Supplementation with Major Royal-Jelly Proteins Increases Lifespan

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Supplementation with Major Royal Jelly Proteins Increases Lifespan, Feeding and Fecundity in Drosophila Xiao-xuan Xin, Yong Chen, Di Chen, Fa Xiao, Laurence D. Parnell, Jing Zhao, Liang Liu, Jose M. Ordovas, Chao-Qiang Lai, and Lirong Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00514 • Publication Date (Web): 07 Jul 2016 Downloaded from http://pubs.acs.org on July 7, 2016

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Journal of Agricultural and Food Chemistry

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Supplementation with Major Royal Jelly Proteins Increases

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Lifespan, Feeding and Fecundity in Drosophila

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Short Title

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MRJP supplementation increases Drosophila lifespan

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Authors:

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Xiao-xuan Xin,†

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Jing Zhao, § Liang Liu,§

Yong Chen,†

Di Chen,†

Jose M. Ordovas,‡

Fa Xiao,†

Laurence D. Parnell,‡

Chao-Qiang Lai, *‡

Li-rong Shen*†

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†

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Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou, 310058,

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China;

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‡

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Aging at Tufts University, Boston, MA 02111, United States;

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§

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States

Department of Food Science and Nutrition, Fuli Institute of Food Science, Zhejiang

Nutritional Genomics Laboratory, JM-USDA Human Nutrition Research Center on

Department of Statistics, The University of Georgia, Athens, GA 30602, United

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All author names and Affiliations

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Xiao-xuan Xin, Graduate student of Department of Food Science and Nutrition,

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Zhejiang University, Hangzhou, 310058, China. e-mail: [email protected]

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Yong Chen, Graduate student of Department of Food Science and Nutrition, Zhejiang

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University, Hangzhou, 310058, China. e-mail: [email protected] 1

ACS Paragon Plus Environment


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Di Chen, Graduate student of Department of Food Science and Nutrition, Zhejiang

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University, Hangzhou, 310058, China. e-mail: [email protected]

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Fa Xiao, Graduate student of Department of Food Science and Nutrition, Zhejiang

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Laurence D. Parnell, Scientist of Nutritional Genomics Laboratory, JM-USDA

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Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111,

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United States. e-mail: [email protected]

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Jing Zhao, Graduate student of Department of Statistics, The University of Georgia,

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Athens, GA 30602, United States. e-mail: [email protected]

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Liang Liu, Associate Professor of Department of Statistics, The University of

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Georgia, Athens, GA 30602, United States. e-mail: [email protected]

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Jose M. Ordovas, Professor of Nutritional Genomics Laboratory, JM-USDA Human

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Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, United

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States. e-mail: [email protected]

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Chao-Qiang Lai, Scientist of Nutritional Genomics Laboratory, JM-USDA Human

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Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, United

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States. e-mail: [email protected]

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Li-rong Shen: Professor of Department of Food Science and Nutrition, Zhejiang

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*The e-mail address of the corresponding author 2


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e-mail: [email protected], [email protected]

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Telephone and fax numbers: 86-571-88982167

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ABSTRACTS: The major royal jelly proteins (MRJPs) are the main constituents

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responsible for the specific physiological role of RJ in honeybee. Male and female

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Drosophila flies were fed diets containing either no MRJPs (A), or casein (B) at

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1.25% (w/w) of diet, or MRJPs at 1.25% (C) , 2.50% (D ) or 5.00% (E). Diets B, C,

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D and E increased mean lifespan by 4.3%, 9.0%, 12.4% and 13.9% in males, and by

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5.8%, 9.7%, 20.0% and 11.8% in females when compared to diet A, respectively. The

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diet supplemented with 2.50% MRJPs seems to have the optimal dose to improve

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both physiological and biochemical measures related to aging in both sexes.

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Interestingly, lifespan extension by MRJPs in Drosophila was positively associated

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with feeding and fecundity, and up-regulation of CuZn-SOD and the Egfr-mediated

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signaling pathway. This study provides strong evidence that MRJPs are important

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components of RJ for prolonging lifespan in Drosophila.

