Praca oryginalna Original paper
Dietary restrictions, and many other non-genetic nutrition factors, may change the lifespan and gene expression (24, 28, 45). Among other ways, organisms respond to these factors through TOR (target of rapa-mecin) signaling (8, 42). AKT is a positive regulator of TOR, while PTEN is its negative regulator. The lifespan of organisms, including insects, can be extended by the inhibition of TOR (1, 39). Burzyński (4, 5) found that phenylacetylglutaminate (PG) down-regulates AKT and up-regulates PTEN in humans. Another naturally occurring compound, 4-4-phenylbutyrate (PB), which is a histone deacetylase inhibitor (16, 36), is metabolized to phenylacetate (PN) and PG in the human body (32). PG affects global methylation, and PN is a promoter demethylating agent (4). PG was initially isolated from urine, but, similarly to PB,
it has also been found in plants and dairy products (5, 6). Both PB and PG have been applied in clinical trials (oncology, gerontology; 5, 16). Kang et al. (20) also found that PB increases the lifespan of Drosophila.
In contrast to Drosophila, honeybees (Apis
mel-lifera) show age-related phenotype variety and can
extend their lifespan 10-fold or shorten it by 90% in response to social and nutritional factors (1, 27, 28). Therefore honeybees may provide a unique model for experimental gerontology and for gene-expres-sion studies. However, further studies on honeybee biochemistry and genetics are necessary (26), since, although many gene expression regulating pathways are conservative, there are also inter-species differences, e.g. JH affected the expression of vitellogenin and lifespan in opposite ways in fruit flies and honeybees
Changed gene expression and longevity in honeybees
(Apis mellifera) fed with phenylbutyrate- and
phenylacetylglutaminate-supplemented diet
STANISŁAW R. BURZYŃSKI, JERZY PALEOLOG*, SONALI PATII,
ELWIRA ILKOWSKA-MUSIAL, GRZEGORZ BORSUK*, KRZYSZTOF OLSZEWSKI*, SRIDAR V. CHITTUR**, VAIJAYANTI GUPTA***, RINKU SARANGI***, ANETA STRACHECKA*
Burzyński Clinic/Burzynski Research Institute, Houston, TX, USA *University of Life Sciences, Lublin, Poland
**Center for Functional Genomics and Department of Biomedical Sciences, University at Albany, Rensselaer, NY, USA ***Strand Life Sciences, Carlsbad, CA, USA
Burzyński S. R., Paleolog J., Patii S., Ilkowska-Musial E., Borsuk G., Olszewski K., Chittur S. V., Gupta V., Sarangi R., Strachecka A. Changed gene expression and longevity in honeybees (Apis mellifera) fed with phenylbutyrate- and phenylacetylglutaminate-supplemented diet
Summary
4-4-Phenylbutyrate (PB), present in plants and royal jelly (RJ), plays a vital part in honeybee longevity. Phenylacetylglutaminate (PG) is a metabolite of PB. The aim of this research was to study changes in gene expression in honeybee workers fed with PB+PG-supplemented diet (in 50% sucrose syrup) by gene array and pathway analysis of their brains, and to perform cage longevity studies. The control group received sucrose syrup only. The statistical analysis of microarray results was performed with Genespring and LimmaGUL. Functionally related genes were identified with the help of Gene Ontology in Gostat. A detailed pathway analysis was conducted by importing annotated gene lists to the PathwayArchitect. The most significant results were noted in chromatin remodeling, the TCA cycle, glutathione system (up-regulation), and Notch signaling (down-regulation), which may contribute to increased longevity. These results suggest that PB and PG increase the expression of genes that play an important part in development and life extension. The study of the longevity of caged honeybees consisted in feeding larvae and worker bees with PB+PG-supplemented diet, and comparing them with non-supplemented controls. In three longevity experiments, the bees that were given PB+PG at the larval stage lived longer. This points to a long-term epigenetic effect, which has not been observed in earlier studies.
(10). Royal jelly (RJ) induces epigenetic changes which contribute to the lifespan of honeybees (13, 19, 22). This corresponds with our finding that PB is one of the ingredients of RJ (17 mg/g; HPLC, Mass Spec; Burzyński Clinic Lab).
