CR reduces exercise oxidation

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Hi All,

The title of the below pdf-available paper cited for its Medline abstract and

pdf

excerpts below seems to present the message of the paper. Quantifying the data

gives the degree to which CR seems to reduce the oxidation caused by exercise,

which

in this case is rat swimming. In the methods, the authors even said that they

used

hair dryers to dry the rats after they swam.

Aydin C, Ince E, Koparan S, Cangul IT, Naziroglu M, Ak F.

Protective effects of long term dietary restriction on swimming exercise-induced

oxidative stress in the liver, heart and kidney of rat.

Cell Biochem Funct. 2005 Sep 5; [Epub ahead of print]

PMID: 16143963

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve & db=pubmed & dopt=Abstra\

ct & list_uids=16143963 & query_hl=3

.... Sixty male, Sprague-Dawley rats were assigned as either dietary restricted

on

every other week day (DR) or fed ad libitum (AL) groups, and each group was

further

subdivided into sedentary, endurance swimming exercise training (submaximal

exercise) and exhaustive swimming exercise (maximal exercise) groups. Animals in

the

submaximal exercise group swam 5 days/week for 8 weeks, while maximal exercise

was

performed as an acute bout of exercise.In parallel with the increase in the

intensity of the exercise, the degree of lipid peroxidation and protein

oxidation

were increased in both the DR and AL groups; however the rate of increase was

lower

in the DR group. Reduced glutathione (GSH), glutathione peroxidase (GSH-Px) and

glutathione reductase (GR) enzyme activities were lower in the DR group than in

the

AL group. In parallel with the increase in exercise intensity, GSH and GR enzyme

activities decreased, whereas an increase was observed in GSH-Px enzyme

activity. In

conclusion, the comparison between the DR and AL groups with the three swimming

exercise conditions shows that the DR group is greatly protected against

different

swimming exercise-induced oxidative stress compared with the AL group.

INTRODUCTION

Since the initial reports by McCay and Crowell,

which showed that underfed rats lived longer than rats

fed ad libitum, many studies have been performed

revealing that underfeeding without malnutrition

increases both the maximum and mean life spans of

laboratory rodents. Dietary restriction (DR) is the

only experimental manipulation that has been shown

to retard ageing, to reduce disease, health risks and the

incidence and progression of tumours. Confirma-tory

results have been obtained both in studies started

at weaning and in studies when caloric restriction

was initiated after the onset of adulthood.

During exercise, free radicals may be produced in

excess of the body’s natural defence. Strenuous

exercise increases the whole body and tissue oxygen

consumption up to 20 fold, which then elevates elec-tron

leakage from the mitochondrial transport system

and disturbs the intracellular pro-oxidant and antioxi-dant

homeostasis. This unfavourable condition is a

serious threat to the cellular antioxidant defence

system with diminished reserves of antioxidant

vitamins and glutathione. On the other hand in obese,

sedentary humans, it has been observed that a combi-nation

of a hypocaloric diet and exercise decreases the

risk of coronary heart disease more efficiently than the

hypocaloric diet or exercise alone.

A variety of antioxidants scavenges reactive

oxygen species (ROS) and prevent oxidative damage

to biological structures. Glutathione dependent

antioxidant systems play a fundamental role in the

cellular defence against reactive free radicals and

other oxidant species. The primary defence against

oxidative stress in the cell rests with antioxidants,

including glutathione reductase (GR), reduced

glutathione (GSH) and glutathione peroxidase

(GSH-Px). Glutathione is the most abundant non-protein

thiol found in virtually all mammalian cells

and has important roles in cellular antioxidant

defences. The most important of these functions is

to remove hydrogen peroxide and organic peroxides.

These toxic oxygen species may be detoxified via

reduction by GSH-Px,and GSHisconverted to oxi-dized

glutathione (GSSG) in the process. In turn,

oxidized GSH is reduced by glutathione reductase

(GR) in the presence of NADPH. Therefore, a

decrease in the level of GSH indicates an increased

production of free radicals.

DR modulates the effects of oxidative stress by

reducing the production of superoxide and hydroxyl

radicals and by inhibiting lipid peroxidation or

by enhancing antioxidative defences by increasing

the production of superoxide dismutase (SOD), cata-lase

(CAT) and glutathione peroxidase (GSH-Px)

activity and expression in different tissues. ...

