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Royal jelly ameliorates diet-induced obesity and glucose intolerance by promoting brown adipose tissue thermogenesis in mice
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Obesity Research & Clinical Practice (2016) xxx, xxx—xxx
Royal jelly ameliorates diet-induced obesity and glucose intolerance by promoting brown adipose tissue thermogenesis in mice Takeshi Yoneshiro a, Ryuji Kaede a, Kazuki Nagaya a, Julia Aoyama a, Mana Saito a, Yuko Okamatsu-Ogura a, Kazuhiro Kimura a, Akira Terao a,b,∗ a
Laboratory of Biochemistry, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan b School of Biological Sciences, Tokai University, Sapporo, Hokkaido 005-8601, Japan Received 16 September 2016 ; received in revised form 16 December 2016; accepted 19 December 2016
KEYWORDS Royal jelly; Brown adipose tissue; Obesity; Diabetes mellitus; Hepatic steatosis
Summary Introduction: Identification of thermogenic food ingredients is potentially a useful strategy for the prevention of obesity and related metabolic disorders. It has been reported that royal jelly (RJ) supplementation improves insulin sensitivity; however, its impacts on energy expenditure and adiposity remain elusive. We investigated anti-obesity effects of RJ supplementation and their relation to physical activity levels and thermogenic capacities of brown (BAT) and white adipose tissue (WAT). Methods: C57BL/6J mice were fed under four different experimental conditions for 17 weeks: normal diet (ND), high fat diet (HFD), HFD with 5% RJ, and HFD with 5% honey bee larva powder (BL). Spontaneous locomotor activity, hepatic triglyceride (TG) content, and blood parameters were examined. Gene and protein expressions of thermogenic uncoupling protein 1 (UCP1) and mitochondrial cytochrome c oxidase subunit IV (COX-IV) in BAT and WAT were investigated by qPCR and Western blotting analysis, respectively. Results: Dietary RJ, but not BL, suppressed HFD-induced accumulations of WAT and hepatic TG without modifying food intake. Consistently, RJ improved hyperglycemia and the homeostasis model assessment-insulin resistance (HOMA-IR). Although dietary RJ and BL unchanged locomotor activity, gene and protein expressions of UCP1 and COX-IV in BAT were increased in the RJ group compared to the other experimental groups. Neither the RJ nor BL treatment induced browning of WAT.
∗ Corresponding author at: School of Biological Sciences, Tokai University, 5-1-1-1 Minamisawa, Minami-ku, Sapporo, Hokkaido 005-8601, Japan. Fax: +81 11 571 6904. E-mail address: [email protected] (A. Terao).
http://dx.doi.org/10.1016/j.orcp.2016.12.006 1871-403X/© 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
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T. Yoneshiro et al. Conclusion: Our results indicate that dietary RJ ameliorates diet-induced obesity, hyperglycemia, and hepatic steatosis by promoting metabolic thermogenesis in BAT in mice. RJ may be a novel promising food ingredient to combat obesity and metabolic disorders. © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction The pandemic of obesity has spurred a need for effective therapies to prevent and treat metabolic complications such as type 2 diabetes mellitus and nonalcoholic fatty liver disease. Although decreasing food intake and increasing physical activity constitute logical ways to tip energy balance toward weight loss, sustained interventions are rather difficult to achieve owing to poor adherence of lifestyle changes [1]. Identification of nutrients or food components, which are able to stimulate energy expenditure and thermogenesis, is potentially useful in developing strategies for the prevention and treatment of obesity and related metabolic disorders [2]. Previous findings that dietary supplementation of royal jelly (RJ), a honey bee product, improves oxidative stress, inflammation, lipid metabolism, and insulin sensitivity [3—5] suggest that this food component is a possible ingredient for preventing obesity-related metabolic disorders. However, effects of RJ on thermogenesis and adiposity have not been determined and thus mechanisms by which RJ ameliorates insulin resistance have not been fully understood. Because the prevalence of brown adipose tissue (BAT) [6—8] and its contribution to sympatheticallyactivated nonshivering thermogenesis [9,10] have been widely appreciated in humans, increasing BAT thermogenesis may serve as a novel approach to modulate energy balance [11—13]. We and others previously reported that thermogenic food ingredients, i.e. capsaicin analogs (capsinoids) found in non-pungent type of hot pepper, increase energy expenditure through the activation of BAT thermogenesis in mice [14,15] and in humans [16,17]. Moreover, capsinoids and cold exposure ectopically induces brown-like (beige or brite) adipocyte formation in certain white fat depots through an increased half-life of PR domain containing 16 (PRDM16) [18], a dominant transcriptional regulator of brown/beige adipocyte development [19]. It is assumed that capsinoids act on transient receptor potential (TRP) channels on sensory neu-
Table 1 Amino acid composition of royal jelly (RJ) and bee larva (BL). (g/100 g)
Lyophilized RJ
Lyophilized BL
Arginine Lysine Histidine Phenylalanine Tyrosine Leucine Isoleucine Methionine Valine Alanine Glycine Proline Glutamate Serine Threonine Aspartate Tryptophan Cystine
2.08 3.50 1.09 1.70 1.66 2.89 1.84 1.03 2.10 1.21 1.30 2.36 3.98 2.31 1.72 6.88 0.49 0.40
2.47 3.29 1.33 1.96 2.42 3.73 2.27 1.01 2.73 2.64 2.60 3.94 7.62 2.14 1.90 4.47 0.68 0.48
rons in the gastrointestinal tract and enhance efferent discharges of sympathetic nerves connecting to BAT, thereby increasing thermogenesis [14,20,21]. Considering a fact that hydroxydecenoic acid (HDEA) and hydroxydecanoic acid (HDAA), unique fatty acids in RJ, are capable to activate TRP channels [22], it seems conceivable that the beneficial effect of RJ on insulin sensitivity could be mediated by the activation of BAT and/or browning of WAT. To test this hypothesis, we examined the effects of dietary supplementation of RJ and honey bee larva (BL) which has similar nutritional composition to RJ but lacks HDEA and HDAA (Table 1), on thermogenic capacities of various adipose tissue and analyzed their relation to body fat-reducing effects. As BAT and beige fat thermogenesis is largely dependent on uncoupling respiration by mitochondrial uncoupling protein 1 (UCP1) [23—25], we assessed adipose thermogenic capacity by measuring expression levels of UCP1
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and mitochondrial marker cytochrome c oxidase subunit IV (COX-IV).
blot analysis. Blood samples were immediately centrifuged (5 min, 2500 × g, 4 ◦ C) and plasma was frozen at −80 ◦ C until biochemical analysis.