Scarcity of data on the response to PB and PG treatment in A. mellifera, which is a potential model organism, prompted us to study the effects of PB+PG- -supplemented diet on gene expression and longevity in honeybees. We tested concentrations of PB+PG that were similar to those applied in humans and higher than those used for Drosophila (20). Unlike fruit flies, mammals tolerate high doses of PB/PB (16, 32), but there is no information about PB/PB toleration for honeybees.
Material and Methods
Gene Array and Pathway Analysis. PB and PG were
manufactured synthetically in Burzynski Research Institute, and microarray analyses were carried out in the laboratory of Dr. Gene Robinson, University of Illinois (the Honeybee Oligoneuceotide Microarray from the University of Illinois; http://genome.cshlp.org/content/12/4/555.abstract). Worker bees from the first group (super-sisters) were kept in 4 cages, 40 bees per cage (i.e.160 individuals in 4 biological rep-licates) for one week and fed with a 20 mg/mL solution of PB+PG (1:1) in 50% sucrose syrup. Workers from the second, control group (4 cages; 40 bees per cage) were fed with pure sucrose syrup (50%). After one week, RNA was extracted from the head of each bee by using a combined Trizol-RNeasy extraction protocol. It is known that 80% of the genes of the entire bee genome are expressed in the brain (2) and that the insect nutrition status is monitored by the brain (1). RNA from five samples was pooled for use in the microarray experiment. Twenty-four arrays were used for the 8 cages (8 cages × 3 array replicates; i.e. technical/ experimental replicates). The 15 µg of pooled RNA was converted into aminoallyl-labeled cDNA and labeled with Cy3 or Cy5 for use in the arrays. Scans were done with an Axon 4000B scanner and Genepix v.6.01 software (40). Statistical analysis was done at the University at Albany, using Genespring and LimmaGUI. Pathway functional anal-ysis was done at Strand Life Sciences by Gene Ontology on significant gene lists with the use of GOstat, importing annotated gene lists to the PathwayArchitect (PArch) and finally matching entities from significant gene lists against all known pathways in the KEGG and pre-packaged path-ways in the PArch for Drosophila. The statistical methods were described in detail by Grozinger et al. (15).
Honeybee longevity study. The tests were performed
at the University of Life Sciences in Lublin. Two artifi-cially made, (see 31) identical colonies were used. The larvae combs in colony 1 were sprayed with 15 mg/ml of each PB and PG solution in 0.9% natrium chloratum pro inj. (treatment 1) for six subsequent days after emergence, whereas pure 0.9% natrium chloratum pro inj. was used in colony 2 (treatment 2). Young larvae consume most food via the cuticle, as well as per os. Then, the combs with sealed brood were incubated (+32°C; RH = 70%), the emerged
one-day-old workers were caged (wooden cage; 13 × 13 × 6 (cm) + feeders + ventilation slots; bees were not allowed to fly) and placed in a conditioned chamber (+28°C; RH = 60%), where four treatments were performed (15 cages/50 bees per treatment). Treatment 1a – workers obtained from treatment 1 were given 50% sucrose syrup with 7.5 mg/mL PB and 7.5 mg/mL PG (PB+PG). Treatment 1b – workers obtained from treatment 1 were given the syrup without PB+PG. Treatment 2a – workers obtained from treatment 2 were given the syrup with PB+PG. Treatment 2b – workers obtained from treatment 2 were given the syrup without PB+PG (control). Dead bees were counted and removed from each cage every day. This procedure was repeated three times in experiments 1, 2, and 3. The age of the caged bees was recorded when 75%, 50% and 25% (quartiles for longevity) of them remained alive in the cage (L75%, L50%, L25%, respectively). Autopsies were performed on some of the dead bees from each cage.
Results
Gene Array and Pathway Analysis (array data set; http://www.beespace.uiuc.edu/BeeArray). Several genes involved in chromatin remodeling (Tab. 1A.), as well as genes involved in cell division and endocytic recycling, intercellular signaling, lysosome/Golgi com-plex, immunity, and RJ production were substantially up-regulated (Tab. 1B, C, D, E, F). The pathway analy-sis was performed for Notch, Hedgehog, tricarboxylic acid cycle, and glutathione system pathways.
The Notch pathway was overall down-regulated in the bees fed with PB+PG-supplemented diet even though some of the changes were not confirmed statisti-cally (Tab. 1G). This is because several key molecules of this pathway (Delta, Notch, Mastermind, Suppressor of Hairless and Deltex) were suppressed, whereas repressors (Serrate and Arrestin) and co-repressors (Groucho and Deadpan), and especially SEL-1, were up-regulated. The Hedgehog (Hh) pathway is involved in development, stem cell maintenance, and cancers. However, the bees fed with PB+PG did not differ significantly from the control bees regarding the Hh elements (Tab. 1H).
