The effect of aerobic exercise on relative leukocyte telomere length in male Sprague-Dawley rats given a high fat-diet

There is an increasing number of studies showing that physical Background activity and aerobic exercise have a positive effect on telomere length. Some studies also show that dynamics of telomere length is influenced by various environmental factors such as lifestyle and diet. However, the association between exercise and diet with telomere length is still questionable. The aim of this study was to examine the effects of aerobic physical exercise on relative telomere length changes in high fat-diet condition in rat animal models. : This study was an   experimental study using twelve Methods in vivo Sprague-Dawley male white rats (12-month-old). Subjects were evenly and randomly divided into two groups (n=6): (1) high fat-diet fed control group; (2) high fat-diet fed and aerobic exercise treatment group. Aerobic exercise was conducted using animal treadmill with intensity of 20 m/min, 5 days/week. At weeks 4 and 8, relative telomere length was compared with week 0 control group, using q-RT-PCR. : Lengthening of relative telomere length was observed in both control Results and treatment groups at weeks 4 and 8, when compared to week 0 control group. The lengthening in the control group was much greater than the treatment group. : Excessive increase of relative telomere length was seen in high Conclusions fat-diet conditions. Aerobic exercise for 8 weeks suppresses excessive increase of relative telomere length in high fat-diet conditions.


Introduction
Obesity is a global problem that is associated with high mortality and morbidity.Several studies has shown that obesity increases the risk of cardiovascular disease, renal impairment, diabetes, and even certain cancers [1][2][3] .Meanwhile, the World Health Organization (WHO) showed that in the last 4 decades obesity rates have increased 10 times worldwide 3 .This phenomenon is worrisome because there is an increasing number of people who have a high risk of developing various diseases associated with obesity.Some studies show that there is a strong association between the incidence of obesity with the level of physical activity and high fat diet intake.Individuals with low physical activity levels are known to have a higher risk of developing obesity 4,5 .Physical activity is also known to have a role on lowering blood glucose levels, improving homeostasis in people with diabetes mellitus, decreasing production of oxidative stress, lowering triglyceride levels in the body, increasing endogenous antioxidants, and can also maintain telomere length, thus reducing cardiovascular metabolic disease risk [6][7][8] .
The role of physical activity in decreasing the risk of cardiovascular metabolic mortality and morbidity is thought to be mediated by maintaining the length of telomeres 9 .A study performed by Goglin et al. demonstrates the association between telomere shortening and increased 5-year mortality in patients with acute coronary syndromes.This study also showed that a decrease in telomere shortening rate would be followed by a decrease in mortality rate 10 .
The rate of shortening of the telomere can be suppressed through a healthy lifestyle such as healthy diet and physical activity 9,11 .Certain types of food have been shown to have a correlation with telomere length.A Mediterranean-rich diet of olive oil (38% of total energy as fat) has been observed to maintain telomere length 12 .In contrast, a high western type diet of sugar and red meat is associated with shortening of the telomere 12 .
The aim of this study was to explore the effect of aerobic physical exercise on telomere length under high fat-diet conditions to provide information for further research.

Experimental animals
Twelve male Sprague-Dawley rats (Rattus norvegicus), weighing 250-450g, aged 12-13 months, were obtained from Central Animal Facility (Bogor Agricultural University) and divided into two groups: control and trained high fat diet.They were acclimatized for 1 week in a controlled room temperature of 24+1°C, with a 12-hour light/dark cycle, and access to food pellets and filtered water ad libitum to adapt to the new environment.They were housed in plastic cages (50×34×25 cm), two animals in each cage.All protocols used in this experiment received approval from the Ethical Animal Care and Use Committee of Faculty of Medicine Universitas Indonesia with approval number 164/UN2.F1/ETIK/2017.

Study design
Before the beginning of the study all rats were acclimatized with high fat-diet for 10 weeks, consisting of 19% fat, 24% protein, and 47.77% carbohydrate.After 10 weeks of acclimatization, the rats were evenly and randomly assigned using a random number into two groups (n=6 per group): (1) the control group (without aerobic exercise) and (2) the treatment group (with aerobic physical exercise).The treatment group received aerobic exercise for 8 weeks.During 8 weeks of intervention, both treatment and control groups were still given a high-fat diet.
Aerobic exercise was conducted using an animal treadmill, with a speed of 20 m/min for 20 minutes, 5 days/week, every morning around 6 am until 8 am.The intervention was carried out at the Biochemistry and Molecular Laboratory, Faculty of Medicine, Universitas Indonesia.All aerobic exercise protocols were supervised by experienced researchers.

