The purpose of this study was to investigate the effects of treadmill exercise on oxidative stress in the hippocampal tissue and mitochondrial dynamic-related proteins in rats fed a long-term high-fat diet (HFD).
Obesity was induced in experimental animals using high fat feed, and the experimental groups were divided into a normal diet-control (ND-CON; n=12), a high fat diet-control (HFD-CON; n=12) and a high fat diet-treadmill exercise (HFD-TE; n=12) group. The rats were subsequently subjected to treadmill exercise (progressively increasing load intensity) for 8 weeks (5 min at 8 m/min, then 5 min at 11 m/min, and finally 20 min at 14 m/min). We assessed weight, triglyceride (TG) concentration, total cholesterol (TC), area under the curve, homeostatic model assessment of insulin resistance, and AVF/body weight. Western blotting was used to examine expression of proteins related to oxidative stress and mitochondrial dynamics, and immunohistochemistry was performed to examine the immunoreactivity of gp91phox.
Treadmill exercise effectively improved the oxidative stress in the hippocampal tissue, expression of mitochondrial dynamic-related proteins, and activation of NADPH oxidase (gp91phox) and induced weight, blood profile, and abdominal fat loss.
Twenty weeks of high fat diet induced obesity, which was shown to inhibit normal mitochondria fusion and fission functions in hippocampal tissues. However, treadmill exercise was shown to have positive effects on these pathophysiological phenomena. Therefore, treadmill exercise should be considered during prevention and treatment of obesity-induced metabolic diseases.
Obesity is defined as a low-grade systemic inflammatory state, and it is known to induce various physiological changes such as inflammation, oxidative stress, mitochondrial dysfunction, and apoptosis
In energy metabolism, reactive oxygen species (ROS) are inevitably generated, but the hyperglycemia or insulin resistance induced by obesity leads to continuous production of ROS, which is a major cause of increased oxidative stress. The accumulation of ROS within mitochondria is generally known to be reduced by antioxidant enzymes such as catalase and superoxide dismutase (SOD) as part of homeostasis maintenance
Mitochondria are vital intracellular organelles responsible for biological oxidation in most eukaryotic cells. As cells forms constantly change, mitochondrial dynamics are regulated through repeated fusion (where the length of mitochondria increases) and fission (where mitochondria split into two)
Since mitochondria play a pivotal role in intracellular energy metabolism, its form and function are regulated through continuous cycles of fusion and fission
Physical activity has recently been recognized as an effective way to enhance mitochondrial function, and exercise has been reported to improve damaged mitochondrial dynamics in various disorders. Fealy et al.
As previously mentioned, reducing obesity through exercise has been reported to have a positive effect on energy metabolism, and numerous studies have investigated the role of exercise in improving pathological physiology in peripheral tissues. However, there are currently an extremely limited number of studies on the role of exercise and obesity in nerve cell mitochondrial dynamics. Thus, this study aims to examine the changes in the factors related to nerve cell mitochondrial dynamics in the brain, when long-term, high-fat diet obesity-induced lab animals perform treadmill exercise.
This study was approved by the Institutional Animal Care and Use Committee at H University prior to performing the experiments (KNSU-IACUC-2017-01). The lab animals were 8-week old male Sprague Dawley rats purchased from KOATECH, Co., Ltd and reared until 24 weeks in an animal lab at H University (temperature 22±2 ℃, humidity 50±5 %, and a 12 h light-dark cycle). When the rats reached 24 weeks of age, obesity was induced in some rats (n = 24) with high-fat feed (D12492; carbohydrate: 20 %, fat: 60 %, protein: 20 %) purchased from the Central Lab, Animal Inc., for 20 weeks, while they were allowed liberal intake and supply of water. The lab animals were categorized into three groups: normal diet-control (ND-Con, n=12), high fat diet-control (HFD-Con, n=12), and high fat diet-treadmill exercise (HFD-TE, n=12).
