ORIGINAL ARTICLE

The Central Sirtuin 1/p53 Pathway Is Essential for the

Orexigenic Action of Ghrelin
Douglas A. Velásquez,1,2 Gloria Martínez,1 Amparo Romero,1,2 María J. Vázquez,1,2 Katia D. Boit,1,2,3
Iria G. Dopeso-Reyes,4 Miguel López,1,2 Anxo Vidal,1 Ruben Nogueiras,1,2 and Carlos Diéguez1,2

OBJECTIVE—Ghrelin is a stomach-derived peptide that increases class O (FoxO1), and cAMP-responsive element–binding
food intake through the activation of hypothalamic AMP-activated protein (pCREB), and leading to the final activation of
protein kinase (AMPK). However, the molecular mechanisms initi- agouti-related peptide/neuropeptide Y (AgRP/NPY) neu-
ated by the activation of the ghrelin receptor, which in turn lead to rons. However, the molecular mechanisms occurring after
AMPK activation, remain unclear. Sirtuin 1 (SIRT1) is a deacetylase GHS-R1a activation and before AMPK phosphorylation are
activated in response to calorie restriction that acts through the completely unknown. Ghrelin is the only gut peptide with
tumor suppressor gene p53. We tested the hypothesis that the orexigenic properties in rodents and humans; thus, the
central SIRT1/p53 pathway might be mediating the orexigenic ac- ghrelin system is uniquely positioned as a drug target for
tion of ghrelin.
the treatment of cachexia. The current study tested the
RESEARCH DESIGN AND METHODS—SIRT1 inhibitors, such hypothesis that the central sirtuin 1 (SIRT1)/p53 pathway
as Ex527 and sirtinol, and AMPK activators, such as AICAR, were might be mediating the orexigenic action of ghrelin.
administered alongside ghrelin in the brain of rats and mice (wild- SIRT1 is a NAD+-dependent deacetylase that acts on
type versus p53 knockout [KO]). Their hypothalamic effects on important tumor suppressors like p53. In addition to their
lipid metabolism and changes in transcription factors and neuro- biologic actions on cancer, SIRT1 and p53 are also impor-
peptides were assessed by Western blot and in situ hybridization.
tant in several metabolically relevant tissues. SIRT1 con-
RESULTS—The central pretreatment with Ex527, a potent trols divergent metabolic pathways in adipose tissue (7),
SIRT1 inhibitor, blunted the ghrelin-induced food intake in rats. liver (8), pancreatic b cells (9), and skeletal muscle (10),
Mice lacking p53, a target of SIRT1 action, failed to respond to mainly through the regulation of rate-limiting enzymes in-
ghrelin in feeding behavior. Ghrelin failed to phosphorylate volved in glucose and lipid metabolism. Recent reports
hypothalamic AMPK when rats were pretreated with Ex527, as have shown that central SIRT1 also regulates energy and
it did in p53 KO mice. It is noteworthy that the hypothalamic
SIRT1/p53 pathway seems to be specific for mediating the glucose homeostasis (11–17). On the other hand, p53 is
orexigenic action of ghrelin, because central administration of activated by the lack of nutrients through the activation of
AICAR, a potent AMPK activator, increased food intake in p53 AMPK, and p53 senescence activity contributes to the de-
KO mice. Finally, blockade of the central SIRT1 pathway did not velopment of insulin resistance (18).
modify ghrelin-induced growth hormone secretion. Because the molecular mechanisms that link the effects
of the ghrelin/GHS-R1a system to AMPK are unknown, the
CONCLUSIONS—Ghrelin specifically triggers a central SIRT1/
p53 pathway that is essential for its orexigenic action, but not for
current study tested the hypothesis that the hypothalamic
the release of growth hormone. Diabetes 60:1177–1185, 2011 SIRT1/p53 pathway might be mediating the orexigenic
action of ghrelin.

