure 2B) and full
nuclear translocation of GFP-tagged HDAC5 within 30 min (Fig-
ure 2D). Similar results were observed with forskolin, another
cAMP-inducing compound (Figures S2B and S2C). No such
effect was observed for GFP alone or the nonphosphorylatable
a permanent nuclear localization identical to wild-type HDAC5
localization following glucagon or FSK treatment (Figure S2D).
Subcellular fractionationcorroboratedthat under basal
Figure 1. Class IIa HDACs Are Regulated by LKB1-Dependent Kinases and Metformin Treatment in Liver
(A) Clustal alignment of class IIa HDACs showing sequence conservation on established phosphorylation sites matching the optimal AMPK motif.
(B) Primary mouse hepatocytes or mouse liver lysates infected with adenoviruses bearing indicated shRNAs and immunoblotted with indicated antibodies
(full description of antibody generation and validation in Extended Results).
(C) Lysates of HepG2 or Huh7 cells transfected with indicated siRNA pools and treated with either 2 mM phenformin or vehicle for 1 hr and subjected to
(D) Immunoblot of lysates from murine livers from LKB1+/+or LKB1lox/loxmice deleted for hepatic LKB1 and treated with either 250 mg/kg metformin or saline
alone for 1 hr.
See also Figure S1.
Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc. 609
conditions in primary hepatocytes, endogenous class IIa HDACs
are predominantly cytoplasmic and translocate fully into the
nucleus following glucagon or forskolin treatment (Figure 2C).
Class IIa HDACs Are Required for Expression
of Glucagon-Induced Gluconeogenic Genes
These findings indicate that class IIa HDACs in liver may be
acting as fasting-induced modulators of transcription. Knowing
that glucagon induced their nuclear translocation, we hypothe-
sized that their direct involvement in control of transcription
should occur acutely following hormone treatment. We therefore
performed transcriptional profiling analysis in primary hepato-
cytes to define the genes whose expression is altered by forsko-
lin in a manner that is suppressed by HDAC4/5 shRNAs.
Contrary to our initial expectations that the class IIa HDACs
would act as fasting-induced transcriptional repressors, among
the genes regulated by forskolin, we observed more genes
whose expression was attenuated when HDAC4/5 were
Figure 2. Glucagon Induces Dephosphorylation and Nuclear Translocation of Class IIa HDACs in Hepatocytes
(A) Liver lysates from C57Bl/6J mice either fasted for 18 hr and/or then refed for 4 hr (left panel) or fasted for 6 hr or fed ad libitum (right panel).
(B) Primary mouse hepatocytes treated with 100 nM glucagon or vehicle for indicated times, lysed, and immunoblotted with indicated antibodies.
are representative of three independent experiments for each panel.
(D) Primary mouse hepatocytes infected with adenovirus expressing GFP-HDAC5 WT treated with 100 nM of either glucagon or vehicle (media) for indicated
times and analyzed by confocal microscopy.
See also Figure S2.
610 Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc.
depleted via shRNA (heatmap of 15 representative genes
selected from the top 50 HDAC4/5-regulated genes shown in
Figure 3A; top 25 HDAC4/5-regulated genes shown in Fig-
ure S3A; full dataset GEO submission GSE20979).
Strikingly, the single most-regulated gene on the entire array
following knockdown of class IIa HDACs was the catalytic
subunit of G6Pase (G6pc), a rate-limiting enzyme for gluconeo-
genesis and glycogenolysis (Figure S3A). In addition to G6pc,
gluconeogenic genes PEPCK (Pck1) and Fbp1 was similarly
attenuated when HDAC4/5 were depleted. Several of the
HDAC4/5-regulated genes from the array are known to be
FOXO and/or CREB target genes, and we further validated their
HDAC regulation by qPCR (Figure 3B). We next examined
whether the effect of HDAC4/5/7 knockdown on transcription
of these loci could be observed on a reporter consisting of
2.2 kb of the human G6pc promoter driving luciferase expres-
sion. Similar to the effect on endogenous G6pc mRNA expres-
sion, shRNA-mediated depletion of HDAC4/5/7 inhibited the
induction of luciferase from the G6pc promoter following for-
skolin treatment in hepatocytes (Figure 3C, top panel), compa-
rable to loss of CRTC2 expression, which is needed for
CREB-dependent transactivation of the G6pc promoter. In
addition, overexpression of constitutively nuclear nonphosphor-
ylatable S259A/S498A HDAC5 mutant resulted in a modest but
reproducible increase in basal G6pc reporter activity even in the
absence of forskolin and further potentiated the effect of forsko-
lin-mediated induction. In contrast, HDAC4/5/7 depletion did
not alter forskolin induction of a CRE-luciferase reporter
composed of three tandem copies of the CREB DNA-binding
consensus motif compared to the effect of CRTC2 shRNA (Fig-
ure 3C, bottom panel). Consistent with the results in hepato-
cytes, depletion of HDAC4/5/7 in vivo resulted in attenuation
of G6pc promoter activity but had no effect on the CRE-lucif-
erase reporter in murine liver (Figure 3D, data not shown). No
significant changes in protein levels of CREB, PGC-1a,
CRTC2, Foxo1, or Foxo3 were seen with HDAC4/5/7 knock-
down (Figure S3B).