The HFD-TE group was acclimatized with pre-training for 30 min a day for five days, on a rodent treadmill (DJ2-242, Daejong Instrument Industry Co. Seoul, Korea) fixed at a 0 % gradient. After pre-training was complete, the main exercise was carried out five days a week for eight weeks. Fixing the gradient at 0 %, the animals were given the Maximal Graded Moderate to Intensive Exercise Program suggested in Kang et al. (Initial 5 min at 8 m/min, then 5 min at 11 m/min, and finally 20 min at 14 m/min)
Twelve hours following the 8-week treadmill exercise, plasma glucose was measured from the tail of the rats in fasting state. Next, 1 mL triple distilled water and 0.3 mL glucose were mixed to prepare a 30 % glucose concentrate, which was administered to the rats at 1 mL∙kg-1. Blood was then taken from the rat tail five times (0 min, 30 min, 60 min, 90 min, and 120 min) and using a blood glucose monitoring device (Gluco-CardⅡ, Daichi Kagaku. Co., Kyoto, Japan), plasma glucose was measured and the reaction area for total glucose secretion (area under the curve; AUC0-120, mg/dl-1·min-1) was determined.
Twenty four hours after the 8-week’s treadmill exercise, the rats were anesthetized by intraperitoneal injection of Rompun/Zoletil mixture (2:1, 10 mg/kg), and to estimate protein expression, brain tissue, blood, and abdominal adipose tissue were removed from seven rats. The extracted brain tissue was separated into the cerebral cortex and hippocampus, which were rapidly cooled in liquid nitrogen and stored in a -80 ℃. After collection, blood from the heart was centrifuged to obtain serum for analyzing glucose and insulin. The abdominal adipose tissue was removed by incision and washed with cold physiological saline, and after removing the moisture with gauze, was weighed. For immunohistochemical staining, five rats were anesthetized, their thoracic cavities were opened, and 50 mM phosphate-buffered saline was injected through the left ventricle for 10 min, and perfused with 4 % paraformaldehyde (PFA) fixative dissolved in 100 mM phosphate buffer. After perfusion, the brain was removed and placed in 4 % PFA for 4-h fixation at 4 ℃. The fixed brain tissue was left to settle for two days in 30 % sucrose solution. Rodent Brain Matrix (RBM-4000C, 1 mm coronal section, ASI Instruments, Inc, USA) was used to extract regions of the cortex and hippocampus (from –4.88 mm to –1.76 mm from bregma) from the fixed brain tissue (whole brain). A freezing microtome (Leica, Nussloch, Germany) was then used to prepare a 40 μm thick serial coronal section.
The blood collected by cardiac perforation was centrifuged (FLETA-5 centrifuge, Hanil Biomed Inc. Korea) and the serum was collected. The glucose, insulin, triglyceride (TG), and total cholesterol (TC) concentrations were analyzed by Green Cross Corp. The HOMA-IR index was calculated from the concentrations of glucose and insulin according to the following equation: HOMA-IR = fasting serum glucose (mmol/L) × fasting serum insulin (μU/Ml)/22.5.
The Mitochondria Extraction Kit (IMGENEX Corporation, San Diego, CA, USA) was used to isolate mitochondria. One mL homogenizing buffer was added per 100 mg of hippocampal tissue, followed by 10-min centrifugation at 4 °C and 3,000 rpm, the supernatant was removed and centrifuged again for 30 min at 4 °C at 12,000 rpm. The centrifuged supernatant (cytosolic fraction) was isolated as cytosol, while the remaining pellet was mixed thoroughly with 1 mL suspension buffer and centrifuged again for 10 min at 4 °C and 12,000 rpm. The supernatant was removed, the pellet was resuspended in 1 mL suspension buffer, and after another 10-min centrifugation at 4 °C and 12,000 rpm, the supernatant was removed and the resulting pellet was dissolved in 1 mL Complete Mitochondrial lysis buffer for 30 min at 4 °C. The separated mitochondrial extract was centrifuged for 5 min at 4 °C and 12,000 rpm, and the supernatant (mitochondria fraction) was collected.
