The Effects of Two Exercise Modalities on Novel Cardiovascular Risk Factors in Overweight Women with Type 2 Diabetes: a Randomized Controlled Trial

Background: The present study aims at investigating the effects of different exercise modalities (sprint interval training (SIT) and aerobic plus resistance training (A+R)) on novel cardiovascular risk factors (lipid accumulation product (LAP), Framingham risk score (FRS), the metabolic syndrome severity scores (Mets score), visceral adipose index (VAI), body adipose index (BAI), triglyceride-glucose index (TyG index), triglyceride glucose-waist circumference (TyG-WC), triglyceride glucose-waist circumference (TyG-BMI), HOMA β-cell, atherogenic index of plasma (AIP), and estimated glucose disposal rate (eGDR)) in overweight women with type 2 diabetes. Methods: Fifty-two overweight females with type 2 diabetes (T2D) (aged 45-60 years, BMI > 30, hemoglobin A1c (HbA1c) ≥ 6.5%) were assigned to either SIT (n = 17), A+R training (n = 17), or control groups (n = 18). Intervention consisted of SIT or A+R training for 10 weeks. Results: There were no significant changes in Mets in the SIT group after 10 weeks (p = 0.187). In addition, there were significant changes (improvements) in LAP (p < 0.001) and VAI (p = 0.002), FRS (p = 0.001), TyG index (p = 0.005), TyG-BMI (p = 0.012), TyG-WC (p < 0.001), AIP (p < 0.001), and eGDR (p = 0.001) in A+R training group after 10 weeks, as compared to the baseline. Conclusions: The results illustrated that exercise training modality independent of the mode of exercise training could be an effective intervention to improve some novel cardiovascular risk factors in women with type 2 diabetes.

developing CVD and stroke [1], and diabetes-related cardiovascular complications are a major cause of morbidity and mortality, in comparison to metabolic dysregulation [2]. In addition, the prevalence of obesity increases in the middle of life and decreases upon aging; women are more likely to be overweight than men [3]. There is strong evidence suggesting that T2D confers a stronger excess CVD risk in women than in men. There are sex-specific pathophysiological differences in metabolic syndrome that could be potentially due to their adverse risk-factor profile [4].
Many researchers have focused on identifying and introducing a large number of novel combined biomarkers to predict the risk scores of CVD, aiming at evaluating and monitoring responses to therapeutic strategies. Several risk prediction equations useful in the primary prevention of CVD at the individual and clinical level have been developed in recent years. It has been shown that lipid accumulation product (LAP), Framingham risk score (FSI), atherogenic index of plasma (AIP) [5], the metabolic syndrome severity scores (Mets score) [6], visceral adipose index (VAI) [7], body adipose index (BAI) [8], triglyceride-glucose index (TyG index) [9], triglyceride glucose-waist circumference (TyG-WC) [10], triglyceride glucose-BMI (TyG-BMI) [10], HOMA β-cell [11] and estimated glucose disposal rate (eGDR) [12] are strong markers predicting the risk of coronary heart diseases. It has also been found that only controlling changeable risk factors (such as age, sex, smoking, blood pressure and total cholesterol, triglyceride, and waist and hip circumferences) can be efficient for preventive treatments in patients with diabetes [13].
Exercise has long been recognized as an important management strategy in T2D patients [14]. However, adequate support is not still available to show the influence of exercise interventions on other CVD-related risk factors. According to Canadian Diabetes Association (CDA) guidelines, the main lifestyle interventions for reducing the risk of CVD must include improvements in glycemic control, blood lipid levels reduction, blood pressure control, quality of life, and other diabetes-related coexisting complications [15].
A sprint interval training (SIT) protocol is 4-6 repeated cycling Wingate tests (4-6 × 30 s WT), interspersed by 2 minutes of recovery. Recent researches have shown that very brief SIT bouts (less than10 min/session) have similar adaptations compare to longer SIT protocols and moderate-intensity training. Several studies have demonstrated that SIT is equally or more effective than moderate-intensity continuous training for improving many health-related factors such as VO 2 max, altered used substrate during exercise [16], resting metabolic rate [17], mitochondrial function capacity [18], insulin resistance [18], and body composition [18,19] and cardiorespiratory fitness [20] in obese subjects. In addition, some researchers illustrated that high intensity training may improve resting energy expenditure greater than moderate-intensity training [21,22]. In contrast, high intensity training has 24 hours energy expenditure similar to continuous exercise despite reduced time commitment [22].
There is not enough study has directly compared these types of training (very brief SIT protocol vs combined training) and measured changes in cardiovascular risk scores. There is not enough information, however, regarding these novel cardiovascular risk scores and exercise training in individuals with type 2 diabetes. Therefore, the purpose of this study was to compare the effects of two exercise modalities on novel cardiovascular risk factors in overweight women with type 2 diabetes. The two training program differed obviously with respect to volume and intensity of training (SIT involved 15 min/session, whereas combined training consisted of 50 min/session high intensity training. We hypothesized that compared to control group, SIT and combined training would similarly improve in cardiometabolic risk factors based on novel equations.

