RESEARCH ARTICLE

Aging and exercise affect the level of protein acetylationand SIRT1 activity in cerebellum of male rats

Orsolya Marton • Erika Koltai • Csaba Nyakas •

Tibor Bakonyi • Tania Zenteno-Savin •

Shuzo Kumagai • Sataro Goto • Zsolt Radak

Received: 4 February 2010 / Accepted: 27 April 2010 / Published online: 14 May 2010

� Springer Science+Business Media B.V. 2010

Abstract Aging is associated with a gradual decline

in cognitive and motor functions, the result of complex

biochemical processes including pre- and posttransla-

tional modifications of proteins. Sirtuins are NAD?

dependent protein deacetylases. These enzymes mod-

ulate the aging process by lysine deacetylation, which

alters the activity and stability of proteins. Exercise

can increase mean life-span and improve quality of

life. Data from our laboratories revealed that 4 weeks

of treadmill running improves performance in the

Morris Maze test for young (4 months, old) but not old

(30 months, old) male rats, and the exercise could not

prevent the age-associated loss in muscle strength

assessed by a gripping test. The positive correlation

between protein acetylation and the gripping test

suggests that the age-dependent decrease in relative

activity of SIRT1 in the cerebellum impairs motor

function. Similarly to the acetylation level of total

proteins, the acetylation of a9 -tubulin is also increased

with aging, while the effect of exercise training was

not found to be significant. Moreover, the protein

content of nicotinamide phosphoribosyltransferase,

one of the key enzymes of NAD biosynthesis,

decreased in the young exercise group. These data

suggest that aging results in decreased specific activity

of SIRT1 in cerebellum, which could lead to increased

acetylation of protein residues, including a9-tubulin,

that interfere with motor function.

Keywords Exercise � SIRT1 �Acetylation/deacetylation � Aging �Protein stability

Introduction

Aging is an unavoidable process, which takes place in

all species in an organ dependent manner. A number

of theories have been proposed to explain the nature of

the aging process (Goto 2009; Harman 1998; Ljubun-

cic and Reznick 2009), but the free radical theory, as

proposed by Harman (1969), is the most accepted. It is

clear that reactive oxygen species (ROS) are impor-

tant regulators of physiological signaling pathways,

and the cellular redox state shifts towards an oxidized

milieu with aging (Droge 2002; Droge and Schipper

2007). This shift affects the NAD/NADH ratio, and

the availability of NAD as an energy substrate could

O. Marton � E. Koltai � C. Nyakas � T. Bakonyi �S. Goto � Z. Radak (&)

Faculty of Physical Education and Sport Science,

Research Institute of Sport Science, Semmelweis

University, Alkotas u. 44, TF, Budapest, Hungary

e-mail: radak@mail.hupe.hu

T. Zenteno-Savin

CIBNOR—Planeacion Ambiental y Conservacion,

La Paz, Mexico

S. Kumagai

Graduate School of Human-Environment Studies,

Kyushu University, Kasuga, Fukuoka 816-8580, Japan

123

Biogerontology (2010) 11:679–686

DOI 10.1007/s10522-010-9279-2

significantly modulate the activity of NAD dependent

enzymes, such as poly (ADP-ribose) polymerase and

sirtuins. Indeed, our recent study showed that nico-

tinamide phosphoribosyltransferase (NAMPT), one of

the key enzymes synthesizing NAD, decreased paral-

lel with NAD levels in skeletal muscle of aged rats

(Koltai et al. 2009). Moreover, the specific activity of

SIRT1 also declined with aging. Sirtuins have been

suggested to be powerful regulators of post-transla-

tional protein structure by the deacetylation lysin

residues, which generally curb the activity of proteins.

