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Abstract

The lipidome of royal jelly (RJ) consists of medium-chained (8–12 carbon atoms) free fatty acids. We present herein a liquid chromatography-high resolution mass spectrometry (HRMS) method that permits the determination of RJ fatty acids and at the same time the detection of suspect fatty acids. The method allows for the direct quantification of seven free fatty acids of RJ, avoiding any derivatization step. It was validated and applied in seven RJ samples, where the major RJ fatty acid trans-10-hydroxy-2-decenoic acid (10-HDA) was found to vary from 0.771 ± 0.08 to 0.928 ± 0.04 g/100 g fresh RJ. Four additional suspect fatty acids were simultaneously detected taking advantage of the HRMS detection.

2.4. Analysis of RJ Samples

Seven different RJ samples, which were purchased from the local market, were analyzed. The extracted ion chromatograms (EICs) of the analytes in a standard solution (A), as well as in a representative RJ sample (B) are presented in . The isobaric 10-hydroxydecanoic acid and 3-hydroxydecanoic acid are well separated and are eluted at 2.37 and 3.25 min, respectively.

2.4. Analysis of RJ Samples

The concentrations of the seven analytes in seven RJ extracts (in triplicate) are summarized in . represents the multi sample analysis data. The contents of free fatty acids are expressed as g of fatty acid per 100 g of fresh RJ. 10-HDA was found to be the most abundant, as expected, and its content ranged from 0.771 ± 0.08 to 0.928 ± 0.04 g/100 g fresh RJ. These results are in accordance with literature taking into account that the content of 10-HDA depends on the origin of the jelly and the characteristics of the bee. The levels of 10-HDA have been found to vary from 0.75 to 2.54 by Genc and Aslan using column liquid chromatography [], and from 0.8 to 3.2 by Ferioli et al. using either CZE or HPLC [].

Abstract

Honeybees produce royal jelly (RJ) from their cephalic glands. Royal jelly is a source of nutrition for the queen honey bee throughout its lifespan and is also involved in fertility and longevity. Royal jelly has long been considered beneficial to human health. We recently observed that RJ delayed impairment of motor function during aging, affecting muscle fiber size. However, how RJ affects skeletal muscle metabolism and the functional component of RJ is as of yet unidentified. We demonstrate that feeding mice with RJ daily prevents a decrease in myofiber size following denervation without affecting total muscle weight. RJ did not affect atrophy-related genes but stimulated the expression of myogenesis-related genes, including IGF-1 and IGF receptor. Trans-10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), two major fatty acids contained in RJ. After ingestion, 10H2DA and 10HDAA are metabolized into 2-decenedioic acid (2DA) and sebacic acid (SA) respectively. We found that 10H2DA, 10HDAA, 2DA, and SA all regulated myogenesis of C2C12 cells, murine myoblast cells. These novel findings may be useful for potential preventative and therapeutic applications for muscle atrophy disease included in Sarcopenia, an age-related decline in skeletal muscle mass and strength.

3.1. pRJ Attenuates Denervation-Induced Skeletal Muscle Atrophy

To examine the effect of pRJ on skeletal muscle atrophy, C57BL/6 mice were fed on control or pRJ diets for four weeks, and then muscle atrophy was induced by denervation. Daily feeding of pRJ for 1 month had no significant effect on total body weight (A) or loss of total tibialis anterior muscle weight induced by denervation (B). However, pRJ prevented the decrease in skeletal muscle fiber diameter following denervation (C,D). In order to determine the mechanism by which pRJ prevents the decrease in muscle fiber size, we next compared the expression levels of atrophy, proliferation, or skeletal muscle differentiation genes. qPCR and Western blotting analysis showed that pRJ did not alter the expression of catabolic genes such as Atrogin-1 (A and 2K), Muscle ring finger protein 1 (MuRF1) (B), or Forkhead box O-1 (Foxo-1) (C). However, pRJ increased the expression of proliferation and differentiation-related genes such as Cyclin E1 (D), Cyclin A2 (E), or Myogenin (G). Protease-treated RJ had no significant effect on the upregulation of Mych1 (H), Myhc2 (), Myhc4 (), or Myhc7 (). Protease-treated RJ stimulated the upregulation of IGF-1 (I) and IGF receptor (IGFR) (J) and phosphorylation of AMPK (K).