Materials and methods
Biochemical analysis
Animals Male C57BL/6J mice aged 3 weeks (CLEA Japan Inc., Hamamatsu, Japan) were housed in plastic cages (three per cage) and maintained in an airconditioned room in the Association for Assessment and Accreditation of Laboratory Animal Care International approved animal facility at 22 ± 2 ◦ C with a 12-h light—dark cycle (lights on 07:00—19:00) [26]. The experimental procedures performed on animals were approved by the Animal Care and Use Committee of Hokkaido University (Sapporo, Japan). All efforts were made to minimize the number of animals used and any pain or discomfort experienced by the mice.
Experimental design and treatments The mice were allowed to the laboratory environment with free access to laboratory normal chow diet (ND) and tap water for a week. Then, the mice were divided into four groups and provided with the following diets for 17 ± 0.3 weeks: (i) normal chow diet (ND group, n = 8), (ii) high-fat diet (HFD group, n = 11), (iii) HFD added with 5% lyophilized RJ powder (Yamada Bee Company, Inc., Okayama, Japan) (RJ group, n = 11), and (iv) HFD added with 5% lyophilized BL powder (Yamada Bee Company, Inc.) (BL group, n = 11). The ND and HFD were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). The compositions and macronutrient contents of the diets are listed in Tables 2 and 3. Body weight and food intake in each cage were measured twice a week and energy intake was calculated. Spontaneous locomotor activity was monitored using an infrared sensor (Biotex, Kyoto, Japan) 10 weeks after the feeding of the diets. At the time of sacrifice, mice were fasted for 6 h and anesthetized by isoflurane, and arterial blood was collected from the abdominal aorta with heparinized tubes. After sacrificing the mice by cervical dislocation, the liver, gastrocnemius skeletal muscle, and fat pads of interscapular BAT, inguinal (iWAT), and gonadal white fat (gWAT) were quickly removed and weighed. One-side tissue specimens of fat pads were transferred into RNAlater Stabilization Solution (Life Technologies Inc., Carlsbad, CA, USA) for RNA analysis. Another side tissue specimen was transferred into liquid nitrogen for Western
Plasma glucose, triglycerides (TG), non-esterified fatty acid (NEFA), and hepatic TG were measured by means of commercial kits (Glucose CII-test, Triglyceride E-test, NEFA C-test; Wako Pure Chemical Industries, Osaka, Japan). An ELISA assay kit (Mouse Insulin ELISA Kit; Morinaga Institute of Biological Science, Yokohama, Japan) was employed for the analysis of plasma insulin. Insulin sensitivity was evaluated with the homeostasis model assessment of insulin resistance (HOMA-IR) calculated as (fasting insulin × fasting glucose/22.4) [27].
RNA analysis Total RNA was extracted according to the manufacturer’s protocol using RNAlater Stabilization Solution. Ucp1 and Cox-IV mRNA levels were measured quantitatively by real-time polymerase chain reaction (PCR) using respective cDNA fragment as a standard and expressed as relative to actin (ActB) mRNA level. Briefly, 2 g of total RNA was reverse transcribed with an oligo(dT) 15-adaptor primer and Moloney murine leukemia virus reverse transcriptase (Life Technologies). Real-time PCR was performed on a fluorescence thermal cycler (LightCycler system; Roche Diagnostics, Mannheim, Germany) using SYBR Green PCR kit (Roche Diagnostics). Primer sequences used were as follows: 5 -GGC CTC TAC GAC TCA GTC CA-3 and 5 -TAA GCC GGC TGA GAT CTT GT-3 for mouse Ucp1, 5 -AGG TGT CCC AAA GAA GCT GT-3 and 5 ACA GAA GTG CTT GAG GTG GT-3 for mouse Cox-IV, 5 -AAG TGT GAC GTT GAC ATC CG-3 and 5 -GAT CCA CAC AGA GTA CTT GC-3 for mouse ActB.
Western blot analysis Adipose tissue specimens were homogenized in RIPA Buffer (Nacalai Tesque, Kyoto, Japan). After centrifugation at 800 × g for 10 min at 4 ◦ C, the obtained supernatant was used to determine the content of UCP1 and COX-IV by Western blot analysis. Equal amounts of protein (5 g for BAT, 30 g for iWAT/gWAT) were separated on 13.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidine fluoride membranes (Immobilon; Millipore, Bedford, MA, USA). After blocking the membrane with 5% skimmed milk, it was incubated with a primary anti-rat UCP1 antibody, kindly provided by Dr.
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T. Yoneshiro et al. Table 2
Composition of the experimental diets. ND
Maltodextrin (g/100 g) ␣-Corn starch (g/100 g) Sucrose (g/100 g) Lard (g/100 g) Soy oil (g/100 g) Casein (g/100 g) Cellulose (g/100 g) Vitamin mix (g/100 g) Mineral mix (g/100 g) L-Cysteine (g/100 g) Choline bitartrate (g/100 g) Lyophilized RJ (g/100 g) Lyophilized BL (g/100 g)
Table 3
6.0 16.0 5.50 33.0 2.00 25.6 6.61 1.00 3.50 0.36 0.25 0 0
HFD + 5% RJ 5.7 15.2 5.23 31.4 1.90 24.3 6.28 0.95 3.33 0.36 0.24 5.0 0
HFD + 5% BL 5.7 15.2 5.23 31.4 1.90 24.3 6.28 0.95 3.33 0.37 0.24 0 5.0
Nutritional compositions of the experimental diets.
Protein (g/100 g) Fat (g/100 g) Carbohydrate (g/100 g) Fiber (g/100 g) Energy (kcal/100 g) PFC ratioa a
39.7 13.2 10.0 7.0 — 20.0 5.00 1.00 3.50 0.30 0.25 0 0
HFD
ND
HFD
HFD + 5% RJ
HFD + 5% BL
24.2 4.5 51.9 4.0 345 28:12:60
23.0 35.0 25.3 0.7 506 18:62:19
21.9 33.3 24.0 0.6 501 19:60:21
21.9 33.3 24.0 0.6 504 19:61:20
PFC ratio, energy ratio for protein (P), fat (F), carbohydrate (C).
Teruo Kawada (Kyoto University, Kyoto, Japan), or with a bovine COX-IV antibody (Molecular Probes, Eugene, OR, USA) overnight at 4 ◦ C. The bound antibody was visualized with an enhanced chemiluminescence system (Amersham, Little Chalfont, Bucks, UK) using horseradish peroxidase-linked goat anti-rabbit (Zymed Laboratories, San Francisco, CA, USA) or goat anti-mouse immunoglobulin (Jackson Immunoresearch Laboratories, West Grove, PA, USA).