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KEYWORDS: Drosophila, major royal jelly proteins, lifespan, fecundity and

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feeding , anti-oxidation activity, Egfr-mediated signaling pathway

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INTRODUCTION

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Royal jelly (RJ) is a secretion from the hypopharyngeal and mandibular glands

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of 5-15 day old worker honeybees (Apis mellifera) and is used to feed the queen

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throughout her life as well as the larvae of workers during the first three days after

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emergence.1,2,3 The exclusive feeding of a honeybee larva with RJ induces its

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development into a fertile and long-lived adult queen (average lifespan of 1-2 years,

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and a maximal lifespan of 8 years), whereas larvae that develop into worker bees

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(average lifespan of 15-38 days during the spring and summer and 150-200 days in

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the winter, and a maximal lifespan of 320 days) are fed with a mixture of RJ, honey

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and pollen, while drone larvae receive no RJ (average lifespan of 21-32 days during

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the spring and summer, the only time they are produced).4,5 Therefore, it is widely

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agreed that RJ determines the developmental fate of a female larva.2 Similar change,

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that lower concentration of RJ (10%-40%) increased body size in both sexes of

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Drosophila was also reported.6 Chemical analysis has shown that RJ contains various

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components: water 60-70% (w/w), crude protein 12-15%, carbohydrate 10-16%,

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lipids 3-6%, vitamins, salts and free amino acids.1,7 Owing to the belief that RJ exerts

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on humans similar effects as it does on the honeybee queen, RJ has been used widely

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in medicinal products, functional foods and cosmetics in many countries.8 Numerous

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experiments in animal models and cell culture have suggested that RJ possesses

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several biological and pharmacological effects, such as antibacterial,9 anti-tumor,10

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anti-inflammatory,11,12 and anti-fatigue activity.13 Moreover, supplementation of RJ

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in the diets of human volunteers significantly reduced both total cholesterol and 5



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low-density lipoprotein cholesterol.14 Although RJ has been considered a traditional

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supplement for longevity in parts of Europe and Asia, speculation remains about its

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potential ability to delay aging and to restore in the elderly some biological functions

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associated with its anti-oxidation properties.15, 16

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To search for the ‘queen determinator’, numerous studies have been conducted

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to identify RJ substitutes that can induce queen differentiation.17 For instance, a

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higher sugar level and the appropriate ratio of fructose to glucose in the food induced

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the effect of increasing the proportion of larvae developing into queens.18 In addition,

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Spannhoff et al.(2011) demonstrated that the caste switch in honeybees may be driven

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epigenetically by the histone deacetylase inhibitor activity of 10-hydroxy-2-

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decenoic-acid (10-HDA) leading to increased transcription.19. Conversely, Leimar et

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al. (2012) argued that a single determinator is unlikely to trigger caste determination

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in honeybees.20 These observations suggest that the specifics of the ‘queen-inducing

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agent’ still remain undetermined. However, recent evidence suggests that one or more

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proteins with specific properties in RJ may function as the ‘queen-inducing agent.21

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Proteins are the major components of RJ and constitute nearly 50% of its dry

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matter.22 Although some properties of RJ have been attributed to its lipids, such as

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10-HDA, a key marker for estimating fresh quality and authenticity of RJ.23 many of

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the proven bioactivities of RJ could also arise from its proteins.3 The crude proteins

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of RJ are divided into water-soluble and water-insoluble proteins.22 More than 80% of

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total RJ proteins are the major royal jelly proteins (MRJPs), which include nine

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members: MRJP 1-9 (49-87 kDa). The glycosylation and phosphorylation of the 6


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MRJP family is functionally important to help RJ achieve its biological functions.3 ,24,

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25

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55-57 kDa constitutes of 45% water-soluble proteins, and is the most abundant protein

