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Vol.:(0123456789)1 3
Neurochem Res (2018) 43:153–161 DOI
10.1007/s11064-017-2369-7
ORIGINAL PAPER
Comparing the Effects of Melatonin with Caloric
Restriction in the Hippocampus of Aging Mice:
Involvement of Sirtuin1 and the FOXOs Pathway
Anorut Jenwitheesuk1 · Seongjoon Park2 ·
Prapimpun Wongchitrat3 · Jiraporn Tocharus4 ·
Sujira Mukda1 · Isao Shimokawa2 ·
Piyarat Govitrapong1,5,6
Received: 23 April 2017 / Revised: 25 July 2017 / Accepted: 26
July 2017 / Published online: 2 August 2017 © Springer
Science+Business Media, LLC 2017
control, CR, Mel and CR+Mel treated groups. The Mel and CR+Mel
were treated with melatonin 10 mg/kg, daily, sub-cutaneously
for 7 consecutive days. Mel treatment upregu-lated the mRNA
expression of Sirt1, FOXOs (FoxO1 and FoxO3a) target genes that
regulated the cell cycle [e.g., cyclin-dependent kinase inhibitor
1B (p27)], Wingless and INT-1 (Wnt1) and inducible signaling
pathway protein 1 (Wisp1) in the aged mouse hippocampus. CR
treatment also showed the similar actions. However, the mRNA
expres-sion of Sirt1, FoxO1, FoxO3a, p27 or Wisp1 did not alter in
the CR+Mel group when compared with CR or Mel group. Melatonin
could not produce any additive effect on the CR treatment group,
suggesting that both treatments mim-icked the effect, possibly via
the same pathway. NPY which mediates physiological adaptations to
energy deficits is an essential link between CR and longevity in
mice. In order to focus on the role of Npy in mediating the effects
of mela-tonin, the gene expression between NpyKO and WT male mice
were compared. Our data showed that, in the absence of Npy,
melatonin could not mediate effects on those gene expressions,
suggesting that Npy was required for mela-tonin to mediate the
effect, possibly, on life extension.
Keywords Caloric restriction · Melatonin ·
Sirtuin1 · FoxO1 · Neuropeptide Y · Aging
AbbreviationsADAM10 A disintegrin and metalloprotease domain
10AgRP Agouti-related peptideAMPK Adenosine monophosphate-activated
protein
kinaseCART Cocaine and amphetamine regulated transcriptCBP
CREB-binding proteinCR Caloric restriction
Abstract It has been suggested that age-related
neu-rodegeneration might be associated with neuropeptide Y (NPY);
sirtuin1 (SIRT1) and forkhead box transcrip-tion factors O
subfamily (FOXOs) pathways. Melatonin, a hormone mainly secreted by
the pineal gland, is another anti-aging agent associated with the
SIRT1-FOXOs path-way. This study aimed to compare the effects of
melatonin (Mel) and caloric restriction (CR) on the expression of
Sirt1, FoxO1, FoxO3a and FOXOs target genes in the aging mouse
hippocampus. Neuropeptide Y-knockout (NpyKO) and wild-type (WT)
male mice aged 19 months were pre-viously treated either with
food ad libitum or CR for 16 months. WT old animals
were divided into four groups:
Isao Shimokawa and Piyarat Govitrapong have contributed equally
to this work.
