The main finding of this study is that the oral royal jelly administration had a significant positive effect on inducing the increase in maximal activity of mitochondrial enzyme by endurance training in the soleus muscle, which mainly consists of type I and IIA fibers, while no significant effect of RJ treatment on mitochondrial adaptation was observed in the plantaris and TA muscles, which predominantly consist of type IIB/IIX fibers. Acute RJ treatment and endurance exercise additively increased the phosphorylation of AMPK and ACC, a downstream substrate of AMPK, in the soleus muscle, while no effect of acute RJ treatment was noted in the plantaris and TA muscles.
As the maximal activity of CS, which catalyzes the rate-limiting step of the tri-carboxylic acid (TCA) cycle, is strongly associated with the mitochondrial content in skeletal muscles [
21], it is generally used as an indicator of mitochondrial oxidative capacity. In the
plantaris and
TA muscles, the endurance training had a significant main effect on the maximal CS activity. However, no effect of RJ treatment was observed in these muscles. On the other hand, we found that the RJ treatment concomitant with the endurance training increased the maximal CS activity in the
soleus muscle, while no change was observed in the control training group. We observed a similar trend in the maximal activity of β-HAD, which catalyzes the rate-limiting step of fatty acid β-oxidation. These results suggest that the endurance exercise was enough to induce mitochondrial enzymatic adaptation in fast-twitch fiber dominant muscles, while it was contrary in the high-oxidative muscles. A previous study showed that the increase in the mitochondrial enzymatic activity by chronic stimulation-induced contraction was greater in the low-oxidative muscles compared to the high-oxidative muscles [
12]. Therefore, the possibility exists that the high-oxidative muscles have a higher threshold to induce mitochondrial adaptation than the low-oxidative muscles. In the present study, although the endurance exercise induced significant positive effects on the phosphorylated states of the signaling proteins involved in mitochondrial biogenesis in the
soleus, additional stimulation by RJ treatment was needed to induce the increase in the maximal mitochondrial enzymatic activities.
Some components contained in RJ such as amino acids/proteins [
6,
8], particularly leucine [
7], and 10-hydroxy-2-decenoic acid [
9,
10,
11] which is the unique fatty acid present in RJ have been reported to induce the activation of AMPK, a major mediator of the mitochondrial biogenesis, in skeletal muscles, or the myotubes. Meanwhile, RJ contains several components that have been suggested to have antioxidant property: 10-hydroxydecanoic acid; free amino acids, such as proline, cystine, and cysteine; flavonoids; and phenolic compounds [
22,
23,
24]. Previous studies suggested that oxidative stress plays an important role in AMPK activation in skeletal muscle and then the use of antioxidants might affect AMPK activation [
25,
26]. Moreover, mitochondrial oxidative stress has been suggested to be involved in calcium handling [
27] and influence an activation of Ca
2+/calmodulin-dependent kinase kinase (CaMKK), which is one kind of upstream kinases of AMPK [
28]. Therefore, we examined the effects of a single dose of RJ and endurance exercise on the activation of the AMPK signaling cascade. In the soleus, RJ treatment additively increased the phosphorylation levels of AMPK and ACC concomitant with endurance exercise. We also measured the phosphorylation status of p38 MAPK, an activator of mitochondrial biogenesis as well as AMPK, and there was no effect of RJ treatment in the
soleus. Collectively, the effect of RJ treatment on the endurance training-induced increase in the maximal activities of mitochondrial enzymes in
soleus seems to be mediated by the AMPK signaling amplification. On the other hand, no effect of RJ treatment was observed on the phosphorylation levels of AMPK signaling cascade proteins in
plantaris and
TA muscles. Therefore, the effect of RJ treatment on the activation of AMPK signaling was specific to the soleus muscles, which predominantly consist of type I and type IIA muscle fibers. Regarding amino acids/proteins, a previous study that was focused on investigating the effect of oral casein peptide administration indicated results similar to the present study. They reported that casein peptide treatment increased the phosphorylation in the AMPK regulatory site in the soleus, while no effect was observed in the
plantaris [
6,
8]. However, the mechanism behind the effect of these nutrients on the differential activation levels of AMPK activation in different skeletal muscles with distinct fiber type composition has not yet been elucidated. Leucine is one of the potential activators of AMPK in skeletal muscles [
7]. A previous study showed that the uptake of glutamate, which indicates a simultaneous efflux against leucine, was lower in the soleus compared with those in
flexor digitorum brevis and
epitrochlearis muscles, which are predominantly made of fast-twitch fibers [
29]. As the intracellular glutamine contributes to the leucine uptake as an exchange material, thus the lower glutamine uptake in the
soleus would not induce leucine uptake into muscles. Moreover, a recent study demonstrated that the expression level of the L-type amino acid transporter 1 (LAT1), which is thought to be a primary transporter of neutral amino acids including branched chain amino acids (leucine, valine, isoleucine), was higher in the type II fiber than in the type I fiber [
30]. Therefore, it is quite unlikely that the observed differential response to RJ treatment in
soleus,
plantaris, and
TA muscles is due to the difference in amino acids transport into the cells (particularly leucine). To date, no study has compared the effect of 10-hydroxy-2-decenoic acid and other components of RJ on AMPK activation in different muscle fibers. Further studies are warranted to elucidate the mechanism behind the differential response to the RJ components in different skeletal muscles with discrete types of muscle fibers.
In the present study, a positive effect of RJ treatment on endurance training-induced mitochondrial adaptations was observed in the mice
soleus muscle, which is mainly composed of type I (around 35–45%) and type IIA (around 35–50%) fibers [
13,
14,
15]. In human
vastus lateralis muscles, which is frequently used in human studies, the percentage of type I and type IIA fibers correspond to those in the mice soleus muscle [
31,
32]. Therefore, the possibility exists that effect of RJ treatment combined with endurance training might be observed in human skeletal muscles. We provided mice with 1.0 mg/g body weight of RJ for three weeks. It is recommended that the dose of drug or nutrient is converted across animal species based on the body surface area [
33]. According to the calculation method proposed by Reagan-Shaw et al. [
33], an estimated human equivalent dose of RJ used in the present study is 81.1 mg/kg body weight (4.9 g for 60 kg person). This amount of RJ intake corresponds to that used in the previous study, in which elderly people received RJ for one year [
34]. In that study, no side effect of RJ intake was reported. To our knowledge, no study has examined the effect of RJ treatment on endurance training-induced mitochondrial adaptations in human. Further research is needed to elucidate whether three weeks of RJ treatment with endurance training shows beneficial effect for inducing mitochondrial adaptations in human.