This paper presents the morphometric responses and training performance outcomes of a high-intensity interval training (HIIT) protocol in a Sprague-Dawley rat model of diet-induced obesity. The purpose of this protocol was to maximize exercise intensity and determine the physiologic responses to HIIT in lean and obese rats.
Compared to continuous-moderate or low-intensity training, high-intensity interval training (HIIT) is a more time-efficient alternative method that results in similar physiologic benefits. This paper presents a HIIT protocol that can be used to assess various health markers in a Sprague-Dawley rat model of diet-induced obesity. Female Sprague Dawley rats aged 21 days old were randomly assigned to the following groups: control (CON, n = 10), exercise-trained (TRN, n = 10), high-fat diet (HFD, n = 10), and high-fat diet/exercise training (HFD/TRN, n = 10). The control diets consisted of commercial laboratory chow with 10% kilocalories (kcal) from fat (3.82 kcal/g), and the high-fat diets (HFD) consisted of 45% kcal from fat (4.7 kcal/g). The animals had ad libitum access to their assigned diet throughout the study. After an 8 week diet induction period, the exercise cohorts completed four HIIT sessions per week for 8 weeks. Each HIIT session consisted of 10 intervals of 1 min sprints/2 min rest using a rodent treadmill with a motor-driven belt. After the 8 weeks of training, the animals were sacrificed for tissue collection. The results revealed no differences in the distance run between the TRN and HFD/TRN groups, and the training speed steadily increased over the duration of the study, with a final running speed of 115 cm/s and 111 cm/s for the TRN and HFD/TRN groups, respectively. The weekly caloric intake was decreased (p < 0.05) in the TRN group relative to the CON group but increased (p < 0.05) in the HFD/TRN group relative to the HFD group. Lastly, the animals on the HFD had greater (p < 0.05) adiposity, and the trained animals had reduced (p < 0.05) adiposity relative to controls. This protocol demonstrates an efficient method to evaluate the effects of HIIT on various physiologic outcomes in a diet-induced obesity model.
Obesity and comorbid conditions, such as cardiovascular diseases, metabolic diseases, and cancer, continue to be some of the most serious, costly, and preventable of all health outcomes. Currently, over one-third of adults in the United States and more than 1.6 billion adults worldwide are classified as obese according to their body mass index (BMI; defined as weight in kilograms divided by the square of height in meters)1. Obesity as a disease results from a genetic predisposition, environmental exposures, and a breakdown in the normal mechanisms regulating energy intake and energy expenditure2. As the human and financial costs of the obesity epidemic continue to rise, there has been an intensified focus on trying to understand the mechanisms involved in energy balance and the effects of diet and exercise in combating metabolic disease.
Previous studies have demonstrated that exposure to highly palatable, energy-dense diets stimulates overeating in rat models3. Ad libitum access to highly palatable diets drives excessive weight gain as a result of increased caloric intake4. Studies have also shown that exercise can modulate appetite and improve the sensitivity of satiety signaling in obese individuals5. It is theorized that this recovery of the sensitivity of satiety signaling with exercise is partially mediated through the impact of exercise training on the reactivity of the central and peripheral tissues to leptin, a key adipocyte-derived regulatory hormone that suppresses appetite and stimulates energy expenditure5. While these studies have investigated a variety of exercise protocols, there is no clear consensus on which intervention is superior6,7. There is some evidence to suggest that high-intensity interval training (HIIT), which involves repeated bursts of strenuous exercise interwoven with intervals of recovery, may improve appetite regulation more than other forms of exercise, such as moderate-intensity continuous exercise training (MICT), vigorous-intensity continuous training, or voluntary physical activity8. However, there are gaps in knowledge surrounding the intersectionality of high-intensity interval training, diet, and appetite regulation.
Previous studies have also demonstrated that exercise is a powerful mediator of inactivity-related comorbidities, particularly from the perspective of alterations in muscle and adipose tissue9,10,11. It is hypothesized that these compositional changes lead to the promotion of an anti-inflammatory state that may be responsible for the amelioration of disease risk seen with exercise12. Myokines, which are cytokines, other small proteins, and proteoglycan peptides released from skeletal muscle during muscular contractions have been posited as moderating the anti-inflammatory outcomes associated with physical activity. In contrast, adipokines, cell-signaling molecules produced by adipose tissue, have been shown to primarily play a more deleterious role and contribute to the promotion of an inflammatory state13,14,15,16. While there is significant evidence demonstrating that the compositional alterations seen with MICT promote positive health outcomes, less work has been done to evaluate the potential benefits of HIIT17,18.