Subjects
In current study, we conducted a single-blind randomized clinical trial according to CONSORT statement [23]. The trial protocol was approved by the ethics committee of Shahrekord University (SKU) and registered in Iranian registry of clinical trial (IRCT) (code: 2017090419995N9). 150 voluntary pre-menopausal women with T2D were invited to take part in present study according to some criteria such as fasting blood glucose (FBG) ≥ 126 mg/dl, HbA1c ≥ 6.5%, [24], being sedentary or irregularly active (do not exercise 30 minutes or more twice per week of moderate to vigorous activity) [25], body mass index (BMI) 25 -35 kg/m 2 , and had not lost or gained weight greater than 5 kg during the last 6 months. The exclusion criteria of present study were resting blood pressure more than 160/100 mmHg, severe hypertriglyceridemia triglycerides ≥ 500 mg/dl, serious cardiovascular complications, musculoskeletal disorders, hormonal imbalance, sever kidney and liver failure.
A sample size between 10 and 20 can provide an 80 % statistical power to differentiate the effects of the SIT against the combined exercise training and show a mean difference of 2 % after a 10-week training. Based on expected dropout rate of 20%, the final sample size was considered for each group of 17 people. Every participant provided the written informed consent. Only 54 of the 150 population had study criteria (Fig.1). The eligible subjects were explained and informed orally and in writing about study protocol and procedure and the possible risks and benefits of this study. Researchers assured that all answers were kept confidential.
A concealed randomization (block size of four) was performed by an independent investigator assistant in this trial using a computer-generated random number sequence. Subjects were stratified according to age, primary BMI and HbA1c level. Sequential treatment allocations were enclosed in numbered, sealed and distributed by this research assistant in the groups after the initial assessment. Subjects were randomly assigned to SIT group (n = 17), A + R group (n = 17) or control group (n = 18) (Fig.1).

Exercise training program
Both exercise training interventions (10-week combined aerobic and resistance training and SIT training) were performed in the morning 2 hours after eating breakfast. Subjects exercised three times per week for 50 minutes, according to American College of Sports Medicine (ACSM) guidelines [26], and trainings progressed in duration and intensity.
Trainings were supervised by expert exercise physiologists at a clinical gym. Postprandial glucose levels were tested before and after exercise sessions to see whether the subjects could safely begin exercising. Because blood glucose levels could also run high during or after SIT through increasing glucose-raising hormone levels, when blood glucose level before exercise was less than 5.5 mmol/l (in subjects receiving insulin) or less than 6.6 mmol/l (in non-insulin users), subjects could eat a meal with 25 g carbohydrate and 7 g protein [27].

Combined exercise training (aerobic training plus resistance training):
Through an aerobic exercise program, subjects were free to train on a treadmill or ergometer exercise machine.
Aerobic workout progressed from 20 minutes / session at 60 % of maximum heart rate (MHR) intensity per week in weeks 1-2 to 30 minutes / session at 70% MHR in the weeks 3-10. The heart rate was controlled (Polar Oy, Kempele, Finland) to adjust the load to achieve desired heart rate. To individualize aerobic training, intensity of training was increased personally. Furthermore, resistance training was done at one set of 15 repetitions in weeks 1-2. Then intensity of resistance training was increased from 2-3 sets of 12 to 10 repetitions in the weeks 3-10 (Table 1) [28]. All resistance trainings exercises were performed on machine-based resistance training equipment and included major muscles such as bilateral triceps pushing down, bilateral biceps curling, bending over the lateral pull down, bench pressing, leg pressing.