However, a number of studies suggest that deacety-

lation can also induce the activity of proteins (Csiszar

et al. 2009a, b; Guarente 2007; Iwanaga et al. 1991;

Libert et al. 2008; Nakagawa et al. 2009). Deacety-

lation of histone residues has been suggested to result

in compact chromatine and the inhibition of tran-

scription (Feige and Auwerx 2008; Jung-Hynes and

Ahmad 2009), while some data suggest that deacet-

ylation of PGC1-alpha induces mitochondrial biogen-

esis (Canto and Auwerx 2009). Moreover, p53,

PPAR-gamma, tubulin, and NF-jB, have been shown

to be the subject of sirtuin dependent deacetylation.

Hence, it is not surprising that sirtuins could regulate

the aging process by the modulation of tumor

development, alteration of metabolism, inflammation

etc (Kaeberlein 2008; Kim and Um 2008; Knutson

and Leeuwenburgh 2008).

Post-mitotic tissues, such as skeletal muscle and

brain, are more sensitive to the aging process due to a

limited capacity for cellular renewal. The aging

process of different brain regions has been suggested

to be different, and the cerebellum might be the most

resistant to aging (Boukhtouche et al. 2006; Rodrigo

et al. 2005; Rossi et al. 2006). One of the reasons

behind this could be the special function of the

cerebellum, which plays a crucial role in motor

control as it integrates information that relates to

coordination from different brain regions and sensory

systems. Moreover, in contrast to other brain regions,

the cerebellum does not generate self-sustaining

patterns, and thus allows rapid processing of feed

forward signaling (Ito 2006; Szentagothai 1971).

Regular physical exercise is known to alter the

resistance to oxidative stress, and to modulate the

level of antioxidant enzymes and oxidative damage in

the brain, which could alter the rate of aging and

maintain brain function (Radak et al. 2001a, b, 2008).

Besides the effect of exercise on ROS levels, exercise

could interfere with the level and activity of sirtuins

(Csiszar et al. 2009a, b; Danz et al. 2009), which

might be a potential route to modulate the aging

process. The present study was carried out to test the

hypothesis that exercise training prevents the age-

associated decrease in the level and activity of SIRT1

in the cerebellum.

Methods

Twelve, 6 months old and 12 30 months old male

Wistar rats were selected for the study and divided

into young control (YC), young exercised (YE), old

control (OC) and old exercised (OE) groups. The

study was carried out according to the requirements

of The Helsinki Declaration and approved by the

local ethics committee.

The exercise program included moderate running

on a rodent treadmill for 4 weeks, four times a week,

with a gradually increasing duration starting from

20 min in the 1st week to 40 min in the last week.

The running intensity was set at 60% of the VO2max

(ranged between 10 and 25 m/min).

The animals were sacrificed 2 days after the last

exercise session to avoid the metabolic effects of the

final exercise session. The cerebellum was quickly

excised, weighted, frozen in liquid nitrogen and

stored at -80�C degree.

Morris maze and gripping tests

Brain function was evaluated by the Morris Maze Test

on 5 consecutive days (four trials per day). A platform

of 6 cm in diameter was placed in the centre of the

northwest quadrant of a circular pool of 60 cm in

height and 100 cm in diameter and 1 cm below the

surface of the water. The water was 22–23�C through-

out training and testing. During a given training trial,

each rat was introduced into the pool at one of four

possible starting points (north, south, west, and east)

and allowed a period of 60 s to find the platform. The

order of starting points varied in a pseudo-random

manner for each rat every day. Twenty-four hours after

the final training trial, a probe test was conducted in

which each rat was allowed to swim for 60 s in the pool

with the platform removed.

680 Biogerontology (2010) 11:679–686

123

The forelimb strength of the animals was assessed

by using a gripping test on the pole as described by

Yang et al. (2004).