* Isao Shimokawa [email protected]
* Piyarat Govitrapong [email protected];
[email protected]
1 Research Center for Neuroscience, Institute
of Molecular Biosciences, Mahidol University, Salaya,
Thailand
2 Department of Pathology, Nagasaki University School
of Medicine and Graduate School of Biomedical
Sciences, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan
3 Center for Research and Innovation, Faculty
of Medical Technology, Mahidol University, Salaya,
Nakon Pathom 73170, Thailand
4 Department of Physiology, Faculty of Medicine,
Chiang Mai University, Chiang Mai 50200, Thailand
5 Center for Neuroscience and Department
of Pharmacology, Faculty of Science, Mahidol University,
Salaya, Thailand
6 Chulabhorn Graduate Institute, Chulabhorn Royal Academy,
Kamphaeng Phet 6 Road, Lak Si, Bangkok 10210, Thailand
http://crossmark.crossref.org/dialog/?doi=10.1007/s11064-017-2369-7&domain=pdf
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DKK1 Wnt inhibitor Dickkopf WNT signaling path-way inhibitor
1
FOXOs Forkhead box transcription factors and the O subfamily
GAPDH Glyceraldehyde 3-phosphate dehydrogenaseIGF-1 Insulin-like
growth factor-1Mel MelatoninNAD Nicotinamide adenine
dinucleotideNAMPT Nicotinamide phosphoribosyltransferaseNF-κB
Nuclear factor-κBNMNAT Nicotinamide/nicotinic acid
mononucleotide
adenylyltransferaseNPY Neuropeptide YNrf2 Nuclear factor
(erythroid-derived 2)-like 2POMC Anorexigenic
proopiomelanocortinp27 Cyclin-dependent kinase inhibitor 1Bp300 E1A
binding protein p300p53 Tumor suppressor protein 53PGC-1α
Peroxisome proliferator-activated receptor
gamma coactivator 1-alphaPI3K Phosphoinositide-3-kinasePOMC
ProopiomelanocortinPPAR-γ Peroxisome proliferator-activated
receptor
gammaSAMP8 Senescence-accelerated mouse prone 8SAMR1
Senescence-accelerated-resistant mouseSIRT SirtuinWisp1 Wn1
inducible pathway protein1WNT Wingless-type
Introduction
Caloric restriction (CR) has been shown to delay the onset and
severity of brain diseases associated with aging and to extend the
functional health span of important functions, such as cognition.
Reduced caloric intake has enhanced longevity and prevented
age-related diseases in rodents and monkeys. High calorie diets
have been associated with the risk of Alzheimer’s disease;
therefore, it has been proposed that a low calorie diet might
afford protection against neu-rodegenerative diseases [1–5].
However, the underlying mechanisms of how CR could protect the
brain from aging have not been clearly elucidated.
The neuroprotective effects of CR might result from pathways
that respond to energy status, including the sir-tuin1 (SIRT1)
[6–8] and forkhead box transcription factors and the O subfamily
(FOXOs) [9, 10]. The FOXOs tran-scription family is one of the
targets of SIRT1. FOXO1 and FOXO3a are expressed in the hippocampus
and play an important role in modulating hippocampal neuronal
home-ostasis [11, 12]. CR inhibits insulin-like growth factor-1
(IGF-1) signaling, which negatively regulates the FOXO
transcription factor [13, 14].
Neuropeptide Y (NPY), a neuropeptide from the hypo-thalamic
arcuate nucleus, is elevated by CR. Aging reduces levels of NPY in
the brain and in response to fasting. NPY is present in the
hippocampus and plays a role in learning and memory; thus, the
possible involvement of this peptide in the pathogenesis of
neurodegenerative diseases, such as Alzheimer’s disease [15] and
Parkinson’s disease, has been suggested [16]. The beneficial
effects of CR might result from two pathways that respond to low
energy status: NPY [17] and SIRT1 [7].
Melatonin, a hormone mainly secreted by the pineal gland, is
another anti-aging agent that could potentially be applied in the
treatment of neurodegenerative diseases [18, 19]. This hormone was
recently found to be associated with the SIRT1-FOXOs pathway.
Melatonin preserved the rela-tive protein levels of SIRT1 in
senescence-accelerated mice (SAMP8) [20–22] and in the hippocampus
of total sleep-deprived rats [23]. In addition, melatonin plays a
potent protective role against brain injury via the actions of
pro-tein kinase B (AKT) [24] and FOXOs [25].
The present study aimed to compare the effects of CR and
melatonin on the expressions of the Sirt1, FoxO1, FoxO3a and the
FOXOs target genes, including cyclin-dependent kinase inhibitor 1B
(p27) in the hippocampus of aging mice. In addition, Npy knockout
(NpyKO) mice were used to determine whether NPY was required to
regulate the melatonin SIRT1/FOXOs pathway.