Cycling sprint interval training:
The cycling SIT consisted of exercising on a standard electro-mechanically braked cycle ergometer (Ergomedic 894E Peak Bike, Monark EB; Varberg, Sweden). Each cycling SIT session began with a 5 min warm-up at 50 Watt, followed by 4×30 second cycling "all-out" intensity intervals at the breaking wattage of the individual, interspersed with 2 min of recovery at 50 Watt and finished with 4 minutes of cool-down. Based on the performance and the perceived effort of each subject who completed three sessions at the first session of the SIT, the Wattage increased by 10%. On the other hand, for those participants who were not capable of maintaining the required intensity (120 rpm for any interval), wattage was decreased by 10%. Furthermore, during the 10 weeks of SIT, wattage was adjusted upward in 10% increments to ensure that allout and maximum effort was being exerted during each training session if a subjects had completed 3 intervals by maintaining more than 120 rpm on two consecutive training sessions [29].
Usual care control group: Subjects in control group received usual diabetes care and Farsi-translated diabetes recommendations. They were not given exercise counselling and were asked to maintain habitual activity levels during the study period.

Blood Analysis
Before training protocol and 48 hours after the last session of training program within 12 hours of the fasting state 10 cc blood samples from the antecubital vein were collected.

Novel cardiovascular risk scores
-To estimate cardiovascular risk factor score, we applied the Framingham risk score equation [36]. This equation considers gender, age, systolic blood pressure, smoking status, total cholesterol, HDL-C, and diabetes mellitus status to calculate the percentage likelihood of a coronary disease over a 10-year period [37].

Statistical analyses
Data from 10 subjects who did not participate in post-test assessment were excluded, and only available data of 42 subjects who had completed the pre and post evaluations was analyzed.
To test normality distribution of data a Kolmogorov-Smirnov test was used. Both the Kruskal-Wallis test (when data was not normally distributed) and Analysis of Variance (one-way ANOVA) were used to compare baseline characteristics of the groups.
We conducted a paired t-test in order to compare baseline versus post-training measures and to find the differences between all groups one-way ANOVA followed by a Fisher's Least Significant Difference (LSD) post hoc test was used. Statistical significance was accepted when p < 0.05.

Results
10 subjects were excluded from the final analysis as they did not take part in the post-test assessment. Therefore, only the available data of 42 subjects with an average age of 55.7 ± 7.9 (drop out 19.2%), who had completed the pre and post assessment was analyzed.
An average adherence rates of 78% in SIT and 82% in A+R groups were for the training sessions. 27 subjects were treated with oral diabetes medications, 20 insulin injections, and 5 insulin injections and oral medications. The results show that there were no significant differences in the baseline variables between randomized groups except FBS (p= 0.021) and HbA1c (p= 0.005) (table 1).

Adverse events
There were no reports of any serious medical adverse events related to exercise training were identified and reported during interventions and testing sessions. However, most subjects reported muscle soreness in their legs during SIT (76%) and A+R trainings (82%). At the baseline, One-Way ANOVA showed no differences between the combined, the SIT and the control groups for body mass, % body fat, BMI, WHR, FBG, and HbA1c (Table 1).    In addition, one-way ANOVA test showed that there were significant differences between In addition, there was a significant reduction in fasting blood glucose in the SIT (p <0.001) after 10 weeks. Furthermore, paired t tests showed that there was a significant decrease in the insulin level in combined training (p < 0.001) and SIT (p < 0.001) groups after the 10 week intervention Furthermore, ANOVA test showed that there were no significant differences in FBG levels (F = 1.853, p = 0.171). However, there were significant differences between groups in insulin (F = 3.622, p = 0.036) and HOMA-IR (F = 5.511, p = 0.008).
Post hoc Tukey analysis test showed significant differences in LAP between SIT and control groups (p = 0.05), VAI between SIT and combined training groups (p = 0.035) and SIT and control groups (p = 0.001), FRS between SIT and combined training groups (p = 0.018) and SIT and control groups (p = 0.001), TyG index between SIT and control groups (p = 0.035), TyG-WC index between SIT and control groups (p = 0.001), and eGDR between SIT and control groups (p = 0.001).