G
hrelin is the only known endogenous signal RESEARCH DESIGN AND METHODS
stimulating adiposity and feeding (1–3). At the Animal models. Male Sprague-Dawley rats (8 weeks old, 200–250 g) and C57/B6
mice (8 weeks old) were housed in air-conditioned rooms (22–24°C) under
hypothalamic level, ghrelin activates AMP- a 12/12-h light/dark cycle and fed standard chow. p53-null (8–10 weeks old,
activated protein kinase (AMPK), causing rele- mixed background C57BL/6J and 129/Sv) mice were described previously (19).
vant changes in hypothalamic mitochondrial respiration Homozygous wild-type (WT) and knockout (KO) mice were originated from
and production of reactive oxygen species (4–6), altering heterozygous mating, so only littermate WT and KO animals were compared in
the expression of transcription factors Bsx, Forkhead box each experiment. Animals were treated and killed when they were 12 to 14
weeks of age before any sign of morbidity resulting from tumor development
occurred. Animals were killed by decapitation between 1000 and 1200 h. An-
imal experiments were conducted in accordance with the standards approved
From the 1Department of Physiology, School of Medicine—Instituto de Inves- by the Faculty Animal Committee at the University of Santiago de Compostela,
tigaciones Sanitarias (IDIS), University of Santiago de Compostela, Santiago and the experiments were performed in agreement with the Rules of Laboratory
de Compostela, Spain; 2CIBER Fisiopatologia de la Obesidad y Nutricion Animal Care and International Law on Animal Experimentation.
(CIBERobn), University of Santiago de Compostela, Santiago de Compostela,
Spain; 3Catarinense Institut of Environmental Research and Human Develop- Nutritional status. Rats (n = 8/group), were assigned to one of the following
ment, Capivari de Baixo, Santa Catarina, Brazil; and the 4Department of Cell groups: fed ad libitum, deprived of food for 48 h, and fasted during 48 h and
Biology and Ecology, University of Santiago de Compostela, Santiago de refed during 24 h. All animals had free access to tap water.
Compostela, Spain. Implantation of intracerebroventricular cannulae. Rats were anesthetized
Corresponding authors: Ruben Nogueiras, [email protected], and Car- by an intraperitoneal injection of ketamine (100 mg/kg body wt)/xylazine
los Diéguez, [email protected]. (15 mg/kg body wt). Mice were anesthetized by an intraperitoneal injection of
Received 8 June 2010 and accepted 25 January 2011. tribromoethanol (480 mg/kg; Sigma-Aldrich, St Louis, MO). Intracerebro-
DOI: 10.2337/db10-0802 ventricular cannulae were implanted stereotaxically in rats (20) or mice (21),
This article contains Supplementary Data online at http://diabetes.
diabetesjournals.org/lookup/suppl/doi:10.2337/db10-0802/-/DC1. as described previously.
Ó 2011 by the American Diabetes Association. Readers may use this article as Intracerebroventricular treatments. Rats received an intracerebroventric-
long as the work is properly cited, the use is educational and not for profit, ular administration of 5 mL of vehicle or ghrelin (5 mg; Bachem, Bubendorf,
and the work is not altered. See http://creativecommons.org/licenses/by Switzerland). For the inhibition of SIRT1, we used two potent specific inhib-
-nc-nd/3.0/ for details. itors of SIRT1: Ex527 (1 to 5-10 mg in a total volume of 5 mL; Tocris

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 SIRT1/p53 MODULATES GHRELIN

