CR Promotes Mitochondrial Biogenesis by Inducing Expression of Nitric oxide

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http://www.sciencemag.org/cgi/content/full/310/5746/314

Calorie Restriction Promotes Mitochondrial Biogenesis by Inducing the

Expression of eNOS

Enzo Nisoli,1,2* Cristina Tonello,1 lisa Cardile,1 Valeria Cozzi,1

Renata Bracale,1 Tedesco,1 Sestina Falcone,1,3 Alessandra

Valerio,1 Orazio Cantoni,4 Emilio Clementi,1,3,5 Salvador Moncada,6

Michele O. Carruba1,2

Calorie restriction extends life span in organisms ranging from yeast

to mammals. Here, we report that calorie restriction for either 3 or

12 months induced endothelial nitric oxide synthase (eNOS) expression

and 3',5'-cyclic guanosine monophosphate formation in various tissues

of male mice. This was accompanied by mitochondrial biogenesis, with

increased oxygen consumption and adenosine triphosphate production,

and an enhanced expression of sirtuin 1. These effects were strongly

attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a

fundamental role in the processes induced by calorie restriction and

may be involved in the extension of life span in mammals.

1 Integrated Laboratories Network, Department of Preclinical Sciences,

Luigi Sacco Hospital, Milan University, 20157 Milan, Italy.

2 Istituto Auxologico Italiano, 20149 Milan, Italy.

3 Eugenio Medea Scientific Institute, 23842 Bosisio Parini, Italy.

4 Istituto di Farmacologia e Farmacognosia, University of Urbino

" Carlo Bo, " 61029 Urbino, Italy.

5 Stem Cell Research Institute, Dipartimento di Biotecnologie,

Ospedale San Raffaele, 20132 Milan, Italy.

6 Wolfson Institute for Biomedical Research, University College

London, London WC1E 6BT, UK.

* To whom correspondence should be addressed. E-mail: enzo.nisoli@...

Calorie restriction (CR) extends life span in numerous organisms from

yeast (1) to rodents and possibly primates (2). In mammals, CR delays

the onset of age-associated diseases including cancer,

atherosclerosis, and diabetes (3). A decrease in oxidative damage has

been proposed as a mechanism (4); however, a lack of correlation

between reactive oxygen species (ROS) production and life span was

recently reported in Drosophila (5). Furthermore, increasing evidence

suggests that SIRT1, the mammalian ortholog of the SIR2 gene that

mediates the life-extending effect of CR in yeast (1, 6), is a key

regulator of cell defenses and survival in mammals in response to

stress (7).

Eight-week-old male wild-type mice were fed either ad libitum (AL) or

with a CR diet (food provided on alternate days) for 3 or 12 months

(8). Mice maintained on a CR feeding schedule consume 30 to 40% fewer

calories over time compared with animals fed AL, have a lower body

weight (fig. S1), and are known to have an extended life span (9). At

3 months of treatment, the amounts of mitochondrial DNA (mtDNA, a

marker of mitochondrial content), the expression of peroxisome

proliferator-activated receptor–{gamma} coactivator 1{alpha}

(PGC-1{alpha}), nuclear respiratory factor–1 (NRF-1), and

mitochondrial transcription factor A (Tfam) [master regulators of

mitochondrial biogenesis (10)], and expression of cytochrome c oxidase

(COX-IV) and cytochrome c (Cyt c) (two mitochondrial proteins involved

in cell respiration) were higher in white adipose tissue (WAT) and

many other tissues of CR mice when compared with AL mice (Figs. 1A and

2A). This was consistent with increased mitochondrial biogenesis and

mitochondrial gene expression (11–13).

Fig. 1. CR induces mitochondrial biogenesis in WAT of wild-type (wt)

but not eNOS–/– mice through eNOS expression and cGMP formation. (A

and D) PGC-1{alpha}, NRF-1, Tfam, Mfn1, and Mfn2 mRNA were analyzed by

means of quantitative reverse transcription polymerase chain reaction

(RT-PCR); COX IV, Cyt c, and eNOS proteins were detected by immunoblot

analysis. (Insets) WAT mtDNA (gel shows a representative experiment

with two mice per group). The relative values were obtained by

densitometric analysis, with those measured in the AL mice taken as

1.0. (B and E) O2 consumption and (C and F) cGMP concentrations in

WAT. Each experiment (n = 10) was repeated at least three times.