Statistical analysis Data were expressed as the means ± SEM and analyzed using IBM SPSS Statistics 17.0 (IBM Japan, Tokyo, Japan). Body weight was analyzed using two-way repeated-measures analysis of variance (ANOVA). Differences in variables between the groups were assessed by one-way ANOVA or Kruskal—Wallis test, as appropriate, with post hoc tests by Tukey’s method. Pearson correlation coefficient was used to determine a correlation between the variables. A P value ≤0.05 was considered as statistically significant.
Results Body and tissue weights At the beginning of the study (0 week), the initial body weights of mice assigned for ND (18.5 ± 0.6 g), HFD (17.8 ± 0.6 g), RJ (17.8 ± 0.8 g), and BL (18.3 ± 0.4 g) groups were almost the same. From week 2, body weights for the HFD, RJ, and BL groups were higher than that for the ND group (Fig. 1A). Compared with the HFD group, the RJ group, but not the BL group, showed decreased body weight from week 12 to the end of the study. Two-way repeated-measures ANOVA revealed significant time effect (P < 0.001), diet effect (P < 0.001), and the interaction effect between time and diet (P < 0.001). Body weight gain of the RJ group (13.7 ± 1.3 g) was significantly higher than those of the ND group (6.1 ± 0.7 g, P < 0.01), but lower than those of the HFD (18.0 ± 1.6 g, P = 0.15) and BL groups (21.8 ± 1.7 g, P < 0.01; Fig. 1B). The feeding of HFD significantly increased fat pad weights of iWAT, gWAT, adiposity index (WAT % of body weight), and BAT compared with the ND group (Fig. 1C—F). The HFD-induced gains of WAT weights
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Fig. 1 Dietary supplementation of RJ, but not of BL, reduces HFD-induced fat accumulation in WAT and liver. (A) Body weight. (B) Body weight gain. (C) Inguinal WAT weight. (D) Gonadal WAT weight. (E) Adiposity index as %body weight of WAT. (F) Interscapular BAT weight. (G) Gastrocnemius muscle weight. (H) Liver weight. (I) Hepatic TG content. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
were decreased by the RJ supplementation (P < 0.05 for iWAT, P < 0.01 for gWAT, P < 0.01 for adiposity index), while BAT weight did not change significantly. In contrast, the BL supplementation had no effect on HFD-induced fat accumulation. Gastrocnemius muscle weight was similar between the four groups (Fig. 1G). Liver weight tended to increase in the HFD (+16%) and BL groups (+22%) compared with the ND group (Fig. 1H), while the increment was marginal in the RJ group (+4%). Notably, while hepatic TG content was doubled in the mice of HFD and BL groups compared with mice of the
ND group (Fig. 1I), dietary RJ completely canceled HFD-induced accumulation of TG in the liver. These observations collectively demonstrate that RJ, but not BL, protects against diet-induced obesity and hepatic steatosis in mice.
Plasma levels of glucose, insulin, TG, and NEFA Compared with the ND group (6.07 ± 0.18 mmol/L), plasma fasting glucose level was significantly higher in the HFD, RJ, and BL groups (P < 0.01, Fig. 2A). The
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Fig. 2 The protective effects of RJ administration on HFD-induced hyperglycemia, hyperinsulinemia, and hyperlipidemia. (A) Fasting glucose level. (B) Fasting insulin level. (C) HOMA-IR. (D) Correlation between body weight and HOMA-IR. (E) Fasting TG level. (F) Fasting NEFA level. Data are means ± SEM. *P ≤ 0.05, **P < 0.01, ***P < 0.001.
RJ supplementation, however, markedly reduced HFD-induced hyperglycemia (P < 0.001). The plasma fasting insulin level was notably increased in the HFD and BL groups compared with the ND group (P < 0.05; Fig. 2B) whereas dietary RJ relieved HFD-induced hyperinsulinemia. HOMA-IR was remarkably elevated in the HFD (P < 0.01) and BL groups (P < 0.05) compared with the ND group, whereas that of the RJ group was not significantly different from the ND group (Fig. 2C). While the plasma TG level did not differ significantly between the groups (Fig. 2E), the NEFA level of the RJ, but not the BL, group was notably lower than that of the HFD group (P < 0.05; Fig. 2F). HOMA-IR was sig-
nificantly and positively correlated with final body weight (r = 0.815, P < 0.001; Fig. 2D), body weight gain (r = 0.830, P < 0.001), iWAT (r = 0.721, P < 0.001) and gWAT weights (r = 0.826, P < 0.001), and liver weight (r = 0.338, P < 0.05), but not with skeletal muscle weight (r = −0.032), suggesting that body fat accumulation is a predominant cause of the blunted insulin sensitivity.
Food intake and locomotor activity Daily food intake of the ND, HFD, RJ, and BL groups did not differ significantly (Fig. 3A). Calculated daily energy intake was significantly higher in the
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7 HFD, RJ, and BL groups by 31—38% than in the ND group (P < 0.05; Fig. 3B). The RJ group ingested the same amount of dietary calories as the HFD and BL groups (Fig. 3B). Spontaneous locomotor activity was higher during the dark phase than during the light phase owing to the circadian effect in all diet groups (Fig. 3C). Neither HFD nor RJ/BL supplementation affected the 24-h cumulative spontaneous locomotor activity (Fig. 3D). Thus, the beneficial effects of RJ can scarcely be accounted for by the changes in energy intake and physical activity thermogenesis.
Thermogenic UCP1 expression in adipose depots
Fig. 3 Anti-obesity effects of RJ are independent on the food intake and spontaneous locomotor activity. (A) Daily mean food intake. (B) Daily mean energy intake. (C) Locomotor activity during light and dark phases. (D) Cumulated spontaneous locomotor activity. Data are means ± SEM. ***P < 0.001.
Given that RJ rescued from HFD-induced obesity and insulin resistance without modifying energy intake and physical activity thermogenesis, we hypothesized that this food ingredient may stimulate metabolic thermogenesis. To this end, the expressions of the thermogenic molecule UCP1 and mitochondrial COX-IV in BAT were examined. In the interscapular BAT, Ucp1 mRNA expression was significantly increased in the RJ group compared with the ND mice, whereas those of the HFD and BL groups were not different significantly from the ND group (P < 0.05, Fig. 4A). The RJ mice also exhibited the highest Cox-IV mRNA expression in BAT between the four experimental groups (ND vs HFD P = 0.05; Fig. 4A). Notably, the protein contents of UCP1 and COX-IV in BAT significantly increased in the RJ group compared with the ND (P < 0.001) and HFD groups (P < 0.05; Fig. 4D—F). A close positive correlation between UCP1 and COX-IV protein contents in BAT (r = 0.649, P < 0.05) was observed only in the RJ group (Fig. 4G), indicating a parallelism between UCP1 induction and mitochondrial biogenesis by RJ. Despite the apparent effects on BAT thermogenesis, dietary RJ resulted in no significant change in UCP1 and COX-IV expressions in iWAT (Fig. 4B, D—F), indicating a marginal effect of RJ on browning of WAT at least in our experimental conditions. As expected, we did not observe any effect from RJ on UCP1 and COX-IV expressions in gWAT (Fig. 4C, D—F) because it is known to be resistant to beige/brite fat enrichment.