Experimental Procedure
Animals and Experimental Design
Npy-knockout (NpyKO) and wild type (WT) mice were breed and
raised in animal facilities of Center for Frontier Life Sciences at
Nagasaki University as described in our previous study [26].
Animals were handled according to the guidelines approved by the
Ethics Review Committee for Animal Experimentation at Nagasaki
University. All mice were housed in 12:12 h light/dark cycle
in an ambi-ent temperature of 22–25 °C under specific
pathogen-free conditions. Animals were fed ad libitum with a
Charles River-LPF diet (Oriental Yeast Co. Ltd., Tsukuba, Japan).
NpyKO and WT mice with 3 months of age were used for
experimental procedures. Animals were fed with the dietary regimens
either normal receive food ad libitum or caloric restriction
(CR) throughout the experiments for 16 months. The CR-treated
mice were received a food allot-ment according to previous study
[27], consisting of 70% of the mean daily food intake of the normal
group every day, 30 min before the lights were turned off.
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At 19-month-old, mice were groups to test whether melatonin
could increase Sirt1 and FoxOs expression in the hippocampus. WT
mice were divided to four groups: a con-trol group was continued to
receive food ad libitum, CR-treated group, melatonin-treated
group (Mel) and CR and melatonin-treated group (CR+Mel). NpyKo mice
which normally show a phenotype to reduce in a food intake [28] was
continued to receive food ad libitum and were divided to 2
groups: a control group and Mel group. In the Mel and CR+Mel group,
10 mg/kg of melatonin (Sigma-Aldrich, St Louis, MO, USA) were
administered daily via subcutane-ous injection for
7 consecutive days. Control and CR group animals received
vehicle injections at the same time. All mice were sacrificed, and
hippocampal brain tissues were dissected, collected and stored at
−80 °C until assayed. mRNAs of Sirt1, FoxO1, FoxO3a and
selected genes involved in cell cycle arrest (p27) and FOXO3a
modulation (Wisp1) were determined.
RNA Isolation and Reverse Transcription
Total RNAs were extracted from the hippocampus of dif-ferent
groups of mice by using RNeasy tissue kit (Qia-gen, Valencia, CA,
USA) according to the manufacturer’s instructions. RNA
concentrations were determined by UV spectrophotometry at a
wavelength of 260 nm. The qual-ity of RNA was evaluated as
the densitometric ratio of 28S and 18S ribosomal RNA by
electrophoresis. The extracted RNA was reverse-transcribed using a
Bio-Rad reverse transcriptase reagent kit (Bio-Rad, Hercules, CA,
USA), according to the manufacturer’s instructions. The cDNA
solution was then stored at −20 °C until use.
Quantitative Real‑time PCR (qPCR)
The relative gene expression was performed by using SYBR-based
real-time PCR. Commercially available, prevalidated primer sets for
FoxO1, FoxO3a, p27 and Gapdh were purchased from the TAKARA Perfect
Real Time support system (TAKARA BIO Inc., Shiga, Japan)
as described in our previous report [29]. The other primer pairs
for Sirt1 and Wisp1 were purchased from Takara Bio Inc. See
Table 1 for all primers used details. ThunderBird SYBR qPCR
Mix (TOYOBO Co. Ltd., Osaka, Japan) was used for PCR amplification,
and the reactions were per-formed according to the manufacturer’s
protocol. Gapdh was used as an endogenous control and non-template
con-trols were systematically included to check for contamina-tion.
The amplification, data quantification, and analysis were performed
on an ABI PRISM 7900HT Sequence Detector (Applied Biosystems,
Foster City, CA, USA).
Statistical Analysis
The data are expressed as the means ± SEM. Signifi-cance was
assessed using one-way analysis of variance (ANOVA), followed by
the Tukey–Kramer tests using GraphPad Prism version 5. P values
less than 0.05 were considered significant.