MATERIALS AND METHODS

.... Animals and diet

.... (60 male Sprague–Dawley rats) used

in the study were 1 year old. ... commercial laboratory chow diet

at 08:00 daily (MBD Laboratory Animal Food Com-pany,

Gebze, Kocaeli, Turkey). The composition of

the diet was as follows: protein 18% (min), lipid

2.5% (min), fiber 4% (max), ash 5.5% (max), nitrogen

free extract 57.0% (max), metabolic energy 2650 kcal/

kg (min), water 13% (max) plus various amino acids,

minerals and vitamins (data obtained from the

supplier).

For the experiment, two main groups (dietary

restricted [DR] and ad libitum [AL], n=30 rats each)

were assigned and were kept at the above-mentioned

centre under similar conditions. In the DR group, ani-mals

were fed on Monday, Wednesday and Friday

mornings, and the food hoppers were removed the fol-lowing

morning. Previous studies have shown that rats

and mice maintained on such an every-other-day feed-ing

schedule will consume fewer calories over time

and live longer than animals fed ad libitum.

AL rats were given more food then they consumed

daily, so that food was available to animals at all

times. This feeding practice was carried on for 6

months. The increase in the body weights of sedentary

animals was monitored by weighing each animal bi-weekly.

After 6 months of dietary restriction, animals of the

DR and AL groups were divided into three subgroups

as follows: sedentary, endurance swimming exercise

training and exhaustive swimming exercise groups.

Each group had 10 animals.

.... RESULTS

The monthly changes in the body weights of the DR

and AL fed rats are presented in Figure 1. Data show

that body weight was significantly lower in DR rats

compared with their AL counterparts. The mean body

weight of DR sedentary rats at the end of 8 months

was only 72% of the body weight of their AL counter-parts.

Table 1 shows the lipid peroxidation levels in liver,

heart and kidney of rats in different groups.

Table 1. Effects of swimming exercise on lipid peroxidation level in the organs

of

rats fed dietary restricted (DR) and ad libitum (AL)

.....................................................

----nmol/g tissue----

Tissue Group----Sedentary Endurance swimming Exhaustive swimming----ANOVA F p

----exercise training exercise----

...............................................

Liver

DR 2.49 0.18 2.81 0.15 3.17 0.30 2.246 0.128

AL 3.65 0.23a 5.17 0.31b 6.96 0.52c 16.446 0.001 tp 3.870** 7.096*** 6.056***

Heart

DR 2.59 0.29a 2.58 0.15a 3.58 0.18b 7.019 0.004

AL 3.42 0.31a 4.01 0.29a 5.43 0.32b 10.862 0.001 tp 1.917 4.535*** 4.761***

Kidney

DR 3.14 0.37ac 2.71 0.16a 3.75 0.21bc 4.423 0.023

AL 4.35 0.43a 4.77 0.22a 6.25 0.35b 9.049 0.001 tp 2.118 7.664*** †***

.................................................

Details of the statistical analysis are described in Materials and Methods. The

values are presented as mean SEM of 8–10 animals assayed

in duplicate.

The level of significance between DR and AL groups in the same column: **p

<0.01;

***p <0.001.

a, b, c: For each exercise group, different letters in the same row show

statistical

differences.

†: The statistical analysis was done with Mann–Whitney U-Test.

Feeding the DR diet lowered the LP levels in all organs regard-less

of the exercise levels. In the comparison of the

DR and AL sedentary groups, the LP levels of DR rats

was significantly lower (p <0.01) only in the liver. In

the submaximal and maximal exercise groups, the LP

levels were lower (p <0.001) in all the tested organs

of DR animals. When compared with the sedentary

group, only the LP levels in liver tissue of AL animals

showed a significant increase in the submaximal exer-cise

group. In the comparison of the sedentary and

maximal exercise groups, while in the AL fed animals

all tissues showed higher levels of LP in maximum

exercised animals, in DR animals an increase was

seen only in the heart tissue of maximum exercised

animals.

In the comparison of the effect of diet restriction on

protein carbonyl levels (Table 2),

Table 2. Effects of swimming exercise on protein oxidation level in the organs

of

rats fed dietary restricted (DR) and ad libitum (AL)

.................................................

----nmol carbonyl/mg protein----

Tissue Group----Sedentary Endurance swimming Exhaustive swimming----ANOVA F p

----exercise training exercise----

.................................................