Given the effects on the G6pc reporter, we next used chro-
matin immunoprepitation (ChIP) to examine whether endoge-
nous HDAC4 or HDAC5 may be recruited to the G6pc promoter
following glucagon treatment. As seen in Figure 3E, endogenous
HDAC4 and HDAC5 were immunoprecipitated in a glucagon-
inducible manner with a proximal promoter region of the G6pc
promoter containing the FOXO and CREB consensus-binding
sites (Vander Kooi et al., 2003). In the absence of glucagon, no
association of HDAC4 or HDAC5 was observed with this region
above background, or with nonspecific distal upstream or
internal regions (Figure 3E; Figure S3C). shRNA confirmed the
specificity of the ChIP signal at the G6pc and Pck1 loci
of the other rate-limiting
Class IIa HDACs Control Acetylation of FOXO
Transcription Factors via Class I HDAC3
Given the association of HDAC4 and HDAC5 with the G6pc
promoter following glucagon, we investigated whether the pres-
ence of class IIa HDACs may be modulating the acetylation of
one of the transcription factors or transcriptional coactivators
required for G6pc induction following glucagon. To further inves-
tigate whether class IIa HDACs may be affecting the acetylation
of these transcription factors, we tested whether they physically
associate. We found significant coimmunoprecipitation of
FOXO1 or FOXO3 with HDAC5 following forskolin treatment
(Figure 4A, Figure S4A, data not shown). Consistent with this
interaction, we observed both endogenous Foxo1 and endoge-
nous HDAC4 to be nuclear following forskolin treatment of
primary hepatocytes (Figure 4B).
Foxo1 is acetylated on Lys242, 245, 259, 262, 271, and 291 by
the histone acetyltransferases p300 and CBP, which reduces its
ability to bind DNA (Brent et al., 2008; Matsuzaki et al., 2005).
Using acetylation site-specific
FOXO1 or FOXO3 acetylation in primary hepatocytes treated
with shRNAs against the class IIa HDACs. Acetylation of Foxo1
and Foxo3 was dramatically increased as measured with anti-
acetyl Lys259/262/271 Foxo1 antibody (Figures 4C and 4D),
whereas histone 3 Lys9/Lys14 acetylation remained unchanged.
Identical results were observed with an acetylation-specific
antibody to the nearby Lys242/245 sites in Foxo1 (Matsuzaki
et al., 2005) (Figure S4C). Importantly, adenoviral-mediated
knockdown of HDAC4/5/7 in mouse liver led to increased acet-
ylation of endogenous FOXO1 (Figure 4E). Acetylation of FOXO
has been reported to reduce its DNA binding, making it more
accessible for Akt and related inactivating kinases (Jing et al.,
2007; Qiang et al., 2010). Consistent with the increase of acety-
lation, knockdown of HDAC4/5/7 in hepatocytes led to an
increase in phosphorylation of endogenous Foxo1 and Foxo3
245 directly disrupts its ability to bind DNA (Matsuzaki et al.,
2005; Brent et al., 2008), we examined the association of
Foxo1 with gluconeogenic promoters. Glucagon treatment
resulted in increased ChIP of endogenous Foxo1 with the
G6pc and Pck1 promoters, which was attenuated by HDAC4/
5/7 shRNA, consistent with increased FOXO acetylation and
loss of DNA binding (Figure 5A).