Finally, cardiovascular disease is well-established as the leading cause of morbidity in humans and is highly correlated with obesity, diet, and physical activity1. This protocol provides an efficient way in which to train rodents for the evaluation of the effects of cardiovascular training on numerous systems. In particular, cardiac hypertrophy is a marked adaptation that occurs with cardiovascular exercise. This hypertrophy allows for more robust cardiac contractions and delivery of blood and oxygen to the exercising tissues. Previous research suggests that high-intensity exercise is more likely to induce cardiac hypertrophy than moderate-intensity exercise19.
This protocol helps fill the gaps in the literature by providing an approach for examining the effects of HIIT on appetite regulation, compositional changes (thus, myokine and adipokine changes), and cardiovascular adaptations in a murine model of diet-induced obesity. Further, the performance-based increases in intensity maximize the training outcomes and ensure that animals are not adapting to the exercise training and approaching a moderate intensity later in the training protocol.
The overall goal of this method is to maximize the exercise effort and identify phenotypic changes in Sprague-Dawley rats in response to HIIT, diet-induced obesity, and the interaction of these stimuli. This protocol is unique compared to other techniques due to its ability to maximize the effort throughout the training period, even with increases in the skill and fitness levels of the rats. It also allows for the simultaneous analysis of exercise and obesity, rather than solely focusing on one or the other. Specifically, this study intended to test the following hypotheses. (1) Exercise speeds may increase throughout the training, and the distance covered by rats in the TRN group may be larger than in the HFD/TRN group20. (2) The average weekly caloric intake of the trained rats may be greater than controls, and this may be evident within each diet cohort21. (3) The average daily gain in mass may be greater in control rats than exercised rats, and control rats may have higher fat mass at sacrifice21. (4) The mass of the heart and liver may be larger in the HFD/TRN rats versus the TRN rats19.
All procedures described in the present study followed the Guide for the Care and Use of Laboratory Animals, 8th Edition. The experimental design was approved by the Office of Research and Sponsored Programs (ORSP) under the Institutional Animal Care and Use Committee (IACUC) 2019-5 at the West Virginia School of Osteopathic Medicine. Refer to the Table of Materials and Table 1 for additional details about all the materials used in this protocol. A general outline of the protocol timeline is displayed in Figure 1.
1. Experimental design
2. HIIT training protocol
3. Statistical analysis
Figure 2 demonstrates that the training performance increased over the duration of the protocol. The final running speeds of the TRN and HFD/TRN groups were 115 cm/s and 111 cm/s respectively. The total running distance did not differ between differ between the TRN and HFD/TRN groups (Figure 3).
The average weekly feed intake for the animals on the control diet was higher (p < 0.0001) than for those on the high-fat diet (103 g/week ± 1.0 g/week vs. 91 g/week ± 1.0 g/week, respectively). The average weekly feed intake was also greater (p < 0.001) in trained groups than the non-trained groups (98 g/week ± 1.3 g/week vs. 92.2 g/week ± 1.0 g/week, respectively). When looking at the interactions, the CON versus TRN groups did not differ from each other but had greater (p < 0.05) weekly intake than the HFD/TRN group, which ate more (p < 0.05) than the HFD group (Figure 4). When translating feed intake to kcal intake, the animals on the high-fat diet had a higher (p < 0.0001) caloric intake than those on the control diet (430 kcal/week ± 4.6 kcal/week vs. 396 kcal/week ± 3.7 kcal/week, respectively). This resulted in differences (p < 0.05) in the weekly caloric intake among all four groups, with the HFD/TRN group showing the greatest weekly caloric intake, followed by the HFD, CON, and TRN groups sequentially (Figure 5).
Body weight did not differ between groups until week 8 of the feeding period, when the HFD and HFD/TRN groups reached a greater (p < 0.05) mass than the CON and TRN groups (293 g ± 10.1 g and 298 g ± 13.1 g vs. 270 g ± 8.6 g and 264 g ± 6.8 g, respectively). The HFD and HFD/TRN groups remained heavier (p < 0.05) than the CON and TRN groups for the remainder of the study (reaching 332 g ± 14.4 g, 347 g ± 16.3 g, 304 g ± 10.3 g, and 304 g ± 10.1 g for the HFD, HFD/TRN, CON, and TRN groups, respectively). The average daily gain (ADG) was greater (p < 0.05) in the trained versus non-trained animals over the exercise portion of the study (0.8 g/day ± 0.11 g/day vs. 0.5 g/day ± 0.09 g/day, respectively), and there were no differences in ADG between the CON versus HFD groups over this period. Together, this resulted in greater (p < 0.05) ADG in the HFD/TRN group than in the HFD group and no differences between the CON and TRN groups (Figure 6) over the training period. However, the 8 week training period did not induce a difference in weight between the HFD/TRN and HFD groups (347 g ± 16.3 g vs. 331.5 g ± 14.4 g, respectively).