Liver

DR 1.86 0.26 1.97 0.12 2.30 0.17 1.426 0.260

AL 2.21 0.32a 4.71 0.27b 7.80 0.41c 61.688 0.001 tp 0.845 †*** †***

Heart

DR 1.22 0.19 1.62 0.14 1.92 0.27 2.803 0.081

AL 1.55 0.21a 2.37 0.21b 2.42 0.23b 4.265 0.027 tp 1.146 2.966** 1.397

Kidney

DR 2.63 0.24 2.99 0.22 3.04 0.36 0.608 0.552

AL 3.63 0.26a 3.93 0.21a 2.89 0.21b 5.839 0.009 tp 2.796* 3.032** 0.347

...................................................

Details of the statistical analysis are described in Materials and Methods. The

values are presented as mean SEM of 8–10 animals assayed

in duplicate.

The level of significance between DR and AL groups in the same column: *p <0.05;

**p

<0.01; ***p <0.001.

a, b, c: For each exercise group, different letters in the same row show

statistical

differences.

†: The statistical analysis was done with Mann–Whitney U-Test.

effects of dietary restriction on exercise-induced oxidative stress

lower levels of pro-tein carbonyl were seen in the kidney in the sedentary

group, in all tissues in the submaximal exercise group

and in the liver tissue in the maximal exercise group in

DR animals. An increase was seen in the liver and

heart protein carbonyl levels in AL animals when

compared with the DR animals after submaximal

and maximal exercise.

As shown in Table 3,

Table 3. Effects of swimming exercise on GSH level in the organs of rats fed

dietary

restricted (DR) and ad libitum (AL)

..........................................................

----mmol/g tissue----

Tissue Group----Sedentary Endurance swimming Exhaustive swimming----ANOVA F p

----exercise training exercise----

..........................................................

Liver

DR 15.95 1.14a 15.10 0.82a 10.82 0.81b 8.033 0.002

AL 26.68 1.06a 17.03 0.41b 12.51 0.88c 76.256 0.001 tp 6.801*** 1.952 1.394

Heart

DR 6.42 0.42a 5.39 0.37a 3.79 0.25b 11.360 0.001

AL 8.92 0.56a 7.70 0.51a 5.78 0.40b 10.078 0.001 tp 3.590** 3.713** 3.993***

Kidney

DR 9.09 0.51 8.27 0.54 7.27 0.40 2.755 0.084

AL 14.72 1.11a 13.37 0.50a 7.17 0.38b 36.858 0.001 tp †*** 6.686*** 0.174

...........................................................

Details of the statistical analysis are described in Materials and Methods. The

values are presented as mean SEM of 8–10 animals assayed

in duplicate.

The level of significance between DR and AL groups in the same column: *p <0.05;

**p

<0.01; ***p <0.001.

a, b, c: For each exercise group, different letters in the same row show

statistical

differences.

†: The statistical analysis was done with Mann–Whitney U-Test.

feeding the DR diet was asso-ciated with decreased liver, kidney (p <0.001) and

heart (p <0.01) GSH levels in the sedentary group,

and decreased heart (p <0.01) and kidney

(p <0.001) levels in submaximal exercise and

decreased heart levels (p <0.001) in maximal exer-cise

groups. In the investigation of the effect of exer-cise

on GSH levels, a decrease was observed with

increasing exercise level regardless of diet.

As shown in Table 4, DR feeding was associated

with lower GSH-Px activity in the sedentary animals.

Table 4. Effects of swimming exercise on GSH-Px activity in the organs of rats

fed

dietary restricted (DR) and ad libitum (AL)

...........................................................

----mmol NADPH/min/g tissue----

Tissue Group Sedentary----Endurance swimming Exhaustive swimming----ANOVA F p

----exercise training exercise----

............................................................

Liver

DR 5.87 0.39a 5.91 0.22a 7.35 0.39b 6.254 0.007

AL 8.18 0.62a 10.53 0.37b 15.73 0.85c 34.189 0.001 tp 3.214** 10.923***

8.572***

Heart

DR 5.65 0.35a 6.30 0.29a 8.09 0.63b 7.877 0.002

AL 6.76 0.35a 8.58 0.35b 12.60 0.83c 24.936 0.001 tp 2.193* 4.954*** 4.208***

Kidney

DR 8.81 0.38a 9.23 0.40a 11.59 1.00b 5.396 0.012

AL 10.86 0.67a 9.83 0.39a 17.45 0.67b 52.362 0.001 tp 2.726 1.053 4.935***

................................................................

Details of the statistical analysis are described in Materials and Methods. The

values are presented as mean SEM of 8–10 animals assayed in duplicate.

The level of significance between DR and AL groups in the same column: *p <0.05;

**p

<0.01; ***p <0.001.

a, b, c: For each exercise group, different letters in the same row show

statistical

differences.