Bioscience, St. Louis, MO) (22) and sirtinol (1 to 5-10 mg in a total volume of Biosciences) at room temperature for 4 to 6 days for AgRP and NPY and for 21
5 mL; Tocris Bioscience) (23) before ghrelin administration. For the experi- days for Bsx. The slides were developed in Kodak D-19 developer (Eastman
ments involving only two groups (vehicle versus ghrelin), the vehicle was Kodak Co., Rochester, NY), fixed (Kodak fixer), and counterstained with
saline. For the experiments involving SIRT1 inhibitors, the vehicle was DMSO, methylene blue.
because Ex527 and sirtinol were both diluted in DMSO. Mice received an intra- To compare anatomically similar regions, the slides were matched ac-
cerebroventricular administration of vehicle, ghrelin (5 mg), or AICAR (3 mg; cording to Paxinos and Watson (26). The slides from control and treated
Sigma-Aldrich A9978) in a total volume of 2 mL. For the experiments involving animals at each treatment time were always exposed to the same autoradio-
vehicle versus ghrelin and vehicle versus AICAR, the vehicle was saline. graphic film. All sections were scanned, and the specific hybridization signal
We used the same dose of ghrelin for both rats and mice because this dose was quantified by densitometry using the Molecular Analyst digital imaging
has been demonstrated to be effective in both species (2). We used eight rats system (Bio-Rad Laboratories Inc., Richmond, CA). The optical density of the
per group, and the experiments were repeated at least twice. Rats were killed hybridization signal was determined and subsequently corrected by the optical
by cervical dislocation. Hypothalami were dissected and stored at 280°C until density of its adjacent background value. For this reason, a rectangle with the
further processing. same dimensions in each case was drawn enclosing the hybridization signal
Western blotting. Hypothalami were homogenized in ice-cold lysis buffer over each nucleus and over adjacent brain areas of each section (back-
containing 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EGTA, 1 mmol/L EDTA, 1% ground). We used 16 to 20 sections for each animal (4–5 slides, 4 sections/
Triton X-100, 1 mmol/L sodium orthovanadate, 50 mmol/L sodium fluoride, 5 slide). The mean of these 16 to 20 values was used as the densitometry value
mmol/L sodium pyrophosphate, 0.27 mol/L sucrose, 0.1% 2-mercaptoethanol, for each animal.
and Complete protease inhibitor cocktail (1 tablet/50 mL; Roche Diagnostics, Plasma growth hormone response. Chronic intracerebroventricular and
Mannheim, Germany). Homogenates were centrifuged at 13,000g for 10 min at intracardiac cannulae were implanted under ketamine/xylazine anesthesia, as
4°C, supernatants were removed, and aliquots were snap-frozen in liquid ni- described above. After surgery, the animals were placed directly in isolation
trogen. Hypothalamus lysate (40 mg) was subjected to SDS-PAGE on 6% poly- test chambers for 5 days and were given free access to regular rat chow and tap
acrylamide gels and electrotransferred on a polyvinylidene fluoride membrane. water. On the day of the experiment, blood samples (0.3 mL) were withdrawn at
Membranes were blocked for 1 h in TBS-Tween 20 (TBST: 50 mmol/L Tris- the appropriate times. The animals (n = 7–9 rats/group) received vehicle,
HCl [pH 7.5], 0.15 mol/L NaCl, and 0.1% Tween 20) containing 5% skimmed milk ghrelin (12 nmol/kg i.v.), Ex527 (1 mg i.c.v.), or Ex527 + ghrelin.
or 3% BSA (for pAMPK Thr172 and pACC Ser79) and probed for 16 h at 4°C in Hormone assays. Plasma growth hormone (GH) concentrations were de-
TBST, 5% skimmed milk, or 3% BSA (for pAMPK Thr172, pACC Ser79, SIRT1, termined by double-antibody radioimmunoassay using materials supplied by
and acetyl-p53-Lys379) with the appropriate dilution of the indicated antibodies the National Hormone Pituitary Program, as described previously (27). Values
(acetyl-CoA carboxylase [ACC]: 1:1500; pACC: 1:2000; AMPKa1: 1:1000; are expressed in terms of the GH reference preparation (GH-RP-2). The intra-
AMPKa2: 1:1000; pAMPK: 1:2000; b-actin (loading control): 1:2000). ACC was and interassay coefficients of variation were 7 and 10%, respectively.
detected using horseradish peroxidase (HRP)-conjugated–coupled streptavi- Statistical analysis and data presentation. The experiments constituted by
