Maximal exercise test is a useful method for physical capacity and oxygen consumption determination in streptozotocin-diabetic rats

Diabetes is a great public health problem associated with high morbidity and mortality. Frequently, this condition is accompanied by other disorders, such as coronary heart disease, hypertension, dyslipidemia and obesity [1]. One consequence of this metabolic condition is the reduced exercise capacity that has been commonly observed in diabetic patients. This reduction is probably dependent on various physiological factors, such as neuromuscular activity, hemodynamics, and respiratory mechanics and oxygen consumption [2].

Maximum oxygen consumption (VO₂ max) test is highly reproducible, and it is considered a "gold standard" for functional capacity assessment in athletes and sick individuals. This parameter expresses the functional health of the cardiovascular, pulmonary, and skeletal muscle systems [3]. In addition, it provides important prognostic information that is useful for identifying potential candidates for cardiac transplantation [3, 4]. VO₂ max test also provides objective data, which is helpful in formulating appropriate exercise prescription [5, 6]. However, sometimes VO₂ max measurements have limited use in animal experimentation because of the high cost of the gas analyzer equipment [7] and its time-consuming characteristic.

Maximum and submaximum exercise tests (ET) have been considered safe and well tolerated. This method implies simple measures of functional capacity, and it has been used in clinics, physical qualification programs, and cardiac rehabilitation programs [8, 9]. The assessment of cardio-respiratory responses to acute and chronic exercise, using different methods (as used in humans), has been a common practice in the investigation of animal models.

In experimental practice, VO₂ max measurement is a noninvasive tool that has great value in the study of functional capacity of animals. Therefore, maximum ET, in which animal runs to exhaustion on a graded load treadmill, has been used by our group as an alternative method of evaluation of physical capacity, and it is also used for physical training prescription in health and pathological conditions [6, 10‐16].

Streptozotocin (STZ)-induced diabetes has been largely used in the study of the role of exercise training in diabetic-induced cardiovascular and autonomic dysfunction [6, 14, 16]. However, the relationship between the speed of ET and VO₂, as well as the accuracy of ET in estimating cardio-respiratory capacity remains unclear in STZ-diabetic rats. Therefore, the purpose of the present investigation was to evaluate the relationship between the speeds of ET and both VO₂ and VO₂ reserve (VO₂ res) and to validate the equations to predict the VO₂ and VO₂ res based on speeds of ET in control and STZ-diabetic rats.

The worldwide epidemic of diabetes that have emerged with the dawning of the 21st century has shown to be a major public heath problem, having struck developed countries as well as those still developing [1]. The diabetes induced by streptozotocin has been a largely used in animal models to study diabetic-induced cardiovascular and autonomic dysfunction [5, 18‐20].

It is well known that laboratory animals are constantly used in exercise physiology and cardiovascular rehabilitation research. However, the measurement of oxygen consumption in small animals is, many times, a limitation factor for research in this area, not only because of the high costs of gas analyses systems [7, 21], but also because of two other important factors. First, the VO₂ measurement must be carried out in a separate place from the rest of the laboratory to prevent alterations in the concentration of gases around the environment during data acquisition, and second, it is time-consuming because VO₂ stabilization can take some time in rats. Moreover, the closed environment of the respiratory chamber may provoke one more stress situation for the experimental animal.

Although various instruments for human VO₂ and VO₂ reserve acquisition exist, to our knowledge, the respiratory chamber is the only one permitting continuous direct monitoring of VO₂ in laboratory animals [17].

Studies of our laboratory have been conducted to investigate the benefits of exercise training in STZ-diabetic rats [6, 14, 16] and others pathological conditions [10, 13, 15], using the exercise training prescription based on maximal exercise test. In the present study, we have demonstrated that STZ-diabetes reduces VO₂ max, VO₂ res max and maximum running speed on ET. Furthermore, we also obtained significant correlations between VO₂ and ET speed, VO₂ reserve and ET speed and VO₂ max and maximum ET speed in control and STZ-diabetic rats.

Accurate monitoring of VO₂ consumption healthy or sick subjects is of great interest to medical research [22, 23]. Researches have been wide for indirect forms of estimating VO₂ max [7, 24, 25]. In 1954, Astrand & Ryhming [24] published a nomogram to predict VO₂ max from submaximal pulse rates (120 to 170 bpm). Some years later, this nomogram was modified, and it is still being used today [26]. Balke & Ware [27], in 1959, developed a new protocol, and established a formula to calculate VO₂ based on the speed and on treadmill slope [28]. In addition, recently, Maldonado-Martin et al. [29] demonstrated a good correlation between VO₂ peak and the 6-minute walking test (6-MWT) in heart failure patients. However, these authors also concluded that when VO₂ (peak) is predicted from equations using 6-MWT, the result is a substantial variability. Consequently, it should not be used in older HF patients, in which an accurate determination of functional capacity is essential. Currently, VO₂ max, for both health men and women, can be estimated according to the formulas adopted by the American College of Sports Medicine [30].