Triple asterisks indicate P < 0.001, and single asterisk, P < 0.05,

compared with AL-fed mice. Error bars indicate SEM. [View Larger

Version of this Image (39K GIF file)]

Fig. 2. CR induces mitochondrial biogenesis in different tissues of wt

mice through eNOS expression and cGMP formation. (Large bar graphs)

PGC-1{alpha}, NRF-1, Tfam, Mfn1, and Mfn2 mRNA were analyzed by means

of quantitative RT-PCR with gene-specific oligonucleotide probes. COX

IV, Cyt c, and eNOS proteins were detected by immunoblot analysis.

(Top images) MtDNA (gel shows one representative experiment from one

mouse per group). The relative values were obtained by densitometric

analysis, with those measured in the AL mice taken as 1.0. (Top small

bar graphs) O2 consumption and (bottom small bar graphs) cGMP

concentrations. Each experiment (n = 10 animals) was repeated at least

three times. Triple asterisks, P < 0.001, and single asterisk, P <

0.05, compared with AL-fed mice. Error bars indicate SEM. [View Larger

Version of this Image (45K GIF file)]

To confirm that CR increases mitochondrial function, we measured

oxygen consumption and expression of mitofusin (Mfn) 1 and 2 [the

mitochondrial transmembrane guanosine triphosphatases crucial to the

mitochondrial fusion process and metabolism (14, 15)]. These

parameters were higher in several tissues, particularly in WAT, of CR

than in AL animals (Figs. 1, A and B, and 2). This suggests that CR

induces mitochondrial biogenesis with increased respiration and

expression of genes crucial for the dynamic fusion processes required

for oxidative function. We then investigated whether the increase in

respiration was associated with an increase in adenosine triphosphate

(ATP) synthesis and found that CR increased ATP concentrations in WAT

(0.025 ± 0.001 nmol/mg tissue in CR mice compared with 0.018 ± 0.002

nmol/mg tissue in AL mice, P < 0.001, n = 4 animals) and in other

tissues (table S1). Similar results were obtained in mice treated for

12 months. Thus, the molecular changes induced by CR occur early and

are long-lasting, consistent with the early onset and persistent

effect of CR on life span (9).

Nitric oxide (NO) generated by eNOS increases mitochondrial biogenesis

and enhances respiration and ATP content in various mammalian cells by

acting through its second messenger, 3',5'-cyclic guanosine

monophosphate (cGMP) (11, 16). We investigated whether eNOS and cGMP

play a role in the mitochondrial biogenesis induced by CR. The

expression of eNOS, unlike neuronal and inducible NOS, was higher in

CR than in AL mice (Figs. 1A and 2) and was accompanied by higher

concentrations of cGMP (Figs. 1C and 2) in WAT and in several other

tissues. The increased serum concentrations of nitrite and nitrate (an

index of NO production) and plasma cGMP in obese subjects exposed to

CR in controlled weight loss trials (17, 18) are consistent with our

findings.

To verify the role of eNOS in the mitochondrial biogenesis induced by

CR, we fed 8-week-old male eNOS null-mutant (eNOS–/–) mice either an

AL or a CR diet for 3 months (Fig. 1, D to F, and fig. S2, A to C). In

particular, mtDNA content and PGC-1{alpha}, NRF-1, Tfam, Mfn1, and

Mfn2 mRNA amounts, although different from those in wild-type animals,

were not significantly greater in CR eNOS–/– mice compared to in AL

eNOS–/– animals. Moreover, COX IV and Cyt c expression did not

increase significantly in CR animals except in WAT and brain, where

these parameters increased to a much lesser extent than those in

wild-type animals (Fig. 1D and fig. S2A). Thus, CR was unable to

induce significant mitochondrial biogenesis in a number of tissues of

eNOS–/– mice, including WAT. To confirm this, we measured oxygen

consumption (Fig. 1E and fig. S2B) and cGMP (Fig. 1F and fig. S2C) and

ATP concentrations (table S1) in WAT and other tissues (table S1) of

both CR and AL eNOS–/– animals. These parameters also did not increase

significantly as a result of CR in knock-out compared to in wild-type

mice. AL eNOS–/– mice displayed greater feed efficiency (body weight

gain per food intake) than their wild-type counterparts (11),

suggesting that both energy expenditure and oxidative metabolism are

partly NO-dependent. The CR wild-type mice showed lower feed

efficiency values than AL wild-type animals (0.295 ± 0.023 compared

with 0.488 ± 0.028, respectively; P < 0.001, n = 10 animals), whereas

there was no difference between CR eNOS–/– mice and AL eNOS–/– animals

(0.67 ± 0.025 and 0.654 ± 0.019, respectively; n = 10 animals). Thus,

the CR-induced increase in oxidative metabolism appears to be blunted

in the absence of eNOS expression in mammals.