Several studies have suggested the class IIa HDACs are
catalytically inactive due to critical amino acid substitutions
within the catalytic residues (Lahm et al., 2007; Schuetz et al.,
2008). In other contexts where class IIa-associated deacetylase
activity was detected, it was attributed to class IIa HDAC
association and recruitment of active class I HDAC family
member HDAC3 and its coregulators Ncor1/SMRT(Ncor2)
(Fischle et al., 2002). Consistent with this possibility, we
observed that overexpressed HDAC5 and HDAC3 coimmuno-
precipitated in a forskolin-dependent manner in HEK293 cells
and endogenous HDAC3 and FOXO1 coimmunoprecipitated
with GFP-HDAC5 from hepatocytes in a glucagon-dependent
manner (Figure 5B; Figure S5A). Moreover, we found that re-
combinant HDAC4 or HDAC5 were unable to stimulate in vitro
deacetylation of FOXO1, unlike recombinant HDAC3/Ncor
complex (Figure 5C; Figure S5B). The ability of HDAC3 to cata-
lyze in vitro deacetylation of FOXO was dependent on its associ-
ation with Ncor (Figure 5C), as previously reported in other
deacetylase assays (Fischle et al., 2002; Gre ´goire et al., 2007).
Consistent with these findings, treatment of cells with the class
I/II HDAC inhibitor trichostatin A (TSA) results in increased
Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc. 611
Figure 3. Class IIa HDACs Are Required for the Induction of Gluconeogenic Genes and Associate with the G6pc Locus following Glucagon
(A) Microarray data analysis on genes induced by forskolin in primary mouse hepatocytes and whose expression is altered due to depletion of HDAC4 and 5
(HDAC) but not scrambled (scram) control shRNA. Cells were treated with 10 mM forskolin or vehicle (DMSO) for 2 or 4 hr as indicated. Duplicate samples are
shown for each condition. Gene expression shown relative to scrambled shRNA cells treated with vehicle for 2 hr. FOXO-regulated targets (Dong et al., 2006;
Dansenetal.,2004; Renaultetal.,2009; Paiket al.,2009)(#)or CREB-regulated targets (Zhang etal.,2005)(*)asindicated. Rate-limitinggluconeogenic enzymes
highlighted in red. Representative 15 of the top 50 HDAC4/5-regulated genes shown.
(B) qRT-PCR from primary hepatocytes of FOXO target genes whose FSK-induced expression is attenuated following depletion with HDAC4/5/7 shRNAs.
Expression relative to cyclophilin. n = 9, *p < 0.01.
612 Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc.
FOXO1 acetylation (Figure S5C), as reported previously (Brunet
et al., 2004).
To further examine whether HDAC3 may mediate FOXO
deacetylation in concert with HDAC4/5 in hepatocytes, we
looked at whether HDAC3 similarly associated with the same
this association was regulated by glucagon. ChIP experiments
revealed that endogenous HDAC3 bound to both the G6pc
and Pck1 promoters only following glucagon treatment, and
this association was abolished when HDAC4/5/7 were depleted
(Figure 5D), in contrast to its association with the promoter of the
housekeeping gene TFIIB. Taken altogether, these findings sub-
stantiate the model that following glucagon, class IIa HDACs
translocate into the nucleus where they recruit HDAC3 to the
toward FOXO, promoting its activation and induction of these
gluconeogenic gene promoters.
Suppression of Class IIa HDACs Alters Organismal
G6Pase is a rate-limiting enzyme of both gluconeogenesis and
glycogenolysis (Hutton and O’Brien, 2009), and mutations in
glucose-6-phosphatase (G6pc) result in Glycogen Storage
Disease Type I in humans (GSD Type I or Von Gierke’s disease)
characterized by aberrant glycogen storage and hypoglycemia,
a phenotype also mimicked in genetic mouse models of G6pc
deletion (Salganik et al., 2009; Peng et al., 2009). Given the
we sought to examine the effect of their loss in the intact mouse
liver. Similar to mice lacking G6pc or Foxo1 (Matsumoto et al.,
2007),mice expressing shRNAs againstHDAC4 or HDAC5alone
in liver give rise to increased glycogen accumulation as visual-
ized by Periodic acid-Schiff (PAS) stain in both fasting and refed
mice (Figure 6A). The most significant effect on glycogen
accumulation was observed when HDAC4, HDAC5, and
HDAC7 were all simultaneously knocked down (Figure 6A; quan-
tified in Figure S6A). We also observed that loss of HDAC4/5/7
modestly lowered blood glucose levels in B6 mice on a normal
diet, and importantly, overexpression of nonphosphorylatable
constitutively nuclear HDAC5 led to a modest increase in blood
glucose in these mice (Figure S6B). B6 mice expressing hepatic
HDAC4/5/7 shRNA also showed improved glucose tolerance in
a glucose tolerance test (GTT) (Figure S6C). Gain and loss of
class IIa HDAC function in fasted B6 mice correlated with
changes in G6Pase mRNA levels (Figure 6B), similar to the
effects observed on the G6pc reporter in hepatocytes (upper
panel of Figure 3C).
