The effects of high intensity interval training induced H₂O₂, Nrf2 changes on antioxidants factors in type 2 diabetes

Abstract

Introduction

Nuclear factor erythroid-2-related factor 2 (Nrf2), is an inducible transcription factor that reduced in type 2 diabetes(T2D) and increases oxidative stress and then stimulating antioxidant expression. The purpose of this RCT was to investigate the effects of HIIT induced H2O2, Nrf2 changes on Glutathione Peroxidase (GPx), Glutathione Reductase (GR), Catalase (CAT) and superoxide dismutase (SOD) in T2D.

Methods

Thirty-three male patients with T2D were randomly divided in 2 groups including to 12-weeks HIIT (10 rotations for 60 seconds (10 × 60s) set in constant watt mode at a pedal cadence of 80–100 revolutions/min) or a non-exercise control group. Nrf2, H₂O₂ and GPx, GR, Cat and SOD protein were measured in response to 12 weeks HIIT.

Results

Levels of Nrf2 and H₂O₂ showed high levels in HIIT with respect to control subjects after 12 weeks’ interventions (HIIT). Fasting plasma glucose (FPG) (p = 0.001), postprandial plasma glucose (PPG) (p = 0.001), glycated hemoglobin, (HbA1c) (p = 0.041), plasma total cholesterol (Tc) (p = 0.028), plasma triglyceride (TG) (p = 0.001), Na (p = 0.001), creatine kinase (Ck) (p = 0.035), alkaline phosphatase (ALP) (p = 0.025), hematocrit (Hct) (p = 0.008) and Cortisol (p = 0.001) were statistically significant in the T2D + HIITgroup. After 12 weeks’ interventions (HIIT), control group the Plasma CAT levels (p = 0.001) were found to be higher in HIIT group compared to control group.

Conclusion

Our results provide evidence that HIIT causes an increase in oxidative stress levels, which ultimately the body responds to increased antioxidant levels. Therefore, it is suggested that these indicators (HIIT and H2O2, Nrf2) can be considered as a therapeutic target for type 2 diabetic patients.

Introduction

Type 2 Diabetes (T2D) is a condition caused by irregular release of insulin, glucose and lipid metabolism abnormalities and hyperglycemic appearance [1, 2]. T2D is a significant public health concern that is predisposed to significantly elevated cardiovascular disease and extreme nephropathy, neuropathy and retinopathy-related morbidity and mortality [3]. Previous studies showed that hyperglycemia contributes in reactive oxygen species (ROS) formation, contributing eventually to oxidative stress in a multitude of organisms [4]. The redox imbalance that acts a central role in the development and progression of T2D and its complications is caused by a rise in the amount of ROS or the lack of an acceptable possible contributor from endogenous antioxidants [5].

In T2D, oxidative stress is implicated in the development of pancreatic b-cell disorders and insulin resistance in organs or tissues, such as the liver, adipose tissues, and skeletal muscle. Nuclear factor-erythroid-related factor 2 (NRF2) plays a crucial role. In pancreatic b cells, stimulation of NRF2 reduces ROS levels and inhibits inflammation and serves to protect pancreatic b cells. Moreover, through monitoring the FGF21 mechanism, gluconeogenesis, and AMPK signals, NRF2 influences insulin resistance [6].

Evidence has been indicated that NRF2 serves a crucial function in the modulation of detoxification and antioxidant stress activity of enzymes via Nrf2-mediated gene expression. NRF2 protect the human body against environmental stress. It has also been recently realized that NRF2 operates to preserve metabolic homeostasis. [6]. Antioxidant reaction and detoxification processes directly lead to the defense of pancreatic b-cells regulated by NRF2. Furthermore, NRF2 generates anti-inflammatory features, retains mechanisms of protein breakdown, and inhibits pancreatic b cells from stress-mediated cell transformation [6].

Evidence shows that the energy expenditure of the muscular system raises oxygen intake to a degree 10–20 times beyond that of the remainder during the method of physical exercise; this causes a rise in the amount of ROS within the muscle fibers [7]. Exercise-mediated ROS has an impact on signaling pathways like Nuclear Factor xB (NFF) and Mitogen-Activated Protein Kinase (MAPK) which, when activated, induces antioxidant caused by the mitochondrial superoxide dismutase (MtSOD) and mitochondrial glutathione peroxidase enzymes, furthermore as gas Synthase (NOS) and better Catalase (Cat) function [8]. The stimulation of the enzymatic endogenous antioxidant system (EAS), that modulates enzymes including Glutathione Peroxidase (GPx), Glutathione Reductase (GR), Catalase (CAT), and non-enzymatic EAS, including glutathione and alpha lipoic acid, among others, is induced in thanks to the increase in ROS throughout regular exercise, particularly when it’s not exhaustive [9].