Results
Melatonin, CR and NpyKO in Sirt1 Expression
We compared the Sirt1 mRNA between young and old mice and found
that Sirt1 mRNA was downregulated by aging (P < 0.05)
(Fig. 1a). Therefore, we explored the effects of melatonin
and CR on Sirt1 mRNA expression in the hippocampus obtained from
old mice. The Sirt1 mRNA expression in the hippocampus was compared
with that among old mice. Both the CR group and the Mel group
significantly (P < 0.05) increased Sirt1 mRNA in old mice,
compared with the old control group (Fig. 1b). Sirt1 mRNA
expression in all groups of the NpyKO mice were significantly (P
< 0.05) lower than that in the old WT control group. Ten
milligrams per kilogram of mela-tonin did not alter Sirt1 level in
the CR+Mel group com-pared with CR group and Mel group of WT mice.
The Sirt1 mRNA level in the NpyKO Mel-treated group showed no
Table 1 Primers used details Gene name GenBank accession no.
Takara primer set ID
FoxO1 NM_019739.3 MA072856FoxO3a NM_019740.2 MA075309p27
NM_001293757.1 MA099459Gapdh NM_001289726.1 MA050371
Primer sequenceSirt1 NM_001159589.2 Forward CAG ACC CTC AAG CCA
TGT TTGAT
Reverse TTG GAT TCC TGC AAC CTG CTCWisp1 NM_018865.2 Forward GTC
CTG AGG GTG GGC AAC AT
Reverse GGG CGT GTA GTC GTT TCC TCT
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significant difference, compared with old NpyKO control mice.
NpyKO mice treated with melatonin had lower Sirt1 mRNA than WT mice
treated with melatonin (P < 0.001) (Fig. 1b).
Melatonin, CR and NpyKO in FoxOs Expression
Because FoxO1 is expressed in the hippocampus, and several lines
of evidence have shown that FoxO1 was
downregulated by aging. In WT mice, both the CR group and the
Mel group significantly increased FoxO1 mRNA in the hippocampus,
compared with the old control group (P < 0.01) (Fig. 2A).
FoxO1 mRNA levels in all groups of the NpyKO mice were
significantly lower than in the old WT mice (P < 0.05). In
CR+Mel group, FoxO1 mRNA expression did not alter compared with CR
group and Mel group of WT mice. Melatonin did not affect FoxO
mRNA
Fig. 1 Sirt1 mRNA expression in young and old mouse
hippocam-pus. a Sirt1 mRNA was compared between young and old
wild-type (WT) mice. b The effect of melatonin on Sirt1 mRNA
expression in the hippocampus of old WT mice on a normal diet and
caloric restric-tion (CR). Npy-knockout (NpyKO) and WT mice were
treated with melatonin (Mel) dosages at 10 mg/kg daily for
7 consecutive days and saline via a subcutaneous route. The
bars represent means ± standard errors of 4–5 mice. Graphs
represent percentages of the young (a) and old mice (b), &
denotes significant difference with P < 0.05 com-pared with the
WT young control group, * denotes significant dif-ference with P
< 0.05 compared with the WT old control group, ### denotes
significant difference with P < 0.001 compared with the WT
melatonin-treated group
Fig. 2 The effects of melatonin on FoxO1 (a) and FoxO3a (b)
expressions in the hippocampus of old mice on a normal diet and
caloric restriction (CR). Wild-type (WT) and Npy-knockout (NpyKO)
were treated with melatonin dosages at 10 mg/kg daily for
7 consecu-tive days and saline subcutaneously. The WT mice
were divided into ad libitum as control group, CR, Mel and
CR+Mel group. NpyKo mice were divided into ad libitum as
control group and Mel group. The bars represent means ± standard
errors of 4–5 mice. Graphs rep-resent the percentage of the old
mice. (*, ** denote significant dif-ference with P < 0.05 and P
< 0.01, respectively, compared with the WT old control group,
and ### denotes significant difference with P < 0.001 compared
with the WT melatonin-treated group)
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expression in NpyKO mice and had lower mRNA levels than WT mice
treated with melatonin (P < 0.001) (Fig. 2a).
We determined the FoxO3a mRNA expression in the animals treated
with CR or melatonin because FoxO3a is associated with brain
apoptotic pathways and age-related neurodegeneration. In WT mice,
both the CR group and the Mel group significantly increased FoxO3a
mRNA expression, compared with the old control group (P < 0.01)
(Fig. 2b). FoxO3a mRNA levels in all NpyKO groups were
significantly lower than that in the old WT group (P < 0.05).