Discussion Although previous findings demonstrated that RJ improves lipid metabolism and insulin sensitivity, the target organ of the thermogenic effects as well as anti-obesity effects have not been determined.
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Fig. 4 Dietary RJ elevates BAT thermogenic capacities by UCP1 induction and mitochondrial biogenesis. (A—C) Ucp1 and Cox-IV mRNA expressions normalized to ActB mRNA levels in BAT (A), iWAT (B), and gWAT (C). (D) Western blot analysis for UCP1, COX-IV, and ActB. (E—F) Densitometric quantitation of UCP1 protein expression (E) and COX-IV protein expression (F). (G) Relationship between UCP1 and COX-IV contents in BAT. (H) Proposed mechanism for anti-obesity effects of RJ. Data are means ± SEM. *P ≤ 0.05, ***P < 0.001.
Please cite this article in press as: Yoneshiro T, et al. Royal jelly ameliorates diet-induced obesity and glucose intolerance by promoting brown adipose tissue thermogenesis in mice. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.12.006
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Effects of Royal jelly In the present study, we examined the effects of dietary supplements of RJ and BL on adiposity, glucose homeostasis, and BAT thermogenesis in mice fed HFD. Our major findings were (1) dietary RJ suppressed HFD-induced fat accumulation in WAT and liver, and ameliorated hyperglycemia and hyperinsulinemia, (2) BAT thermogenic capacity was increased by RJ and associated with body fat reduction and improved glucose homeostasis regardless of energy intake and physical activity level, and (3) dietary BL had no apparent effect on HFD-induced changes in body weight, blood glucose and insulin, and BAT thermogenesis. Our results clearly showed that RJ supplementation significantly reduced body fat and hepatic TG contents in HFD-fed mice. This would seem inconsistent with an earlier study showing an insignificant change in body weight after RJ treatment [28]. This discrepancy may be owing to differences in the strain of mice and the diet composition. In our study, the obesity-prone C57BL/6J mice were fed HFD to facilitate the investigation of potential anti-obesity effects of RJ, whereas in the earlier study, a senescence mouse model, SAMR1, was fed a standard lab chow [28]. Because there was little difference between the amount of RJ powder added to the experimental diets of ours and the earlier study, 5% w/w and 4% w/w, respectively, the ingestion dose may not be a major cause for the discrepant results. However, the difference in the duration of the RJ treatment may explain the discrepancy because the treatment period in our study was ∼17 weeks, which was two times longer than that in the earlier study (9 weeks) [28]. If correct, a long-term treatment may be effective in achieving the anti-obesity effects of RJ. Indeed, we observed a significant reduction in body weight after 12 weeks of starting the RJ treatment. Despite the significant reduction of body fat and hepatic TG in the RJ group, we observed no notable difference in energy intake, spontaneous locomotor activity, or skeletal muscle mass between the HFD, RJ, and BL groups, suggesting that observed body fat reduction by RJ was independent of energy intake and physical activity thermogenesis. Consistent with previous findings that HFD and cafeteria feedings result in BAT hyperplasia [29], UCP1 protein content in BAT increased in the HFD, RJ, and BL groups compared with the ND group. The most interesting results in our study was that the UCP1 protein content in BAT was significantly higher in the RJ group than in the HFD and BL groups even though these three mice groups consumed comparable amounts of HFD. As UCP1 protein content is the most relevant parameter for BAT thermogenic
9 capacities [23], these data indicate that dietary RJ elicited UCP1-medited thermogenesis and, thereby, instigates body fat reduction. It should be also emphasized that COX-IV content in BAT was significantly increased by dietary RJ, suggesting increased mitochondrial content, which is positively related to UCP1 induction. UCP1 localizes in the mitochondrial inner membranes, and the mitochondrial biogenesis and dynamics, i.e., increased fission and/or decreased fusion, are crucial for BAT thermogenesis [24]. Therefore, it is highly possible that RJ synergistically enhances BAT thermogenesis by inducing UCP1 expression and increasing mitochondrial content. In contrast to BAT, neither UCP1 nor COX-IV was increased by dietary RJ in iWAT and gWAT, implying no browning of WAT. Thus, our results correctly indicate that the decreased energy balance characterized by the reduced body fat and hepatic TG content were attributable to the activation and recruitment of BAT by RJ. Because the RJ used contained a number of bioactive substances including various types of free amino acids, proteins, carbohydrates, lipids, and vitamins, the compounds responsible for the observed effect of RJ are not known at present. It should be noted that dietary supplementation with amino acids, particularly arginine, protects against diet-induced obesity [2,30,31], and arginine might be able to stimulate BAT through the nitric oxiderelated mechanisms [2,21]. However, although both RJ and BL have similar amino acid composition, only dietary RJ increased BAT thermogenesis. Of note, the RJ used in this study contains the unique fatty acids HDEA and HDAA, which account for 3.8% and 0.6% of the lyophilized RJ, respectively (data provided by Yamada Bee Company, Inc.), and these fatty acids are capable of stimulating coldsensitive TRP in vitro [22]. Moreover, it has been reported that orally administered TRP channel agonists (capsinoids and allyl isothiocyanate) increase BAT thermogenesis through the activation of the sympathetic nervous system (SNS), thereby reducing body fat accumulation [13,14,17]. Therefore, it is speculated that the activation of TRP-SNS-BAT axis by HDEA/HDAA is one of the likely mechanism by which RJ reduced body fat and improved glucose homeostasis (Fig. 4H). In summary, our results suggest that dietary RJ suppresses diet-induced obesity, insulin resistance, and hepatic steatosis through the activation of BAT in mice. Since acceptable human interventions aimed at activating BAT has been limited, dietary RJ may serve as a safe and efficient nutritional treatment for obesity.
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Conflict of interest [12]
A.T. has been funded by the Yamada Bee Company, Inc. The other authors declare no conflict of interest.
[13]
[14]
Acknowledgments [15]
The authors thank Emeritus Professor Masayuki Saito (Hokkaido University) for the helpful discussion and critical reading of the manuscript. This work was supported in part by a Yamada Research Grant and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15K07140).