The function of H2O2 was assessed not only as an OS predictor, but instead as a messenger transmitting redox equilibrium signals at various cellular sites. By way of enzymatic processes or at the transcriptional level, modulation of the redox equilibrium may be impaired. H2O2 activates the function of a wide range of transcriptional factors in mammals, including AP-1, Nrf2, CREB, HSF-1, HIF-1, TP53, NF-kB, NOTCH, SP1, and SCREB-1 [10, 11]. Redox balance regulates a wide range of basic biological processes in biological systems, involving inflammation, muscle contraction, proliferation, apoptosis, tumor formation, circadian rhythm, and aging, among many others. Because of the influence of physical activity and bioactive substances, experiments have shown the induction of Nrf2 and the antioxidant cytoprotector method [11, 12].

In addition, it has been reported H2O2 increases during the cellular differentiation of myocytes as a result of the metabolic requirements needed by the mechanism, which not only acts as a signal, but also regulates the development of glutathione and the decreased index of glutathione (GSH)/oxidized glutathione (GSSG) by activating the Nrf2-GCL/GR-GSH pathway [13].

While the health effects of daily exercise have been well illustrated, few individuals obey these physical activity guidelines. “Lack of time” is sometimes listed as one of the predominant issues relative to daily exercise [14]. Aerobic training was therefore planned to be high-intensity interval training (HIIT). Latest findings have demonstrated the effects of low-volume HIIT training in contrast with continuous and moderate-intensity aerobic activity [15]. In this respect, in rats with myocardial infarction (MI), Kai-lo et al. observed that eight weeks of HIIT decreased oxidative pressure through rising levels of SOD and GPX. A dramatic rise in the production of anti-oxidant SOD and GPX enzymes in lymphocytes following HIIT was found by Fisher et al., but this rise was not significant [16, 17]. Marcel Pimenenta et al., demonstrated that in ovariectomized rats, HIIT decreases oxidative stress in the skeletal muscle [18] .

It is therefore difficult to create a link between exercise training and ROS, because the types, intensity and length of the training, and also the clinical context of the person, must be taken into consideration. Nevertheless, while the antioxidant protector mechanism complex mediated by Nrf2 is not yet fully understood, it is known to be effective in responding to stimuli such as exercise. The goal of this randomized controlled trial was to examine the effect 12-week intervention (HIIT) on H2O2 and Nrf2 and antioxidant (GPx, GR and CAT) responses.

Methods

Subjects

The cohort composed of 33 male patients with T2D (in two groups; first group, HIIT; second group, Control). At the outset of the research, both HIIT and control groups were not pursuing any physical fitness regimen. The Department of Physical Education and Sports Sciences, Isfahan University, enrolled all the participants. Participants were fully aware of the research and were requested to fill a consent forms document. The research was accepted by ethical committee of Isfahan college of Medical Sciences. The inclusion criterion for the choice of diabetic patients is at least 5 years of disease duration and metformin treatment alone or in combination with repaglinide or gliclazide. Existence of physiological imbalances (HbA1c ≥ 7.5), elevated neuropathy, high-level vasculopathy, cutaneous ulcers, and insulin therapy were withdrawal requirements.

Visiting evaluation

Once the consent forms were registered by volunteers, a medical records checklist and the updated Historical Recreation Physical Activity survey were performed for lifelong physical exercise measurement. Height, weight, waist circumference and resting blood pressure were assessed and a 12-lead supine resting EKG was collected to rule out any heart irregularities which must prohibit a peak aerobic ability exercise from being conducted by individuals. The body composition was evaluated using a Lange skinfold caliper with a seven-site skinfold. Utilizing standardized formulas for men and women defined by age, body density was determined. For measurements of final excess weight, the body fat percent formula by Siri et al. was used. In order to avoid intertester heterogeneity, all skinfold measurements were conducted by the same investigator. Restriction conditions is as follows for all participants, HIIT and control groups: obesity (BMI ≈ 25–29.9), major coronary disorders (ischemic cardiac dysfunction or heart problems), chronic obstructive pulmonary disease, serious hypertension (diastolic blood pressure≈ 88, systolic blood pressure 155), In the 6 months preceding, smoke patterns, substance misuse, RX therapies, or prognostics. Following consent form, Blood samples (EDTA or heparin) were obtained shortly before and simultaneously after the 12-month training program. The HIIT group performed the interventions for 12 months. The control group did not engage in regular exercise during this time.