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in RJ.26 MRJPs are thought to be the major factor responsible for the specific

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physiological role of RJ in honeybee queen development, as MRJPs are rich in

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essential amino acids similar to vertebrate ovalbumin and casein.27 MRJPs are also the

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main constituents involved in the physiological actions of RJ, including cell

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proliferation, cytokine suppression and antimicrobial activity.9, 28 Review of MRJPs

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for honeybees hymenopteran insects revealed that MRJPs have important functions

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for insect development and not just a nutritional value for developing honeybee

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larvae .29

MRJP1, a glycoprotein of 432 amino acid residues with a molecular weight of

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All MRJPs that comprise of 400-578 amino acids and share 111 conserved

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amino acids are highly homologous to each other, with pair-wise sequence identities

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ranging from 47 to 74%. Because the five proteins MRJP 1-5 represent up to 82-90%

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of MRJPs in RJ, it has been suggested that these five members have mainly a

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nutritional function.27 MRJP1 is also known as royalactin, apalbumin 1, or D III

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protein, a 57 kDa protein that accounts for more than 45% of MRJP content in RJ. It

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is a weakly acidic glycoprotein and forms an oligomeric complex.3, 21, 26 Among the

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other MRJP members, MRJP2, MRJP3, MRJP4 and MRJP5 are glycoproteins of 49

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kDa, 60-70 kDa, 60 kDa and 80 kDa, respectively. Whereas MRJPs 2-5 are basic

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proteins, MRJP 7 is an acidic protein. Asparagine-linked (N-linked) glycosylation,

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phosphorylation and methylation are also thought to be important post-translational 7



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factors imparting various functions onto MRJPs.30 Many important biological

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processes are mediated by the involvement of glycoproteins in cell adhesion, cell

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differentiation, cell growth, and immunity. 31

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Most functional studies with MRJP1 have been performed in model organisms.

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MRJP1 appears to possess growth-factor-like activity, including promoting DNA

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synthesis, proliferation and suppression of apoptosis in rat hepatocytes.12. MRJP1 and

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MRJP2 were demonstrated to stimulate mouse macrophages to release TNF-alpha,32

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while MRJP3 modulated immune responses in mice.33 The glycosylated MRJP2 have

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antibiotic activity against bacteria, P. larvae.2 In honeybee, feeding royalactin, the

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monomeric form of MRJP1, to larvae shortened their developmental time, and

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increased body mass and ovary size at adult emergence. Royalactin acted on

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epidermal growth factor receptor (EGFR) signaling to induce queen differentiation in

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honeybee larvae.20 Moreover, over-expression of royalactin in Drosophila led to the

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accelerated development, larger body size, increased fecundity, and extended

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lifespan.21 Royalactin increases not only lifespan (i.e., mean lifespan by

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26.8%-33.9%), but also healthspan (i.e. swimming activity by 20%) of the nematode,

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Caenorhabditis elegans, a non-insect species in the absence of other RJ constituents.34

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Recently we have extracted MRJPs from RJ and stored it as a freeze-dried form, and

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demonstrated that the MRJPs in freeze-dried form was biologically active as an

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alternative to fetal bovine serum in culturing human cells.35 But it is not clear if

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feeding biologically active MRJPs in freeze-dried form extends lifespan in both

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Drosophila and nematode. Royalactin could increase not only lifespan (increase mean 8


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lifespan by 26.8%-33.9% in the media contained royalactin 0.04-1.6 µg/mL) but also

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healthspan (enhances swimming activity by 20% in the medium contained 1.7 µg/mL

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royalactin) of the nematode, Caenorhabditis elegans, a non-insect species in the

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absence of other RJ constituents.34 But it is not clear if feeding biologically active

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MRJPs extends lifespan in both Drosophila and nematode.