Hippocampal tissue from isolated brain tissues was homogenized using lysis buffer and a homogenizer. It was then centrifuged for 15 min at 4 ℃ and 13,000 g, and the total protein content in the supernatant was quantified by the Bradford method. Protein (30 μg) was separated by electrophoresis in SDS-Polyacrylamide gel (7%, 10%), transferred to PVDF membrane (Amersham, Arlington Heights, IL, USA), blocked in 1× TBS-T solution containing 3 % BSA for 1 h at room temperature. The proteins and the corresponding primary antibody (
Antibody | Source | Vender | Catalog No. |
---|---|---|---|
Mfn1 | mouse monoclonal | Santa Cruz | sc-166644 |
Mfn2 | mouse monoclonal | Santa Cruz | sc-100560 |
OPA1 | mouse monoclonal | Santa Cruz | sc-393296 |
Drp1 | mouse monoclonal | Santa Cruz | sc-271583 |
Fis1 | mouse monoclonal | Santa Cruz | sc-376447 |
CS | mouse monoclonal | Santa Cruz | sc-390693 |
gp91 phox | rabbit polyclonal | Santa Cruz | sc-20782 |
SOD-2 | mouse monoclonal | Santa Cruz | sc-133134 |
Catalase | mouse monoclonal | Santa Cruz | sc-271803 |
β-Actin | rabbit polyclonal | Santa Cruz | sc-47778 |
Mfn1; Mitofusin1; OPA; Optic atrophy 1; Drp1; Dynamin-related protein 1; Fis; Mitochondrial fission 1; CS; Citrate synthase; gp91 phox; Glycoprotein 91 phagosome oxidase; SOD-2; Superoxide dismutase-2.
For the five rats to be analyzed by immunofluorescence assay, the brain tissue samples from each group (5 rats) were washed three times for 10 min using 0.01 M PBS according to the free-floating method. Then, each sample was placed in a beaker containing 0.01 M sodium citrate for 60 min for incubation at 90 °C, followed by blocking with 10 % normal donkey serum for 60 min. After blocking, the primary antibody (gp91phox) was added and the sample was left overnight for 12 h at 4 ℃, then washed three times for 5 min using 0.01 M PBS. The sample was then reacted with the secondary antibody (Alexa-488 conjugated donkey anti-mouse; 1:200 dilution, Jackson Immunochemicals, West Grove, PA, USA) for 2 h and washed four times, and each sample was moved to a slide and sealed using mounting solution (Vector Laboratories, Burlingame, CA, USA). The slides were imaged using an immunofluorescence microscope (Leica Microsystems, TCS SP8, Germany).
All data obtained in this study were used to generate descriptive statistics (mean ± SEM) using the 18.0 SPSS program for Windows. Intergroup differences were analyzed by one way ANOVA, and when a significant intergroup difference was found, the Bonferroni method was used as the post hoc test. Here, the statistical level of significance was set as α=0.05.
The changes in weight based on high-fat diet and treadmill exercise were measured at weeks 24, 43, 44, and 51, and the results are presented in
The area under the curve of glucose (AUC), HOMA-IR, and AVF/bodyweight, obtained from testing the plasma lipids TG, TC, and OGTT, is given in
Metabolic parameters | ND-CON | HFD-CON | HFD-TE |
---|---|---|---|
TG (mg/dl) | 97.00±2.59 | 130.00±2.43 | 124.00±4.38 |
TC (mg/dl) | 108.29±5.15 | 150.00±3.48 | 120.71±2.94 |
AUC (mg/dl∙min) | 43416.25±2004.77 | 57106.25±1045.13 | 46520.00±2046.74 |
HOMA-IR | 1.74±0.16 | 5.89±0.27 | 3.80±0.34 |
AVF/body weight (%) | 5.48±0.17 | 8.82±0.19 | 6.17±0.06 |
Values are presented as means ± SE, ND-CON; Normal diet-control, HFD; High fat diet, TE; Treadmill exercise, TG; Triglyceride, TC; Total cholesterol, AUC; Area under the curve, HOMA-IR; Homeostasis model assessment-insulin resistance, AVF; Abdominal visceral fat. #Denotes statistical difference from the ND-CON group. *Denotes statistical difference from the HFD-CON group (p<0.05).
