2.2. Experimental Design
After a week of the adaptation period, the rats were divided into eight equal groups containing seven rats each. The initial body weights of the rats were similar among groups (214.53 ± 4.67, p
> 0.05). The rats were fed either (1): a standard diet (Table 1
) as a control with no exercise or Carnipure®
Tartrate (CT) supplementation at all (C0) or (2): a control diet supplemented with 200 mg/kg CT with no exercise (C200) or (3): a control diet supplemented with no CT but with exercise (E
) or (4): a control diet supplemented with 25 mg/kg CT with exercise (E+C25) or (5): a control diet supplemented with 50 mg/kg CT with exercise (E+C50) or (6): a control diet supplemented with 100 mg/kg CT with exercise (E+C100) or (7): a control diet supplemented with 200 mg/kg CT with exercise (E+C200) or (8): a control diet supplemented with 400 mg/kg CT with exercise (E+C400). Table 1
shows the composition of the control diet fed to the rats. CT is a special quality of L-carnitine, which is produced via chemical synthesis. Lonza Consumer Health Inc., Morristown, NJ, USA provided CT. CT and placebo (physiological saline) were administered orally via gavage every day before exercise during the experimental period (6 weeks).
The rats were subjected to treadmill exercise on a motorized rodent treadmill (Commat Limited, Ankara, Turkey). The treadmill contained a stimulus grid at the back end of the treadmill, which gave an electric shock when the animal placed its paw on the grid. The apparatus had a 5-lane animal exerciser utilizing a single belt unit divided with walls suspended over the tread surface. To eliminate the diurnal variations, all exercise tests were applied during the same time of the day. A week of adaptation was provided as pre-training practice for the animals to get familiar with the treadmill equipment and handling. In doing so, the rats in the exercise training groups were accustomed to treadmill exercise over 5 days such that: (i) 1st day, 10 m/min for 10 min, (ii) 2nd day, 20 m/min for 10 min, (iii) 3rd day, 25 m/min for 10 min, (iv) 4th day, 25 m/min for 20 min, and (v) 5th day, 25 m/min for 30 min. Upon an adaptation of a week toward the treadmill system for the novel and stress impacts, the rats in treadmill exercise groups ran on the treadmill at 25 m/min for 45 min/per day and 5 days per week for 6 weeks, according to the protocol described by Sahin et al. [5
]. The exercise model was chosen because the exercise model is the most common procedure to carry out animal exercise training from weeks to months at 45 min–1 h/day and 5 days/week. In addition, the exercise model provides adaptations to the cardiovascular system including physiological remodeling of the heart representative with increased O2 consumption, improvement of cardiac contractile function, and calcium handling [30
]. The chosen long-term animal exercise model fits an effective program to benefit both healthy subjects and individuals with cardiovascular risk [31
At the end of each exercise session, exhaustion time and average distance run were recorded. Endurance capacity was measured using treadmill running to fatigue on the last day of week 6 by compelling the rats to run on a motorized treadmill. This process is defined as the inability of the rat to maintain an appropriate pace despite continuous hand prodding for 1 min. After this application, the rats were removed from the treadmill. The rats were deemed to be fatigued when it was no longer able to continue to run on the treadmill as judged by the rat spending >50% of the time or >30 consecutive seconds on the electrical stimulus.
Body weights were measured in rats using a scale after 6 weeks of exercise. The body mass index (BMI) was then calculated based on the formula: BMI (g/cm2) = body weight (g)/length (cm)2.
At the end of the experiment, all rats were subjected to overnight fasting and blood samples were taken from decapitated animals via cervical dislocation via anesthesia. In addition, visceral fat was carefully removed from the carcass and weighed. This procedure was carried out immediately after the last exercise session. Blood samples, collected by gel biochemical tubes, and serum samples were taken and centrifuged at 4 °C at 3000× g for 10 min in a chilled centrifuge. In addition, the tissues obtained from the animals were stored in a deep freeze at −80 °C until analysis.
2.3. Biochemical Analysis
Serum concentrations of glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine, total cholesterol, and triglyceride were assayed using a portable automated chemistry analyzer (Samsung LABGEO PT10, Samsung Electronics Co., Suwon, Korea). The rat lactate assay kit (Cayman Chemical Co., Ann Arbor, MI, USA) was used to measure the serum lactate concentrations through enzyme-linked immunosorbent assays (ELISA, Elx-800, Bio-Tek Instruments Inc., Winooski, VT, USA). ELISA (MyBioSource, San Diego, CA, USA) was also used in measuring serum myoglobin concentrations. The intra-and inter-assay coefficients of variation for lactate and myoglobin kits were both <13%. An enzymatic cycling method using commercial kits (MyBioSource, San Diego, CA, USA) was applied in assaying serum total and free L-carnitine concentrations. Acyl-carnitine was then calculated as subtracting free L-carnitine from the total L-carnitine amounts. A parameter of the oxidative stress indicator includes MDA concentrations assayed by a fully automatic High-performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) equipped with a pump (LC-20AD), an ultraviolet-visible detector (SPD-20A), an inertsil ODS-3 C18 column (250 × 4.6 mm, 5 m), a column oven (CTO-10ASVP), an autosampler (SIL-20A), and a degasser. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were determined using the commercially available kits (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s procedure.
2.4. Statistical Analysis
The data were statistically analyzed by one-way ANOVA using the SPSS statistical program (IBM, SPPS Version 21; Armonk, New York: IBM Corp). Differences between the groups were achieved by Tukey’s correction to control for false positives, which were generated by the multiple comparisons performed in the analyses. In addition, p < 0.05 was considered statistically significant. Data were reported as mean and standard deviations in tables. The data in the figures are presented as the median, minimum, and maximum values.