Discussion
To our knowledge, this study could be regarded as the first randomized controlled trial (RCT) comparing the effects of different modes of exercise modalities on novel cardiovascular risk factors among women with diabetes type 2. The major novel finding from the this study was that 10 weeks of SIT in previously inactive women with diabetes This study demonstrated that sprint interval training was a more potent stimulus than combined aerobic and resistance training in improving some cardiovascular factors (LAP, VAI, FRS, TyG, TyG-WC and eGDR), though it failed to significantly improve Mets, TyG-BMI and AIP, as compared with combined training. Additionally, SIT and combined training did not lead to more powerful significant changes in body composition in terms of body mass, BMI, WHR, and body fat percentage in women suffering from diabetes.
In the present study, the LAP index was significantly improved after two exercise interventions. The association between exercise training and LAP index has not been studied before. The LAP index is an accurate index for predicting the risk of developing The different improvements of interventions on VAI, a reliable marker of visceral fat distribution, can be gender dependent, reflecting that fat distribution associated with cardiovascular risk in this study was independent of body composition changes.
Furthermore, the independence of body composition change from -improvement in VAI could be associated with the increased physical activity volume and also, the improvement of physical fitness parameters [40], as seen in this study (data not shown).
Thus, exercise training led to a significant reduction in VAI, which pointed out the potential additive effect of exercise training on WC in T2D women. It seems that exercise program that is aerobic, in contrast to resistance training and/or combined interventions, could alter adipose tissue metabolism and regional visceral adipose tissue depot loss, possibly by mobilizing free fatty acids from visceral adipose tissue at different abdominal regions [41]. Several inconsistent findings have been found regarding how interval training can reduce abdominal fat mass in T2D men [42,43]. Comparison with our results in women participating in this shows that there are some gender differences in the effects of exercise modalities on some biomarkers of CVD risk factors. For examples, women had greater reductions in body fat than men; women could decrease hsCRP level more than men by following exercise intervention through sex differences in body fat distribution and sex hormones [44]; women could also reduce sympathetic nervous activity more than men, showing that there are greater changes in arterial stiffness and endothelial function in women than in men [44].No differences were found in MetS Z-score changes between SIT and combined training. In addition, there were also no significant changes in MetS Zscore from the baseline to post-exercise training in all groups. Furthermore, the beneficial effects of exercise on the MetS Z-score were achieved without concomitantly altering body composition. It has been shown that fat mass changes are not correlated to MetS Z-score [45]. Interestingly, we did not see any significant reduction in body composition in all groups. It seems that exercise-dependent changes in body composition or more precisely, changes in body mass, body fat percentage and WHR are not important factors in reducing the cardiovascular risk score [45].
In this study, we showed that no significant change in the MetS Z-score would be possible in the absence of change in body composition. The strengths of present study were the use of the novel Mets Z-score to evaluate the effects of different exercise modalities.
Confirming the present results, Gates illustrated that 16 weeks of aerobic training did not change the Mets Z-score [35]. In addition, Johnson et al. did not show the superiority of SIT, as compared with moderate intensity training, in overweight/obese subjects [46]. Furthermore, Earnest et al. [54] showed similar reductions in the MetS Z-score in high intensity training and moderate training in overweight males [47]. Because of methodological differences across studies, such as differences in gender, age, health, weight and physical fitness status, medication, mode and intensity of exercise and duration of the training program [48], drawing general conclusions is difficult [49]. It could be speculated that both trainings might not induce improvements in the metabolism, metabolic capacity, and body composition [49,50]. The hypothesis of the study was that the regular SIT and combined training could reduce the risk of CVD in type 2 diabetes. It has also been proven that both exercise modalities could be recommended for type2 diabetes patients. This finding is consistent with the results obtained by Ramos et al, who found that low-volume HIIT could be as effective as moderate-intensity continuous training in the reduction of the MetS Z-score [51]. In addition, Fisher and colleague demonstrated that both high intensity interval training and continuous moderate intensity training are associated with improvements in cardiovascular risk factors (body fat percent, cholesterol, VLDL, HDL, triglycerides and VO2peak) in overweight men [52]. Furthermore, the results of the present study showed that applying regular SIT and combined training could reduce risks of CVD for 10 years. This improvement may result from the decreased systolic and diastolic blood pressure and improvements of lipid profile [53], as shown in our study.
Significant improvements in systolic and diastolic blood pressure and lipid profile might be because of these reductions in FRS [53]. The FRS reduction results were in agreement with those obtained by Amin-Shokravi et al., [53] and Tulley et al., [54]. It seems that the severity of applied exercise training in this study was sufficient to bring about changes in FRS.
Perhaps the most striking and novel finding form the present work was the similar improvements in TyG, TyG-BMI and TyG-WC indices. Although few studies have investigated the relationships between TyG indices and CVD risk [10,55], none of them has yet explored the effects of different exercise modalities on TyG indices. It has been shown that TyG index is a reliable predictor to identify insulin resistance and diabetes type 2 [9,56]. One major advantage of the TyG index is the use of fasting glucose and triglyceride parameters. It seems that the TyG index predominantly shows muscle insulin resistance, while HOMA-IR mainly reflects liver insulin resistance [57]. Several candidate mechanisms have been suggested to explain the effect of exercise training on TyG indices in patients with diabetes type 2. It might be postulated that improvement in insulin resistance, as seen in our study, may explain improvements of TyG-indices in women with diabetes type 2 [9]. Our results demonstrate that a surprisingly small amount of SIT can be as effective as a large volume of moderate combined training for improving TyG, TyG-BMI and TyG-WC indices.
As shown by the previous studies, exercise training is an effective anti-atherosclerosis intervention [58,59] . Some novel findings from the present study were the similar improvements in atherogenic index of plasma in SIT and combined training. Both training protocols were capable of reducing AIP in women with diabetes type 2. As AIP was calculated based on TG and HDL-C levels, it was expected that any changes in TG and HDL-C following exercise training would result in the change in AIP. But, the findings also showed that vigorous intensity interval training and combined aerobic plus resistance training did not significantly change the levels of HDL and TG. The present study showed that even without changes in HDL and TG levels, both modalities could serve as an appropriate stimulus to reduce the atherogenic risk. It could be speculated that LDL particle size may explain the exercise training related mechanisms involved in the reduction of the atherogenic risk [58,59].
Several studies recently have shown that SIT appears to be a very time-efficient exercise regime that shares many of the metabolic adaptations as traditional endurance exercise training does [60][61][62].
However, combined exercise training had a more considerable total duration of exercise and calorie consumption, as compared with the time when each type of training was done alone [63]. Despite the fact that such patients tolerate this exercise mode, it seems that by following this exercise prescription, more calories could be consumed.
It has been shown that lower eGDR score was associated with the prevalence of T2D and increased diabetes-related vascular complications (the use of the Estimated Glucose Disposal Rate as a Measure of Insulin Resistance in an Urban Multiethnic Population with Type 1 Diabetes). This study showed that eGDR was decreased by following two exercise modalities in 2DM patients. This effect of exercise intervention on eGDR has not been described previously. It seems that the decrease of eGDR by following exercise training could be caused by the reduction of HbA1C. Decline in HbA1C reflects an adaptation in glucose hemostasis. The novel aspect of our study was that we investigated the utility of eGDR in the assessment of exercise-induced training effects on insulin sensitivity in the patients with T2DM. The results of this study showed that both protocols had a positive effect on eGDR in the patients with type 2 diabetes, and the change in HbA1c levels at the exercise program. MThe most important was the fact that subjects receiving the SIT training program showed significantly greater improvement in eGDR levels over the 10 week period, as compared to the control group. One limitation of the present RCT was that SIT protocols had also to be evaluated in terms of safety in women living with T2D though exercise training, though it was generally quite safe [64].