Evaluation parameters of metabolic

Fasting plasma glucose (FPG) serum biochemistry parameters, postprandial plasma glucose, total cholesterol, high-density lipoprotein cholesterol, plasma triglycerides, total protein, albumin, urate, potassium, sodium, creatinine, urea, aminotransferase aspartate, aminotransferase alanine, g-glutamyl transferase, creatine kinase, and alkaline phosphatase is determined with colorimetric methods, using commercial kits (Abbott, Abbott Park, Ill., USA), with the Architect c8000 analyzer (Abbott). The chemiluminescence assay (Diagnostic Products Corporation, Los Angeles, Calif., USA; minimum detection value 15 pg·mL-1) determined cortisol and thyroid stimulating hormones. The hemograms were immediately carried out on the Coulter STKS apparatus (Coulter Electronics Inc., Hialeah, Fla., USA).

Concentrations of Nrf2 blood circulation and H2O2 levels

In compliance with the recommended schedule, circulatory concentrations of plasma Nrf2 were calculated using the Human Nuclear Factor Erythroid 2-related factor 2 (NFE2L2) ELISA package (Cusabio, MD, USA). Briefly, samples together with the set criteria were incubated for 2 hr. in pre-coated 96-well plate accompanied by Biotin-antibody incubation for 1 hr. After incubation all the wells were washed and incubated with HRP-avidin for 1 hr. followed by TMB substrate for 15 min in the dark. The reaction was stopped by addition of stop solution and plate was read at 450 nm with wavelength correction at 540 nm using microplate reader (Infnite 1000, Tecan, Switzerland). H2O2 measurement: 48 hours before the beginning of the first training program and again after 12 weeks of high intensity interval training, 5 ml of blood was extracted from the Antecubital vein by the researchers. Individuals were fasting for 12 hours. Samples taken were subsequently sent to the laboratory for plasma centrifuge separation and frozen at 70 °C. Samples were sent to the medical diagnostic laboratory for plasma H2O2 level examination (Eliza kit, Glory Company, USA) and calculation with a precision of 1 micro mole/litter.

Concentrations of glutathione peroxidase (GPx), glutathione reductase (GR) superoxide dismutase (SOD), and catalase (Cat)

For analysis of antioxidants, 10 mL of EDTA, sodium heparin and serum were obtained from blood samples. Both serum tests were allowed to coagulate and then, at 4 °C for 15 min at 2000 g, serum and plasma were isolated by centrifugation. For salivary NO, lipid peroxidation product, sulfhydryl groups, and CAT activity, the samples were stored at −80 °C until analysis; all other parameters were determined on the day of collection. All samples were analyzed in duplicate and then averaged. For GPx, GR, and CAT, the samples were stored at −80 °C before analysis; all other parameters were calculated on the day of processing. All samples were duplicately analyzed and then averaged. After a reduction in absorbance at λ = 340 nm for 3 minutes attributed to NADPH oxidation to NADP+, the catalytic activity of GR was assessed in the presence of GSSG. The coefficient of molar extinction is ε = 6.22 × 103 cm − 1 M − 1. Catalase activity was calculated by calculating a reduction in the level of hydrogen peroxide (10 mM) in a newly formulated solution at 240 nm.20 Glutathione peroxidase (commercial ELISA kit (Stabiofarm, China)) was tested at 340 nm through tertbutyl hydroperoxide inactivation by the Glutathione/NADPH/ Glutathione Reductase method. Glutathione Reductase was calculated in a reaction medium containing buffered DPTA (diethylenetriaminepentaacetic acid) and 1 mM oxidized glutathione at 340 nm through the oxidation rate of NADPH.

HIIT program (12 weeks)

Study participants performed HIIT exercises three days (Saturday, Monday and Wednesday) a week. The training sessions consisted of three sessions per week for 12 weeks (3 × 12 = 36 sessions). The type of HIIT exercise included activity on the ergometer cycle (Monark 828E, made in Sweden). Each session was performed by warming up on an ergometer cycling for 3 minutes. Pedal intensity was considered when warming up to 50 watts. After warming up, the participants performed the main activity. The main activity consisted of 10 rotations for 60 seconds (10 × 60s (set in constant watt mode at a pedal cadence of 80–100 revolutions/min. Individual workloads were selected to elicit a heart rate of 90% HRmax during the intervals. The rest time between intervals was 60s pedaling at 50 watts. After completing 10 intervals and resting between them, participants also completed the cooling activity for 3 minutes by pedaling at 50 watts. Consequently, under an overall time commitment of 75 minute per week, the training regimen included an overall of 30 minutes of high-intensity workout. Checking by Post. Information was acquired over a 24-h duration beginning 48 hours after the final training program (Fig. 1). The food was regulated to be the same as pre-training.