din (Amersham Biosciences, Little Chalfont, U.K.). two groups: Sprague-Dawley rats and mice (WT and p53 KO) treated with
Detection of proteins was performed using HRP-conjugated secondary ghrelin or AICAR and analyzed using a nonparametric Mann–Whitney test. In
antibodies and an enhanced chemiluminescence reagent (Amersham Bio- the experiments constituted by four groups (Sprague-Dawley rats treated with
sciences). We used 8 to 12 hypothalami per experimental group. Acetyl-p53- vehicle, ghrelin, Ex527, and Ex527 + ghrelin, and vehicle, ghrelin, sirtinol, and
Lys379 was obtained from Cell Signaling (Danvers, MA). ACCa, pACCa-Ser79, sirtinol + ghrelin), the data were analyzed by two-way ANOVA, followed by
AMPKa1, and AMPKa2 were obtained from Upstate Biotechnology (Teme- a post hoc multiple comparison test (Tukey’s test). Data are expressed as
cula, CA); pAMPKa-Thr172 from Cell Signaling; fatty acid synthase (FAS), mean 6 SEM and analyzed using PASW Statistics 18.0 software (SPSS Inc.,
pCREB, and FoxO1 from Santa Cruz Biotechnology (Santa Cruz, CA); and Chicago, IL). A value of P , 0.05 was considered as being significant.
b-actin from Abcam (Cambridge, U.K.), as described previously (6).
For the blotting assays, the experiments constituted by two groups: Sprague-
Dawley rats and mice (WT and p53 KO) treated with ghrelin or AICAR and RESULTS
analyzed using a nonparametric Mann–Whitney test. In the experiments con-
stituted by four groups (Sprague-Dawley rats treated with vehicle, ghrelin, Regulation of SIRT1 by nutritional status. Rats fasted
Ex527, and Ex527 + ghrelin, or with vehicle, ghrelin, sirtinol, and sirtinol + during 48 h exhibited a loss of body weight, whereas the
ghrelin), the data were analyzed by two-way ANOVA, followed by a post hoc refeeding during 24 h partially led to a substantial recovery
multiple comparison test (Tukey’s test). (Fig. 1A). Acetyl-p53 levels, a marker of SIRT1 activity in
In situ hybridization. Coronal hypothalamic sections (16 mm) were cut on
vivo (28), were decreased in the hypothalamus of fasted
a cryostat and immediately stored at 280ºC until hybridization. For AgRP,
NPY, and Bsx mRNA detection, we used the specific antisense oligodeox- rats (F2,18 = 3.651, P , 0.05), whereas those levels were
ynucleotides (Table 1). These probes were 39-end–labeled with [35S]deoxy- similar to baseline in rats after refeeding (Fig. 1B). Be-
ATP using terminal deoxynucleotidyl transferase. The specificity of the probes cause ghrelin is a key player for increasing feeding be-
was confirmed by incubating the sections with an excess of the unlabeled havior, we tested the hypothesis that central ghrelin
probes, as reported previously (24,25). In situ hybridizations were performed administration might stimulate hypothalamic SIRT1 activ-
as reported previously (24,25).
ity. As previously reported, ghrelin increased food intake
The frozen sections were fixed with 4% paraformaldehyde in phosphate
buffer (0.1 mol/L, pH 7.4) at room temperature for 30 min. They were dehy- after 2 h (F1,13 = 24.161, P , 0.001; Fig. 1C) and 6 h (F1,13 =
drated using 70%, 80%, 90%, 95%, and 100% ethanol for 5 min each. The hy- 36.14, P , 0.001; Fig. 1E) (29) and decreased hypothalamic
bridization was done overnight at 37°C in a moist chamber. The hybridization acetyl-p53 levels after 2 h (F1,13 = 7.915, P , 0.01; Fig. 1D)
solution contained 5 3 105 (AgRP and Bsx) or 1 3 106 cpm (NPY) per slide of and 6 h (F1,13 = 7.645, P , 0.05; Fig. 1F).
the labeled probe, 43 standard saline citrate (SSC), 50% deionized formamide, Blockade of the SIRT1/p53 pathway blunts the orexi-
13 Denhardt’s solution, 10% dextran sulfate, and 10 mg/mL sheared, single-
genic action of ghrelin. We next studied the functional
stranded salmon sperm DNA.
Afterward, the hybridization sections were sequentially washed in 13 SSC relevance of these findings by assessing whether the
at room temperature, four times in 13 SSC at 42ºC (30 min/wash), and once in pharmacologic blockade of SIRT1 activity might regulate
13 SSC at room temperature (1 h) and then rinsed in water and ethanol. Fi- the orexigenic action of ghrelin. First, we observed that
nally, the sections were air-dried and exposed to Hyperfilm b-Max (Amersham the central injection of Ex527, a potent inhibitor of SIRT1