As hepatic deletion of LKB1 leads to the loss of HDAC4/5/7
phosphorylation (Figure 1D), HDAC4/5/7 will be constitutively
nuclear in LKB1?/?livers, potentially contributing to increased
gluconeogenic gene expression. To examine whether constitu-
tive activation of HDAC4/5/7 may play a role in the hypergly-
cemia of hepatic LKB1 knockout mice, we combined a model
of inducible loss of hepatic LKB1 in mice with subsequent intro-
duction of adenoviral shRNA against HDAC4/5/7. We utilized
liver-specific inducible Cre recombinase transgenic mice (Imai
et al., 2000) crossed to the LKB1 conditional floxed knockout
mice. Consistent with previous results of Cre-mediated LKB1
loss, tamoxifen-induced loss of hepatic LKB1 led to a doubling
of fasting blood glucose levels within 10 days post-administra-
tion. Subsequent loss of HDAC4/5/7 in these mice led to remark-
able suppression of the LKB1-dependent elevation in blood
glucose (Figure 6C). Immunoblotting confirmed that LKB1 and
HDAC4/5 expression were attenuated and that in the absence
of LKB1 expression in liver, HDAC4 and HDAC5 were basally
hypophosphorylated (Figure 6E). We next looked at the expres-
sion levels of FOXO-regulated genes in the context of class IIa
HDAC loss in this mouse model. Indeed, in addition to G6pc
and Pck1, the expression of several FOXO target genes was
significantly elevated in the LKB1?/?livers compared to
livers and was subsequently reduced following
HDAC4/5/7 shRNA but not scrambled shRNA (Figure 6D, data
Class IIa HDACs Are Required for Hyperglycemia
in Diabetic Mouse Models
Given that insulin resistance associated with the metabolic
syndrome is known to result in FOXO-dependent increases in
gluconeogenesis (Gross et al., 2009), we sought to more broadly
examine whether deregulation of HDAC4/5/7 function may
contribute to hyperglycemia in widely used mouse models of
type 2 diabetes and whether targeting their inactivation would
be sufficient to restore glucose homeostasis in this setting. First,
weutilized the ob/ob and db/db mouse models deficientin leptin
signaling and deregulated for insulin signaling and treated these
mice with either scrambled control or HDAC4/5/7 shRNAs as
above. Reduction of class IIa HDAC expression in these diabetic
mouse models also led to a substantial decrease in fastingblood
glucose levels (Figure 7B; Figure S7A), paralleling loss of HDAC
expression (Figure S7B). To more fully characterize this
response, we performed GTTs and pyruvate tolerance tests
(PTTs) on db/db cohorts treated with control or HDAC4/5/7
shRNAs. Loss of the class IIa HDACs significantly lowered
fasting blood glucose levels and improved glucose tolerance in
db/db mice (Figures 7A and 7C). Next we examined whether
the class IIa HDACs were also involved in regulating hepatic
blood glucose in a high fat diet-induced diabetes mouse model,
which is thought to be more representative of human type 2
icant reduction of fasting blood levels and improved glucose
(C) Ad-G6pc-luc activity (top panel) or CRE-luc activity (bottom panel) in primary hepatocytes expressing indicated adenoviruses and treated with vehicle or
10 mM forskolin for 4 hr as indicated. Representative of four independent experiments. n = 6, *p < 0.007.
(D) G6pc-luc activity in 18 hr fasted mice expressing Ad-scrambled or Ad-HDAC4/5/7 shRNAs. Results representative of three independent experiments and
quantified using the Living Image 3.2 program. n = 6, *p < 0.002.
(E) Endogenous HDAC4 or HDAC5 chromatin immunoprecipitation (ChIP) with primers against indicated regions of the murine G6pc promoter at the times
indicated following 100 nM glucagon treatment. n = 4, *p < 0.05.
Data are shown as mean ± SEM with Student’s t test. See also Figure S3.
Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc. 613
Figure 4. Class IIa HDACs Control FOXO Acetylation
(A) HEK293T cells transfected withMYC-FOXO1 and GFP-HDAC5 as indicated, treated with 10 mMforskolin or vehicle for 1 hr and immunoprecipitated with anti-
myc tag antibody.
(B) Primary hepatocytes treated for 1 hr with vehicle or 10 mM forskolin and endogenous FOXO1 and HDAC4 were detected by immunocytochemistry.
