Effect of Ergonomic Exercises in Modifying Sirtuin 1 Gene Expression in Obese Down Syndrome Patients

Article history: Received on: 16/05/2017 Accepted on: 14/07/2017 Available online: 30/09/2017 The current study was conducted to investigate the effect of ergonomic exercises on modifying SIRT1 gene expression in obese Down syndrome patients. Forty obese Down syndrome patients (IQ level ˃ 60) were included in current study. Their age were ranged from 14 to 18 years old, their body mass index (BMI) was ranged from 30kg/m 2 to 39.9 kg/ m 2 , These patients were divided randomly and equally into two groups GI composed of twenty obese patients with down syndrome. They were received indoor ergonomic exercises two times / week for 3 months plus balanced diet and GII composed of twenty obese patients with Down syndrome. They were received balanced diet only. Data were obtained from each patient for BMI and SIRT1 level. Measurements were performed before study and after three months. Statistical analysis revealed that there was reduction of BMI within both groups after treatment in comparison to pre-treatment (p>0.05). Ergonomic exercises with balanced diet has a significant effect in reduction BMI and waist circumference (p; 0.0001* & 0.019*, respectively). Fold change as indicator for SIRT1 gene expression was changed in GII and GI respectively (40%, 27.27%). These results indicated that BMI and waist circumference reduced with exercises which may be an effective tool in modifying gene expression in Down syndrome obese patients.


INTRODUCTION
Down syndrome (DS) children have typical characteristic facial features, physical growth delays and mild to moderate intellectual impairment.This genetic disorder results from the presence of a piece or even an extra copy of chromosome 21 which means those DS patients have 47 chromosomes.95% of DS cases have meiotic non-disjunction that result in trisomy 21.Robertsonian translocation and mosaic type are the other types of DS.Children with DS are shorter than normal age matched individuals and may have health problems including; immune dysfunction, leukemia, gastrointestinal abnormalities, heart and vision diseases, Alzheimer's disease, hypothyroidism and diabetes mellitus (Aburawi et al., 2015).Furthermore, Down syndrome has a higher risk for developing obesity.Childhood obesity is accompanied with health problems throughout life that may slow or reverse other health gains for children with DS.In addition, Obesity is a stigmatizing condition and can be another characteristic that identifies those children as different (Koebnick et al., 2012).The primary care providers can help children and adolescents with DS those are at higher risk to be obese by establishing an early screening and management plan to avoid or treat weight gain (Murray and Ryan-Krause, 2010).Sirtuin 1(SIRT1) stands for sirtuin (silent mating type information regulation 2 homolog) 1.It is also known as NAD+-dependent deacetylase sirtuin 1.This protein encoded by the SIRT1 gene in human.SIRT1 enzyme deacetylates proteins that contribute to cellular regulation.It also affects the activity of both members of the PGC1-alpha/ERR-alpha complex, which are essential metabolic regulatory transcription factors (Zhao et al., 2014;Schug and Li, 2011).
SIRT1 is activated during calorie restriction which may be related to energy balance through glucose or lipid metabolism and insulin signaling.Therefore, SIRT1 suggested playing a significant role in the pathophysiology of visceral obesity.In addition, SIRT1 expression levels were negatively correlated with body mass index, waist circumference, and abdominal visceral fat area.The significant association between abdominal visceral fat accumulation and SIRT1 gene expression in circulating peripheral blood mononuclear cells (PBMCs) suggests that SIRT1 may be a new therapeutic target for the prevention of disease related to obesity, especially visceral obesity (Lee et al., 2013;Nasrin et al., 2009).
Essential factors to be consider before taking the decision for operation including the patient's; mental status, BMI, eating habits, health conditions related to obesity, and previous stomach surgeries (Mechanick et al., 2013).
Aerobic exercises are exercises having an adequate oxygen supply to the working groups of muscles without exhaustion.A wide variety of physical activities considered as exercises including yard work and many recreational activities such as dancing, tennis, basketball, and golfing.Also the traditional aerobic exercises (e.g., walking, running, bicycling, and swimming), stretching, and resistance training are considered to improve flexibility and muscle strength (Green and Smith, 2017).
Therefore this current study is an attempt to evaluate the effect of ergonomic exercises on modifying SIRT1 gene expression in obese Down syndrome.

Subjects
Forty obese Down syndrome patients were included in current study.Their IQ level was greater than 60 .their ages were ranged from 14 to 18 years.Their body mass index was between 30 Kg/m² And 39.9 Kg/m² (moderate obesity).They were free from any pathological disorders or previous surgery which may affect the study and application of treatment modalities.The subjects were divided equally and randomly into two groups: Group I; In this group, twenty obese patients with Down syndrome were received a balanced diet and indoor ergonomic exercises therapy (each session was 30 minutes, 2 times per week for three months) and Group II; In this group, twenty obese patients with Down syndrome were received balanced diet only.Weight was determined, height was measured, then the body mass index was calculated where: Body mass index (BMI) = body weight (Kg)/ height (m) 2 (Ellulu et al., 2014).Skin fold was measured by using Electronic skin caliper and abdominal circumference was recorded for each patient.