Data collection
Blood was collected from both groups after an overnight fasting.All animals were anesthetized intraperitoneally with a ketaminexylazine (KX) solution before blood was taken.Approximately 1 ml of whole blood was taken from the sinus orbitalis on week 0, week 4 and week 8. Genomic DNA in leukocytes was extracted from peripheral blood.DNA isolation were then performed using DNA isolation kit (GeneAll® ExgeneTM Clinic SV mini).Relative telomere length from isolated DNA were measured on a real-time PCR detection system using a Quantitative PCR method.Kit used for qPCR were pipettes (and tips), optical PCR plates and caps, and master mix.The type of taq used was AmpliTaq Gold DNA polymerase.The model number/ name of the PCR machine was Applied Biosystems 7300.The cycling conditions used were 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min, followed by a dissociation (or melt) curve.The primer sequences were as follows: • Telo F: CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGG TTTGGGTT The primers were obtained from rodent (GenScript®).Relative telomere length was calculated using the formula of 2 -ΔΔ Ct13 .

Statistical analysis
All statistical analysis was performed using SPSS 20 for Windows.Because the distribution of data is not normal, the data was assessed using a nonparametric Kruskal-Wallis test.

Results
The characteristics of the animals are shown in Table 1.All the animals were in good condition throughout the length of study.
There was no significant difference in age, body weight, Lee index and telomere length between control and treatment groups.
There was an increase in relative telomere length at weeks 4 and 8 compared to week 0 in both groups.At week 4, the relative telomere length of the control group (2.231) did not differ much with the treatment group (1.802) when compared to week 0 of control group.At week 8, there was a progressive increase of relative telomere length in both groups compared to week 0 and week 4. Relative telomere length increase in week 8 of the control group was much higher (178.62)compared to week 8 treatment group (74.86) (Figure 1; Table 2).

Discussion
One important structures located at the ends of the linear chromosomes is the telomere.In human cells, they are composed of TTAGGG repeats and a number of proteins.Their function is to protect the integrity and stability of the DNA 14 .
Many studies showed that telomere length is influenced by a number of factors 9,12,15 .Sedentary lifestyle, high blood glucose levels, and increased percentage of body fat have a negative influence on telomere length.The underlying mechanism have been suggested as being mediated through oxidative stress and inflammation 16 .
Exercise as a lifestyle intervention has been associated with longer leukocytes telomere length 15 .A study by Cherkas on 2401 subject showed that telomere length has a direct relation with increased level of physical activity 17 .An observation of physical activity and telomere length by Du et al. on 7813 adult women concluded that moderate and vigorous intensity activity increased telomere length compared with least active women 18 .Ludlow et al. studied the effect of physical activity on telomere length in three different groups: sedentary, moderate and overtraining.The result showed a positive effect on telomere length in the moderate group 19 .
To date, to our knowledge, very few studies have investigated the relative effect of a specific diet on telomere length.Cassidy et al. found that total fat intake was only inversely associated with leukocytes telomere length and higher polyunsaturated fatty acids (PUFA) intake, specifically linoleic acid intake, was inversely associated with leukocytes telomere length 11 .Li et al. found that there was no difference in telomere length between consumption of fish oil-rich diet and soy oil-rich diet 20 .Kiecolt-Glaser et al. compared telomere length between subjects with n-6 PUFA and n-3 PUFA supplementation.They found that telomere length is longer in subject with omega 3 or n-3 PUFA supplementation (high in fish oil) compared to n-6 PUFA supplementation 21 .
Results from our current study showed a lengthening of relative telomere length in both groups in week 4 and week 8.This was in contrast with studies showing telomeres usually shortened with age 9 ., but there are also studies which indicate that in vivo, telomere may shorten or elongate, and leukocyte telomere length may fluctuates within months 22,23 .
In general, preserved telomere length and lengthening of telomere are considered as something good because it is thought to play an important role in extending the biological age of cells 12,21,24 .
Nevertheless, studies have also shown that telomere lengthening can be an initial response that arises after exposure to low doses of various carcinogenic chemicals in vitro and in experimental animals 23 .Zhang et al. concluded from their study that subject with longer telomere length had a higher risk of getting lung cancer, and this was especially true for men 25 .
The positive associations between high fat-diet conditions and telomere length is difficult to explain because a high fat-diet is associated with increased risk of various diseases.Telomeres generally shorten with age, thus, the discovery of telomer elongation in the provision of high-fat diet can be regarded as something that deviates from normal condition.Therefore, the positive associations between high fat-diet conditions and telomere length observed in this study are notable.
Telomere will shorten at each cell division.Telomeres that elongate excessively in both groups in this study may indicate a prolonged period before apoptosis, and this could indicates a change from normal cell function.Currently, the implications of excessive telomere lengthening are still unknown.Our result shows that aerobic exercise can act as a barrier to progressive changes that occur in the relative telomere length caused by a high-fat diet condition.Modulation of oxidative stress in the   body is one possible mechanism that may explain how aerobic exercise resist relative telomere changes.Aerobic exercise upregulate genes that encode various antioxidant enzymes.
Several studies shows that regular physical exercise increase the body's endogenous antioxidant activity and thus increase body's resistance to oxidation events 15,26 .