In both exercise groups, GSH-Px activity was lower in

DR animals, and an increase was observed with the

increasing exercise intensity regardless of diet.

Table 5 shows the GR enzyme activity of tissues in

the sedentary, submaximal and maximal exercise

groups.

Table 5. Effects of swimming exercise on GR activity in the organs of rats fed

dietary restricted (DR) and ad libitum (AL)

...............................................

----mmol NADPH/min/g tissue----

Tissue Group----Sedentary Endurance swimming Exhaustive swimming----ANOVA F p

----exercise training exercise----

...............................................

Liver

DR 2.00 0.22a 2.86 0.15 b 1.03 0.11c 29.009 0.001

AL 2.77 0.20a 1.90 0.17b 1.12 0.13c 22.554 0.001 tp 2.498* 4.079*** 0.456

Heart

DR 2.60 0.24a 2.46 0.12a 1.80 0.22b 3.606 0.043

AL 3.44 0.24a 2.09 0.24b 1.49 0.19b 17.906 0.001 tp 2.419* 1.433 1.330

Kidney

DR 3.03 0.25a 2.96 0.20a 1.81 0.14b 9.624 0.001

AL 4.29 0.26a 2.46 0.18b 1.40 0.17c 46.406 0.001 tp 3.372** 1.741 1.743

....................................................

Details of the statistical analysis are described in Materials and Methods. The

values are presented as mean SEM of 8–10 animals assayed

in duplicate.

The level of significance between DR and AL groups in the same column: *p <0.05;

**p

<0.01; ***p <0.001.

a, b, c: For each exercise group, different letters in the same row show

statistical

differences.

DR feeding reduced the liver, heart (p <0.05) and kidney (p <0.01) GR enzyme

levels

in sedentary animals. A decrease was observed with

the increasing exercise intensity in both DR and AL

groups, except for the values in the liver of DR

animals.

DISCUSSION

The current study determined the effect of DR on the

oxidative stress and antioxidant enzyme systems

accompanying different intensities of swimming exer-cise

in rats. It was observed that long term DR may

have substantially positive effects on the oxidative

stress and antioxidant enzyme systems.

Swimming was chosen as a suitable model since it

is a natural behaviour of rodents. The method causes

less mechanical stress and injury, and leads to a better

redistribution of blood flow among tissues without

significant variations in cardiac output and heart rate

which in turn may minimize the magnitude of injury

caused due to the generation of ROS.

It has been shown that in Sprague–Dawley rats, the

body weight increases constantly with age from wean-ing

to senility. Throughout the experiment, AL

sedentary rats presented significantly higher body

weights than DR sedentary rats. It is known that rats

fed ad libitum ingest more energy than needed and

that leads to increased body weight, which is mainly

due to fat deposition. However in the DR sedentary

rats, the body weight decreased for the first month,

increased to the initial level by the second month

and then stabilized at a level that was slightly less than

the initial weight, reflecting an adaptive mechanism to

dietary restriction in adult rats.

Our analysis revealed significant effects of both diet

and exercise intensity on liver, heart and kidney LP

levels. In the investigation of the effect of dietary

restriction on LP at various organs, we found that

although the LP levels were lower in all tissues of diet-ary

restricted sedentary rats than in ad libitum fed

sedentary rats, the only significant difference was

observed in the liver. The particular protective effect

of dietary restriction on liver has been documented

previously by - et al., although the

underlying mechanism is not fully understood. It

was suggested that rats on a calorie restricted diet

show faster clearance of LP in liver.

Feeding the DR had a positive effect on oxidative

stress as observed by the lowered LP values in both

endurance and exhaustion exercise groups compared

with those of the AL groups. The effect was more

striking in the endurance and exhaustion exercise

groups than in the sedentary group. It has been sug-gested

that low caloric diet consumption leads to

decreased oxidative damage to lipids, protein and

DNA by altering the rate of free radical produc-tion,

possibly via an increase in the efficiency of

mitochondrial function, that is decreasing the amount

of free radicals they produce without making signifi-cant

changes in the amount of energy produced.

On the other hand DR seems to improve the ability to

remove reactive substances, damaged macromolecules

and LP in liver. Some researchers have suggested

that the whole body metabolic rate, and hence the rate

of free radical generation that arises from cellular meta-bolism,

would be similar in DR and AL rats. The protec-tive

role is thus due to the increased production of

antioxidants. Our findings however, show that both

the LP and PO levels, as well antioxidant enzyme levels,

were lower in DR rats.