Ten milligrams per kilogram of melatonin treatment did not
significantly alter FoxO3a expression in the CR animals, compared
with CR or Mel group of WT mice. FoxO3a mRNA levels in the NpyKO
Mel-treated group showed no significant difference, compared with
old NpyKO controls. NpyKO mice treated with melatonin had lower
FoxO3a mRNA than WT mice treated with mela-tonin (P < 0.001)
(Fig. 2b).
Melatonin, CR and NpyKO in Cell Cycle Arrest
Signals
The cell cycle arrest of p27 in FOXOs target genes was examined
for FOXOs activity. In WT mice, both the CR group and the Mel group
significantly increased p27 mRNA expression in old mice, compared
with old control mice (P < 0.05, P < 0.01, respectively)
(Fig. 3). In CR+Mel group, p27 mRNA expression did not alter
compared with CR group and Mel group of WT mice. p27 mRNA levels in
the NpyKO Mel-treated group showed no significant differ-ence,
compared with the old NpyKO controls (Fig. 3).
Melatonin, CR and NpyKO in Wisp1 Expression
Because melatonin and CR increased FoxO3a mRNA expression, which
was modulated by the Wnt/β-catenin pathway, the CR or 10
mg/kg melatonin significantly (P < 0.01) increased Wisp1 mRNA
expression in old WT mice. Wisp1 mRNA levels in the Mel-treated
NpyKO mice showed no significant difference, compared with the old
NpyKO controls. However, Wisp1 mRNA expression in both group the
NpyKO mice was significantly higher than that in the old WT
controls (P < 0.05) but was signifi-cantly lower than all of the
CR, Mel and CR+Mel group of WT mice (P < 0.001). NpyKO
Mel-treated mice had lower Wisp1 mRNA than WT mice treated with
melatonin (P < 0.001) (Fig. 4).
Discussion
The present study found that 10 mg/kg melatonin treat-ment
for 7 days attenuated the reduction in Sirt1 expres-sion in the
hippocampus of aged mice. Many studies have
demonstrated that melatonin increased SIRT1 expression in a
variety of models [21, 23]. The supportive effect of melatonin on
SIRT1 acts via the nicotinamideadenine dinu-cleotide (NAD) system.
Melatonin preserves NAD lev-els under oxidative stress. Oxidative
stress plays a crucial role in cognitive impairment in aging model
such as klotho mutant mice. Klotho mutant mice have an extremely
short lifespan and develop cognitive impairment [30]. Melatonin
decreases glutathione/oxidized glutathione (GSH/GSSG) ratio,
phospho-ERK expression, Nuclear factor (eryth-roid-derived 2)-like
2 (Nrf2) nuclear translocation, Nrf2 DNA-binding activity, and GCL
mRNA expression in the hippocampi of klotho mutant mice via
melatonin receptor type 2 (MT2 receptor) [31]. When melatonin was
incu-bated with PC12 cell line in an oxidative environment, the
oxidation was reduced, and melatonin donated an electron to NAD
radical, resulting in the recreation of NAD [32]. CR induced the
expression of the lifespan-regulating pro-tein SIRT1 in lower
organisms such as yeast and worms to higher mammalian animals [8,
33, 34]. Like melatonin, CR induced an increase in SIRT1 by
maintaining the nor-mal energetic nicotinamide adenine dinucleotide
(NAD) system [35–37] and cAMP responsive-element binding protein
(CREB) [14]. In yeast, CR decreased the levels of
Fig. 3 The effects of melatonin on p27 expression in the
hippocam-pus of old mice on a normal diet and caloric restriction
(CR). Wild-type (WT) and Npy-knockout (NpyKO) were treated with
melatonin dosages at 10 mg/kg daily for 7 consecutive days and
saline subcu-taneously. The WT mice were divided into ad
libitum as control group, CR, Mel and CR+Mel group. NpyKo mice were
divided into ad libitum as control group and Mel group. The
bars represent means ± standard errors of 4–5 mice. Graphs
represent percentages of old mice. *, ** denote significant
difference P < 0.05 and P < 0.01, respectively, compared with
the WT old control group, ## denotes significant difference with P
< 0.01 compared with the WT mela-tonin-treated group
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NADH or nicotinamide, leading to the activation of Sir2, a
homolog of mammalian SIRT1 [38, 39]. In addition, nico-tinamide
phosphoribosyltransferase (NAMPT) was recently found to be required
for CR-induced-SIRT1 [40–42].