TABLE 1
Antisense oligonucleotides for in situ hybridization analysis
mRNA GenBank accession number Sequence
AgRP AF206017 59-CGACGCGGAGAACGAGACTCGCGGTTCTGTGGATCTAGCACCTCTGCC-39
BSX XM_001064837 59-CCTCAACGGCTTGGGCTTGTGTAGCAGAATGTCC-39
NPY M20373 59-AGATGAGATGTGGGGGGAAACTAGGAAAAGTCAGGAGAGCAAGTTTCATT-39

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 D.A. VELÁSQUEZ AND ASSOCIATES

FIG. 1. Effects of nutritional status on hypothalamic SIRT1. A: Fasting for 48 h caused a significant (P < 0.001) decrease in body weight, whereas
refeeding during 24 h partially recovered the weight loss. B: Hypothalamic acetylated p53 levels decreased in fasted rats and recovered in refed
rats. Effects of intracerebroventricular ghrelin injection (5 mg/rat) after 2 h on food intake (C), and hypothalamic acetylated p53 levels (D).
Effects of intracerebroventricular ghrelin injection (5 mg/rat) after 6 h on food intake (E), and hypothalamic acetylated p53 levels (F). Values
were normalized to those of the internal control b-actin, and the results are expressed as arbitrary units. Mean values were obtained from six
animals per group. Values are the mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

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activity, increased hypothalamic acetyl-p53 levels at dif- It is important to note that even though pAMPK levels
ferent doses (1, 5, and 10 mg; Supplementary Fig. 1A), in- were increased, pACC did not reach statistical signifi-
dicating that this compound decreased hypothalamic SIRT1 cance, probably because of the different kinetics of phos-
activity in vivo. We then centrally administered ghrelin, phorylation of both enzymes (6). Furthermore, the higher
Ex527, and Ex527 + ghrelin to the rats. We found that expression of the transcription factors FoxO1 (F3,27 =
ghrelin increased food intake at 2 h (data not shown) and 2.509, P , 0.05), pCREB (F3,26 = 3.668, P , 0.05; Fig. 2D),
6 h (F3,27 = 12.282, P , 0.001; Fig. 2A), but when the SIRT1 and Bsx (F3,26 = 3.526, P , 0.05; Fig. 2E and F) and the
inhibitor was administered 20 min before ghrelin, the neuropeptides NPY (F(3,26) = 4.362, P , 0.05) and AgRP
orexigenic action of ghrelin was markedly blunted after 6 h (F3,26 = 3.33, P , 0.05; Fig. 2E and F) in the hypothalamic
(Fig. 2A). Because ghrelin increases food intake through arcuate nucleus induced by ghrelin was also abolished
its effects on hypothalamic fatty acid metabolism (5,6), we when the SIRT1 inhibitor was coadministered. When we
next assessed the levels of several key enzymes for the used sirtinol, another inhibitor of SIRT1 activity, results
synthesis of lipids. We found that 6 h after an intrace- were similar to those obtained with Ex527 (Supplementary
rebroventricular ghrelin injection, pAMPK levels were in- Fig. 1).
creased (F3,28 = 2.455, P , 0.05), but ACC levels were Tumor suppressor protein p53 is a substrate of SIRT1,
decreased (F3,27 = 6.045, P , 0.01). Those effects were and it is found hyperacetylated in SIRT1 KO mice (30).
abolished when the SIRT1 inhibitor was coadministered Because a recent report showed that p53 is involved in
(Fig. 2B and C). energy metabolism and homeostasis (31), we next inquired