(C) Immunoblot showing amounts of acetylated FOXO (Ac-Lys259/262/271) from primary hepatocytes transduced with adenoviruses expressing Foxo3 and
indicated shRNAs. Total cell lysates were blotted with indicated antibodies.
(D) Primary hepatocytes were transduced with adenoviruses expressing FOXO3 or GFP-FOXO1 and indicated shRNA-expressing adenoviruses. FOXO
immunoprecipitates were immunoblotted with indicated antibodies.
614 Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc.
tolerance when depleted for HDAC4/5/7 in the liver (Figures 7D
and 7E), indicating that the class IIa HDACs play a critical role
in controlling hepatic glucose homeostasis.
We report here that class IIa HDACs are critical components of
the transcriptional response to fasting in liver, shuttling into the
nucleus in response to glucagon. Once nuclear, they bind to
the promoters of gluconeogenic target genes and mediate their
transcriptional induction, at least in part through promoting
deacetylation and activation of FOXO transcription factors
(Figure 7F). These findings illuminate a mechanism by which
ular basis for how FOXO mediates effects of both fasting
hormones and insulin on hepatic glucose production (Matsu-
moto et al., 2007). Consistent with this, hepatic knockdown of
class IIa HDACs in vivo results in lowered blood glucose and
altered glycogen storage, phenocopying hepatic deficiency of
Foxo1 in mice (Matsumoto et al., 2007), as well as the G6pc defi-
ciency in mice and human Glycogen Storage Disease Type I
(GSDI) patients (Salganik et al., 2009; Peng et al., 2009).
Thus, fasting may promote FOXO activation by a two-pronged
mechanism wherein loss of insulin signaling results in dephos-
phorylation of the Akt sites in FOXO, allowing its re-entry into
the nucleus, while glucagon-induced dephosphorylation of the
class IIa HDACs results in their nuclear translocation and deace-
tylation of nuclear FOXO, enhancing FOXO DNA-binding activity
and association with gluconeogenic gene promoters. Whether
deacetylation of FOXO is involved in the function of class IIa
HDACs in other tissues remains to be seen. Class IIa HDACs
are best appreciated for roles in transcriptional repression of
muscle differentiation through modulation of the MEF2 family
of transcription factors (Haberland et al., 2009). Interestingly,
FOXO family members have been shown to work in concert
with MEF2 family members in cardiomyocytes (Creemers
et al., 2006), and the only transcription factor that HDAC3 has
been previously reported to deacetylate is MEF2 itself (Gre ´goire
et al., 2007). These findings suggest a possible coordinated
regulation of FOXO and MEF2 by a class IIa HDAC—HDAC3
deacetylase complex. Importantly, it is likely that there might
be additional nonhistone targets whose acetylation is controlled
by class IIa HDACs. Notably, the effects of class IIa HDAC
proteins on FOXO acetylation and induction of catabolic gene
expression following hormones are conserved in Drosophila as
described in an accompanying paper (Wang et al., 2011).
Although our studies demonstrate a key role for class IIa
HDACs in the control of FOXO acetylation following glucagon
in liver, FOXO has also been previously shown to be a target of
SIRT1 in a number of cell types, particularly defined in muscle
(Canto ´ et al., 2009). As shown here for class IIa HDACs, SIRT1
activity in liver is also thought to be increased following fasting.
It is notable, however, that in previous reports, SIRT1 levels are
not increased rapidly following fasting (Rodgers et al., 2005),
though it is possible that SIRT1 may also be controlled post-
translationally as well. SIRT1 has been shown to control gluco-
neogenesis and other hepatic processes though a number of
downstream targets (reviewed in Houtkooper et al., 2010).
Future studies will be required to fully delineate the contexts
and relative contributions of Sirtuins versus the class II HDAC/
HDAC3 complex in the control of FOXO acetylation in liver and
other tissues (see Extended Discussion for further details).
Notably, our findings suggest that both the CRTC family of co-
activators and the class IIa HDACs are coordinately regulated in
liver by the opposing activity of LKB1-dependent kinases stimu-
lating 14-3-3 docking and cytoplasmic sequestration and
glucagon-induced signals promoting dephosphorylation and
nuclear import. How might glucagon mediate these effects?
AMPK, SIK1, and SIK2 (Screaton et al., 2004; Hurley et al., 2006;
phosphorylating the class IIa HDACs. It also remains possible
that PKA actively stimulates a phosphatase such as calcineurin
and this achieves the efficient nuclear translocation of CRTC
and HDAC proteins in parallel. By promoting the simultaneous
activation of a positive regulator of CREB-dependent transcrip-
tion (CRTCs) and a positive regulator of FOXO-dependent
transcription (HDAC4/5/7), glucagon further promotes the
elements including the gluconeogenic enzymes.
