Nitric oxide and CR life extension effects

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Hi All,

There are just three comments to make after reading the for a new CR paper's

text.

a. Alternate day feeding equals CR:

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).

b. In agreement with Rae sentiments and reviewing the evidence of the

greater metabolism over a lifetime with CR:

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).

21. E. J. Masoro, B. P. Yu, H. A. Bertrand, Proc. Natl. Acad. Sci. U.S.A. 79,

4239

(1982).

c. The longevity implications of the study in showing the mechanism for:

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).

24. E. Dere et al., Genes Brain Behav. 1, 204 (2002).

25. S. Cook et al., Swiss Med. Wkly. 133, 360 (2003).

http://tinyurl.com/dkh66

Common gas could be elixir of life

Nitric oxide governs calorie reduction mechanism (ANSA)

- Rome, October 13 -

A common gas could be the elixir of life, Italian scientists hope. Working with

mice, the team found that molecules of the gas, whose scientific name is nitric

oxide (NO), boosted cell energy. Combined with a reduction in calorie intake,

this

slowed down the ageing processes in a group of test mice. Another group of mice

from

which the gas's gene had been removed showed no such beneficial effects from a

reduction in calories consumed. The scientists in the new study, which has been

published in the prestigious journal Science, concluded that NO is a key

mediator of

human ageing. It has a direct impact on the calorie restriction mechanism that,

on a

molecular level, plays a crucial role in keeping cells alive longer.

The pdf-available article is:

Calorie Restriction Promotes Mitochondrial Biogenesis by Inducing the Expression

of

eNOS

Enzo Nisoli, Cristina Tonello, lisa Cardile, Valeria Cozzi, Renata Bracale,

Tedesco, Sestina Falcone, Alessandra Valerio, Orazio Cantoni, Emilio

Clementi,

Salvador Moncada, and Michele O. Carruba

Science 310, (5746): 314-317, 14 October 2005

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.

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– coactivator 1 (PGC-1), 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).

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).

Table S1 ATP levels (nmol/mg tissue) in tissues from either AL or CR wild-type

(wt)

and eNOS -/- mice.

===============

wt eNOS^-/-

AL CR AL CR

==============

WAT 0.018±0.002 0.025±0.001*** 0.0152±0.001*** 0.0147±0.002

BAT 0.325±0.085 0.370±0.096* 0.2150±0.075*** 0.2240±0.063

Liver 0.769±0.068 0.795±0.023 0.4850±0.069*** 0.4915±0.065

==============

Steady-state levels of ATP were measured as described.

WAT, white adipose tissue

BAT, brown adipose tissue.

Each experiment was performed twice; n = 4 animals per group.

***, P < 0.01 and *, P < 0.05 vs. AL wt mice.

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, 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.

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).

Al Pater, PhD; email: old542000@...

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