Fig. 1.

Study design. Participants (T2D) were randomized to the 12 weeks HIIT intervention or without HIIT. HIIT training program was showed in this chart. Data were collected for a 24–72-h period starting and 48 h after the final training session

Results

The mean and SD of the anthropometric data of the volunteers is seen in Table 1 before the experiment. The features of the treatment assignment such as age, diagnostic period, BMI, and gender of HIIT group were no significantly different between the two groups. For all patient characteristics tested, all participants had identical parameters at the time of study admission.

Table 1.

Physiological and Anthropometrics features for participants in study

Characteristic	HIIT	control	P
n (male)	16	17	–
Age (years)	57.2 ± 2.5	58.4 ± 3.3	0.517
Body weight (kg)	90.2 ± 7.9	87.7 ± 6.0	0.423
BMI (kg/m2)	27.6 ± 2.2	28.2 ± 1.6	0.635
Fasting plasma glucose (mg·dL–1)	163.32 ± 51.37	159.17 ± 43.43	0279
Fasting serum insulin (pmol/l)	90.1 ± 12.5	88.3 ± 14.7	0.675
HbA1c (%)	7.66 ± 1.15	7.60 ± 1.81	0.863
Plasma triacylglycerols (mg·dL–1)	157.90 ± 86.62	155.52 ± 72.15	0.801
GDR, basal (mg min-1 m-2)	71.6 ± 4.2	72.4 ± 5.2	0.625
GDR, insulin (mg min-1 m-2)	165.5 ± 22.3	162.3 ± 18.9	0.201
Diabetes duration (years)	5.2 ± 0.2	5.5 ± 0.5	0.855

Glucose Disposal Rates (GDR), Data are presented as mean ± SEM. * P < 0.05 considered statistically significant

For certain clinical features tested in Table 2, fasting plasma glucose (FPG) (p = 0.001), postprandial plasma glucose (PPG) (p = 0.001), glycated hemoglobin, (HbA1c) (p = 0.041), plasma total cholesterol (Tc) (p = 0.028), plasma triglyceride (TG) (p = 0.001), Na (p = 0.001), creatine kinase (Ck) (p = 0.035), alkaline phosphatase (ALP) (p = 0.025), hematocrit (Hct) (p = 0.008) and Cortisol (p = 0.001) were statistically significant (post vs pre) in the HIIT group, but other factors were not statistically significant in the HIIT and control groups.

Table 2.

Medical variables at baseline and after Post12week HIIT intervention

variables	Sub-variables	HIIT			Control
		Baseline	Post12week	p value	Baseline	Post12week	p value
Glycemic parameters	FPG (mg·dL–1)	163.32 ± 51.37	131.28 ± 43.11*	0.001*	159.17 ± 43.43	157.28 ± 43.11	0.765
	PPG (mg·dL–1)	180.33 ± 59.03	146.76 ± 65.73*	0.001*	176.29 ± 43.15	178.40 ± 35.19	0.664
	HbA1c (%)	7.66 ± 1.15	7.12 ± 1.37*	0.021*	7.60 ± 1.81	7.57 ± 1.39	0.834
Lipid profile	TC (mg·dL–1)	176.09 ± 43.71	153.27 ± 54.36*	0.001*	175.64 ± 23.42	176.29 ± 35.44	0.845
	HDL-Chl (mg·dL–1)	50.42 ± 6.25	47.93 ± 7.04	0.452	52.29 ± 7.83	51.12 ± 8.15	0.769
	LDL-Chl (mg·dL–1)	108.77 ± 20.03	104.25 ± 36.18	0.649	107.37 ± 16.97	108.36 ± 20.37	0.902
	TG (mg·dL–1)	157.90 ± 86.62	127.75 ± 68.71*	0.001*	155.52 ± 72.15	154.71 ± 80.46	0.712
Kidney function markers	Na (mEq·L–1)	145.45 ± 3.25	122.46 ± 23.12*	0.001*	147.23 ± 15.37	145.81 ± 17.83	0.693
	K (mEq·L–1)	4.55 ± 1.04	4.40 ± 1.37	0.824	4.49 ± 1.08	4.50 ± 1.25	0.866
	Urea (mg·dL–1)	37.23 ± 9.91	36.9 ± 6.14	0.796	37.17 ± 8.67	37.73 ± 8.42	0.648
	PCr (mg·dL–1)	0.93 ± 0.09	0.91 ± 0.12	0.826	0.93 ± 0.25	0.92 ± 0.46	0.798
Liver function markers	AST (U·L–1)	13.55 ± 6.76	13.29 ± 7.63	0.834	14.01 ± 8.22	13.92 ± 7.29	0.692
	ALT (U·L–1)	32.46 ± 25.13	31.4 ± 53.03	0.789	33.08 ± 15.17	32.9 ± 26.91	0.792
	γ-GT (U·L–1)	33.16 ± 25.67	30.12 ± 35.11	0.124	32.57 ± 48.38	33.61 ± 53.72	0.802
	CK (U·L–1)	220.12 ± 153.03	114.20 ± 126.17*	0.001*	218.31 ± 99.65	222.15 ± 100.60	0.697
	ALP (U·L–1)	111.49 ± 26.73	103.56 ± 27.73*	0.026*	109.55 ± 36.27	108.80 ± 45.53	0.795
Hematological profile	RBC (×106·mm–3)	5.01 ± 1.03	4.90 ± 1.15	0.297	5.10 ± 1.22	5.03 ± 2.01	0.625
	Hgb (g%)	14.73 ± 2.05	14.43 ± 2.11	0.756	13.96 ± 2.67	14.26 ± 2.52	0.789
	Hct (%)	45.07 ± 3.99	40.47 ± 4.27*	0.001*	43.44 ± 5.08	42.73 ± 4.90	0.802
Hormones	TSH (mU·L–1)	1.50 ± 1.19	1.55 ± 1.73	0.634	1.49 ± 0.87	1.52 ± 0.98	0.630
	Cortisol (μg·dL–1)	14.17 ± 4.23	10.17 ± 4.46*	0.001*	13.73 ± 4.34	13.60 ± 4.76	0.856

