1. Introduction
Carbohydrates, lipids and proteins are three classes of molecules utilized as fuel, or to make up bodily structures. Among these classes, protein is a vital nutrient that holds our bodies together. Compared with protein, carbohydrates and lipids are more flexible for use as energy sources. Compared with the storage capacity for carbohydrates (which is limited to ~5 g glucose in blood circulation and ~100 g or ~500 g glycogen in skeletal muscle or liver), the upper limit for human lipid store as fat seems to be unrestricted [
1]. A person weighing 70 kg, possessing 15% body fat percentage, is capable of completing >30 marathon races while utilizing stored fat [
2]. Therefore, due to limited carbohydrate reserves but an abundant lipid reserve, coaches and elite athletes are wondering if there is an effective way to enhance fat utilization. Investigations into this started decades ago, and continue to the present day [
3,
4].
Excess adipose tissue may cause health problems, whilst excessive bodyweight can be a problem in some events that are divided by weight, e.g., Taekwondo [
4]. Moreover, participants of extremely strenuous aerobic events, such as marathon runners, usually display very low body fat percentage (6.4 ± 1.3%, for males, according to a survey conducted in elite athletes in America) [
5]. Therefore, for athletes, only pursuing an abundant lipid reservoir may be unrealistic, making the ability to utilize fat effectively more important.
An attempt to increase dietary fat and enhance fat utilization proved ineffective in a human trial of young men provided with a fat supplement (987 ± 55 kcal/day) over 36 h, which did not alter 24 h energy expenditure (2783 ± 232 kcal/day vs. 2820 ± 284 kcal/day), and fat oxidation rate (fat oxidation 1032 ± 205 kcal/day vs. 1042 ± 205 kcal/day) [
6]. However, a high-fat diet (HFD) combined with exercise enhanced fat oxidation, according to previous studies [
7,
8,
9]. In studies conducted in both trained and untrained subjects, training increases fat oxidation in human subjects and reduces the reliance on carbohydrate as an energy source during submaximal exercise tests [
7,
8,
9]. Researchers also report that HFD adaption, including keto-adaption, may enhance endurance capacity in trained cyclists and rodents [
10,
11]. To date, many studies have made it clear that an HFD is a two-sided coin. For example, a one-month HFD was reported to cause increased muscle mitochondrial biogenesis and insulin resistance (IR) at the same time [
12]. In another study conducted in rodents, a one-month alternate-day HFD increased mitochondrial enzyme activities and protein content; however, a reduced oxidative capacity was also observed in a short period, e.g., genes of the electron transport chain, mitochondrial carrier proteins and mitochondrial biogenesis [
13,
14]. Clinical evidence shows that non-obese subjects are capable of adjusting fat oxidation in response to increased fat intake within a week, even when energy balance is sustained [
15]. Therefore, long-term traditional HFD is challenging when employed as a fat-loading tactic, since it may cause a weight burden for athletes, along with health problems. That is why we employed a low-carbohydrate, high-fat, ketogenic diet in our previous study [
10].
In our previous study, we reported that an eight-week low-carbohydrate, high-fat, ketogenic diet enhanced exercise capacity, but the mechanisms remained unclear. In the previous study, body fat content was not altered significantly by a ketogenic diet (KD), although the average weight of the KD mice dropped dramatically in the feeding period. Therefore, we postulated whether another form of fat reserve contributed to this enhancement. Intramuscular triacylglycerol (IMTG) is a special way for skeletal muscle to store lipids. During exercise, IMTG may constitute up to 20% of total energy turnover, thus contributing significantly to adenosine triphosphate (ATP) synthesis during exercise [
16]. However, abnormal or excessive fat deposition in skeletal muscle may also induce insulin resistance [
17]. Regulating intramuscular lipolysis is crucial for energy supply during exercise. Amati and colleagues reported that well-trained athletes exhibit higher levels of IMTG and diacylglycerol (DAG), together with a well-preserved sensitivity to insulin, indicating lipolysis may be enhanced during exercise [
18]. Ketone bodies and fatty acids are produced or released from the liver and adipose tissue, where other organs may then employ the metabolites as an energy source during fasting, exercise or other medical conditions. It is reported that working muscles have an increased capacity to extract ketone bodies from circulating blood during exercise [
19]. However, metabolic profiles of different muscle fiber types are reported to be different, which may be attributed to their function [
20,
21]. Therefore, it is reasonable to presume that enhanced fat utilization, (including fatty acid mobilization from adipose tissue and IMTG), transportation, and oxidation by different muscle fibers together with ketone body utilization can be directly linked to exercise capacity. In this present study, we studied the rate-limiting enzymes during FAO, to see whether an eight-week KD enhanced FAO and contributed to exercise capacity.