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Using our established techniques of quantitatively extracting soluble and

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biologically active RJ proteins from fresh RJ, here we show the effects of MRJPs on

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lifespan extension, feeding and fecundity in Drosophila and add details of the

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mechanisms of action. In addition, to determine the potential anti-oxidation effects of

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MRJPs, we measured superoxide dismutase (SOD) activity, malondialdehyde (MDA)

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levels, and expression of age-related genes in flies fed diets supplemented with

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MRJPs.

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Materials and Methods

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Reagents. Fresh RJ was obtained from Hangzhou Biyutian Baojianpin Co.,

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Ltd (Hangzhou, China) and stored at -40°C until use. Kits to assay MDA, SOD

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and protein standard were purchased from Nanjing Jiancheng Bioengineering

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Company (Nanjing, China). Membra-Cel MD-34 was purchased from Shanghai

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Zeheng Biological Technology Co., Ltd (Shanghai, China).

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Preparation of MRJPs. RJ proteins were extracted with phosphate buffered

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saline (PBS) for 24 h at 4°C according to previous report.35, 36 A crude protein

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extract was recovered by centrifugation at 12,000-g for 30 min at 4°C. After 24 h, 9



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the supernatant was dialyzed three times against distilled water at 4°C using a

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14400 Da cutoff dialysis membrane, followed by lyophilization. The protein

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concentration of lyophilized product was determined by the Bradford method

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using bovine serum albumin as standard. The presence of proteins in the

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lyophilized product was analyzed by SDS-PAGE.

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Fly husbandry. Wild-type Canton-S flies were maintained at 25°C and 65%

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humidity on a 12 h light: 12 h dark cycle and were reared on various mediums in

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this experiment. Base fly diet was prepared according to the traditional corn-yeast

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medium (105 g corn, 75 g sucrose, 40 g yeast, 7.5 g agar, 10 mL propionic acid

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mixed with hot water to make 1000 mL diet). Casein-supplemented and

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MRJP-supplemented diets were prepared by dissolving 1.25 g casein, 1.25 g, 2.50

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g or 5.00 g lyophilized product into 100-g base medium, respectively. Therefore,

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one negative control group (base diet group), one positive control group

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(casein-supplemented diet group) and three test groups (MRJPs-supplemented diet

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groups) were prepared.

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Lifespan assay. Newly emerged flies of the same age were collected under

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light anesthesia and housed at a density of ~30 males or ~30 females per vial. Ten

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vials (~300 flies) were set up for each treatment with three replicates for each

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treatment. On every Tuesday and Friday of each week flies were transferred to

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fresh medium until no survivors remained, and the number of dead flies was

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recorded for each transfer.

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Feeding assay. The method of using Blue No.1 dye as food tracer to quantify 10


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food intake, as described.37, was employed. Briefly, newly emerged male or

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female flies were kept in the vials containing various diets. At the age of 3 days,

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groups of either 20 male or 20 female flies were transferred to 1% (w/w) agar for

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24 h. Next, flies were exposed to the dyed food containing 0.5% (w/w) Blue No. 1

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for 4 h in the darkness. After this feeding period, the flies were frozen on dry ice

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immediately. Flies were then homogenized in 1mL PBS (140 mMNaCl, 2.7

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mMKCl, 10 mM Na2 HPO4, 1.8 mM KH2PO4, pH 7.3) and centrifuged (12,000-g )

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for 2 min. A 0.9-mL sample was taken immediately from the supernatant and

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brought to a final volume of 1.5 mL with phosphate buffer. The solution was

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centrifuged again for 2 min and the supernatant was transferred to cuvettes.

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Absorbance was measured at 625 nm. The absorbance measured for supernatants

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from flies fed normal food was subtracted from the absorbance of the supernatant

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from blue food-fed flies. The net absorbance reflected the amount of food

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ingested.

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Female fecundity assay. Within the first 8 h of emergence, adult flies were

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collected and each vial was set up with a density of 1 female and 1 male.

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Fecundity (number of eggs laid by each female) was evaluated for 10 days since

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the first fly emerged. A total of 10 vials with the same test food were tested for

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each group (treatment).

