Endogenous carbohydrate stores are limited and thus sufficient to fuel only a few hours of continuous, submaximal (70–80% maximal oxygen uptake) exercise. Muscle and liver glycogen depletions are associated with the onset of fatigue and impairment of exercise performance [1
]. Meanwhile, body fat deposits are large and represent a vast source of fuel for exercise. Therefore, enhancement of fat oxidation leads to glycogen sparing and has been suggested to improve endurance exercise performance [2
]. It is well known that chronic endurance exercise training enhances the capacity for muscle and whole-body fat oxidation [3
]. In addition, long-term intake of a high-fat diet additively enhances training-induced fat oxidation capacity [7
]. In particular, a very high-fat and extremely low-carbohydrate diet, known as the ketogenic diet, enhances the capacity to convert fat to ketone bodies in the liver and utilize them in skeletal muscle [10
]. Ketone bodies have been suggested to be more energy-efficient substrates than fatty acids [12
]. Therefore, the ketogenic diet might be an effective dietary strategy to improve athletic performance in endurance events, especially in ultra-endurance events, such as ultra-marathon and triathlon races [15
], and it has recently received much attention from athletes [16
However, previous studies have demonstrated that extremely low-carbohydrate, very high-fat ketogenic diets induce substantial increases in pyruvate dehydrogenase kinase 4 (PDK4) content in skeletal muscle [18
], which is a negative regulator of glycolytic flux [19
]. Physiological adaptation to a ketogenic diet therefore diminishes carbohydrate utilization capacity during exercise [7
]. In addition, ketogenic diets reduce muscle and liver glycogen levels as a consequence of their extremely limited carbohydrate content [21
]. Thus, it has been suggested that low-carbohydrate ketogenic diets may not be suitable for athletes who engage in prolonged bouts of exercise involving high-intensity exertion, during which carbohydrates are utilized as a major energy substrate [22
Medium-chain fatty acids (MCFAs), which consist of chains of 8–10 carbon atoms, have several unique properties in comparison with long-chain fatty acids (LCFAs). Unlike the majority of other dietary fats that are rich in LCFAs, medium-chain triglycerides (MCTs), which are composed exclusively of MCFAs, are hydrolyzed rapidly and the resultant MCFAs are absorbed directly by the liver through the portal vein [23
]. MCFAs are more easily oxidized in the liver because their intramitochondrial transport does not require a carnitine palmitoyl transferase system [25
], which is a rate-limiting step in mitochondrial β-oxidation. These characteristics make MCFAs a more ketogenic substrate than LCFAs in the liver. Therefore, incorporating MCTs into ketogenic diets, instead of long-chain triglycerides (LCTs), may allow for the consumption of more carbohydrate content and less fat content while preserving ketosis and enhancing ketone body utilization capacity without upregulating muscle PDK4 expression and thus inhibiting muscle carbohydrate metabolism. If MCTs have this effect, then MCT-containing ketogenic diets might be a valuable dietary strategy for athletes who require the ability to perform both high-intensity and endurance exercises.
The purpose of this study was therefore to examine the effects of long-term intake of a ketogenic diet containing MCTs and relatively more carbohydrate on endurance training–induced adaptations in metabolic enzymes in rat skeletal muscle tissue and to compare these effects with those observed under a conventional ketogenic diet composed exclusively of LCTs.
Ketogenic diets are widely used as a weight loss strategy because they can potently suppress appetite and reduce energy intake [31
]. Although, in the present investigation, the LKD-fed rats had similar total energy intake relative to the CON group rats, they showed significantly lower body weight and total intra-abdominal fat mass. As shown in Table 3
, the LKD group had lower food efficiency, suggesting that LKD intake may prevent weight gain possibly through an increase in energy expenditure but not a decrease in energy intake. In the present investigation, we observed a further significant decrease in total intra-abdominal fat mass in the MKD group, especially in the EX-MKD group (the MKD intake and endurance training additively decreased total intra-abdominal fat mass) (Table 3
). It has been well documented that MCT intake has body fat–lowering effects because it stimulates diet-induced thermogenesis and energy expenditure [33
]. In addition, Ooyama et al. reported that MCT ingestion suppresses subsequent food intake in rats, possibly owing to an increase in hepatic ATP content [35
]. Our results showing that the MKD group exhibited significantly lower total energy intake as well as lower food efficiency confirmed the findings of previous studies and provided additional evidence that the MKD is a more effective dietary strategy to control body weight and body fat.