*=P < 0.05 considered statistically significant. Values are expressed as means ± SD. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TC, plasma total cholesterol; CK, creatine kinase; FPG, fasting plasma glucose; g-GT, g-glutamyl transferase; HbA1c, hemoglobin A1c; Hct, hematocrit; HDL, high-density lipoprotein; Hgb, hemoglobin; LDL, low-density lipoprotein; PCr, plasma creatinine; PPG, postprandial plasma glucose; RBC, red blood cells; TG, plasma triglyceride; TSH, thyroid-stimulating hormone

Table 3 shows that statistically significant correlated the levels of Nrf2 alterations with CAT (r = 0.496, p = 0.017), SOD (r = 0.529, p = 0.001), GR (r = 0.428, p = 0.032), GSH (r = 0.623, p = 0.001) and H2O2(r = 0.553, p = 0.001) in HIIT. Also, the data indicated that statistically significant correlated the levels of H2O2 alterations with CAT (r = 0.431, p = 0.031), SOD (r = 0.458, p = 0.020), GR (r = 0.419, p = 0.038), GSH (r = 0.536, p = 0.001) in HIIT.

Table 3.

Relationship between indicators in the post-test stage

Correlation	CAT	SOD	GR	GSH-Px	H2O2
Nrf2	0.496*	0.529*	0.428*	0.623*	0.553*
H2O2	0.431*	0.485*	0.419*	0.536*

P < 0.05 considered statistically significant

After 12 weeks interventions (HIIT), Fig. 2A depicts the Plasma CAT levels (p = 0.001) were found to be higher in HIIT subjects when compared to control subjects. Also, Fig. 2B, C and D demonstrated the Plasma GR levels (p = 0.001), GSH levels (p = 0.001) and SOD levels (p = 0.001) were found to be higher in HIITsubjects when compared to control subjects.

Fig. 2.

Exercise Training effects on (A) catalase (CAT); (B) Glutathione Reductase (GR); (C) glutathione peroxidase (GSH-Px) (D) superoxide dismutase (SOD) activity. P < 0.05 considered statistically significant

Levels of Nrf2 and H2O2 in Fig. 3A and B showed high levels in HIIT with respect to control subjects after 12 weeks interventions (HIIT).

Fig. 3.