3.2. pRJ Stimulates Myoblast Proliferation

Protease-treated royal jelly treatment for 48 hours increased the number of cells (A,B) as well as the expression of cell-cycle-related genes (C–F). Furthermore, Ki67 immunostaining showed that pRJ treatment increased the number of Ki67-positive proliferative cells (G,H). Trans-10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid are two fatty acids specifically occurring in RJ. Trans-10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid can be metabolized into 2DA and SA, respectively []. To determine whether RJ-derived fatty acids and/or their metabolic products affect proliferation, C2C12 cells were treated with the indicated compounds for 24, 48, and 72 hours, after which cell numbers were quantified by WST-8. As shown in A, 10H2DA, 10HDAA, 2DA, and SA all significantly increased cell proliferation (A). These RJ-related fatty acids also increased the protein levels of cell cycle genes such as Cyclin D1 and Cyclin A2 (B).

3.3. pRJ Stimulates Myoblast Differentiation

Finally, we examined the effect of pRJ and RJ-related fatty acid products on myoblast differentiation. C2C12 cells were induced to differentiate in the presence or absence of pRJ. Myosin heavy-chain immunostaining showed that pRJ treatment led to an increase in myotube formation compared to control treatment cells (A,B). Furthermore, pRJ treatment elevated the expression level of muscle differentiation genes such as MyoD, Myogenin, Mck, or Myhc1 (C–H). C3H10T1/2 cell model is a mouse embryonic fibroblast cell line with myogenic potential. C3H10T1/2 cells, however, do not normally express MyoD, a master regulator of myogenesis []. To evaluate the effect of pRJ on MyoD function, we overexpressed MyoD in C3H10T1/2 cells and then treated the cells with or without pRJ. qPCR analysis revealed that pRJ treatment enhanced the induction of Myogenin and Myhc1 induced by MyoD (I,J). Treatment with 10H2DA, 10HDAA, 2DA, and SA increased myotube formation (A,B). The treatment of cells with these RJ-related fatty acids increased the protein levels of myogenic differentiation marker genes such as Myhc and myogenin (C).

4. Discussion

In this study, we demonstrate that feeding mice daily with pRJ prevents a decrease in myofiber size following denervation. In a previous study, we showed that pRJ affects muscle fiber size in elderly mice without changing total muscle weight []. Our current findings did not conflict with this study. Muscle weight during pathological conditions such as obesity, type 2 diabetes, or age-related Sarcopenia can be affected by the infiltration of adipose and/or connective tissue []. In our study, we did not quantify the infiltration of adipose or connective tissue into the muscle tissue, even though pRJ treatment tended to increase muscle weight. It may be interesting to explore the effect of RJ on the infiltration of muscle by adipose and connective tissue. Our data showed that daily feeding with pRJ did not decrease the expression levels of atrophy-related genes such as Atrogin-1, Murf1, or Foxo-1 although our previous work showed that pRJ feeding decreases atrophy-related gene expression during aging []. This discrepancy might be explained by differences in the relative extent of atrophy induced by denervation in comparison to aging. Nevertheless, differences between the two experimental models may be helpful to clarify the effect of RJ on skeletal muscle metabolism.

4. Discussion

We observed that RJ treatment stimulated both proliferation and differentiation. RJ stimulates cell proliferation and increases the size of Myhc positive fibers in primary satellite cells isolated from aged mice via upregulation of IGF-1 and IGFR []. In this study, RJ treatment significantly increased expression of IGF-1 and IGFR, suggesting that the IGF1-Akt pathway may contribute to the phenotype. In in vivo experiments, RJ treatment did not increase the expression levels of cyclin D1, whereas RJ treatment in vitro strongly increased cyclinD1. Currently, it is difficult to explain this discrepancy. Furthermore, we could not reveal which cell types are proliferating and differentiating in vivo following RJ treatment. This will be an important issue to resolve in future studies.