FIG. 2. Pharmacologic blockade of SIRT1 blunts the orexigenic action of ghrelin. A: Effects of intracerebroventricular ghrelin injection (5 mg/rat),
Ex527 (1 mg/rat), and ghrelin + Ex527 on food intake after 6 h. Hypothalamic protein levels of pAMPKa, AMPKa1, AMPKa2, pACC, ACCa, FAS
(B and C), pCREB, FoxO1 (D), and Bsx, NPY, and AgRP (E and F) after 6 h of ghrelin and Ex527 injection. Values were normalized to those of the
internal control b-actin, and the results are expressed as arbitrary units. Mean values were obtained from six animals per group. Values are the
mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

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 D.A. VELÁSQUEZ AND ASSOCIATES

whether p53 could be a mediator of SIRT1-dependent between WT and p53 KO mice regarding the expression of
effects of ghrelin. For this purpose, we treated WT and AMPKa1, AMPKa2, or FAS (Fig. 3C and D). It is note-
p53 KO mice with intracerebroventricular ghrelin, fol- worthy that we found that 6 h after its central injection,
lowing the same protocol as that described above. The ghrelin increased pAMPK levels in WT mice (F1,12 = 4.466,
p53 KO mice did not show alterations in body weight, P , 0.05) but failed to do so in p53 KO mice (Fig. 3C and D),
food intake, fat mass, or nonfat mass compared with WT suggesting that p53 is an essential mediator of ghrelin
littermates (Supplementary Fig. 2A–D). As expected, actions on AMPK. The levels of pAMPK and pACC are
central ghrelin administration increased food intake in correlated in normal conditions; however, we found that the
WT animals, whereas identical intracerebroventricular hypothalamic levels of pACC are downregulated in p53 KO
ghrelin treatment in p53 KO animals had no effect on food mice (F1,12 = 8.576, P , 0.01) but not in WT mice (Fig. 3C
intake after 2 (Fig. 3A) or 6 h (Fig. 3B). and D).
We next assessed the levels of several key enzymes for Although we do not have a clear explanation for these
the synthesis of lipids. No principal differences were found results, it seems that ghrelin is able to activate ACC when

FIG. 3. Mice lacking p53 do not respond to ghrelin injection. Effects of intracerebroventricular ghrelin injection (5 mg/mouse) on food intake after
2 h (A) and 6 h (B) in WT and p53 KO mice. Hypothalamic protein levels of pAMPKa, AMPKa1, AMPKa2, pACC, ACCa, and FAS after 6 h of ghrelin
injection (C and D). Values were normalized with to those of the internal control b-actin, and the results are expressed as arbitrary units. Mean
values were obtained from six animals per group. Values are the mean 6 SEM. *P < 0.05, **P < 0.01.