Exercise Training effects on (A) Nuclear factor erythroid 2-related factor 2 (Nrf2)); (B) hydrogen peroxide(H2O2) P < 0.05 considered statistically significant

Discussion

Workouts is one of the key effective resources used to boost fitness. Plenty of the beneficial effects focus on pathways for triggering exercise responses based on redox-dependent cell signaling. ROS and other electrophiles produced by workout serves as essential signaling molecules that contribute to strengthen physiological role and safe modifications [9]. Indeed, the advantageous effect of exercise on metabolic parameters and its effects on the production of reactive oxygen species is largely dependent on the method, speed, length and energy demands of the exercise, oxygen intake levels, and the mechanical stress on the tissues [19]. In this study, we examined the effect of high intensity interval training induced Nrf2 and H2O2 on concentrations of GPx, GR, SOD and Cat responses in type 2 diabetes. This research evaluated the hypothesis that high intensity interval training (repeated high and low workout periods) would modify T2D Nrf2 and H2O2 changes? To our knowledge, this is the first randomized control study to measure reactions to chronic exercise training (Especially HIIT) in T2D men, as well as the impact of HIIT induced H2O2 and Nrf2 responses on antioxidants.

We found that in the HIIT group, after 12 weeks of activity, the Nrf2 and H2O2 activity were upregulated. Thus, the nitrogen index has almost doubled. While the hydrogen index shows an increase of almost 50%. The role of Nrf2 in the response to OS and the physiological adaptations exhibited during physical training is becoming increasingly clear [11]. In this regard, results of Done et al. [20] study demonstrated that independent of exercise intensity, aerobic exercise activates nuclear Nrf2, although that higher intensity exercise increases glutathione reductase activity. The effect of exercise mode and duration on Nrf2 signaling, as well as the role of intensity in compromised populations, should be investigated in future studies [19]. However, our data support our theory that high-intensity interval exercise would result in a higher Nrf2 response. It’s also likely that the intensity of training has a bigger impact on whole-cell Nrf2 expression than the exercise protocol. Contrary to Duan et al. study, which noted that training duration had a greater effect on this index [21]. Exercise training is a powerful stimulus that supports physiological and metabolic modifications that allow you to deal with the periodic rise in physical work demands, such as alterations in plasticity and cellular remodeling [22]. Modifications like these are the consequence of a complicated interaction among regulator factors and signaling pathways [21]. The endogenous antioxidant defense systems, which are largely controlled by Nrf2, are induced by the OS produced by physical exercise [23]. In this regard, many studies that have demonstrated the induction of Nrf2 and the antioxidant cytoprotector system due to the effect of physical exercise and bioactive compounds [8, 23, 24]. In fact, the nrf2 response to oxidative stress and exercise training will be apparent with more studies in the future [22].

In essence, such changes are the product of a dynamic interaction between regulator variables and mechanisms of signaling. The OS produced by physical activity stimulates endogenous antioxidant protection mechanisms which are largely managed by Nrf2 themselves [23]. Redox effector factor1 (Ref-1) is one of the factors engaged in the antioxidant adaptive mechanism, because it translocates from the cytoplasm to the mitochondria or the nucleus in the presence of OS, particularly H2O2 [11]. This factor has the potential to control the transcription of antioxidant genes such as Nrf2 and others [6]. Its primary function is to bind to oxidized transcription factors (Nrf2, Hypoxia Inducible Factor1, and p53); in the case of Nrf2, the DNA binding domain (Cys-514) contains a cysteine amino acid, which is the Ref-1 binding site for mediating the reaction to the OS [20]. Taken together, the data from our study demonstrate that regular exercise (HIIT) upregulates Nrf2 and H2O2 abundance. To date there have been no exercise intervention studies conducted on Nrf2 and H2O2 in humans. Finally, the evidence suggests that many of the advantages of exercise are mediated by redox activation of Nrf2 and H2O2 signaling.