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Obesity Research & Clinical Practice (2016) xxx, xxx—xxx
Royal jelly ameliorates diet-induced obesity and glucose intolerance by promoting brown adipose tissue thermogenesis in mice Takeshi Yoneshiro a, Ryuji Kaede a, Kazuki Nagaya a, Julia Aoyama a, Mana Saito a, Yuko Okamatsu-Ogura a, Kazuhiro Kimura a, Akira Terao a,b,∗ a
Laboratory of Biochemistry, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan b School of Biological Sciences, Tokai University, Sapporo, Hokkaido 005-8601, Japan Received 16 September 2016 ; received in revised form 16 December 2016; accepted 19 December 2016
KEYWORDS Royal jelly; Brown adipose tissue; Obesity; Diabetes mellitus; Hepatic steatosis
Summary Introduction: Identification of thermogenic food ingredients is potentially a useful strategy for the prevention of obesity and related metabolic disorders. It has been reported that royal jelly (RJ) supplementation improves insulin sensitivity; however, its impacts on energy expenditure and adiposity remain elusive. We investigated anti-obesity effects of RJ supplementation and their relation to physical activity levels and thermogenic capacities of brown (BAT) and white adipose tissue (WAT). Methods: C57BL/6J mice were fed under four different experimental conditions for 17 weeks: normal diet (ND), high fat diet (HFD), HFD with 5% RJ, and HFD with 5% honey bee larva powder (BL). Spontaneous locomotor activity, hepatic triglyceride (TG) content, and blood parameters were examined. Gene and protein expressions of thermogenic uncoupling protein 1 (UCP1) and mitochondrial cytochrome c oxidase subunit IV (COX-IV) in BAT and WAT were investigated by qPCR and Western blotting analysis, respectively. Results: Dietary RJ, but not BL, suppressed HFD-induced accumulations of WAT and hepatic TG without modifying food intake. Consistently, RJ improved hyperglycemia and the homeostasis model assessment-insulin resistance (HOMA-IR). Although dietary RJ and BL unchanged locomotor activity, gene and protein expressions of UCP1 and COX-IV in BAT were increased in the RJ group compared to the other experimental groups. Neither the RJ nor BL treatment induced browning of WAT.
∗ Corresponding author at: School of Biological Sciences, Tokai University, 5-1-1-1 Minamisawa, Minami-ku, Sapporo, Hokkaido 005-8601, Japan. Fax: +81 11 571 6904. E-mail address: [email protected] (A. Terao).
http://dx.doi.org/10.1016/j.orcp.2016.12.006 1871-403X/© 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
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T. Yoneshiro et al. Conclusion: Our results indicate that dietary RJ ameliorates diet-induced obesity, hyperglycemia, and hepatic steatosis by promoting metabolic thermogenesis in BAT in mice. RJ may be a novel promising food ingredient to combat obesity and metabolic disorders. © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction The pandemic of obesity has spurred a need for effective therapies to prevent and treat metabolic complications such as type 2 diabetes mellitus and nonalcoholic fatty liver disease. Although decreasing food intake and increasing physical activity constitute logical ways to tip energy balance toward weight loss, sustained interventions are rather difficult to achieve owing to poor adherence of lifestyle changes [1]. Identification of nutrients or food components, which are able to stimulate energy expenditure and thermogenesis, is potentially useful in developing strategies for the prevention and treatment of obesity and related metabolic disorders [2]. Previous findings that dietary supplementation of royal jelly (RJ), a honey bee product, improves oxidative stress, inflammation, lipid metabolism, and insulin sensitivity [3—5] suggest that this food component is a possible ingredient for preventing obesity-related metabolic disorders. However, effects of RJ on thermogenesis and adiposity have not been determined and thus mechanisms by which RJ ameliorates insulin resistance have not been fully understood. Because the prevalence of brown adipose tissue (BAT) [6—8] and its contribution to sympatheticallyactivated nonshivering thermogenesis [9,10] have been widely appreciated in humans, increasing BAT thermogenesis may serve as a novel approach to modulate energy balance [11—13]. We and others previously reported that thermogenic food ingredients, i.e. capsaicin analogs (capsinoids) found in non-pungent type of hot pepper, increase energy expenditure through the activation of BAT thermogenesis in mice [14,15] and in humans [16,17]. Moreover, capsinoids and cold exposure ectopically induces brown-like (beige or brite) adipocyte formation in certain white fat depots through an increased half-life of PR domain containing 16 (PRDM16) [18], a dominant transcriptional regulator of brown/beige adipocyte development [19]. It is assumed that capsinoids act on transient receptor potential (TRP) channels on sensory neu-
Table 1 Amino acid composition of royal jelly (RJ) and bee larva (BL). (g/100 g)
Lyophilized RJ
Lyophilized BL
Arginine Lysine Histidine Phenylalanine Tyrosine Leucine Isoleucine Methionine Valine Alanine Glycine Proline Glutamate Serine Threonine Aspartate Tryptophan Cystine
2.08 3.50 1.09 1.70 1.66 2.89 1.84 1.03 2.10 1.21 1.30 2.36 3.98 2.31 1.72 6.88 0.49 0.40
2.47 3.29 1.33 1.96 2.42 3.73 2.27 1.01 2.73 2.64 2.60 3.94 7.62 2.14 1.90 4.47 0.68 0.48
rons in the gastrointestinal tract and enhance efferent discharges of sympathetic nerves connecting to BAT, thereby increasing thermogenesis [14,20,21]. Considering a fact that hydroxydecenoic acid (HDEA) and hydroxydecanoic acid (HDAA), unique fatty acids in RJ, are capable to activate TRP channels [22], it seems conceivable that the beneficial effect of RJ on insulin sensitivity could be mediated by the activation of BAT and/or browning of WAT. To test this hypothesis, we examined the effects of dietary supplementation of RJ and honey bee larva (BL) which has similar nutritional composition to RJ but lacks HDEA and HDAA (Table 1), on thermogenic capacities of various adipose tissue and analyzed their relation to body fat-reducing effects. As BAT and beige fat thermogenesis is largely dependent on uncoupling respiration by mitochondrial uncoupling protein 1 (UCP1) [23—25], we assessed adipose thermogenic capacity by measuring expression levels of UCP1
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and mitochondrial marker cytochrome c oxidase subunit IV (COX-IV).
blot analysis. Blood samples were immediately centrifuged (5 min, 2500 × g, 4 ◦ C) and plasma was frozen at −80 ◦ C until biochemical analysis.
Materials and methods
Biochemical analysis
Animals Male C57BL/6J mice aged 3 weeks (CLEA Japan Inc., Hamamatsu, Japan) were housed in plastic cages (three per cage) and maintained in an airconditioned room in the Association for Assessment and Accreditation of Laboratory Animal Care International approved animal facility at 22 ± 2 ◦ C with a 12-h light—dark cycle (lights on 07:00—19:00) [26]. The experimental procedures performed on animals were approved by the Animal Care and Use Committee of Hokkaido University (Sapporo, Japan). All efforts were made to minimize the number of animals used and any pain or discomfort experienced by the mice.