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p53 is not present, suggesting that p53 might also regulate (27) led to the expected increase in plasma GH levels at
the actions of ghrelin on different key enzymes modulating 5, 10, and 15 min (Fig. 5A), whereas the central blockade of
fatty acid metabolism. Further studies analyzing not only SIRT1 did not alter that response (Fig. 5A). Ghrelin ex-
protein levels but also enzymatic activity and lipolysis/ hibited a similar stimulatory effect in both area under the
lipogenesis will be necessary to address this issue. Fur- curve and mean peak GH levels (Fig. 5B and C).
thermore, we detected that central ghrelin injection de-
creased ACCa levels in both WT (F1,12 = 2.844, P , 0.05)
and p53 KO mice (F1,12 = 6.699, P , 0.01; Fig. 3C and D), DISCUSSION
indicating that p53 is not essential for ghrelin-mediated Our current data demonstrate that the hypothalamic SIRT1/
ACC regulation. p53 pathway is crucial for the orexigenic effect of ghrelin.
p53 does not mediate the orexigenic action of AICAR. Pharmacologic inhibition of SIRT1 or genetic depletion of
Central injection of AICAR, a potent activator of AMPK p53 abolish the effects of ghrelin on AMPK and thereby
activity, stimulates food intake in rodents (32). To de- blunt ghrelin-induced effects on transcription factors, in-
termine whether p53 is a crucial player for the orexigenic cluding pCREB, FoxO1, and Bsx, and neuropeptides, such
action of AICAR, we centrally treated p53 KO mice with as NPY and AgRP, leading to a suppression of ghrelin-
AICAR (3 mg) and found a stimulation in food intake after induced food intake.
6 h (F1,12 = 3.542, P , 0.05; Fig. 4A). Hypothalamic pAMPK SIRT1 is a deacetylase that regulates metabolism in
levels were also increased in p53 KO mice treated with multiple peripheral tissues. It has been reported recently
AICAR (F1,12 = 4.479; Fig. 4B and C). Therefore, our data that SIRT1 mRNA is located in metabolically relevant areas
indicate that p53 is not required for the orexigenic action of the mouse neuroaxis, such as pro-opiomelanocortin neu-
of direct AMPK activators. rons, which are critical for energy and glucose homeostasis
The central SIRT1 pathway does not modulate ghrelin- (11). It seems that SIRT1 regulates the central melano-
induced GH secretion. Finally, we assessed whether the cortin system (13) and that the specific lack of SIRT1 in
SIRT1 pathway is mediating other neuroendocrine actions the brain abolishes the higher physical activity induced by
of ghrelin, namely GH secretion. Administration of ghrelin calorie restriction (12). More specifically, the lack of SIRT1

FIG. 4. Central injection of AICAR increases food intake in p53 KO mice. A: Effects of intracerebroventricular AICAR injection (3 mg/mouse) on
food intake after 6 h in p53 KO mice. B and C: Hypothalamic protein levels of pAMPK, pACC, AMPKa1, AMPKa2, ACCa, and FAS after 6 h of
AICAR injection. Values were normalized to those of the internal control b-actin, and the results are expressed as arbitrary units. Mean values
were obtained from six animals per group. Values are the mean 6 SEM, *P < 0.05.

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 D.A. VELÁSQUEZ AND ASSOCIATES