Our data of the present study showed that after 12 weeks of exercise intervention, the CAT index in the HIIT group with exercise showed a significant increase compared to the control group. However, there was a doubling of this index in the post-test phase of the HIIT group. This increase was also observed in the post-test phase compared to the control group. Regarding glutathione reductase index, the findings of this study showed that after 12 weeks of HIIT, a threefold increase in GR was observed. in the post-test stage these changes were also observed in HIIT group Compared with control group. The findings of the present study confirmed that GPx index showed a significant increase compared to the pretest in HIIT group. In the post-test phase, the HIIT group showed a significant increase compared to the control group, so that this increase showed almost 150%. Regarding the superoxide dismutase index, the findings showed that this index showed a greater increase compared to other indices, so that this increase was more than three times compared to the pre-test. These results showed that the HIIT group showed a 300% increase compared to the control group in the post-test phase. Indeed, the results of the present study showed that an increase in HIIT group induced the levels of Nrf2 and H2O2 led to an increase in antioxidant (CAT, GR, GPx, SOD) production. Herein Pittaluga et al. [4] reveals for the first time that moderate exercise training is not only effective in boosting redox homeostasis by increasing endogenous antioxidant defenses in both stable and diabetic patients, but also in lowering susceptibility to oxidative DNA damage and lipid peroxidation levels in diabetic patients [4]. Poblete Aro et al. [25] pointed out that Despite the fact that both training groups (HIIT compared to moderate intensity continuous training) show improvements over markers of lipid profile and fitness, high intensity interval training has shown to be more effective in the normalization of oxidative stress, impacting positively on the concentration of pro-oxidant markers and antioxidants in patients with T2D [25]. Although Done et al. [20] have shown that aerobic exercise activates Nrf2 in young men, irrespective of training intensity, that high intensity exercise demonstrated a greater effect on increasing GR activity which could indicate improved redox potential [19]. Numerous studies have been performed on the effect of regular (low intensity) exercise on NRF2 signaling and antioxidants. Asghar et al. [26] showed that after 6 weeks of training (6-weeks Treadmill; 5 days/Wk, 60 min/day, 10 m/ min, 15% grade.) in rat (Fischer), the NRF2 increased and led to an increase in SOD [26]. George et al. [27] indicated that 16 weeks of training (12-weeks Treadmill; 5 days/wk., 60 min/day, 12 m/ min, 15% grade) led to NRF2 and antioxidant increase [27]. Kumar et al. reported that after 15-days of swimming (6 days/wk., 30–60 min/day, swim to exhaustion on final exercise day), an increase in SOD and NRF2 was observed [28]. [29] indicated that 6-week treadmill (6-weeks Treadmill; 7 days/wk., 50 min/day, 10 m/min, 7% grade, the NRF2 increased, increased in GSR, G6PD, CAT) led to an increase in NRF2, GSR, G6PD and CAT. Sun et al. showed that 8-week treadmill (8-weeks Treadmill; 6 days/wk., 60 min/day, 20 m/ min, 5% grade/ the NRF2 increased/ SOD2, CAT increased) led to an increase in NRF2, SOD2 and CAT [30]; Other studies have shown that NRF2 increased after various exercise training. In most of these studies, the research sample was rat [31–35]. Based on the results of mentioned studies, our data confirm the hypothesis that regular exercise increases the production of NRF2 content. However, it seems that this increase is induced by high-intensity training. As many studies have confirmed in T2D, oxidative stress is involved in progression of pancreatic b cell dysfunction and insulin resistance in peripheral organs or tissues, such as liver, adipose tissues, and skeletal muscle [36]. NRF2 activation decreases ROS levels in pancreatic b cells and suppresses inflammation, and protects pancreatic b cells. In addition, NRF2 improves insulin resistance by regulating FGF211 pathway, gluconeogenesis, and AMPK2 signals [11, 36]. Barari et al. [37] showed that neither saffron extract (SE) nor aerobic training (AT) had a significant effect on GPX levels [37]. Contrary to the results of the present study, Afzalpour et al. investigated the effect of acute resistance and aerobic exercises on GPX levels in healthy active men and found no significant differences in GPX activity between the exercise and control groups following resistance and aerobic exercises [38]. In Barari et al. study, AT+SE led to a significant increase in the amount of GPX in type 2 diabetic men similar to our study [37,42], Piri et al. [39] investigated the effect of SE and AT+SE on concentrations of GPX in streptozotocin-induced diabetic rats and reported that both treatments led to significant increases in GPX activity [39]. Conversely, the results of a study by Kalkhoran et al. [40] on diabetic male rats showed that SE,AT, and AT+SE led to significant decreases in GPX [40]. The diabetic state is associated with decreased Nrf2 content and activity, which plays a key role in diabetic complications. NRF2 signaling dysfunction is implicated in T2D [36, 41]. Evidence indicates that multiple aspects of the Nrf2 signaling pathway are dysregulated in T2D, including: 1) increased expression of the Keap1, 2) increased GSK-3 activity, 3) upregulated nuclear Fyn expression, and 4) possibly decreased acetylation via increased HDAC activity [11, 36]. The activation of the enzymatic Endogenous Antioxidant System (EAS), which regulates enzymes like GPx, GR, and CAT, as well as the non-enzymatic EAS, which includes glutathione and alpha lipoic acid, is caused in response to an increase in ROS during physical training, particularly when it is not thorough. Studies have shown that Exercise-induced OS exerts an impact on signaling pathways such as Nuclear Factor κB (NFκB) and Mitogen-Activated Protein Kinase (MAPK) that, on being activated, the antioxidant response is induced of the Mitochondrial enzymes Super Oxide Dismutase (MtSOD) and GPx, as well as that of Nitric Oxide Synthase (NOS) and greater CAT activity [31–35]. According to the findings of this study, it can be suggested that HIIT increases the production of Nrf2 and H2O2 in large quantities. Therefore, it seems that increasing the intensity of exercise increases oxygen consumption and decreases ATP. Continuing to increase production of AMP, production of PGC-1α increased, then production of Nrf2 increased which production of antioxidants (CAT, GR, GPx and SOD) increased. Also, it can be suggested that high-intensity interval training increases the production of H2O2mitochondria due to the increase in oxygen consumption, which leads to an increase in Nrf2 production. Increasing the production of Nrf2 by affecting nuclear factors increases the production of antioxidant responses. Therefore, it can be said that high-intensity exercise increases antioxidant factors in type 2 diabetic patients, which can weaken many of the abnormalities produced by this disease. According to this medical variable at baseline and after post 12 week HIIT intervention showed that in HIIT group, compared to the control group, most indicators improved. However, in HIIT significant decrease was observed in FPG, PPG, HbA1c, TG, TC, Na, cK, ALP, Hct and cortisol indices. In this study we showed that NRF2 regulates expressions of glucose metabolism as well as antioxidative stress response to HIIT. NRF2 aids in the stabilization of metabolic homeostasis by preserving pancreatic b cells from oxidative stress and reducing insulin resistance. Several factors have been shown to activate NRF2 signaling, and these factors are thought to increase glucose homeostasis. In this study, we have shown that HIIT induced NRF2 and H2O2 which play a role in the glucose metabolism [6]. Finally, NRF2 appears to be a promising therapeutic target for diabetes and associated metabolic abnormalities. In agreement with our data, in adult (50–70 years) controlled-patients with type 2 diabetes mellitus both moderate-intensity continuous training, and high-intensity interval training appear to be equally effective in normalizing lipid profile markers and increased general fitness. However, high-intensity interval training seems to be more effective in reducing oxidative stress markers [25, 41, 42].