The maximum VO₂ values obtained in the present work are in accordance with previous findings for normal adult rats [31]. Likewise, the maximum speed values obtained in the exercise test are considered adequate values for rats and mice [7, 13, 15, 26]. The reduction in VO₂ max observed in diabetic rats can be due to many factors, such as cardiac output limitations [5, 6], reduced peripheral blood flow [32] and problems in skeletal muscle metabolism [33].

The results of the present study provide values of VO₂ for adult Wistar male rats at different treadmill speeds. In the range of treadmill speeds, 3 to 24 m/min for CG and 3 to 19 m/min for DG, VO₂ increased progressively as a function of running speed, and it could be expressed by simple linear equations obtained by linear regression (VO₂ = 1.54 * ET speed + 52.34, for CG; and VO₂ = 1.16 * ET speed + 51.99, for DG). This equation allows VO₂ estimation for one definitive workload, and it also gives the VO₂ max from the maximum ET speed.

The increment in VO₂ as a function of speed (Figure 1) was described in previous studies [34, 35], and other predicted forms were proposed for Wistar-Kyoto and Spontaneously Hypertensive Rats [36]. However, our study has shown that STZ-diabetic rats present different slopes of VO₂ in response to speed increment when compared to control rats. In fact, available data suggests that each experimental rat model needs a specific equation to predict the VO₂ and VO₂ reserve.

In this study, we tested the approximation between measured VO₂ and VO₂ reserve with VO₂ and VO₂ reserve predicted by linear regression equations in study groups. Significant correlations were observed between measured VO₂ and VO₂ reserve with predicted VO₂ and VO₂ reserve variables for CG (r = 0.81 and r = 0.83; p < 0.003) and DG (r = 0.75 and r = 0.75; p < 0.005), respectively. Thus, our study has showed that these equations, which are used to predict VO₂ and VO₂ reserve from ET, are valid for control and STZ-diabetic rats.

In exercise physiology, it is common and appropriate to characterize work intensity as percentage of VO₂ max. In this study, it was observed that 60% of the maximum ET speed (CG: 14 m/min and DG: 12 m/min) corresponded to 83% of VO₂ max and 72% of VO₂ reserve in CG and 88% of VO₂ max and 75% of VO₂ reserve in DG. In animal models, a correlation between ET speed and VO₂ was also established for adult and old C57BL/6J mice. In this study, the authors found that the speed of 12 m/min (~50% of the maximum ET speed) produced a VO₂ equivalent to 76% and 89% of the VO₂ max in adult and old mice, respectively [7]. Furthermore, Wisloff et al. [31] have demonstrated a linear relationship between VO₂ and HR in sedentary and trained rats according to the increment of ET speed. Nevertheless, these authors emphasized that the maximum HR is not reached along with VO₂ max. Instead, it is reached in intensities above VO₂ max. Moreover, this study indicates that values of 90% of the maximum HR correspond to ~80% of the VO₂ max.

It is important to highlight the positive correlations observed in the present study between maximum ET speed and VO₂ max in CG (r = 0.7, p < 0.05) and DG (r = 0.7, p < 0.05). These correlations demonstrate that rats with higher ET performance presented higher VO₂ max. As a result, this finding suggests that a simple ET is capable of detecting differences in the cardio-respiratory capacity of control and diabetic-STZ rats, and it also suggests that the improvement in cardio-respiratory performance (VO₂max) after a period of physical training could be detected by maximum ET. In this aspect, it is important to emphasize that Noakes et al. [37] previously demonstrated that the highest speed reached in maximum ET is a better predictor of performance in laboratory tests when compared to %VO₂ max, VO₂max or racing speed at an extreme lactate threshold among marathon athletes. In our group, we have used ET as an indicator of improvement of exercise capacity after exercise training in diabetic, hypertensive ovariectomized rats and in normal mice [12‐16].

The results of the present study demonstrated that VO₂ can be estimated from the results of ET in control and STZ-diabetic rats. This estimate can be assessed by obtaining equation by linear regression between VO₂ and ET. The correlation between ET speed and VO₂ showed that physical training prescription based on ET is adequate for control and diabetic animals. Furthermore, our data indicates that ET can detect differences in aerobic performance, as the maximum speed achieved in the ET was correlated with the maximum VO₂ in these evaluated experimental models.

Therefore, we concluded that maximal exercise test can be used as an indicator of cardio-respiratory capacity, and it can be useful in further studies that investigate physiological effect of acute or chronic exercise on control and STZ-diabetic male rats.