Western blots

Ten to 50 lg of protein were electrophoresed on 8–

12% v/v polyacrylamide SDS-PAGE gels. Proteins

were electrotransferred onto PVDF membranes. The

membranes were subsequently blocked and after

blocking, PVDF membranes were incubated at room

temperature with antibodies (1:500 #ab53517 Abcam

SIRT1, 1:500 #ab37299 Abcam NAMPT (PBEF),

1:1,000 #ab193 Abcam Acetylated protein, 1:500

#MAB1665 Chemicon VEGF, 1:200 #sc-7159 Santa-

Cruz Cytocrome C, 1:15,000 #T6199 Sigma a9-Tubu-

lin, and 1:2,000 #ab2072 Abcam APP). After

incubation with primary antibodies, membranes were

washed in TBS-Tween-20 and incubated with HRP-

conjugated secondary antibodies. After incubation

with the secondary antibody, membranes were repeat-

edly washed. Membranes were incubated with an ECL

plus reagent (RPN 2132, Amersham) and protein

bands were visualized on X-ray films. The bands were

quantified by ImageJ software, and normalized to

tubulin, which served as an internal control.

Assessment of SIRT1 activity

To measure SIRT1 deacetylase activity, a Cyclex

SIRT1/Sir2 Deacetylase Fluorometric Assay Kit

(Cyclex, CY-1151) was used according to the estab-

lished protocol, including the separation of nuclear

extract. In brief, 10 ll of cytosolic extracts of

cerebellum were mixed with a reaction mixture

(40 ll) containing 50 mM Tris-HCl pH 8.8, 4 mM

MgCl2, 0.5 mM DTT, 0.25 mAU/ml lysyl endpepti-

dase, 1 lM trichostatin A, 20 lM fluoro-substrate

peptide, and 200 lM NAD in microplate. The

samples were mixed well and incubated for 10 min

at RT and the fluorescence intensity (ex. 355 nm, em.

460 nm) was read for 2 h every 10 min and normal-

ized by the protein content.

Statistical analyses

Statistical significance was assessed by one-way

ANOVA, followed by Tukey’s post hoc test. The

significance level was set at P \ 0.05.

Results

The brain performance of the animals, as assessed by

the Morris Maze Test, revealed that the given

exercise load improved the performance of the young

group, but did not effect that of the older animals

(Fig. 1). Exercise, in general, has beneficial outcomes

for brain function, but the exercise load in the present

study may have been too mild to overcome the age-

associated decline in brain performance.

The forelimb strength of the animals, as appraised

by the gripping test, was revealed to show age-

associated decreases. Exercise did not significantly

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

1. day 2. day 3. day 4. day 5. day

Tim

e (s

ec)

Days

Morris-maze test

**

YC

OC

YE

OE

Fig. 1 The figure shows the results of the Morris Maze Test

on young control (YC), old control (OC), young exercise

trained (YE), and old exercise trained (OE) animals. The

animals had to find the platform, which was underwater, and

faster localization of the finding was considered to be a better

result (shorter time in seconds). Values are means ± SE for six

animals per group. * DP \ 0.05, YC vs. YE

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

YC OC YE OE

Tim

e (s

ec)

Gripping test

#

Fig. 2 The relative strength of the animals was appraised by a

gripping test, which decreased significantly as a result of aging.

Exercise training did not affect this performance. Values are

means ± SE for six animals per group. # DP \ 0.05 YC vs. OC

Biogerontology (2010) 11:679–686 681

123

alter gripping time (Fig. 2). The mass of the

cerebellum tended to increase as a result of exercise

training in young animals (YC: 0.52 ± 0.07 g, OC:

0.68 ± 0.06 g, YE: 0.61 ± 0.05 g, OE: 0.62 ±

0.04 g). However, the increase did not reach a level

of statistical significance.

One of the interesting findings of this study was

that the acetylation level of lysin residues increased

with aging in both control and exercise trained groups

(Fig. 3). The relative activity of SIRT1, one of the

key enzymes in protein deacetylation, decreased with

aging (Fig. 4). The acetylation level of a9 -tubulin was

increased with aging and this was true also for the

older exercised group (Fig. 5). This finding is in

consort with the decreased activity of SIRT1. The

correlation between the acetylation of tubulin and

SIRT1 activity was moderately high (r = -0.61,

P = 0.029). The levels of SIRT1 were unaltered by

aging and/or exercise (Fig. 6).

