Effect of moderate exercise and vitamin D3 on adipose tissue SIRT1 proteinin obese rats

Abstract

AimObesity is a chronic metabolic disease affecting almost all physiological functions of the body leading to increased morbidity and mortality. The increased prevalence of obesity has been attributed to several risk factors that contribute to several metabolic dysfunctions. This study was conducted to evaluate the effect of moderate exercise and vitamin D3 on adipose tissue SIRT1 protein level in obese rats.MethodsThe study was carried out on 40 adult male Wister albino rats, divided into Group 1 (control group): 8 rats received a standard rat diet for 12 weeks and Group 2 (obese group): 32 rats received a high fat diet (HFD) for 4 weeks. Group 2 was further subdivided into 4 subgroups each of 8 rats; (a) Control sedentary group (b) Exercise group (c) Vitamin D3 supplement group (d) Exercise plus vitamin D3 supplement group. At the end of 12 weeks, the anthropometric index and adipose tissue SIRT1 protein by ELISA were measured.ResultsHFD results in abnormal anthropometric data and a significant reduction of SIRT1 protein in adipose tissue. In addition to elevation of SIRT1 protein in combined treatment, there was a negative correlation between SIRT1 protein and adiposity index.ConclusionThe combination of VD and exercise improved significantly anthropometric parameters and increased adipose tissue SIRT1 protein.

Keywords

Introduction

Obesity is the outcome of complex relations between environmental, genetic, and epigenetic factors. The two main factors that are thought to contribute to obesity are excessive consumption of high-energy foods and physical inactivity.¹ One potential tactic for the prevention and/or treatment of obesity and its related complications has been to focus on the development and functional alterations of adipose tissues during the obesity process. Obese people frequently have low vitamin D levels, which are associated with a higher risk of obesity.² Sirtuins (SIRT1) is a family of NAD-dependent enzymes that deacetylate different proteins’ lysine residues. It seems that adipose tissue is a significant SIRT1 action site. It has been documented that a high-fat diet (HFD) increased insulin resistance by causing the proteolytic cleavage of SIRT1.³
Exercise is a crucial non-pharmacological tool that has a profoundly positive impact on the body’s many functional systems. Nevertheless, the precise process by which regular exercise improves organ function is still unclear. As a result, this research was performed to explore their effect on SIRT1 protein in adipose tissue in a rat model of HFD-induced obesity.

