The combined effect of high-intensity interval training along with vitamin D3 supplementation on mitophagy factors in heart tissue of rats induced to type 2 diabetes

Document Type : Original Article

Authors

1 Postdoctoral Researcher, Department of Physical Education and Sports Sciences, Faculty of Physical Education and Sports Sciences, University of Kurdistan, Kurdistan, Iran.

2 Associate Professor at Department of Physical Education and Sports Sciences, Faculty of Physical Education and Sports Sciences, University of Kurdistan, Kurdistan, Iran.

Abstract

Extended abstract
Background and Aim: Type 2 diabetes mellitus (T2DM) is a prevalent metabolic disorder characterized by chronic hyperglycemia that disrupts cellular and tissue homeostasis. One major consequence of T2DM is impaired mitochondrial function, which is essential for energy production and oxidative regulation, particularly in the high-energy-demand cardiac tissue, where dysfunction can cause diabetic cardiomyopathy. Mitophagy, a selective autophagy process that removes damaged mitochondria, is crucial for maintaining mitochondrial health. Key regulatory proteins such as PINK1 and Parkin initiate mitophagy, and their downregulation in T2DM leads to mitochondrial accumulation and oxidative stress. Non-pharmacological interventions, such as high-intensity interval training (HIIT) and vitamin D3 supplementation, have shown promise in improving mitochondrial function. HIIT enhances metabolism and aerobic capacity, while vitamin D3, often deficient in T2DM patients, offers anti-inflammatory and mitochondrial protective effects. The present study aimed to investigate the combined effects of HIIT and vitamin D3 supplementation on PINK1 and Parkin expression in cardiac tissue of T2DM Wistar rats, along with associated metabolic and functional outcomes.
Materials and Methods: In this experimental study, forty male Wistar rats (8–10 weeks old; 200–250 g) were obtained from the university’s animal research center. The animals were housed under standard laboratory conditions (temperature of 22±2°C, humidity of 50-60%, and a 12:12-hour light/dark cycle) with free access to water and food. To induce T2DM, the rats were initially fed a high-fat diet (containing 40% fat, 40% carbohydrates, and 20% protein) for 8 weeks. Subsequently, Streptozotocin (STZ) was administered intraperitoneally at a dose of 35 mg per kilogram of body weight. Diabetes was confirmed 72 hours later by measuring a fasting blood glucose level exceeding 250 mg/dL. The diabetic rats were randomly assigned to 5 groups (n=8 per group): a normal control group (NC), a diabetic control group (DC), a diabetic + high-intensity interval training group (D+HIIT), a diabetic + vitamin D3 injection group (D+VD3), and a diabetic combined intervention group (D+HIIT+VD3). The HIIT protocol was performed for 8 weeks, with 5 sessions per week. Each session consisted of a 5-minute warm-up at 10 m/min, followed by 10 intervals of 4-minute high-intensity running at 85-90% of maximum running speed (approximately 20-25 m/min) interspersed with 2-minute active recovery periods at 10 m/min, and concluded with a 5-minute cool-down. The maximum running speed (Vmax) was re-evaluated and adjusted biweekly. Vitamin D3 (10,000 IU/kg) was injected subcutaneously weekly. The D+HIIT+VD3 group received both interventions. Forty-eight hours after the final intervention, rats were anesthetized (Ketamine 80 mg/kg, Xylazine 10 mg/kg), and left ventricular tissue was collected and stored at -80°C. Parkin and PINK1 levels were measured via Western blot. Serum glucose, insulin, vitamin D3, HOMA-IR, body weight, and time to exhaustion (TTE) were assessed. Statistical analyses were conducted using one-way ANOVA with Bonferroni post-hoc tests and two-way ANOVA for TTE. Statistical significance was defined as p<0.05.