Bicycle ergometer
Electronic bicycle ergometer (Universal, made in New York, USA) equipped with pedals, electronic break, adjustable seat, handle bar, display screen and foot straps also provided with programmable control unit.

Program of aerobic training
Each patient was sit on stationary bicycle ergometer with her/his back in relaxed position, before exercises the limit of subject tolerance would be assessed by exercises test which was comprised of 3 minutes control period of unloaded pedaling, followed by an incremental ramp on a cycle ergometer at a rate of 10 W per minute to the limit of subject tolerance.Then the subject cycle was sit at 30 W for warming up, and then the intensity was increased every 60 seconds by 15 W until exhaustion, then the subject cycle was 30 W for cooling down.Duration was for three months, 2 times /week.

Low caloric diet protocol
Patients were beginning adding thicker liquids that are high in protein and low in fat and sugar.They were used highprotein, Low-calorie liquid supplement drinks during this period.Daily caloric intakes were not exceeding 1500 calories.They were drinking 1 to 1.5 liters of water or other non-caloric liquids per day.

Gene expression of SIRT1 gene
Samples of whole blood were collected from each patient in 5-ml sterile RNAase free vaccutainer tubes containing EDTA.Samples were collected two times throughout the study, before starting (pre-test) and after training sessions (post-test).Blood samples were collected on ice and processed within 30 minutes after collection.Total RNA was extracted from whole blood using PureLink RNA Mini Kit (Invitrogen; Thermo Fisher Scientific, USA) according to manufacturer's instructions.RNA extraction was carried out using cooling centrifuge (Labofuge 400R-Heraeus, Germany).RNA concentration and purity were identified using nanodrop spectrophotometer device (2000 C, Thermo Scientific, USA).RNA samples were kept immediately at -80 C until assayed.cDNA synthesis was carried out for each RNA sample in a thermal cycler (T gradient, Biometra, Germany) using High Capacity cDNA Reverse Transcription Kit (Applied biosystem, USA, Foster) according to manufacturer's instructions.Gene expression of SIRT1gene in obese children of the two groups was studied to investigate the effect of ergonomic exercises in SIRT1 gene expression in obese Down syndrome children.Gene expression assay for SIRT1 gene was carried out using customized ready-to-use SIRT1 gene expression assay and TaqMan universal master mix II (Applied biosystem, USA, Foster).Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control.Quantitative real-time polymerase chain reaction (qRT-PCR) (DNA Technology, Moscow, Russia) was applied for quantitative SIRT1 gene mRNA expression (Lee et al., 2013).

Ethical Approval
All procedures performed in the current study involving human participants were in accordance with the ethical standards of the Medical Research Ethics Committee of the National Research Centre-Egypt (MREC, NRC, no.14136), and with 1964 Helsinki declaration and its later amendments.Informed consent was obtained from the parents of each participant included in the study.

STATISTICAL PROCEDURES Data collection
Data were collected two times as follow: before starting (pre-test) and after three months (post-test) including body weight BMI and SIRT 1 gene expression .

Data analysis
Statistical analysis was conducted using SPSS for windows, version 20 (SPSS, Inc., Chicago, IL).The current test involved two independent variables.The first independent variable was the (interventions); between subjects factor which had two levels (group I receiving ergonomic exercises and group II receiving balanced diet only).The second independent variable was the (measuring periods); within subject factor which had two levels (pre & post).In addition, this test involved three tested dependent variables (body weight, waist circumference, and skin fold).Accordingly, 2×2 mixed design MANOVA was used to compare the tested variables of interest at different tested groups and measuring periods.With the initial alpha level set at 0.05.

Body mass index
Table (2) revealed the results for the Body mass index pre-and post-treatment between two groups of the study.There was no significant difference in pre-treatment values.But there was a significant difference in the post-treatment values (P<0.05) .

SIRT 1 gene expression
Table (3) shows median score, U, Z and P-values of the fold change (a fold change is basically a ratio (post-treatment and pre-treatment).It indicates the number of times gene expression has changed in comparison to an original amount) at both groups.In group I, the median score of fold change for gene expression was 0.07205.While in group II, the median score of fold change for gene expression was 0.26375."Mann-Whitney tests" revealed there was no significant difference between the both groups in fold change (U = 39, Z = -1.385,and p = 0.166).

Correlation among fold change and BMI of group I at posttreatment
As illustrated at figure (1) the correlations among fold change at post-treatment and BMI.Waist circumference, skin fold and BMI at group I were studied through the Pearson product moment correlation coefficient.It revealed that there was no significant correlation between fold change and BMI (r= 0.261, p= 0.466).
While, there was positive strong significant correlation between fold change and waist circumference (r= 0.69, p= 0.019*).But, there were no significant correlation between fold change and skin fold (r=0.347,p= 0.295).