As we show that AMPK activation by metformin treatment
leads to increased HDAC4/5/7 phosphorylation and inactivation,
this provides another mechanism by which the widely used type
2diabetes therapeutic serves to suppress hepatic gluconeogen-
esis and lower blood glucose (Shaw et al., 2005). Perhaps most
unexpectedly, the results here suggest that class I and class IIa
HDACs in the liver of type 2 diabetic rodent models actively
contribute to the hyperglycemic phenotype of these animals,
which may result from the critical role of FOXO in hyperglycemia
in these insulin-resistant states. Remarkably, shRNA-mediated
suppression of class IIa HDAC function led to a dramatic reduc-
high fat diet mice, db/db mice, and ob/ob mice. If extended to
human studies, these results suggest that small molecules that
Given the intense ongoing effort in the pharmaceutical industry
to develop HDAC inhibitors as anticancer agents (Witt et al.,
2009),their potentialutility forthetreatmentofmetabolicdisease
Antibodies and Biochemistry
Cell signaling antibodies used: pAMPK, pACC, pRaptor, Raptor, HDAC3,
HDAC4, HDAC5, pHDAC4 Ser246/HDAC5 Ser259/HDAC7 Ser155, pHDAC4
immunoblotted with indicated antibodies.
(F) Primary hepatocytes knocked down for the class IIa HDACs or control scramble shRNAs and total cell lysates were immunoblotted with indicated antibodies.
See also Figure S4.
Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc. 615
Figure 5. Class I HDAC3 Is Recruited by Class IIa HDACs to Deacetylate Foxo
(A) ChIP analysis on primary hepatoyctes transduced with control scramble or HDAC4/5/7 shRNAs and assessed for FOXO1 on G6pc or Pck1 promoters
following 1 hr treatment with 100 nM glucagon. n= 4, *p < 0.05.
(B) HEK293T cells transfected with a FLAG-HDAC3 and GFP-HDAC5 as indicated and treated with forskolin or vehicle for 1 hr and then immunoprecipitated with
anti-FLAG tag antibodies. Immunoprecipitates and input cell lysates were blotted with indicated antibodies.
(C) In vitro deacetylation assays were performed on recombinant GST-FOXO1, preacetylated in vitro with a recombinant fragment of p300. GST-FOXO1
acetylation is detected using the FOXO1 K242/245 acetylation-specific antibody. Recombinant HDAC3 or HDAC3 complexed with Ncor was used at varying
concentrations. Recombinant SIRT1 used as positive control.
(D) ChIP analysis on primary hepatocytes transduced with control scramble or HDAC4/5/7 shRNAs and assessed for HDAC3 association on FOXO-binding sites
within G6pc or Pck1 or the housekeeping TfIIb promoter following 1 hr treatment with 100 nM glucagon. n= 4, *p < 0.05.
Data are shown as mean ± SEM with Student’s t test. See also Figure S5.
616 Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc.
Figure 6. Class IIa HDACs Are Required for Glucose Homeostasis
(A) C57Bl/6J mice infected with indicated shRNAs in liver and fasted for 18 hr and/or then refed for 4 hr. Livers were processed for histology and stained with
hematoxilin and eosin (H&E) or Periodic acid-Schiff (PAS) stain to detect glycogen. Images were taken at 403.
(B) qRT-PCR for G6pc expression from livers of ad lib fed C57Bl/6J mice expressing GFP or GFP-HDAC5-AA or HDAC4/5 shRNAs in liver. n = 9, *p < 0.01,
**p < 0.001, ***p < 0.0001.
(C) Albumin-creERT2LKB1+/+or LKB1lox/loxmice were tamoxifen-treated and subsequently infected with scrambled or HDAC4/5/7 (HDAC) shRNAs. Five days
later, mice were fasted for 18 hr, and blood glucose was measured. Average blood glucose value shown in red. n = 5, **p < 0.001 ***p < 0.0001.
(D) qRT-PCR for FOXO target genes (Igfbp1, Agxt2l1, Mmd2) or Hdac4 (control) from livers of indicated mice from (C). n = 9, *p < 0.01, **p < 0.001, ***p < 0.0001.
(E) Liver lysates from mice in (C) were immunoblotted with indicated antibodies. Asterisk indicates a nonspecific band recognized by the HDAC5 antibody.
Data are shown as mean ± SEM with Student’s t test. See also Figure S6.
Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc. 617
Figure 7. Suppression of Class IIa HDACs Lowers Blood Glucose in Mouse Models of Metabolic Disease
(A) Glucose tolerance test was performed on db/db mice infected with either scramble (scramb) shRNA or HDAC4/5/7 (HDAC) shRNAs. n = 5, *p < 0.02.
(B) db/db mice knocked down with (scramb) shRNA or HDAC4/5/7 (HDAC) shRNAs in liver. Seven days later, mice were fasted for 18 hr and blood glucose was
measured. Average blood glucose value shown in red. n = 5, *p < 0.02.
(C) Pyruvate tolerance test performed on db/db mice injected with either scramble (scramb) shRNA or HDAC4/5/7 (HDAC) shRNAs. n = 5, *p < 0.04.
(D) Seven-month-old B6 mice on a high fat diet (HFD) were treated as in (A). n = 5, *p < 0.03.
(E) Glucose tolerance test was performed on 7-month-old B6 mice on HFD as in (A). n = 5, *p < 0.02.
(F) Model for glucagon-dependent regulation of class IIa HDACs and FOXO. Under fasting conditions, glucagon induces dephosphorylation and nuclear
translocation of class IIa HDACs. Once nuclear, they associate with the G6pc and Pck1 promoters and bind to HDAC3-Ncor/SMRT and FOXO, resulting in
HDAC3-mediated deacetylation and activation of FOXO. Under fed conditions, insulin-dependent activation of the LKB1-dependent kinases SIK1/2 stimulates
phosphorylation and cytoplasmic shuttling of class IIa HDACs. Similarly, following metformin treatment, the LKB1-dependent AMPK activation induces class IIa
HDAC phosphorylation and 14-3-3 binding. In response to glucagon, PKA is activated and directly phosphorylates and inactivates AMPK, SIK1, and SIK2, hence
resulting in loss of HDAC phosphorylation.
Data are shown as mean ± SEM with Student’s t test. See also Figure S7.
618 Cell 145, 607–621, May 13, 2011 ª2011 Elsevier Inc.
Ser632/HDAC5 Ser498/HDAC7 Ser486, SIRT1, LKB1, Foxo1, Foxo3, pFoxo,
CREB, Myc, GST. Millipore antibodies used: LKB1, Histone 3 K9/K14,
Acetyl-Lysine. Santa Cruz antibodies used: Ac-Foxo1, aTubulin, HDAC7.
Abcam antibodies used: HDAC3. Sigma antibodies used: M2 Flag, anti-Flag.
Anti-CRTC2 and PGC-1a previously described (Dentin et al., 2007). All catalog
numbers and buffers described in Extended Experimental Procedures.
DNA Constructs and Adenoviruses
GST-14-3-3, Myc CA-AMPKa2, GFP HDAC5 wild-type (WT), GFP HDAC5
S259A/S498A, and Myc-Foxo1 were described previously (Gwinn et al.,
2008; Berdeaux et al., 2007). FLAG HDAC5 WT, Flag tagged WT HDAC3,
GFP Foxo1, and Myc Foxo1 were obtained from Addgene. FLAG HDAC5
S259A and FLAG HDAC5 S259A/S498A were generated using QuickChange
Site-Directed Mutagenesis kit (Stratagene). For full details on adenoviruses
used and adenoviral construction, see Extended Experimental Procedures.
HEK293T, Huh7, HepG2, C2C12, and U2OS cells were obtained from ATCC.
RNAi SMARTpool human LKB1 (Dharmacon) or RNAi negative control (Invitro-
gen) were used at 20 nM final concentration and transfected using RNAiMAX
transfection reagent (Invitrogen). Knockdowns were carried out for 72 hr. Cells
were treated with 1 mM TSA and/or 10 mM NAM (Sigma). Cells were treated
with 2 mM AICAR (Toronto Research Chemicals) or 2 mM Phenformin (Sigma).
Primary Hepatocyte Treatment and Subcellular Fractionation
Primary hepatocytes were derived from C57BL/6J mice and maintained in
serum-free Media 199. Cells were transduced 24 hr after harvesting. Knock-
down and overexpression studies in hepatocytes were done by infecting cells
at 5 PFUs/cell. All adenoviral shRNA knockdowns were carried out for 72 hr.
For subcellular fractionation, cells were treated as indicated, washed three
times with PBS, and lysed utilizing NE-PER Cell Fractionation Kit (Pierce).