In the SED group, LKD intake elevated plasma βHB concentration by up to ~1.5 mmol/L (Figure 1
A). The LKD-induced ketosis might be mediated, at least in part, by higher expression of hepatic HMGCS2 (Figure 2
), which is a rate-limiting factor in the synthesis of ketone bodies [28
]. In contrast, plasma βHB concentration in the SED-MKD group increased to a level similar to that attained in the SED-LKD group without a significant increase in hepatic HMGCS2 expression (Figure 2
). MCTs are absorbed via the portal vein and transported directly to the liver, where they are rapidly oxidized and converted to ketone bodies [23
]. These unique properties made the MKD treatment more ketogenic, even though it was composed of more carbohydrates (18% of total energy vs. 1% in the LKD treatment) and did not upregulate hepatic HMGCS2 content.
Although both ketogenic diets in combination with endurance training induced further increases in plasma βHB concentration, the EX-MKD group showed significantly lower plasma βHB concentration compared with the EX-LKD group (Figure 1
A). The lower plasma βHB concentration in the EX-MKD group might be caused by either diminished ketogenesis in the liver or enhanced ketolysis in other organs such as skeletal muscle. As shown in Figure 3
, the MKD and endurance training treatments additively increased content of a key ketolytic enzyme, OXCT, in epitrochlearis muscle, resulting in the highest expression in the EX-MKD group. A previous study demonstrated a significant positive correlation between muscle OXCT content and ketone body utilization [29
]. It is therefore more likely that the EX-MKD group could utilize more ketone bodies during training, accounting for their lower plasma βHB concentration compared with the EX-LKD group.
Oxidation of ketone bodies (βHB) yields more ATP per mole of substrate compared with oxidation of the end-glycolytic substrate pyruvate [36
]. In addition, ketone bodies increase the free energy released from ATP hydrolysis by reducing the mitochondrial NAD couple and oxidizing the coenzyme Q couple, thereby increasing the redox span between complex I and complex II of the mitochondrial electron transport chain [37
]. These energetic characteristics of ketone bodies enabled a working, perfused rat heart to increase the efficiency of hydraulic work by ~30% compared with pyruvate [12
]. Based on these findings, ketone bodies are therefore thought to be the most energy-efficient fuel. The MKD treatment, which additively enhanced the endurance training–induced increase in muscle ketolytic capacity, may therefore be a beneficial dietary intervention to improve endurance exercise performance. Previous studies demonstrated the lower adherence to a conventional ketogenic diet, because it consists exclusively of fat with an extremely limited carbohydrate content [38
]. In contrast, the MKD treatment employed in this study could induce beneficial metabolic adaptations, as mentioned above, despite there being a relatively higher carbohydrate content (~18% energy from carbohydrates) and therefore might be a more feasible dietary intervention compared with conventional ketogenic diets containing LCTs.