Given the role of yeast SIR2 protein in life span extension by CR (1,

6), we studied the expression of SIRT1 and found it to be higher in

many tissues (fig. S3) of CR wild-type animals than of AL wild-type

mice, including WAT (Fig. 3A) (19), where SIRT1 triggers lipolysis and

loss of fat (20). SIRT1 mRNA and protein were ~threefold higher in

cultured white adipocytes exposed either to NO donors, such as

(Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2

diolate (DETA-NO) and S-nitrosoacetyl penicillamine (SNAP), or to a

cGMP analog (8 Br-cGMP) than in untreated cells (Fig. 3B) and ~80%

lower in WAT of eNOS–/– mice when compared with wild-type animals

(Fig. 3, C and D). Thus, the expression of SIRT1 in WAT during CR

might be partly mediated by NO acting via cGMP.

Fig. 3. SIRT1 expression is regulated by NO in WAT and white

adipocytes. (A) SIRT1 protein levels in WAT of either AL or CR wt and

eNOS–/– mice. A representative experiment is shown, with means ± SEM

of densitometric measurements performed in 10 animals per group. The

histogram values were obtained by densitometric analysis, with values

measured in the AL mice taken as 1.0. ( SIRT1 protein concentrations

in white adipocytes cultured for 3 days with or without SNAP (100 µM),

DETA-NO (50 µM), and 8 Br-cGMP (3 mM). A representative experiment is

shown, with means ± SEM of densitometric measurements performed in

five separate experiments. The protein concentrations were obtained by

densitometric analysis, with values measured in the untreated cells

© taken as 1.0. (C and D) SIRT1 protein and mRNA expression,

respectively, in WAT of male eNOS–/– mice compared with wt mice. Each

experiment (n = 10 animals) was repeated at least three times. The

relative values of mRNA were obtained by densitometric analysis, with

values measured in wt mice taken as 1.0. Triple asterisks, P < 0.001

and single asterisk, P < 0.05 compared with AL-fed wild-type mice or

untreated cells. [View Larger Version of this Image (35K GIF file)]

To investigate whether the CR-induced SIRT1 expression was dependent

on eNOS-derived NO, we performed immunoblot analysis in WAT of eNOS–/–

mice fed either an AL or a CR diet. In eNOS–/– mice fed a CR diet,

SIRT1 expression was also increased (~30%) in WAT compared with that

of eNOS–/– mice fed an AL diet (Fig. 3A), although the change was much

smaller than that in wild-type animals (~120%, P < 0.001). Similar

results were obtained in the other tissues tested (fig. S3).

Thus, CR induces an increase in eNOS expression, which in turn is

involved in both mitochondrial biogenesis and SIRT1 expression in a

variety of tissues. The enhanced expression of SIRT1 by CR is

consistent with a potential increase in life span. This transcription

factor may be an evolutionarily ancient biological stress response

that slows aging, promoting the mobilization of fat into the blood

from WAT stores (20), the down-regulation of adipogenesis (20), and

the long-term survival of irreplaceable cells (7, 19). The increase in

mitochondrial activity, i.e., in oxidative metabolism, that we see in

CR animals is intriguing in view of the widely accepted hypothesis

that CR increases longevity by slowing metabolism and reducing

mitochondrial ROS and accompanying cellular damage (4). In fact,

metabolic rate normalized to body weight does not decline in CR mice,

and the lifetime metabolic output of these animals is therefore larger

than that of their AL cohorts (21). Respiration actually increases

during CR in yeast (22) and the nematode worm Caenorhabditis elegans

(23). The effects of CR on life span may be independent of excessive

ROS production.

The effects of CR in mammals are complex, affecting many organs and

physiological pathways. Nevertheless, the significantly reduced

effects observed in eNOS–/– animals point to a role for NO in the

response to CR. eNOS–/– mice are characterized by a reduced life span

(24) due to age-related diseases (25). One possibility is that in

wild-type CR animals NO, acting via mitochondrial biogenesis and

expression of SIRT1, increases ß-oxidation and lipolysis. This would

result in a reduction in the accumulation of fat, which is known to

have an impact on life span (26, 27).

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for technical assistance. This work was supported by grants from the

Ministero dell'Istruzione, dell'Università e della Ricerca

cofinanziamento 2003, the Italian Ministry of Health, and the Italian

Association of Cancer Research (AIRC).

Supporting Online Material