The possibility that protein acetylation could have

an impact on physiological function, especially on the

relationship between the gripping test results (marker

of motor function) and the protein acetylation levels

in the cerebellum, was also shown to be moderately

high (r = 0.67, P = 0.031). Though, coincidental

correlation cannot be completely ruled out, we have

also observed that an aging-associated increase in

acetylation of lysine residues had negative correlation

with VO2max in human subjects (Bori et al. unpub-

lished observation).

Although it has been shown that exercise increases

NAMPT levels in skeletal muscle (Koltai et al. 2009),

the current data suggest that exercise decreases

NAMPT levels in the cerebellum of young animals

(Fig. 7). The level of amyloid precursor protein (APP)

was unchanged by aging or exercise, despite the fact

that acetylation of the cleaving enzyme is known to

significantly affect the level of APP (Jonas et al.

2008) (Fig. 8).

The level of cytochrome C was evaluated to

appraise the level of apoptosis, but significant differ-

ences were not found (results not shown).

Discussion

In the present investigation the effects of aging and

mild exercise on SIRT1 activity and acetylation of

proteins in cerebellum were tested. SIRT1 content did

not change with aging. However, the specific activity

decreased significantly, although the applied mild

exercise training was without significant effects on

the activity. Since SIRT1 is a powerful lysin deacet-

ylator, the lysin acetylation levels in cytoplasmic

proteins and the acetylation level of a9 -tubulin, which

is a crucial protein in the structure of microtubules,

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

YC OC YE OE

Rel

ativ

e de

nsit

y

Acetylated protein

α-tubulin 50 kD

YC OC YE OE

64 kD

49 kD

37 kD

82 kD

115 kD 180 kD#

§(A) (B)

Fig. 3 Changes in levels of

acetylated proteins in the

cytoplasmic compartments

of the cerebellum were

assessed by Western blot.

a Shows the densitometric

data and b shows

representative examples for

acetylation from each age

and exercise group. Values

are means ± SE for six

animals per group.# DP \ 0.05, YC vs. OC,§ DP \ 0.05 OC vs. OE

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

400.0

YC OC YE OE

Rel

ativ

e ac

tivi

ty (

avti

vity

/con

tent

) Relative activity of SIRT1

#

Fig. 4 The relative activity of SIRT1 was calculated from the

activity and the protein content of SIRT1. Aging resulted in

decreased activity of SIRT1. Values are means ± SE for six

animals per group. # D YC vs. OC

682 Biogerontology (2010) 11:679–686

123

were also measured. Indeed, protein acetylation has

been suggested to play an important role in the aging

of the nervous system (Mattson 2003). However,

the availability of research data is limited. The

acetylation level of cytoplasmic proteins increased

significantly and a positive correlation was found

between the relative force, and the acetylation levels

of protein (r = 0.71, P = 0.035). Since cerebellum

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

YC OC YE OE

Rel

ativ

e de

nsit

y

Acetylated tubulin

YC OC YE OE

Acetylatedtubulin

α-tubulin

50 kD

50 kD

#

*

§

(A)

(B)

Fig. 5 The acetylation of alpha tubulin increased as a result of aging in the cerebellum of both control and exercise trained rats.

Values are means ± SE for six animals per group. * DP \ 0.05, YC vs. YE, # DP \ 0.05 YC vs. OC, § DP \ 0.05 OC vs. OE

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

YC OC YE OE

Rel

ativ

e de

nsit

y

SIRT1

YC OC YE OE

SIRT1

α-tubulin

120 kD

50 kD

(A)

(B)

Fig. 6 The protein level of

SIRT1 was not altered by

aging or exercise training,

in the cerebellum of rats.

Values are means ± SE for

six animals per group

Biogerontology (2010) 11:679–686 683

123

plays an important role in motor activity, this

relationship indicates that the acetylation/deacetyla-

tion levels of proteins could have a significant impact

on physiological function and observation deserves

further investigation.