Study limitations:
The randomized controlled trial design, the inclusion of two different training programs in the same study, the direct personalized exercise training for all training sessions, and the use of a novel cardiovascular risk score to evaluate the effects of different exercise modalities on the risk of cardiovascular diseases, which provided an increased level of accuracy and sensitivity, were amongst the strengths of the present study. As for the limitations of this study, the small size, significant dropout rate, and supervised exercise only in the experimental groups can be mentioned. Moreover, the non-significant reductions of body composition parameters, which can be attributed to the lack of diet control could be taken one other limitation. This study could yield some insight in selecting the type of exercises that can be more helpful for cardiovascular risk reduction.
However, this exercise schedule may not be suitable for real-life setting as adequacy of exercise cannot be quantified and many patients with type 2 diabetes may not be having the facilities for such exercises. This is also is another limitation of this study.

Conclusions
The results of this research support the idea that Taken together, the results of this research support the importance of SIT and combined training program in improving T2D and novel cardiovascular risk scores, despite the fact that some studies had shown that A+R and SIT interventions could improve glucose homeostasis in women with T2D. Results highlighted that much lower exercise volume involved with SIT, these data also seemingly suggest that training intensity, rather than volume, may be the more critical determinant of the improvement in some novel cardiometabolic-related scores. Results highlighted that exercise training independent of the mode of training (SIT vs. A+R) could be an effective training method to improve some novel cardiovascular risk scores in overweight women with type 2 diabetes. Furthermore, even without weight loss during the course of the 10-week exercise, there was a significant reduction in anthropometric variables, suggesting that weight loss is not mandatory for the healthy body composition. and MMG supervised laboratory exams and data collection. EB and MF analyzed and interpreted the data. EB, MF and SN wrote the first draft of the manuscript. MF edited the paper. All authors contributed to the writing of the paper. All authors read and approved the final manuscript.