Endurance exercise may induce systemic inflammatory response [
22]. According to the above studies, plasma interleukin (IL)-6 may increase over 100-fold after strenuous exercise [
23,
24]. Muscle-derived, exercise-induced IL-6 is reported to have lipolytic properties [
25]. However, whether IL-6 may contribute to keto-adaption, thus enhancing exercise capacity, is unknown. Since our previous report indicated the amount of fatty acid and triglyceride consumption were enhanced in KD mice, we have herein examined the association between IL-6 and enhanced lipolysis. As discussed previously, a KD has been investigated as a low-carbohydrate nutritional approach within athletic nutrition to performance enhancement, but controversial conclusions have been obtained [
10,
26,
27]. In our previous studies, we reported that an eight-week, low-carbohydrate, ketogenic diet increased running time until exhaustion in male C57BL6/J mice, and suggest the mechanism to be an enhanced fat utilization [
10,
27]. In the present study, we have investigated any alteration in the pattern of messenger RNAs related to lipid mobilization, fatty acid utilization and ketone body oxidation, in red slow-twitch and white fast-twitch muscle tissues, and adipose tissue, to ascertain the underlying mechanisms.
2. Materials and Methods
2.1. Mouse Maintenance and Diets
Male C57BL/6J mice (n = 35) were purchased from Takasugi Experimental Animals Supply (Kasukabe, Japan) at 7 weeks of age, and were allowed to adapt to the environment for a week before formal experimentation commenced. Four or five animals were housed together to a cage (27 × 17 × 13 cm) in a controlled environment under a light–dark cycle (lights on at 08:00 and off at 20:00). The experimental procedures were approved and followed the Guiding Principles for the Care and Use of Animals in the Academic Research Ethical Review Committee of Waseda University (10K001). All mice were randomly divided into four groups: chow diet (control: Con), involving a chow diet and a promotion of sedentary behavior (n = 8), chow diet plus exercise (Con + Ex, n = 9. Ex is the abbreviation for exercise), a KD and a promotion of sedentary behavior, (n = 9), and a KD plus exercise (KD + Ex, n = 9). The KD diet (TP-201450) consisting of 76.1% fat, 8.9% protein and 3.5% carbohydrate, 7.342 kcal/g and the chow diet (AIN93G) consisting of 7% fat, 17.8% protein and 64.3% carbohydrate, 3.601 kcal/g) wt/wt were obtained from Trophic (TROPHIC Animal Feed High-tech Co., Ltd., Jiangsu, China). Mice were maintained on ad libitum chow diet or KD, for 8 weeks commencing at 8 weeks of age.
2.2. Endurance Capacity Test Protocol
One week before exhaustive exercise, all mice were accustomed to the treadmill by running at 15 m/min for 10 min. The endurance test was performed on a motorized treadmill (Natsume, Kyoto, Japan). That is, mice in the Con + Ex and KD + Ex groups were subjected to treadmill running at 10 m/min for 15 min, followed by 15 minutes at 15 m/min and then 20 m/min, followed finally by running at 24 m/min and 7% grade until exhaustion. Exhaustion was defined as the inability to continue regular treadmill running despite the stimulation of repeated tapping on the back of the mouse. The running time of exercised mice was recorded. Immediately after the exhaustion, mice were sacrificed under light anesthesia with the inhalant isoflurane (Abbott, Tokyo, Japan). Heparinized blood samples were collected from the abdominal aorta under isoflurane-induced mild anesthesia, whilst tissues and organs were immediately excised and frozen in liquid nitrogen. Plasma was obtained from blood samples by centrifugation at 1500× g for 10 min at 4 °C. These samples were stored at −80 °C until analyses.
2.3. Real-Time PCR
Total RNA was extracted from the gastrocnemius muscle (white, fast-twitch muscle) and soleus muscle (red, slow-twitch muscle) using the RNeasy Fibrous Mini Kit, and from epididymal adipose tissue using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The purity and concentration of total RNA was assessed using the NanoDrop system (NanoDrop Technologies, Wilmington, DE, USA). Total RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. PCR was performed with the Fast 7500 real-time PCR system (Applied Biosystems) using the Fast SYBR® (Applied Biosystems) Green PCR Master Mix (Applied Biosystems). The thermal profiles consisted of 10 min at 95 °C for denaturation followed by 40 cycles of 95 °C for 3 s and annealing at 60 °C for 15 s. 18 s mRNA was used as the housekeeping gene, and the ΔΔCT method was used to quantify target gene expression. All data are represented relative to its expression as fold change based on the values of the Con group.
2.4. ELISA Procedure and Glycerol Assay
Plasma and gastrocnemius IL-6 concentrations were measured using a R&D Mouse IL-6 ELISA Duo set (R&D Systems, Minneapolis, MI, USA) according to the manufacturer’s instructions. Gastrocnemius IL-6 concentration was related to total protein concentration measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Plasma glycerol was measured using the Glycerol Colorimetric Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA).
2.5. Statistical Analysis
Data are presented as means ± standard deviation (SD). A two-way analysis of variance (ANOVA) was performed to determine the main effects of diet and/or exercise. Statistical analysis was done using Graphpad 7.0 (Graphpad, Ltd., La Jolla, CA, USA). When this analysis revealed significant interaction, Tukey’s post hoc test was performed to determine the significance among the means. Associations among variables were analyzed using Pearson’s correlation coefficient. Statistical significance was accepted as p < 0.05.