Endurance exercise training and a high-fat diet intake independently and additively increased the protein content of mitochondrial fatty acid oxidation enzymes and fat oxidation capacity in skeletal muscle [39
]. The high-fat diet-induced upregulation of muscle fatty acid oxidation enzymes is thought to be a result of the activation of a nuclear receptor, peroxisome proliferator-activated receptor (PPAR) β, by elevated blood FFA [41
]. In accordance with the previous findings [42
], the LKD treatment substantially elevated plasma FFA levels with concomitant increases in muscle βHAD protein content, with the EX-LKD group having the highest values (Figure 1
D and Figure 4
). In contrast, the MKD groups did not show such increases in muscle βHAD protein content, suggesting that the MKD treatment enhanced utilization capacity only for ketone bodies, but not fatty acids. Consistent with a previous study showing that even larger amounts of MCT failed to produce detectable increases in plasma FFA [44
], the MKD-fed groups had lower plasma FFA levels, which led to lower muscle βHAD protein content. Because βHAD is a key enzyme in fatty acid β-oxidation and its activity has been shown to be significantly corelated with fatty acid oxidation rate during exercise [30
], the long-term intake of LKD, but not MKD, in combination with endurance training might be a sufficiently strong stimulus to enhance muscle fatty acid utilization capacity. However, although fatty acids are important substrates during endurance exercise, metabolism of fatty acids leads to a reduction in mitochondrial NAD and mitochondrial coenzyme Q, thus causing a decrease in the free energy released from ATP hydrolysis and thereby requiring more oxygen [45
]. Actually, Burke et al. demonstrated that adaptation to a conventional low-carbohydrate, high-fat ketogenic diet impaired exercise economy (causing higher oxygen consumption at the same exercise intensity) and negated the performance benefit of intensified training in elite race walkers [7
]. Thus, athletes may derive little benefit from conventional ketogenic diets consisting of LCTs.
A major concern associated with ketogenic diet intake in athletes is its effects on carbohydrate metabolism in skeletal muscle [22
]. Numerous studies have shown that very low-carbohydrate, ketogenic diets reduce glycolytic enzyme activity and thus diminish carbohydrate oxidation in humans and animals [7
], suggesting that ketogenic diets may impair high-intensity exercise performance, during which carbohydrates are utilized as major energy substrates. Potential mechanisms by which ketogenic diets induced inhibition of glycolytic flux is postulated to be upregulation of PDK4 expression in skeletal muscle [47
]. Consistent with previous finding [18
], we observed a huge increase in PDK4 protein content in epitrochlearis muscle tissue of the LKD-fed rats, in particular in the EX-LKD group (Figure 5
). This result supports the notion that this type of ketogenic diet exerts deteriorating effects on muscle carbohydrate utilization and high-intensity exercise capacity. Because expression of PDK4 as well as βHAD are mediated by PPARβ [48
], the higher plasma FFA levels in the LKD groups might be responsible for the LKD-induced muscle PDK4 expression via activation of PPARβ. In contrast, as shown in Figure 5
, the MKD treatment completely prevented such an increase in muscle PDK4 protein content concomitant with lower plasma FFA levels, although it contained relatively higher amounts of fat and induced ketosis. Our results therefore suggest that long-term intake of ketogenic diets containing MCTs and relatively more carbohydrate may enhance the utilization capacity of ketone bodies, which are particularly energy-efficient substrates, in skeletal muscle tissue without exerting inhibitory effects on muscle carbohydrate metabolism.
Previous studies have reported that muscle and liver glycogen concentrations were lower following consumption of a low-carbohydrate, ketogenic diet [49
], which may be another contributing factor to the ketogenic diet-induced impairment in high-intensity endurance exercise performance. In this study, as a consequence of their lower carbohydrate intake, both ketogenic diet groups had significantly lower muscle and liver glycogen levels (Figure 6
). However, the MKD-fed rats had less reduction in muscle and liver glycogen concentration than did the LKD-fed rats (Figure 6
). These unique characteristics of the MKD treatment, which have reduced inhibitory effects on glycogen content as well as glycolytic enzyme levels, make this diet suitable for athletes who need the ability to perform prolonged high-intensity exercise that relies heavily on energy coming from glycolytic pathways.
This study has several limitations. First, we unfortunately could not measure other blood energy substrates such as plasma glucose and evaluate exercise capacity in humans or in rats. Thus, it remains unclear whether MKD treatments have athletic performance–enhancing effects or not, especially in prolonged high-intensity exercise. Second, this study was performed on only male rats. The results obtained in this study may not be directly extrapolated to female rats as well. Finally, we did not evaluate the effect of intake of LKD containing more carbohydrate at levels equivalent to the MKD in this study. We therefore could not rule out the possibility that consumption of relatively more carbohydrate rather than MCTs intake was responsible for the MKD-induced adaptations. Future extensive studies are required to solve these issues.