After the completion of the training protocol, the tissue retrieval revealed that animals on the HFD had greater (p < 0.05) visceral adiposity than the CON group (25 g ± 2.1 g vs. 19 g ± 1.5 g, respectively), and the exercise-trained animals had reduced (p < 0.05) visceral adiposity relative to the control animals (21 g ± 2.4 g vs. 25 g ± 2.1 g, respectively). The HFD group had a greater (p < 0.05) visceral adiposity than the TRN and HFD/TRN groups (Figure 7). The heart mass was greater in the HFD/TRN group than in the CON, TRN, and HFD groups (p < 0.05; 1.3 g ± 0.2 g vs. 1.1 g ± 0.1 g, 1.1 g ± 0.1 g, and 1.0 g ± 0.1 g, respectively). There were no differences observed in the liver mass among the groups. No differences were identified in the mass of any other organs or tissues.
Figure 1: Study protocol timeline by animal age in days. Please click here to view a larger version of this figure.
Figure 2: HIIT speed throughout the training protocol for the TRN and HFD/TRN animals by session. HIIT was performed on four different days every week for 8 weeks, resulting in 32 training sessions. The mean data per workout are presented. Please click here to view a larger version of this figure.
Figure 3: Average distance covered per sprint in the TRN and HFD/TRN groups throughout the training protocol. HIIT was performed on four different days every week for 8 weeks, resulting in 32 training sessions. The data are presented as mean ± SEM. Please click here to view a larger version of this figure.
Figure 4: Average weekly feed intake of the CON, TRN, HFD, and HFD/TRN cohorts. Data are presented as mean ± standard error of the mean (SEM). a,b,cMeans with different letters differ (p < 0.05). Please click here to view a larger version of this figure.
Figure 5: Weekly caloric intake of the CON, TRN, HFD, and HFD/TRN cohorts. Data are presented as mean ± SEM. a,b,c,dMeans with different letters differ (p < 0.05). Please click here to view a larger version of this figure.
Figure 6: Average daily weight gain in the CON, TRN, HFD, and HFD/TRN cohorts. Data are presented as mean ± SEM. a,bGroups with different letters differ (p < 0.05). Please click here to view a larger version of this figure.
Figure 7: Average visceral fat mass at necropsy. Data are presented as mean ± SEM. a,bGroups with different letters differ (p < 0.05). Please click here to view a larger version of this figure.
Table 1: Compositions of the diets used in the protocol. Please click here to download this Table.
This protocol provides an effective method for examining the effects of HIIT on several health markers in a diet-induced obesity model. The procedure draws from previous studies to allow for a more time-efficient method of examining multiple outcome variables, such as exercise training variables, appetite regulation markers, and invasive analyses of body composition3,7,8,18,23,24. The diet content, duration, and exercise intervention protocol were consistent with prior publications23,24. In this study, commercially available laboratory chow was purchased (see Table of Materials). The laboratory chow for the high-fat and control diets contained the same amount of protein and micronutrients. The carbohydrate and fat content of the diets were modified to provide a safe method of inducing obesity in the experimental group (see Table 1).
The 8 week obesity induction period used in the present study was modeled based on previous research showing significant changes in weight following the provision of commercial laboratory chow consisting of 45% kcal from fat (4.7 kcal/g), which represents the macronutrient breakdown found in the typical western diet23. Additionally, prior studies have demonstrated the effectiveness of an 8 week HIIT protocol on influencing food intake7,8, adipose profiles18,23, and muscle gain18. The results of the protocol described in this study were consistent with previous studies reporting that HIIT impacts appetite regulation, as well as compositional changes in adiposity and muscle mass.
A benefit of this protocol is that it maximizes the intensity of the exercise training in the animals and maintains maximum effort throughout the protocol. As the animals continuously learn how to use the treadmill proficiently and make fitness gains, the speed of the treadmill is increased accordingly relative to their performance. Furthermore, the use of the 5.0% inclination allows for the animals to reach maximum intensity in each session and throughout the protocol more quickly than would be accomplished without using inclination. As a result, the exercise performance is maximized for each workout and for the duration of the protocol.
During the study, one animal was unable to complete the experimental protocol due to illness, resulting in n = 39 animals completing the study, with only n = 9 rats in the HFD cohort. This protocol was initially designed to assess changes in cytokine profiles in response to exercise and diet, and the power analysis resulted greater than 90% power to identify a difference (p < 0.05) in the primary target cytokine (irisin). Future studies using this model should rely on unique power analyses to determine appropriate sample sizes.