Experimental design and treatments The mice were allowed to the laboratory environment with free access to laboratory normal chow diet (ND) and tap water for a week. Then, the mice were divided into four groups and provided with the following diets for 17 ± 0.3 weeks: (i) normal chow diet (ND group, n = 8), (ii) high-fat diet (HFD group, n = 11), (iii) HFD added with 5% lyophilized RJ powder (Yamada Bee Company, Inc., Okayama, Japan) (RJ group, n = 11), and (iv) HFD added with 5% lyophilized BL powder (Yamada Bee Company, Inc.) (BL group, n = 11). The ND and HFD were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). The compositions and macronutrient contents of the diets are listed in Tables 2 and 3. Body weight and food intake in each cage were measured twice a week and energy intake was calculated. Spontaneous locomotor activity was monitored using an infrared sensor (Biotex, Kyoto, Japan) 10 weeks after the feeding of the diets. At the time of sacrifice, mice were fasted for 6 h and anesthetized by isoflurane, and arterial blood was collected from the abdominal aorta with heparinized tubes. After sacrificing the mice by cervical dislocation, the liver, gastrocnemius skeletal muscle, and fat pads of interscapular BAT, inguinal (iWAT), and gonadal white fat (gWAT) were quickly removed and weighed. One-side tissue specimens of fat pads were transferred into RNAlater Stabilization Solution (Life Technologies Inc., Carlsbad, CA, USA) for RNA analysis. Another side tissue specimen was transferred into liquid nitrogen for Western
Plasma glucose, triglycerides (TG), non-esterified fatty acid (NEFA), and hepatic TG were measured by means of commercial kits (Glucose CII-test, Triglyceride E-test, NEFA C-test; Wako Pure Chemical Industries, Osaka, Japan). An ELISA assay kit (Mouse Insulin ELISA Kit; Morinaga Institute of Biological Science, Yokohama, Japan) was employed for the analysis of plasma insulin. Insulin sensitivity was evaluated with the homeostasis model assessment of insulin resistance (HOMA-IR) calculated as (fasting insulin × fasting glucose/22.4) [27].
RNA analysis Total RNA was extracted according to the manufacturer’s protocol using RNAlater Stabilization Solution. Ucp1 and Cox-IV mRNA levels were measured quantitatively by real-time polymerase chain reaction (PCR) using respective cDNA fragment as a standard and expressed as relative to actin (ActB) mRNA level. Briefly, 2 g of total RNA was reverse transcribed with an oligo(dT) 15-adaptor primer and Moloney murine leukemia virus reverse transcriptase (Life Technologies). Real-time PCR was performed on a fluorescence thermal cycler (LightCycler system; Roche Diagnostics, Mannheim, Germany) using SYBR Green PCR kit (Roche Diagnostics). Primer sequences used were as follows: 5 -GGC CTC TAC GAC TCA GTC CA-3 and 5 -TAA GCC GGC TGA GAT CTT GT-3 for mouse Ucp1, 5 -AGG TGT CCC AAA GAA GCT GT-3 and 5 ACA GAA GTG CTT GAG GTG GT-3 for mouse Cox-IV, 5 -AAG TGT GAC GTT GAC ATC CG-3 and 5 -GAT CCA CAC AGA GTA CTT GC-3 for mouse ActB.
Western blot analysis Adipose tissue specimens were homogenized in RIPA Buffer (Nacalai Tesque, Kyoto, Japan). After centrifugation at 800 × g for 10 min at 4 ◦ C, the obtained supernatant was used to determine the content of UCP1 and COX-IV by Western blot analysis. Equal amounts of protein (5 g for BAT, 30 g for iWAT/gWAT) were separated on 13.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidine fluoride membranes (Immobilon; Millipore, Bedford, MA, USA). After blocking the membrane with 5% skimmed milk, it was incubated with a primary anti-rat UCP1 antibody, kindly provided by Dr.
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T. Yoneshiro et al. Table 2
Composition of the experimental diets. ND
Maltodextrin (g/100 g) ␣-Corn starch (g/100 g) Sucrose (g/100 g) Lard (g/100 g) Soy oil (g/100 g) Casein (g/100 g) Cellulose (g/100 g) Vitamin mix (g/100 g) Mineral mix (g/100 g) L-Cysteine (g/100 g) Choline bitartrate (g/100 g) Lyophilized RJ (g/100 g) Lyophilized BL (g/100 g)
Table 3
6.0 16.0 5.50 33.0 2.00 25.6 6.61 1.00 3.50 0.36 0.25 0 0
HFD + 5% RJ 5.7 15.2 5.23 31.4 1.90 24.3 6.28 0.95 3.33 0.36 0.24 5.0 0
HFD + 5% BL 5.7 15.2 5.23 31.4 1.90 24.3 6.28 0.95 3.33 0.37 0.24 0 5.0
Nutritional compositions of the experimental diets.
Protein (g/100 g) Fat (g/100 g) Carbohydrate (g/100 g) Fiber (g/100 g) Energy (kcal/100 g) PFC ratioa a
39.7 13.2 10.0 7.0 — 20.0 5.00 1.00 3.50 0.30 0.25 0 0
HFD
ND
HFD
HFD + 5% RJ
HFD + 5% BL
24.2 4.5 51.9 4.0 345 28:12:60
23.0 35.0 25.3 0.7 506 18:62:19
21.9 33.3 24.0 0.6 501 19:60:21
21.9 33.3 24.0 0.6 504 19:61:20
PFC ratio, energy ratio for protein (P), fat (F), carbohydrate (C).
Teruo Kawada (Kyoto University, Kyoto, Japan), or with a bovine COX-IV antibody (Molecular Probes, Eugene, OR, USA) overnight at 4 ◦ C. The bound antibody was visualized with an enhanced chemiluminescence system (Amersham, Little Chalfont, Bucks, UK) using horseradish peroxidase-linked goat anti-rabbit (Zymed Laboratories, San Francisco, CA, USA) or goat anti-mouse immunoglobulin (Jackson Immunoresearch Laboratories, West Grove, PA, USA).
Statistical analysis Data were expressed as the means ± SEM and analyzed using IBM SPSS Statistics 17.0 (IBM Japan, Tokyo, Japan). Body weight was analyzed using two-way repeated-measures analysis of variance (ANOVA). Differences in variables between the groups were assessed by one-way ANOVA or Kruskal—Wallis test, as appropriate, with post hoc tests by Tukey’s method. Pearson correlation coefficient was used to determine a correlation between the variables. A P value ≤0.05 was considered as statistically significant.