Oxidative damage to proteins is accompanied by an

increased number of carbonyl residues. We

observed that dietary restriction had little effect on

PO in sedentary animals. Youngman et al. and Sohal

et al. reported that the increase of protein carbonyls

with age can be retarded, but not eliminated by DR.

The mechanism underlying the protective effect of

dietary restriction is unclear; it is possible that the

decrease of caloric intake improves mitochondrial

respiration and therefore decreases free radical pro-duction.

Alternatively, DR may decrease the accu-mulation

of damaged proteins by increasing the rate

of their proteolytical degradation. The tissue PO

level was seen to increase in parallel with the increase

in exercise intensity, particularly when the diet is not

restricted. DR was observed to retard dramatically or

partly prevent the exercise associated accumulation of

oxidative damage.

One of the most interesting results from the current

study is that when the effect of dietary restriction on

GSH contents was investigated we observed that DR

animals start with lower GSH and have lower GSH-Px

activity which presumably reflects lower protein

expression levels in sedentary animals. This can be

attributed to the fact that DR groups are undergoing

lower rates of oxidative stress. It has been argued that

dietary restriction inhibits the generation of oxidative

molecules and does not directly increase antioxidant

enzyme activity. Gong et al. reported an apparent

reduction in antioxidant enzyme activity in rat lens

and kidney in response to dietary restriction, presum-ably

due to a decrease in substrate oxidative mole-cules.

Dietary restriction, on the other hand, also

regulates the redox balance by a thiol-reducing system

in which protein-thiol mixed disulphides are formed

from protein thiols. The highest levels of GSH were observed in liver

both in AL and DR sedentary rats and the lowest rates

were observed in heart. Liver synthesizes GSH from

endogenous or dietary amino acids de novo and sup-plies

most of the circulating GSH. On the other hand

the heart is an aerobic organ and has one of the highest

mass-specific oxygen consumption rates in the body;

therefore it can cope with high rates of oxidant forma-tion

and stress.

After endurance exercise training, the GSH level

was decreased in the heart and kidney, but after

exhaustive exercise it was decreased in the heart in

DR animals compared to AL animals. Decreases in

GSH levels that parallel the increase in the severity

of the exercise were reported previously. The

decrease can be attributed to the diminishing GSH pool

and regulation of redox balance and may generally

be expected due to a possible increase in formation

of GSSG and subsequent export of GSSG out of cell.

We observed an increase in LP levels in parallel

with the decrease in GSH levels. The increase and

decrease ratios for these two parameters, respectively,

suggest a strong correlation between them. Existence

of such a correlation is being supported by studies on

long-duration treadmill run performance.

GSH-Px enzyme activities were found to be lower

in DR animals than in AL animals both in sedentary

group and at different exercise intensities. This is

due to the low levels of oxidative injury in DR ani-mals.

Food restriction may reduce free radical damage

at various steps, e.g. by reducing the generation of

ROS affecting the sensitivity of cellular components

to free radical oxidation; the expression of antioxidant

enzymes and antioxidant levels.

An increase in GSH-Px enzyme activity was

observed with the increasing severity of exercise in

both DR and AL animals, where the change in ratios

were more prominent in AL animals. Increased levels

of GSH-Px by exercise intensity are thought to be

one of the adaptation phenomena to efficiently elimi-nate

ROS produced during physical exercise and

minimize damage caused by ROS. In addition, DR

may increase the endogenous levels of NADP^+ and

glucose-6-phosphate dehydrogenase (G6PDH) and

thus, the efficiency of the GSH-Px enzyme system.

At the same time the higher GSH-Px levels in the sub-maximal

AL group compared to sedentary AL likely

reflects altered protein expression levels with 8 weeks

of training, although this did not occur in the DR

group. This is probably because caloric restriction

induces a metabolic reprogramming characterized

by a transcriptional shift towards energy metabolism,

increased biosynthesis and protein turnover.

In previous studies researchers have reported that

dietary restriction has a positive effect on ageing

and that regular and mild exercise minimizes oxida-tive

injury and adapts the body for a maximum bout

of exercise. In this study we found that DR has a posi-tive

effect on oxidative injury in sedentary animals

and at various intensity of swimming exercise. Our

next step will be to investigate the optimum percen-tages

of DR and exercise intensity for the minimal tis-sue

damage, minimized negative effects of ageing and

for maximal, high-quality life span.

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

______________________________________________________

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