In confirmation of these findings, the research results also showed that a significant correlation was observed between H₂O₂ and Nrf2 indices caused by HIIT and antioxidants (CAT, GR, GPx and SOD). This means that with the increase of these indices after HIIT, a significant increase in antioxidant indices was observed. Therefore, it seems that with a high probability, an increase in the production of these indices will have a direct effect on the production of antioxidant indices in type 2 diabetic patients.

Strengths and limitations

The RCT design of this study along with the extremely tight data in the control group strengthens the results and data interpretation. The volunteers’ and research personnel’s commitment are shown by their commitment to the exercise intervention and low dropout (none in the control groups). The study’s main drawback was that it lacked the requisite power to identify gender differences. Instead of restricting the research cohort to the T2D + HIIT, we felt it was necessary to be able to compare the response to the exercise intervention to that seen in the T2D only, and there were no data suggesting sex differences in response. To investigate sex differences in redox signaling responses in humans, future research should involve a sufficient number of subjects. We assume that higher pre-intervention concentrations of Nrf2 were due to increased or abnormal development of ROS or other electrophilic species, and that exercise training helped reduce baseline production of those reactive species.

Conclusions

In humans, studies on Nrf2 and H₂O₂ are limited especially in T2D. Furthermore, increase in H₂O₂ caused increase Nrf2 that regulates a number of cellular processes, including proliferation, differentiation, antioxidants and the inflammatory response. The rise in content or structure of mitochondrial biogenesis is one of the most striking phenotypic modifications in response to physical training. Due to its capacity to induce the activity of cytoprotective components such as antioxidant enzymatic and non-enzymatic systems, detoxifying enzymes, and xenobiotics elimination, the transcriptional factor Nrf2 plays a critical role in the maintenance of homeostasis, especially in instances where OS elevation is involved. Increases in OS caused by physical exercise, which is responsible for producing acute and chronic physiological responses, resulting in various health benefits, is one of the aspects that has been shown to induce Nrf2 expression. This is most probably attributed to the impact on neuronal plasticity, as well as an increase in neurotrophic factors, anti-inflammatory cytokines, and a decrease in pro-inflammatory cytokines that promote neurodegeneration. Animal models have helped researchers better understand nervous system disorders. Finally, it seems that intense exercise training (HIIT) increases oxygen consumption and produces oxidative stress. Increasing these indicators increases H₂O₂ production, which in turn increases Nrf2. Then the antioxidant indices in the body of type 2 diabetic patients increase. Following these increases (CAT, GR, GPx and SOD), glycemic index and lipid index in these patients will improve.

Declarations

Conflict of interests

The authors announce that the publishing of this paper does not involve any conflicts of interest.