Materials and Methods

2.1 Experimental DesignThe present study involved 40 healthy male Wister albino rats aged 2-3 months with body weights from 150-180 g each. Animals were housed in separate cages. They were maintained in normal light and temperature conditions with unrestricted access to food and water. Before the study began, the animals were allowed to adjust to their new housing for a week. All institutional and national guidelines for the care and use of laboratory animals were followed.
All the animals were randomly divided into two groups. Group 1, the normal control group (NC), included 8 rats. They served as healthy controls and received a standard rat diet for 12 weeks. Group 2, the high-fat diet group (HFD), included 32 rats and received HFD for 4 weeks. The HFD was used to make rats obese, and it contained 20 g of fat per 100 g of food (To supply essential fatty acids, use 1 g of soybean oil and 19 g of butter oil).⁴
Then Group 2 was subdivided into 4 subgroups; each of 8 rats. Group 2A: sedentary HFD control group (Sed): rats received HFD for another 8 weeks; Group 2B: Exercise group (Exe): rats received HFD and did swimming exercise for 8 weeks; Group 2C: Vitamin D supplement group (VD): rats received HFD and were given oral VD supplementation (10 mcg/kg/day).⁵ In addition, Group 2D: Exercise plus vitamin D supplement group (combined): rats received HFD, vitamin D (10 mcg/kg/day) for 8 weeks and did swimming exercise for 8 weeks.2.2 Swimming Exercise ProtocolIn the exercised group, rats were subjected to free swimming in a pool filled with 32 ± 1°C water.⁶ The protocol was conducted 5 days/week between 9 and 12 a.m. for 8 weeks. Each swimming session lasted for 30 minutes.⁶2.3 Anthropometric MeasurementThe rats’ weights were measured weekly from the beginning of the experiment. At the end of the experiment body length, weight, body mass index (BMI) and Lee index = cube root of final body weight (g)/length (cm).⁷ Furthermore, after anesthesia (isoflurane 4% inhalation) visceral white adipose tissues from the epididymal, mesenteric, perirenal, and retroperitoneal regions were dissected, weighed and the adiposity index was calculated (total visceral fat/final body weight × 100).⁸2.4 Determination of SIRT1 Protein in Adipose TissueA. Adipose tissue preparation
After removing any remaining blood, visceral adipose tissue was stored at -80 °C after being cleaned with pre-cooling PBS buffer (0.01M, pH=7.4). After homogenizing the frozen tissues in 100 mg tissue/ml cold PBS with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO. USA), the samples were centrifuged for 15 minutes at 12000 xg, and aliquots of the supernatant were taken for ELISA analysis to determine the presence of SIRT1 protein.⁹
B. Determination of adipose tissue SIRT1 protein by ELISA
Adipose tissue supernatant was used to determine adipose tissue SIRT1 protein using Rat SIRT1 ELISA Kit (Catalog No. ER1338, Fine Test, Wuhan, China, e-mail: www.fn-test.com) based on sandwich enzyme-linked immune-sorbent assay technology according to the manufacture instructions. The samples were analyzed in duplicate and SIRT1 protein was expressed in ng/ml. In addition the supernatant of adipose tissue homogenate was used for the estimation of total protein by Lowry’s method using folin phenol reagent with bovine serum albumin as a standard and the samples were analyzed in duplicate.¹⁰
The results of adipose tissue SIRT1 ELISA was normalized to total tissue proteins and expressed in ng/mg protein.Ethical ApprovalThis study was approved by Ethics committee of the Alexandria University (Date: 16.06.2022, Decision No: 0201690).Statistical AnalysisData were analyzed using IBM SPSS software package version 20.0 (Armonk, NY: IBM Corp). To confirm that the distribution was normal, the Shapiro-Wilk test was performed. Measures like mean and standard deviation were used to describe quantitative data. The results were deemed significant at the 5% level. The Post Hoc test (Tukey), one-way ANOVA test, and Pearson coefficient were utilized.

Results

3.1 Anthropometric ParametersA. Comparison between the different studied groups according to body weight in (gm) in week 1, 4 and 12: In week 1 no significant difference between studied groups (P = .885), while in week 4 there was a significant increase in body weight in groups that took HFD in comparison to normal control group (P < .001). In addition, in week 12 exercise and combined groups showed no significant difference when compared to normal control groups and a significant decrease in body weight when compared to the sedentary HFD group.
B. Comparison between the different studied groups according to (Adipose tissue weight, BMI, Lee index and adiposity index): The mean of adipose tissue weight, BMI, Lee index and adiposity index showed a significant difference between studied groups. In addition, exercise and combined groups showed no significant difference when compared to normal control groups and a significant decrease in adipose tissue weight, BMI, Lee index and adiposity index when compared to sedentary HFD group (Table 1).3.2 Adipose Tissue SIRT1 ProteinThere was a significant decrease in SIRT1 protein in the sedentary HFD group in comparison to the normal control group, while no significant difference in exercise and VD groups in comparison to normal control group (Table 2). In addition, there was a significant increase in SIRT1 protein in combined group in comparison to normal control group. Furthermore, there was a significant increase in SIRT1 protein in VD, exercise and combined group in comparison to sedentary HFD group and its level showed no significant difference between VD and exercise groups.
Our study showed that SIRT1 protein had a significant negative correlation with adiposity index (r = -0.415, P = .008) in total sample (n = 40) (Figure 1).