Recent studies have found that CR or melatonin admin-istration
could restore the reduction in FOXO1 expression and activity in
senescence models and during aging [21, 27, 43]. The anti-aging
properties of CR are expected to restore FOXO1 levels during
senescent periods. CR upreg-ulated FoxO1 mRNA in skeletal muscle
and many tissues, including the brain [9]. FOXO1-overexpressing
transgenic mice increased GADD45α and glutamine synthase proteins
in skeletal muscle. Gadd45α, glutamine synthase, and cata-lase are
longevity-related genes [44].
Melatonin might promote FOXOs activities via SIRT1 deacetylation
action and also act by suppressing CREB-binding protein (CBP) and
p300. The acetylation of FOXOs via CBP and p300 decreased DNA
binding also decreased FOXOs activity [45, 46]. Melatonin
suppressed p300 histone acetyltransferase activity and
p300-mediated NF-κB acetylation in the human vascular smooth muscle
cell line CRL1999 [47]. In addition, melatonin was found to
increase the deacetylation of several substrates of SIRT1, such as
p53, peroxisome proliferator-activated receptor γ
coactivator 1α (PGC-1α), FOXO1, a disintegrin and
metal-loprotease domain 10 (ADAM10) and NF-κB in a murine
senescence model [21].
The present study showed that cell cycle arrest p27 mRNA was
induced by CR and melatonin, suggesting that both factors were
beneficial for protecting against neuro-degenerative diseases
during aging. The cyclin-dependent kinase inhibitor p27, which is a
critical determinant of cell cycle progression, is also an
important regulation target of mitogenic signals. In young animals,
the upregulation of these cell cycle arrest molecules has adverse
effects by reducing neurogenesis in the hippocampus [48, 49]. In
con-trast, p27 protects the brain during chronic stress to arrest
the G1 stage, leading to additional time for DNA repair and fewer
cycling cells left to engage in apoptosis [50]. In old animals, the
attempts of neurons to re-enter mitosis in response to external
stimuli can lead to abnormal cell cycle processes and neuronal
degeneration.
The action of FOXO3a could be regulated through the
WNT-β-catenin pathway [51]. Wisp1 is a target gene of the
WNT/β-catenin pathway [52, 53]. WISP1 induced the inhibition of
apoptosis, mediated AKT activation, and enhanced survivin
expression and the inhibition of pro-apoptotic BAX, accompanied by
blocked cytochrome c [53]. WISP1 phosphorylated FOXO3a and
prevented cas-pase degradation [52]. CR and melatonin increase
Wisp1 by enhancing the WNT pathway. CR and melatonin decrease
glycogen synthase kinase (GSK)-3β activity. Increased GSK-3β
activity during aging, results in inactiva-tion of β-catenin by a
phosphorylation process. CR induced increase in insulin-stimulated
GSK-3β (Ser9) phosphoryla-tion in rat muscles [54]. Melatonin
attenuated the increase in phosphorylated GSK-3β in primary
cultures of cerebellar granule neurons [55], in a SAMP8 mice model
[56], and in an Alzheimer’s model [57, 58].
NPY, which mediates physiological adaptation in energy
deficiency, is an essential link between CR and longevity in mice.