This study was primarily designed to examine the physiologic outcomes of HIIT in a rodent model of diet-induced obesity and to maximize the intensity of exercise. This protocol was able to demonstrate variation in ADG and adiposity in response to diet and HIIT (Figure 6 and Figure 7). Future studies could specifically identify endocrine, myokine, and adipokine responses to HIIT. The elucidation of these mechanisms may prove beneficial in the treatment and prevention of obesity and its comorbidities.
This study also demonstrated the impact of diet and HIIT on feed intake. The results indicated that when the animals consumed a high-fat diet, the trained animals consumed more calories than the non-trained animals. In contrast, when the animals ate the control diet, the trained animals consumed less calories than the non-trained animals, demonstrating different appetite regulation responses depending on the composition of the diet. Therefore, strategies for weight loss that utilize HIIT may be less effective for those that simultaneously consume a high-fat diet, as they may be more likely to consume excess calories. In contrast, balanced macronutrient intake during HIIT may promote low calorie intake and, therefore, facilitate weight loss. This model can facilitate research efforts to develop a deeper understanding of the mechanisms behind energy balance and efforts to develop effective weight loss strategies.
Finally, this protocol demonstrated variation in cardiac tissue among the cohorts, reflecting adaptational changes in the body composition in response to diet and exercise training. These data suggest that obesity induction followed by HIIT may predispose individuals to myocardial hypertrophy without any accompanied alterations in hepatic size. Future analyses to determine the mechanisms behind these findings could be useful for investigating myocardial hypertrophy and the metabolic connections between obesity, HIIT, and cardiovascular disease.
The protocol described in this study has several limitations. First, the treadmill used in this study had five lanes, which allowed for five rats to be run at a single time. While this manner of executing the protocol was efficient, it was difficult for a single researcher to attend to each of the animals at once. There were occasions when it was difficult for the treadmill attendant to divide their attention among the multiple animals needing stimulation with bristle brushes. In the future, ensuring that more research personnel are available to assist with the training protocols will be a priority. Additionally, the five-lane treadmill model does not have the capability to measure gas exchange, and, therefore, the aerobic/anaerobic metabolism of the animals during the protocol could not be assessed. The company that provided the rodent treadmill (see Table of Materials) does offer a treadmill with the capability to measure gas exchange, but it is a single-lane treadmill and, therefore, would require significantly greater time and effort. That effort may be worthwhile, however, for investigators who need to measure or control for specific outcomes of indirect calorimetry. Additionally, there is very little evidence available regarding how the shock grid may impact exercise performance, which should be considered when interpreting the results from this model. Lastly, the exercise protocol described in this study was designed with young female Sprague-Dawley rats. Previous studies have shown sexually dimorphic effects, especially regarding HIIT and appetite regulation3,7. Although similar outcomes are anticipated, this protocol did not test animals of different species, ages, sexes, or health outcomes.
In comparison to prior models, this protocol demonstrates a more time-efficient method to evaluate a range of outcome variables. For instance, this protocol was able to identify interactions between HIIT and appetite regulation in a protocol that involved four training sessions per week for 8 weeks, in comparison to prior studies that involved five training sessions per week for 8 weeks24 or even 12 weeks of training8. Additionally, this study design allowed for the analysis of a variety of health markers, such as exercise data, markers of appetite regulation, and body composition. These markers, as well as the heart adaptations to exercise training, represent promising means of evaluating the training adaptations of the cardiovascular system as well. Measures of endothelial function, muscle fiber type composition, and cardiac myocyte hypertrophy could easily be added to further the understanding of these exercise-induced adaptations. Further, this protocol included performance-based escalations in intensity. This design allowed for the maximization of the training outcomes and ensured that the rats did not adapt to the exercise environment and approach a moderate-intensity continuous training model toward the end of the intervention. This is demonstrated in Figure 2; specifically, the sprint speeds of these animals were more than double the speeds achieved in previous publications, which went on to demonstrate many cardiovascular, skeletal muscle, and thermoregulatory adaptations consistent with HIIT interventions25.
The authors have nothing to disclose.
The authors would like to thank Michael Pankey, Chris Butler, and the WVSOM staff for their assistance in the animal care and data collection.
Commercial laboratory chow for control diet | Research Diets Inc., New Brunswick, NJ | D12450H | |
Commercial laboratory chow for high-fat diet | Research Diets Inc., New Brunswick, NJ | D12451 | |
GraphPad Prism software | GraphPad Software Inc., San Diego, CA | ||
Precision Electronic Digital Scale | Ohaus Corporation, Pine Brook, NJ | V11P30 | |
Rodent treadmill | Panlab, Barcelona, Spain | ||
Sprague Dawley rats | Charles River, Durham, NC | ||
Table top anesthesia machine | VetEquip Inc., Livermore, CA | V0557 |