Discussion

Animal obesity brought on by a high-fat diet has been documented in a number of studies, and it is regarded as the most widely used and trustworthy model for researching obesity. In the present study, as expected, a significant increase in anthropometric indices was observed in sedentary HFD control compared to normal control (NC) rats.
In support to our study Madkhali et al proved that, when compared to rats fed a standard diet (SD), rats fed an HFD for 12 weeks had significantly higher body weights, BMIs, Lee indices, and food intake.¹¹ Furthermore, Yang et al found that the body weight, adiposity index were significantly increased in HFD group.¹²
Contradictory to our result Roza et al discovered that, in contrast to age-matched normal diet (ND) rats, the female HFD-treated animals displayed a decrease in body weight during the follow-up (8-week) period, even with higher calorie intake, after the fifth week of life. This result could be due to difference in period of study and animal sex.¹³
In the current study moderate swimming exercise was effective in reducing anthropometric indices as compared to the sedentary HFD control group. Exercise interventions are beneficial for overweight or obese people in terms of lowering body weight, BMI, and body fat percentage, according to a previous meta-analysis of three studies.¹⁴ Lim et al found that anthropometric indices including weight, body fat mass (BFM), BMI, and weight circumference (WC) significantly decreased in older women following a 12-week period of aquatic exercise.¹⁵
In the current study, VD supplementation decreased the anthropometric indices as compared to sedentary HFD control group. Consistent with our study Verma et al found that VD decreases body weight, various adipose tissues weight, BMI, Lee index, as compared to HFD-treated rats.⁵ Sergeev verified that a decrease in the weight of white adipose tissue is correlated with an increase in VD3 intake in a mouse model of induced obesity.¹⁶
A contradictory study by Salehpour et al on 85 women with BMI ≥ 25 kg/m2 showed that VD supplementation (VD3 supplement tablet of 25 μg/d) for 12 weeks did not affect body weight, BMI, and percentage body fat (PBF), this could be due to low dose of VD used in the study.¹⁷
In the current study, the combined group showed a significant reduction in anthropometric indices when compared to the sedentary HFD control group. Kim et al showed that VD supplementation and exercise attenuated body weight gain, abdominal adiposity, and non-alcohol fatty liver (NAFLD) in rats following induction of obesity.¹⁸ Salarinia et al demonstrated that in women with type 2 diabetes, exercise and VD supplementation had positive synergistic effects on the cardiometabolic profile. The 12-week intervention with exercise plus VD resulted in significant reductions in body weight, BMI and body fat mass.¹⁹
In the present study, HFD caused a significant reduction in adipose tissue SIRT1 protein level as compared to NC group. In addition SIRT1 showed significant negative correlation with adiposity index. Consistent to our study, Wei et al discovered that the SIRT1 gene and protein expression are decreased in white adipose tissue (WAT) and brown adipose tissue (BAT) upon the administration of a high-fat non-ketotic diet.²⁰ Another study investigated whether HFD affects the levels of SIRT1. The results of the quantitative RT-PCR analysis of RNA extracted from WAT revealed that mice given an HFD had two times lower levels of SIRT1 mRNA.³
In the current study, exercise and VD showed a significant elevation in SIRT1 protein level in AT as compared to sedentary HFD control group. Liu et al demonstrated that moderate aerobic exercise had positive effects on the liver and kidney functions of diabetic mice. Elevated acetylation of nuclear factor κ (NF-κB) is linked to decreased SIRT1 expression, while SIRT1 expression in the kidney and liver is restored during exercise, which suppresses NF-κB activity.²¹ In addition, Vargas et al stated that physical activity can boost oxidative metabolism, mitochondrial biogenesis, and SIRT1 expression in skeletal muscles.²²
VD up-regulate SIRT1 protein expression through up-regulation of adiponectin and consequent activation of activated mitogen phosphokinase (AMPK).²³ Safarpour et al showed that VD supplementation for 8 weeks increases the serum level of SIRT1 compared to placebo.²⁴ In addition, Chang et al suggested that VD supplementation may help to improve the increased intramyocellular fat deposition and related muscle mitochondrial changes that come with obesity, while also increasing the activity of AMPK and SIRT1.²⁵

Conclusion

HFD induced obesity in rats resulted in abnormal anthropometric parameters. Furthermore, HFD induced a significant decrease in SIRT1 protein in adipose tissue compared to normal rats. In addition, treatment of HFD by exercise or combined treatment (VD+exercise) normalized the anthropometric abnormalities in the treated groups versus sedentary HFD. This treatment also increases SIRT1 protein in adipose tissue of rats.

Declarations

References

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