Tricarboxylic acid cycle (TCA) shows an overall up-regulation in bees fed with PB+PG with a signifi-cant increase in the expression of the mRNA of key molecules of this pathway (Tab. 1I). There is a down -regulation of ketoglutarate dehydrogenase, which may lead to the accumulation of ketoglutarate that can detoxify reactive oxygen species (ROS). Regarding the Glutathione System, the key molecules of the path-way were up-regulated in the bees fed with PB+PG (Tab. 1J). Similarly to Notch, the whole pathway shows an overall up-regulation. In particular, the Ciot gene is up-regulated, and this protein is an essential component of the glutathione system necessary for the synthesis of drosopterins.
Longevity Study. The autopsy of workers that con-sumed PB+PG revealed that their digestive systems
Tab. 1. List of the most important differentially expressed transcripts in the brain of the honeybees fed with the PB+PG-sup-plemented diet compared to the control group (fold change = 1.0)
Pathway Gene tag Transcript Gene Description Fold change
A: Chromatin
Remodeling AM05389 GB12774-PA GB12774 A. mellifera homologue lysine-specific histone demethylase 1(LSD1) 2.30***
AM09997 GB17425-PA GB17425 CG1968 2.50***
AM07768 GB15179-PA GB15179 Npc CG11314 1.42***
AM06658 GB14059-PA GB14059 Nap1 1.25**
AM12694 GB20148-PA GB20148 Pyd3 0.77***
B: Cell Divison and
Endocyti Recycling AM09983 GB17411-PA GB17411 A. mellifera similar to septin-2 (NEDD5) 2.70*** C: Intercellular
Signaling AM03777 GB11153-PA GB11153 A. mellifera similar to regucalcin (RC) (senescence marker protein 30) 2.30* AM09264 GB16685-PA GB16685 A. mellifera similar to CG5880-PA 2.20* AM09197 GB16619-PA GB16619 A. mellifera similar to Neprilison 2 (CG9761 PA) 1.50*** D: Lysosome & Golgi
Complex AM06320 GB13722-PA GB13722 A. mellifera similar to Y4C6B.6 in C. elegans 3.30** AM09997 GB17425-PA GB17425 A. mellifera similar to CG1968 PA, isoform A 2.70*** E: Immunity AM10109 GB17538-PA GB17538 A. mellifera hymenoptaecin, antimicrob. defense 2.70*** F: Royal Jelly AM06388 GB13788-PA GB13788 A. mellifera major royal jelly protein 6 (Mrjp6) 2.70***
AAM063 A. mellifera major royal jelly protein 2 (Mrjp2) 2.70***
G: Notch Pathway AM05998 GB13395-PA GB13395 A. mellifera similar to Sel-1 homolog precursor 2.50***
AM05381 GB12766-PA GB12766 Arr2 1.11***
AM04563 GB11946-PA GB11946 Mam 0.84***
AM10385 GB17813-PA GB17813 Notch 0.91
AM04477 GB15347 Gro (for Anaplasma marginalne) 1.11
AM04032 GB11411 Su(H) 0.84
AM11316 GB18756-PA GB18756 Numb 0.91
AM03867 GB11244 Nicastrin 1.11
AM05081 GB12464 Delta 0.91
AM07745 GB15155-PA GB15155 Serrate 1.25
AM06818 GB14219 Dsh 0.91
AM03395 GB10770 Deltex 0.91
AM04693 GB12076 deadpan 1.11
H: Hedgehog
Pathway AM07170 GB14573 Hh (for Drosophila melanogaster) 1.11
AM08929 GB16349-PA GB16349 Ptc 1.11
AM03008 GB10379 Smo 0.91
AM03640 GB11016-PA GB11016 Cos2 1.11
AM08123 GB15538 Su(fu) 0.91
AM03345 GB10720 CK1 1.11
I: Tricarboxylic Acid Cycle (TCA)
AM03615 GB10992-PA GB10992 ATP-citrate lyase 1.25***
AM05189 GB12573-PA GB12573 Citrate synthase 1.18***
AM07115 GB14517-PA GB14517 Isocitrate dehydrogenase 1.11*
AM05469 GB12855-PA GB12855 α-ketoglutarate dehydrogenase 0.91*
AM08709 GB16129-PA GB16129 Succinate dehydrogenase 1.11
AM06963 GB14365-PA GB14365 Succinyl CoA ligase 1.11
AM03053 GB10425 Succinyl CoA synthase 0.95
AM05105 GB12488 Aconitase 1.11
had pathological changes, irrespective of the larval diet. Moreover, the workers had a shorter lifespan (by 54% to 66% in bees reared from larvae that consumed PB+PG, and by as much as 55% to 85% in bees reared from larvae that didn’t consume PB+PG). Thus, the lifespan of bees emerged from larvae that consumed PB+PG was shortened less (the difference is signifi-cant P < 0.01), which indicates that such a larval diet supplementation positively influenced the lifespan of worker bees. Because significantly shortened lifespan decreased and PB+PG toxicity in bees that consumed PB+PG, detailed results are not presented here. On the other hand, toxic effects were not observed in larvae fed with PB+PG. The longevity of the bees that emerged from the larvae that consumed PG+PB diet increased, particularly in experiments 2 and 3 (Fig. 1). This may confirm long-term diet-induced changes in the expres-sion of possible longevity genes (Tab. 1).