Findings: The induction of T2DM resulted in a significant reduction in the cardiac expression of the mitophagy-related proteins Parkin and PINK1. A one-way ANOVA demonstrated statistically significant differences in the levels of both Parkin (F=77.29, η²=0.89, p=0.001) and PINK1 (F=58.31, η²=0.86, p=0.001) across the experimental groups. Post-hoc Bonferroni analysis specified that Parkin levels in the DC group were 50% lower than those in the NC group (p=0.03). In contrast, all intervention groups exhibited a substantial upregulation of Parkin compared to the DC group. The D+HIIT, D+VD3, and D+HIIT+VD3 groups showed increases of 344%, 394%, and 496%, respectively (p=0.001 for all). Furthermore, the combined intervention group (D+HIIT+VD3) demonstrated Parkin levels that were 34.23% and 20.65% higher than the D+HIIT and D+VD3 groups, respectively (p=0.001), while no significant difference was observed between the D+HIIT and D+VD3 groups (p=0.97) (Table 1).
A similar pattern was observed for PINK1. Expression in the DC group were 36% lower than in the NC group (p=0.01). The interventions successfully elevated PINK1 levels, with the D+HIIT, D+VD3, and D+HIIT+VD3 groups showing increases of 143.75%, 75%, and 240.63% over the DC group, respectively (p=0.001). The combined therapy group (D+HIIT+VD3) also displayed significantly higher PINK1 levels than both the D+HIIT (39.74% higher, p=0.001) and D+VD3 (94.64% higher, p=0.001) groups. Additionally, the D+HIIT group had 39.29% higher PINK1 levels than the D+VD3 group (p=0.002). Concerning glycemic control, one-way ANOVA indicated significant inter-group differences in serum glucose (F=221.5, η²=0.96, p=0.001). Serum glucose in the DC group was 396.90% higher than in the NC group (p=0.001). All intervention groups showed significant reductions compared to the DC group, with the D+HIIT, D+VD3, and D+HIIT+VD3 groups exhibiting 27.56%, 18.83%, and 37.31% lower glucose levels, respectively (p=0.001). The most pronounced effect was seen in the D+HIIT+VD3 group, which had significantly lower glucose than both the D+HIIT (13.51% lower) and D+VD3 (22.76% lower) groups (p=0.001). Similarly, HOMA-IR was 110% higher in DC than NC (p=0.001), but reduced by 66.67%, 47.92%, and 72.92% in D+HIIT, D+VD3, and D+HIIT+VD3 respectively (p=0.001). D+HIIT and D+HIIT+VD3 had 36% and 48% lower HOMA-IR than D+VD3 respectively (p=0.001). Both HIIT and vitamin D3 improved aerobic capacity in diabetic rats; combined group showed 186.39% TTE increase, surpassing vitamin D3 group (p=0.007), but not HIIT group (p=0.065).
Conclusion: The present findings demonstrate that the combination of HIIT and vitamin D3 significantly upregulates the levels of the mitophagy-related proteins Parkin and PINK1 in the cardiac tissue of T2DM rats, thereby enhancing mitochondrial protective mechanisms. This combined intervention not only mitigates T2DM-induced mitochondrial dysfunction but also improves metabolic parameters, including serum glucose, insulin resistance, and vitamin D3 levels, while enhancing functional exercise capacity (TTE) and body weight control. These findings suggest that integrated exercise and nutritional supplement strategies could serve as an effective therapeutic approach for preventing cardiometabolic complications in T2DM. Future studies are warranted to examine the long-term effects and elucidate the molecular mechanisms to better support clinical translation. Overall, this study highlights the potential of pairing HIIT with vitamin D3 supplementation as a complementary strategy in T2DM management.
Compliance with Ethical Guidelines: All experimental procedures were conducted in accordance with ethical guidelines for animal research and were approved by the Ethics Committee of the University of Kurdistan (Approval Code: IR.UOK.REC.1400.015). All steps were carried out with minimal pain and stress to the animals.
Funding: The present study received no financial support from any organization or entity.
Conflicts of Interest: The authors declare no conflicts of interest.

Keywords

References

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Journal of Practical Studies of Biosciences in Sport
Volume 13, Issue 36
December 2025
Pages 8-25

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