Conclusions
Our study showed that exposure to a high fat-diet plays an important role to the emergence of altered telomere length, and aerobic exercise could reduce the progression of the alteration in length.Our results support the hypothesis that leukocyte telomere length is associated with daily dietary intake and physical activities.Further investigation is still needed to explore the mechanism and implications of telomere length changes found in this study.Santoso et al. explore the relationship between a high fat diet, aerobic exercise and telomere length.In this small-scale animal research study, the authors make the unexpected observation that after 8 weeks of high fat diet, telomeres are elongated in circulating blood cells (leukocytes).They observed in aerobically trained animals that the apparent telomere elongation was significantly attenuated compared to a sedentary control.These interesting findings observations call into question the strict dogmatic view that telomere shortening is the rule in pathology.Human epidemiological studies have reported similar discrepancies, where telomere shortening is not always observed in pathological scenarios but rather telomere elongation can also be apparent in disease situations.While these observations are provocative, I have several concerns with the sample size, the model organism, the method used to measure telomere length, and the interpretation of the data.
This study is very positive in terms that it points out the need to perform longitudinal analysis of telomere biology in both human and model organism studies.Repeat sampling will be critical to moving the field of telomere biology and environmental/lifestyle impacts forward.For instance, what happens to telomere biology or cell biology after 12 weeks on a high fat diet following the apparent rapid and massive telomere elongation event at 8 weeks?
The negatives of this study are the model organism of choice, the measurement biochemistry, the data analysis, and the lack of mechanistic insights provided by the authors.
1. Sample size, model organism, and statistical treatment of the data.
While the effect observed in this study is large, the sample size is small.Further the authors utilize a repeated-measures design and do not analyze the data as such.Each rat's telomere length was measured three times.Thus, change in telomere length per individual rat should be reported between each time point and analyzed via a repeated measures analysis.Further, with the assay of choice, 6 animals would be borderline powered enough to detect a meaningful change.What were the authors expectations in terms of a priori predictions on the magnitude of change in telomere length?My hypothesis would have been no or very subtle changes in telomere length for this duration of intervention.What are the speculative mechanisms the authors see occurring?Are the leukocytes entering a stress mode whereby they activate telomerase and elongate telomeres dramatically to try to survive?Could this be the beginning of alternative lengthening of telomeres in these cells?Can the author speculate a bit about what they think might be happening in terms of mechanisms? 1.

3. 4.
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Partly
No competing interests were disclosed.

Competing Interests:
We have read this submission.We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com

Figure 1 .
Figure 1.Comparison of relative telomere length in leukocytes of rats fed a high fat-diet at weeks 0, 4 and 8 between control group (no aerobic exercise) and treatment group (aerobic exercise).

Table 2 . Comparison of relative telomere length in leukocytes of rats fed with high-fet diet at week 0, 4 and 8 between control group (no aerobic exercise) and treatment group
(aerobic exercise).Median (minimum-maximum).