Several studies have suggested a key role for Npy in mediating the
effects of CR. The results of the present study showed that
melatonin could not alter Sirt1, FoxO1, FoxO3a, p27 or Wisp1
expressions in Npy-knockout hip-pocampus. NPY might directly
activate Sirt1 expression or might indirectly affect Sirt1
expression via melatonin. SIRT1 regulated food intake and body
weight by acting on NPY/AGRP and an anorexigenic
proopiomelanocortin (POMC) neurons [59]. Melatonin and NPY have
agonistic effects on each other. NPY is present in high
concentrations in the mammalian pineal gland, mainly localized in
the neu-ronal innervations. The rat pineal gland expresses the gene
coding for Y1- receptor NPY increased melatonin secre-tion due to
the induced Ca2+ increase in rat pinealocytes, and this effect was
inhibited by the Y1-receptor antago-nist BIBP3226 [60]. In
contrast, melatonin increases NPY
Fig. 4 The effects of melatonin on Wisp1 mRNA expression in the
hippocampus of old mice on a normal diet and caloric restriction
(CR). Wild-type (WT) and Npy-knockout (NpyKO) were treated with
melatonin dosages at 10 mg/kg daily for 7 consecutive days
and saline subcutaneously. The WT mice were divided into ad
libi-tum as control group, CR, Mel and CR+Mel group. NpyKo mice
were divided into ad libitum as control group and Mel group.
The bars represent means ± standard errors of 4–5 mice. Graphs
represent percentages of the old mice *, **, *** denote significant
difference with P < 0.05, P < 0.01, and P < 0.001,
respectively, compared with the WT old control group, and ###
denotes significant difference with P < 0.001 compared with the
WT melatonin-treated group
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levels. Prenatal exposure to melatonin increased NPY syn-thesis
in the hypothalamus [61]. Pinealectomy decreased NPY synthesis, and
exogenous melatonin restored NPY levels in the hypothalamic arcuate
nucleus of the rat [62]. Our data showed that melatonin induced
Sirt1 expression in the wild-type, but not in the Npy-knockout
mouse hip-pocampus, suggesting that Npy is essential for melatonin
to upregulate Sirt1 expression. In addition, it also requires
several downstream factors, i.e., FoxO1, FoxO3a, p27 and Wisp1
upregulation.
Conclusion
The present study compared two anti-aging treatments, melatonin
administration and caloric restriction, which are involved with the
SIRT1 and FOXOs pathways (Fig. 5). Both CR and melatonin
significantly upregulated Sirt1, FoxO1 and FoxO3a mRNAs in aged
mouse hippocam-pus. Both upregulated FOXOs target genes including
p27, which plays a role in controlling the cell cycle. Melatonin
could not produce additive actions on CR treatment. The results
suggested that melatonin administration and CR might share the same
regulatory pathways in controlling the SIRT1-FOXOs cascade for
lifespan extension. In addi-tion, our results also indicated that
melatonin regulated the SIRT1-FOXOs pathway required the presence
of NPY. Fur-ther studies need to be fully elucidated to clarify the
effects of melatonin- and CR-mediated life span extension,
par-ticularly the connections between SIRT1 and other longev-ity
factors.
Acknowledgements This study was supported in part by the
Thai-land Research Fund (TRF) (DPG5780001), a Mahidol University
Research Grant to PG and Goho Life Sciences International Funding
to AJ.
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Fig. 5 Summary of the possible mechanisms that explain the
ben-eficial effects of CR and melatonin on SIRT1 and the FOXOs
path-way that protect the aging brain. Both CR and 10 mg/kg
melatonin significantly upregulated Sirt1, FoxO1, and FoxO3a mRNA.
In addi-tion, CR and melatonin also upregulated FOXOs target genes,
includ-ing p27, which plays a role in cell cycle control. Although
melatonin
and CR upregulated FoxO3a expression, melatonin and CR might
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Comparing the Effects of Melatonin with Caloric
Restriction in the Hippocampus of Aging Mice:
Involvement of Sirtuin1 and the FOXOs
PathwayAbstract IntroductionExperimental ProcedureAnimals
and Experimental DesignRNA Isolation and Reverse
TranscriptionQuantitative Real-time PCR (qPCR)Statistical
Analysis
ResultsMelatonin, CR and NpyKO in Sirt1
ExpressionMelatonin, CR and NpyKO in FoxOs
ExpressionMelatonin, CR and NpyKO in Cell Cycle Arrest
SignalsMelatonin, CR and NpyKO in Wisp1 Expression
DiscussionConclusionAcknowledgements References