Therefore, the regulation of hypothalamic SIRT1 activity

by nutritional status is similar to its regulation in several
peripheral tissues (33).
Ghrelin is a stomach-derived hormone that rapidly
increases food intake and body weight (1,29). Its regula-
tion by nutritional status was controversial because the
assays detected both acyl-ghrelin and des-acyl ghrelin and
thus were not specific. Studies using new technologies for
separately detecting both isoforms indicate that circulating
des-acyl ghrelin increases significantly with fasting,
whereas blood acyl-ghrelin levels are not changed over the
course of fasting (34,35). Most of the effects of ghrelin are
exerted through the GH secretagogue receptor 1a (GHS-
R1a) (36), which is expressed in AgRP/NPY neurons in the
hypothalamic arcuate nucleus (37). The orexigenic effect
of ghrelin is mediated by AMPK, a key upstream master
regulator of lipid metabolism (5,6). However, the molecu-
lar events regulating AMPK phosphorylation after the ac-
tivation of the GHS-R1a are unknown.
In the present work, we demonstrate that central ghrelin
administration increases hypothalamic SIRT1 activity, stim-
ulating the deacetylation of p53. More interestingly and
consistent with previous findings (14), the blockade of
central SIRT1 activity abolished the potent orexigenic ef-
fect of ghrelin. At molecular level, the ghrelin-induced
activation of AMPK is prevented when SIRT1 activity is
blocked. The interaction between SIRT1 and AMPK has
been previously shown in vitro, indicating that resveratrol
activates AMPK in neurons (38).
Although pharmacologic experiments based on the ad-
ministration of SIRT1 inhibitors to animal models are
providing important insight into principal effects, targets,
and mechanisms of the SIRT1 system, understanding the
function of its endogenous role requires more sophisti-
cated approaches, such as genetic disruption of SIRT1 or
its substrates. We focused on p53 because this tumor-
suppressor protein is a well-known target of SIRT1 action,
and there is growing evidence of its role on metabolism
and energy balance in peripheral tissues (31). Our results
indicate that p53 is required for the ghrelin-induced food
intake, because ghrelin failed to increase food intake in
p53 KO mice. In accordance with pharmacologic findings,
ghrelin stimulated hypothalamic pAMPK levels in WT mice
but failed to do so in p53 KO mice. On the other hand, it has
been shown that SIRT1 is a target gene of p53 in some but
not all of the tissues (31,39), so it might be possible that p53
mediates the changes in SIRT1 upon ghrelin treatment or
calorie restriction. However, the observed correlation be-
tween acetylated p53 and SIRT1 activity in the hypothala-
mus on different experimental conditions indicate that
p53 is an essential mediator of SIRT1-dependent effects of
ghrelin on AMPK. Mice lacking p53 in specific hypothalamic
FIG. 5. Pharmacologic blockade of SIRT1 does not modify the ghrelin-
areas will be essential to demonstrating which particular
induced GH secretion. A: Effects of intravenous ghrelin injection (12 neuronal circuits are responsible for those actions. How-
nmol/kg), intracerebroventricular Ex527 (1 mg/rat), and ghrelin + ever, the central SIRT1/p53 pathway is not required by di-
Ex527 on plasma GH levels in adult male freely moving rats. Area under rect AMPK activators, because AICAR stimulated food
the curve (AUC) (B) and mean peak GH (C) levels. Mean values were
obtained from six animals per group. Values are the mean 6 SEM. *P < intake in p53-deficient mice. Therefore, these findings cor-
0.05, **P < 0.01. roborate that the central SIRT1/p53 pathway is not associ-
ated with AICAR-induced activation of AMPK.
Finally, our results indicate that ghrelin stimulated GH
in pro-opiomelanocortin neurons causes hypersensitivity release as expected, but the blockade of central SIRT1 did
to diet-induced obesity because of reduced energy ex- not modify GH levels. Therefore, it seems that the central
penditure (17). Concurring with those data, we observed SIRT1/p53 pathway is specifically mediating the ghrelin
that acetylation of p53 in the hypothalamus is decreased orexigenic action and suggests that different neuronal
after food deprivation, indicating that SIRT1 as a deacety- pathways modulate ghrelin-induced food intake and GH
lase increased its hypothalamic activity during starvation. secretion.

diabetes.diabetesjournals.org DIABETES, VOL. 60, APRIL 2011 1183

 SIRT1/p53 MODULATES GHRELIN

FIG. 6. Schematic overview summarizing our proposed model for the molecular mechanisms initiated by the activation of the ghrelin receptor
leading to AMPK activation and finally to an increased feeding behavior. ROS, reactive oxygen species; CPT, carnitine palmitoyltransferase; UCP,
uncoupling protein. (A high-quality color representation of this figure is available in the online issue.)

In summary, we provide a combination of pharmaco- A.V., R.N., and C.D. reviewed and edited the manuscript
logic and genetic evidence to demonstrate that the central and contributed to discussion.
nervous system SIRT1/p53 pathway is essential for the The authors thank Dr. Hector J. Caruncho (University of
orexigenic response to ghrelin (Fig. 6). The molecular path- Santiago de Compostela) for helpful comments, Carmen
way mediating those effects involves alterations in AMPK Cadarso (University of Santiago de Compostela) for sound
activation, which leads to changes in hypothalamic fatty statistical advice, and Luz Casas (University of Santiago de
acid metabolism, and finally, modifies feeding behavior. Compostela) for excellent technical assistance. The authors
thank Dr. Manuel Serrano from the Spanish National Cancer
ACKNOWLEDGMENTS
Research Center (CNIO) for providing p53-deficient mice.
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