The effect of treadmill exercise on CS, gp91phox, SOD-2, and Catalase expression is presented in
The effect of exercise on mitochondrial fusion factors (Mfn1, Mfn2, and Opa1) is given in
The effect of treadmill exercise on mitochondrial fission factors (Drp1 and Fis1) is given in
This study investigated the effects of treadmill exercise on oxidative stress and the fusion and fission of mitochondria in the hippocampal tissue of lab animals, where obesity was induced by a long-term, high-fat diet. The 20-week high-fat diet increased the weight and abdominal visceral fat in the animals while decreasing the level of HOMA-IR, an indicator of insulin resistance, and their ability to control plasma glucose (AUC from OGTT data). It also increased the serum concentrations of TG and TC. However, treadmill exercise was shown to improve these pathological symptoms associated with obesity. The long-term, high-fat diet was also shown to promote the fusion of mitochondria in the hippocampal tissue while reducing their fission, thereby partially disrupting mitochondrial dynamics. Moreover, the high-fat diet led to an increase in oxidative stress due to increased activity of NADPH gp91phox and decreased expression of CS, an indicator of mitochondrial energy metabolism activity. However, treadmill exercise suppressed the oxidative stress by enhancing the expression of antioxidant enzymes, and this improvement alleviated the imbalance in mitochondrial dynamics. A detailed discussion is given below.
The long-term, high-fat diet increased the weight, abdominal visceral fat, and serum TG and TC concentrations in the lab animals. The level of HOMA-IR and its AUC also increased. While conditions like impaired glucose tolerance, hyperlipidemia, or type II diabetes cannot be conclusively demonstrated, their negative influence on energy metabolism can be seen, such as the decrease in the ability to control plasma glucose or the increased incidence of hyperinsulinemia compared to that seen in rats fed a normal diet. However, treadmill exercise reduced the increase in weight due to high-fat diet and improved AUC, HOMA-IR, and abdominal fat content while reducing the concentrations of plasma TG and TC. These results are in concordance with previous studies that reported the effect of exercise on improving the negative influence of obesity on energy metabolism
According to a recent study, oxidative stress plays a critical role in linking obesity to cognitive dysfunction and brain function is sensitive to oxidative stress, which was reported to increase in obesity
Mitochondrial dynamics are a crucial mechanism in the maintenance of mitochondrial homeostasis, and oxidative stress was reported to increase due to unbalanced mitochondrial dynamics in obesity. This study also showed that, in the HFD-Con group, where obesity was induced, the expression of proteins related to mitochondrial fission, Drp1 and Fis1, increased, whereas the expression of proteins related to mitochondrial fusion, Mfn1, Mfn2, and Opa1, decreased. This can be explained by an excessive increase in mitochondrial fusion partially reducing mitochondrial function. Obesity causes an imbalance in mitochondrial fusion and fission, which reduces the mitochondria content. It is also associated with defects in neuronal development, plasticity, and function
To conclude, a long-term high-fat diet induced increased weight in lab animals, a decrease in the ability to control plasma glucose, and hematopathological changes that elevate the oxidative stress in the mitochondria of the brain hippocampal tissue to cause an imbalance in mitochondrial dynamics. However, treadmill exercise improved the pathological changes induced by obesity, so that it may contribute to functional improvement in mitochondria in the hippocampal tissue. Thus, this study suggests the need to consider treadmill exercise in the prevention and treatment of mitochondrial dysfunction in the brain hippocampal tissue and in the alleviation of the pathological changes induced by obesity.
This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF-2017S1A5A8020792)