Results Body and tissue weights At the beginning of the study (0 week), the initial body weights of mice assigned for ND (18.5 ± 0.6 g), HFD (17.8 ± 0.6 g), RJ (17.8 ± 0.8 g), and BL (18.3 ± 0.4 g) groups were almost the same. From week 2, body weights for the HFD, RJ, and BL groups were higher than that for the ND group (Fig. 1A). Compared with the HFD group, the RJ group, but not the BL group, showed decreased body weight from week 12 to the end of the study. Two-way repeated-measures ANOVA revealed significant time effect (P < 0.001), diet effect (P < 0.001), and the interaction effect between time and diet (P < 0.001). Body weight gain of the RJ group (13.7 ± 1.3 g) was significantly higher than those of the ND group (6.1 ± 0.7 g, P < 0.01), but lower than those of the HFD (18.0 ± 1.6 g, P = 0.15) and BL groups (21.8 ± 1.7 g, P < 0.01; Fig. 1B). The feeding of HFD significantly increased fat pad weights of iWAT, gWAT, adiposity index (WAT % of body weight), and BAT compared with the ND group (Fig. 1C—F). The HFD-induced gains of WAT weights
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Fig. 1 Dietary supplementation of RJ, but not of BL, reduces HFD-induced fat accumulation in WAT and liver. (A) Body weight. (B) Body weight gain. (C) Inguinal WAT weight. (D) Gonadal WAT weight. (E) Adiposity index as %body weight of WAT. (F) Interscapular BAT weight. (G) Gastrocnemius muscle weight. (H) Liver weight. (I) Hepatic TG content. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
were decreased by the RJ supplementation (P < 0.05 for iWAT, P < 0.01 for gWAT, P < 0.01 for adiposity index), while BAT weight did not change significantly. In contrast, the BL supplementation had no effect on HFD-induced fat accumulation. Gastrocnemius muscle weight was similar between the four groups (Fig. 1G). Liver weight tended to increase in the HFD (+16%) and BL groups (+22%) compared with the ND group (Fig. 1H), while the increment was marginal in the RJ group (+4%). Notably, while hepatic TG content was doubled in the mice of HFD and BL groups compared with mice of the
ND group (Fig. 1I), dietary RJ completely canceled HFD-induced accumulation of TG in the liver. These observations collectively demonstrate that RJ, but not BL, protects against diet-induced obesity and hepatic steatosis in mice.
Plasma levels of glucose, insulin, TG, and NEFA Compared with the ND group (6.07 ± 0.18 mmol/L), plasma fasting glucose level was significantly higher in the HFD, RJ, and BL groups (P < 0.01, Fig. 2A). The
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Fig. 2 The protective effects of RJ administration on HFD-induced hyperglycemia, hyperinsulinemia, and hyperlipidemia. (A) Fasting glucose level. (B) Fasting insulin level. (C) HOMA-IR. (D) Correlation between body weight and HOMA-IR. (E) Fasting TG level. (F) Fasting NEFA level. Data are means ± SEM. *P ≤ 0.05, **P < 0.01, ***P < 0.001.
RJ supplementation, however, markedly reduced HFD-induced hyperglycemia (P < 0.001). The plasma fasting insulin level was notably increased in the HFD and BL groups compared with the ND group (P < 0.05; Fig. 2B) whereas dietary RJ relieved HFD-induced hyperinsulinemia. HOMA-IR was remarkably elevated in the HFD (P < 0.01) and BL groups (P < 0.05) compared with the ND group, whereas that of the RJ group was not significantly different from the ND group (Fig. 2C). While the plasma TG level did not differ significantly between the groups (Fig. 2E), the NEFA level of the RJ, but not the BL, group was notably lower than that of the HFD group (P < 0.05; Fig. 2F). HOMA-IR was sig-
nificantly and positively correlated with final body weight (r = 0.815, P < 0.001; Fig. 2D), body weight gain (r = 0.830, P < 0.001), iWAT (r = 0.721, P < 0.001) and gWAT weights (r = 0.826, P < 0.001), and liver weight (r = 0.338, P < 0.05), but not with skeletal muscle weight (r = −0.032), suggesting that body fat accumulation is a predominant cause of the blunted insulin sensitivity.
Food intake and locomotor activity Daily food intake of the ND, HFD, RJ, and BL groups did not differ significantly (Fig. 3A). Calculated daily energy intake was significantly higher in the
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7 HFD, RJ, and BL groups by 31—38% than in the ND group (P < 0.05; Fig. 3B). The RJ group ingested the same amount of dietary calories as the HFD and BL groups (Fig. 3B). Spontaneous locomotor activity was higher during the dark phase than during the light phase owing to the circadian effect in all diet groups (Fig. 3C). Neither HFD nor RJ/BL supplementation affected the 24-h cumulative spontaneous locomotor activity (Fig. 3D). Thus, the beneficial effects of RJ can scarcely be accounted for by the changes in energy intake and physical activity thermogenesis.
Thermogenic UCP1 expression in adipose depots
Fig. 3 Anti-obesity effects of RJ are independent on the food intake and spontaneous locomotor activity. (A) Daily mean food intake. (B) Daily mean energy intake. (C) Locomotor activity during light and dark phases. (D) Cumulated spontaneous locomotor activity. Data are means ± SEM. ***P < 0.001.
Given that RJ rescued from HFD-induced obesity and insulin resistance without modifying energy intake and physical activity thermogenesis, we hypothesized that this food ingredient may stimulate metabolic thermogenesis. To this end, the expressions of the thermogenic molecule UCP1 and mitochondrial COX-IV in BAT were examined. In the interscapular BAT, Ucp1 mRNA expression was significantly increased in the RJ group compared with the ND mice, whereas those of the HFD and BL groups were not different significantly from the ND group (P < 0.05, Fig. 4A). The RJ mice also exhibited the highest Cox-IV mRNA expression in BAT between the four experimental groups (ND vs HFD P = 0.05; Fig. 4A). Notably, the protein contents of UCP1 and COX-IV in BAT significantly increased in the RJ group compared with the ND (P < 0.001) and HFD groups (P < 0.05; Fig. 4D—F). A close positive correlation between UCP1 and COX-IV protein contents in BAT (r = 0.649, P < 0.05) was observed only in the RJ group (Fig. 4G), indicating a parallelism between UCP1 induction and mitochondrial biogenesis by RJ. Despite the apparent effects on BAT thermogenesis, dietary RJ resulted in no significant change in UCP1 and COX-IV expressions in iWAT (Fig. 4B, D—F), indicating a marginal effect of RJ on browning of WAT at least in our experimental conditions. As expected, we did not observe any effect from RJ on UCP1 and COX-IV expressions in gWAT (Fig. 4C, D—F) because it is known to be resistant to beige/brite fat enrichment.