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Anti-oxidation assay. A sample (more than 300 flies per sample) of males

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and females of 7-day and 21-day old flies that were fed base and test diets were

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collected, respectively. The samples were frozen in liquid nitrogen and stored at 11



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-80°C for future analysis. MDA levels, total SOD activity, copper/zinc-superoxide

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dismutase (CuZn-SOD) activity and protein content were measured separately by

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using MDA kit (Cat A003-1), SOD kit (Cat A001-2) and Bradford kit (Cat A045-2)

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produced by Nanjing Jianchen Bioengineering Institute. The protocols used were

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provided by the manufacturers. The level of lipid peroxidation was expressed as

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mmol MDA per mg protein. One unit of SOD activity was defined as the amount

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of enzyme required to cause a 50% inhibition and expressed as enzyme units per

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gram of the total protein. For each parameter measured, at least three replicates

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were used for each control and treatment.

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Quantitative RT-PCR analysis. A sample (more than 100 flies per sample) of

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males and females of 1-day and 21-day old flies fed control and test diets were

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collected, respectively. The samples were frozen in liquid nitrogen and stored at

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−80°C for future analysis. Total RNA was extracted from each sample using

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RNAiso Plus (TaKaRa, Japan), and then was synthesized into cDNA with a

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PrimeScrit RT reagent Kit (TaKaRa, Japan) according to the manufacturer’s

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protocol. CuZn-SOD or SOD1, RPS6-p70-protein kinase (S6K), mitogen activation

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protein kinases (MAPK) and epidermal growth factor receptor (Egfr) according to

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Kamakura (2011) were selected for quantitative RT-PCR analysis. Ribosomal

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protein 49 (rp49) expression was used as internal control. All primers listed in Table

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S1 were synthesized by TaKaRa. Mastercycler®eprealplex Real Time System

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(Eppendorf, Germany) and SYBR Premix Ex Taq (TliRNaseH Plus) (TaKaRa,

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Japan) were used in quantitative RT-PCR analysis. The comparative cycle threshold

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(Ct) method was used to analyze the data. Levels of gene expression in all groups

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were expressed as a ratio of the day 1 control group value. All experiments were

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repeated at least three times.

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Statistics. The Kaplan-Meier test was employed to compare differences

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between the survival curves using SPSS 17.0 (Statistical Package for the Social

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Sciences software, SPSS Inc., Chicago, USA). Lifespan analyses were performed

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on

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(http://www.r-project.org/). Survivorships between diets were compared and tested

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for significance with a log-rank test. Survivorship is a cumulative function where

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differences between male and female flies are carried forward to subsequent age

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intervals. The remaining experiments were analyzed by using one-way ANOVA

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with Dunnett's test. Values are expressed as mean ± standard error calculated from

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triplicate samples. The level of p3)

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are given in Tables 1, 2 and 3, respectively. When compared to the basal diet (no

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casein or MRJPs), diets B (1.25% casein), C (1.25% MRJPs), D (2.50% MRJPs) and

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E (5.00% MRJPs) extended mean lifespan of male flies by 4.3%, 9.0%, 12.4%, and 13



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13.9%, respectively. In females (Tables 1 and 2), diets B, C, D and E extended mean

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lifespan by 5.8%, 9.7%, 20.0% and 11.8%, respectively. This suggests

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supplementation of both dietary casein and MRJPs significantly extended mean

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lifespan in Drosophila. Likewise, when compared to flies fed diet B (casein), those

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fed diets C, D and E (MRJPs) demonstrated extended mean lifespan for male flies by

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4.4%, 7.8% and 9.1%, respectively. Similarly, diets C, D and E extended mean

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lifespan for female flies by 3.7%, 13.5% and 5.7%, respectively (Tables 1 and Table

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2). This suggests that supplementation with MRJPs significantly extended mean

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lifespan in Drosophila (p

Supplementation with Major Royal-Jelly Proteins Increases Lifespan

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