Primary hepatocytes were treated with 10 mM forksolin (Sigma), 100 nM
glucagon (Novo Nordisk), 100 mM A769662 (Abbott), or 100 nM insulin (Lilly)
at indicated times.
Primary hepatocytes were stimulated with PBS or 100 nM glucagon and fixed
in 1% formaldehyde. Nuclear extracts were sonicated and precleared with
normal rabbit IgG (Santa Cruz Biotechnology). Chromatin was immunoprecip-
itated with anti-HDAC4 (CST, #2072), anti-HDAC5 (CST, #2082), anti-HDAC3
(Abcam), anti-FOXO1 (A. Brunet), or normal rabbit IgG. Immunoprecipitated
chromatin was decrosslinked, ethanol precipitated, and quantified by SYBR
green quantitative PCR. Recoveries were calculated as percent of input.
Animal Experiments and Procedures
LKB1lox/loxmice (Shaw et al., 2005) were crossed to Albumin-creERT2mice
(Imai et al., 2000). To induce Cre-mediated deletion in Albumin-creERT2
mice, mice were intraperitoneally injected with 1 mg/ mouse of Tamoxifen
(SIGMA) for 5 consecutive days. Ad-Cre-mediated deletion in LBK1lox/lox
mice was done by tail vein injection of 1 3 109PFUs/mouse in 8-week-old
males (Figure 1D). C57BL/6J, db/db, ob/ob, and C57BL/6J high fat diet-fed
mice (60% kcal%, Research Diets Incorporated D12492i) were obtained
from Jackson Laboratories. For metformin experiments, mice were injected
intraperitoneally with 250 mg/kg metformin in 0.9% saline for 1 hr. For basal
blood glucose, mice were fasted 18 hr overnight (o/n) and then glucose was
measured using a glucometer (Bayer). All animal care and treatments were
in accordance with the Salk Institute guidelines for the care and use of animals
(IACUC protocol 08-045). For additional details, see Extended Experimental
mRNA from primary hepatocytes was isolated using RNAeasy (QIAGEN) kit
and reverse transcribed using SuperScript II Reverse Transcriptase. Three
samples/mice were used per condition and qPCR was done in technical trip-
licate for each sample. qPCR reaction was carried out using Syber GreenER
(Invitrogen). AllqPCRresultsarerepresentative ofthreeseparateexperiments.
Comparisons were made using the unpaired Student’s t test. Values represent
the mean ± standard error of the mean (SEM) and are represented as error
bars. Statistical significance as indicated.
Supplemental Information includes Extended Results, Extended Discussion,
Extended Experimental Procedures, and seven figures and can be found
with this article online at doi:10.1016/j.cell.2011.03.043.
M.M.M. performed all cell and biochemistry experiments, designed shRNAs,
generated and large-scale purified all adenoviruses utilized, characterized all
antisera utilized, and with assistance from D.S.V. performed all mouse exper-
iments. M.M.M. and R.J.S. designed the study, analyzed the data, and wrote
the paper. In addition, K.R. in the lab of M.M. performed ChIP in Figure 3, Fig-
ure 5, and Figure S3; P.-D.D. in the lab of R.J.S. assisted with hepatocyte
ysis in Figure 3A. J.G.A. and M.D. in the lab of R.M.E. performed qPCR in Fig-
ure 3B and Figure 6D. We thank A. Fukamizu (U. of Tsukuba) and A. Brunet
(Stanford) for Foxo1 antibodies; M. Karin (UCSD) and P. Chambon (IGBMC)
for the Albumin-creERT2 mice; B. Wang and S. Hedrick for sharing reagents
and assistance; H. Juguilon for technical assistance; D. Shackelford for assis-
tance with the in vivo mouse imaging studies; R. Dentin for initial help with
hepatocytes; L. Gerken for genotyping; R. Kohnz for initial characterization
of Alb-creERT2 LKB1 mice; J. Fitzpatrick for confocal imaging assistance;
and K. Lamia for comments on the manuscript. We apologize to many inves-
duetospacelimitations. M.M.M.wassupportedthroughtheT32CMG training
grant to UCSD/Salk. R.M.E. is funded by the NIH HD027183 and DK062434.
M.M. is funded by the NIH R01 DK049777 and R01DK083834. R.J.S. is funded
by the NIH R01 DK080425 and P01CA120964 and the American Diabetes
Association Junior Faculty Award 1-08-JF-47. We thank the Leona M. and
Harry B. Helmsley Charitable Trust for their generous support.
Received: September 13, 2010
Revised: January 14, 2011
Accepted: March 25, 2011
Published: May 12, 2011
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