The present data show that aging increases the

acetylation level of a9-tubulin in cerebellum, but is

not affected by exercise training. This change in

the acetylation level of proteins is not a general

age-associated process, but is protein-dependent.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

YC OC YE OE

Rel

ativ

e de

nsit

y

NAMPT

*

NAMPT

α-tubulin

55 kD

50 kD

YC OC YE OE

(A)

(B)

Fig. 7 The content of

NAMPT decreased

significantly in the

cerebellum of young

exercised animals. A similar

tendency was observed also,

in aged groups. Values are

means ± SE for six animals

per group. * DP \ 0.05,

YC vs. YE

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

YC OC YE OE

Rel

ativ

e de

nsit

y

APP

α-tubulin

YC OC YE OE

APP 110 kD

50 kD

(A)

(B)

Fig. 8 The content of

amyloid precursor protein

(APP) did not change with

aging or exercise training.

Values are means ± SE for

six animals per group

684 Biogerontology (2010) 11:679–686

123

Increased acetylation of a9-tubulin is believed to be the

disruption of microtube-based transport, which is a

precausative step to synaptic degeneration and has

been associated with Alzheimer disease (Butler et al.

2007). The present data suggest that the age-associated

increase in a9-tubulin acetylation is associated with the

decreased activity of SIRT1. This is a novel observa-

tion and extends the role of sirtuins in the tubulin

acetylation in the brain, since it has been shown that

SIRT2 also deacetylates a9-tubulin (Li et al. 2007).

Acetylation of protein residues has been suggested to

affect the turnover rate of proteins (Sadoul et al.

2008), which sometimes stabilizes a target protein or,

in contrast, enhances its degradation. Tubacin, an

agent which prevents deacetylation of a9-tubulin,

causes an accumulation of polyubiquitinated proteins

and apoptosis thus mimicking the effects of protea-

some inhibitors (Simms-Waldrip et al. 2008). It has

also been shown that SIRT1-induced deacetylation

of Smad protein increases ubiquitination-associated

degradation by a proteasome complex (Kume et al.

2007).

Interestingly, beta-site APP cleaving enzyme 1

acetylation is obligatory to nascent the enzyme,

which then regulates APP levels. However, despite

the changes in protein acetylation levels, the concen-

tration of APP in the present study was not effected

by aging and/or exercise, which is in agreement with

earlier observations (Adlard et al. 2005; Jeong et al.

1997). This finding would further suggest the impor-

tance of the protein specificity of acetylation/

deacetylation.

NAMPT is a crucial enzyme in NAD biogenesis,

which serves as a substrate for sirtuins. Exercise

appears to increase the NAMPT levels in skeletal

muscle (Koltai et al. 2009; Costford et al. 2009) and

decreases them in plasma (Haus et al. 2009). The

present data show that exercise training in young, but

not in old, rats decreases the NAMPT protein levels

in the cerebellum. The decrease in NAMPT levels is

not associated with decreases in the activity of

SIRT1, which might suggest that under the given

experimental conditions, NAD biogenesis is not the

limiting factor of SIRT1 activity in the cerebellum.

The data support and confirm earlier findings that

exercise increases spatial learning, as appraised by

the Morris Maze Test (Albeck et al. 2006; Gomez-

Pinilla et al. 1998; Griesbach et al. 2009; Molteni

et al. 2004). However, no relationship between the

measured biochemical parameters and spatial learn-

ing were found, which is not surprising, since the

cerebellum is not involved in learning and memory.

Moreover, the exercise load might not be powerful

enough to prevent the age-associated decline in brain

function of the aged group, which was 30 months old,

the age considered to be close to maximum in our

animal facilities.

In conclusion, the results indicate that aging and/or

exercise training effect the acetylation levels of

proteins, including alpha-tubulin, which could alter

selected physiological functions. SIRT1 specific

activity decreases with aging and this could impact

the age-associated protein acetylation levels in the

cerebellum.

Acknowledgments We acknowledge the fruitful consultation

with Professor Albert W Taylor. The present work was

supported by Hungarian grants: OTKA K75702, TeT JAP13/

02, ETT awarded to Z. Radak.

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