Correlation among fold change and BMI of group II at post treatment
As at figure (2) the correlations among fold change and BMI at post-treatment.Waist circumference, skin fold and BMI at group II were studied through the Pearson product moment correlation coefficient.It revealed that there was no significant correlation between fold change and BMI (r= -0.099, p= 0.801).As well as, there was no significant correlation between fold change and waist circumference (r= 0.001, p= 0.998).Additionally, there were no significant correlation between fold change and skin fold (r=0.042,p= 0.915).

Role of exercises therapy in controlling obesity
The results of the present study showed that there is a significant difference in BMI in both studied groups pre-and posttreatment.According to (Petridou et al., 2017) low density lipoprotein (LDL) was decreased by increasing of lipoprotein lipase activities.These increased activities cause clearance of residual chylomicron and cholesterol-rich LDL.Cholestryl ester transfer protein control the exchange of triglycerides for cholesterol ester in LDL and HDL.Moreover, weight loss and exercises decrease the level of cholestryl ester transfer protein, may be due to part of this protein is synthesized in adipose tissue.
On the other hand, regular training by using cycle ergometer showed a decrease in abdominal fat and body mass index as a result of decrease in total amount of stored calories (Driss and Vanderwalle, 2013).This decrease in energy stores was attributed to a negative energy balance produced by exercises (Fearnbach et al., 2016).(Fernström et al., 2017) Studies have demonstrated that aerobic fitness is associated with low risk for cardiovascular diseases in Swedish young adults .
Moreover, (Nelson et al., 2014) found that physical activity causes a long term weight loss and decrease body mass index.They revealed that this loss, due to regular physical activity that enhance fat oxidation (Arquer et al., 2010) and partially prevent the age related increase in central body fatness.The present study revealed that ergonomic exercises with balanced diet has a significant effect in BMI reduction.

Modifying SIRT1 gene expression
The results of the current study found that the median score of fold change for SIRT1 gene expression in the study groups I and II (0.07205 and 0.26375, respectively).On the other hand, there was no significant difference between both groups concerning fold change.However, a significant positive correlation was found between waist circumference and fold change of SIRT1 in group I but was not found in group II.According to (Ogorodnikov et al., 2016;Covington et al., 2015) Mechanisms that control gene expression are relatively well understood.The dynamic regulation in the cell control each step in this process.This regulation includes structural changes in the chromatin to make a particular gene accessible for transcription into RNA, splicing of RNA into mRNA, editing and other covalent modifications of the mRNA, translation of mRNA into protein, and, finally, post-translational modification of the protein into its mature, functional form.However, it is not clear that visceral adiposity affects SIRT1 expression or SIRT1 regulates fat distribution.This relationship could be explained on the basis that visceral fat increases free fatty acid flux and inhibit insulin effect, but SIRT1 decreases lipogenesis, increase fatty acid oxidation, affect insulin signaling and protect against oxidative stress.In addition, the relationship between SIRT1 and visceral adiposity may also be explained on the basis of mitochondrial function.Visceral adiposity is associated with mitochondrial dysfunction and diminished glucose tolerance in elderly, but SIRT1 induce mitochondrial activity and biogenesis in several tissues (Lee et al., 2013).
The mechanism by which SIRT1 gene affect obesity could be attributed to its effect on the nuclear peroxisome proliferator-activated receptor (PPARγ).This receptor plays an important role in glucose metabolism and maturation of preadipocytes into mature fat cells.PPARγ in mature fat cells induce the genes that accumulate free fatty acid and synthesis triglycerides.SIRT 1 was found to suppress PPARγ by anchoring to a negative cofactors of this receptor.This process down regulate genes such as adipocyte protein 2 (aP2) in mice.Therefore, mice starvation induced SIRT1 to bind the aP2 promoter in white adipose tissue (WAT), thereby down regulate gene expression, and accelerate mobilization of fat into the blood.This explain the association between decreasing fat storage and increasing lipolysis with up regulation of SIRT1 in differentiated adipose cells (Li, 2013).Moreover, treatment of mice on a high fat diet with resveratrol that is an activator of SIRT1 was shown to reduce weight gain.These findings suggest that SIRT1 affect lipid regulating transcription factors which adapt gene transcription with changes in nutrients (Zhou et al., 2016;Kauppinen et al., 2013).

CONCLUSION
In summary, fold change as indicator for SIRT1 gene expression was changed in the study groups GII and GI (40%,27.27%,respectively).This could be explained by the effect of ergonomic exercise with balanced diet on the reduction in BMI and waist circumference of cases which may affect gene expression in the studied Down syndrome obese patients.
Financial support and sponsorship: Nil.

Fig. 1 :Fig. 2 :
Fig. 1: Scatter plot for the bivariate correlation between fold change and BMI post treatment at group I.

Table 1 :
General and Clinical Characteristics (age and height) of the Study Groups.

Table 2 :
Mean ±SD and p values of body mass index of the study groups.
*Significant level is set at alpha level <0.05, SD: standard deviation, MD: mean difference, p: probability value.

Table 3 :
Median, U, Z, and p values of fold change at both groups.