Discussion Although previous findings demonstrated that RJ improves lipid metabolism and insulin sensitivity, the target organ of the thermogenic effects as well as anti-obesity effects have not been determined.
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Fig. 4 Dietary RJ elevates BAT thermogenic capacities by UCP1 induction and mitochondrial biogenesis. (A—C) Ucp1 and Cox-IV mRNA expressions normalized to ActB mRNA levels in BAT (A), iWAT (B), and gWAT (C). (D) Western blot analysis for UCP1, COX-IV, and ActB. (E—F) Densitometric quantitation of UCP1 protein expression (E) and COX-IV protein expression (F). (G) Relationship between UCP1 and COX-IV contents in BAT. (H) Proposed mechanism for anti-obesity effects of RJ. Data are means ± SEM. *P ≤ 0.05, ***P < 0.001.
Please cite this article in press as: Yoneshiro T, et al. Royal jelly ameliorates diet-induced obesity and glucose intolerance by promoting brown adipose tissue thermogenesis in mice. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.12.006
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Effects of Royal jelly In the present study, we examined the effects of dietary supplements of RJ and BL on adiposity, glucose homeostasis, and BAT thermogenesis in mice fed HFD. Our major findings were (1) dietary RJ suppressed HFD-induced fat accumulation in WAT and liver, and ameliorated hyperglycemia and hyperinsulinemia, (2) BAT thermogenic capacity was increased by RJ and associated with body fat reduction and improved glucose homeostasis regardless of energy intake and physical activity level, and (3) dietary BL had no apparent effect on HFD-induced changes in body weight, blood glucose and insulin, and BAT thermogenesis. Our results clearly showed that RJ supplementation significantly reduced body fat and hepatic TG contents in HFD-fed mice. This would seem inconsistent with an earlier study showing an insignificant change in body weight after RJ treatment [28]. This discrepancy may be owing to differences in the strain of mice and the diet composition. In our study, the obesity-prone C57BL/6J mice were fed HFD to facilitate the investigation of potential anti-obesity effects of RJ, whereas in the earlier study, a senescence mouse model, SAMR1, was fed a standard lab chow [28]. Because there was little difference between the amount of RJ powder added to the experimental diets of ours and the earlier study, 5% w/w and 4% w/w, respectively, the ingestion dose may not be a major cause for the discrepant results. However, the difference in the duration of the RJ treatment may explain the discrepancy because the treatment period in our study was ∼17 weeks, which was two times longer than that in the earlier study (9 weeks) [28]. If correct, a long-term treatment may be effective in achieving the anti-obesity effects of RJ. Indeed, we observed a significant reduction in body weight after 12 weeks of starting the RJ treatment. Despite the significant reduction of body fat and hepatic TG in the RJ group, we observed no notable difference in energy intake, spontaneous locomotor activity, or skeletal muscle mass between the HFD, RJ, and BL groups, suggesting that observed body fat reduction by RJ was independent of energy intake and physical activity thermogenesis. Consistent with previous findings that HFD and cafeteria feedings result in BAT hyperplasia [29], UCP1 protein content in BAT increased in the HFD, RJ, and BL groups compared with the ND group. The most interesting results in our study was that the UCP1 protein content in BAT was significantly higher in the RJ group than in the HFD and BL groups even though these three mice groups consumed comparable amounts of HFD. As UCP1 protein content is the most relevant parameter for BAT thermogenic
9 capacities [23], these data indicate that dietary RJ elicited UCP1-medited thermogenesis and, thereby, instigates body fat reduction. It should be also emphasized that COX-IV content in BAT was significantly increased by dietary RJ, suggesting increased mitochondrial content, which is positively related to UCP1 induction. UCP1 localizes in the mitochondrial inner membranes, and the mitochondrial biogenesis and dynamics, i.e., increased fission and/or decreased fusion, are crucial for BAT thermogenesis [24]. Therefore, it is highly possible that RJ synergistically enhances BAT thermogenesis by inducing UCP1 expression and increasing mitochondrial content. In contrast to BAT, neither UCP1 nor COX-IV was increased by dietary RJ in iWAT and gWAT, implying no browning of WAT. Thus, our results correctly indicate that the decreased energy balance characterized by the reduced body fat and hepatic TG content were attributable to the activation and recruitment of BAT by RJ. Because the RJ used contained a number of bioactive substances including various types of free amino acids, proteins, carbohydrates, lipids, and vitamins, the compounds responsible for the observed effect of RJ are not known at present. It should be noted that dietary supplementation with amino acids, particularly arginine, protects against diet-induced obesity [2,30,31], and arginine might be able to stimulate BAT through the nitric oxiderelated mechanisms [2,21]. However, although both RJ and BL have similar amino acid composition, only dietary RJ increased BAT thermogenesis. Of note, the RJ used in this study contains the unique fatty acids HDEA and HDAA, which account for 3.8% and 0.6% of the lyophilized RJ, respectively (data provided by Yamada Bee Company, Inc.), and these fatty acids are capable of stimulating coldsensitive TRP in vitro [22]. Moreover, it has been reported that orally administered TRP channel agonists (capsinoids and allyl isothiocyanate) increase BAT thermogenesis through the activation of the sympathetic nervous system (SNS), thereby reducing body fat accumulation [13,14,17]. Therefore, it is speculated that the activation of TRP-SNS-BAT axis by HDEA/HDAA is one of the likely mechanism by which RJ reduced body fat and improved glucose homeostasis (Fig. 4H). In summary, our results suggest that dietary RJ suppresses diet-induced obesity, insulin resistance, and hepatic steatosis through the activation of BAT in mice. Since acceptable human interventions aimed at activating BAT has been limited, dietary RJ may serve as a safe and efficient nutritional treatment for obesity.
Please cite this article in press as: Yoneshiro T, et al. Royal jelly ameliorates diet-induced obesity and glucose intolerance by promoting brown adipose tissue thermogenesis in mice. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.12.006
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Conflict of interest [12]
A.T. has been funded by the Yamada Bee Company, Inc. The other authors declare no conflict of interest.
[13]
[14]
Acknowledgments [15]
The authors thank Emeritus Professor Masayuki Saito (Hokkaido University) for the helpful discussion and critical reading of the manuscript. This work was supported in part by a Yamada Research Grant and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15K07140).
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Please cite this article in press as: Yoneshiro T, et al. Royal jelly ameliorates diet-induced obesity and glucose intolerance by promoting brown adipose tissue thermogenesis in mice. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.12.006