Discussion
We found that the lysine-specific histone demethyl-ase-l (LSD1) was substantially up-regulated by PB+PG
feeding (AM05383; Tab. 1A). This indicates an in-creased activity of the enzyme that plays an important role in the determination of cell fate. The up-regulation of additional genes involved in chromatin remodeling, including Nap1, eya, and SGP, indicates that PB+PG consumption may influence the survival and growth of different types of apian cells. LSD1 decreases the expression of Hey1 which is a component of Notch sig-naling (12). Notch receptors trigger sigsig-naling cascades that govern a cell’s differentiation, proliferation, and apoptosis. They prevent neuronal differentiation and constitute a switch from neurogenesis to gliogenesis (7). In this study (Tab. 1G), Notch was down-regulated by PG+PB-supplemented diet.
Septins are important for cytokinesis (33), SNARE proteins (3), chromosome segregation (38), vesicle trafficking, apoptosis, and maintaining the integrity of cells (17). Endocytic recycling is necessary for the terminal steps of cytokinesis (21). Rab 35 GTPase is involved in the incorporation of septin 2 and phos-phatidylinositol-4,5-bis phosphate (PIP2) into the intracellular bridge. SEPT2 and PIP2 are required for Continuation of Tab. 1.
Pathway Gene tag Transcript Gene Description Fold change
J: Glutathione
System AM02962 GB10332-PA GB10332 A. mellifera similar to CG31992-PA, isoform A
2.60*** AM06738 GB14138-PA GB14138 Gutathione peroxidase (PHGPx) 1.15*** AM04989 GB12371-PA GB12371 Microsomal glutathione-S transferase-like (Mgst1) 1.20***
AM03995 GB11372-PA GB11372 Clot 1.25*
AM06970 GB14372-PA GB14372 Glutathione S transferase S1 (GstS1) 1.05
AM12512 GB19965-PA GB19965 Glutathione-S synthase 1 1.10
AM03547 GB10922 Glutathione cysteine ligase catalytic subunit (Gclc) 1.10
AM10785 GB18219-PA GB18219 Gamma glutamyl transferase 0.90
Explanations: Fold change is significant at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). The database Transcript-based; http:// metazoa.ensembl.org/Apis_mellifera/Transcript/Summary was used
Fig. 1. Cage tests on the lifespan of honeybee workers that consumed PB+PG only at the larval stage
Explanations: PB+PG yes: larvae consumed PB+PB. PB+PG no: larvae did not consume PB+PB. L-75%, L50%, L25%: number of days counted from the beginning of the test to the day on which, respectively, 75%, 50%, and 25% of bees were still alive in the cages. Small letters: the difference is significant at p < 0.05
L75% L50% L25% 0 10 20 30 40 Days a b Experiment 1 L75% L50% L25% 0 10 20 30 40 Days a a a b b b Experiment 2 L75% L50% L25% 0 10 20 30 40 Days a a a b b b Experiment 3 PG + PB yes PG + PB no
the completion of cytokinesis (14). We found (Tab 1B) that PB+PG consumption caused a 2.7-fold increase in the expression of AM09983, which encodes a protein analogous to Septin-2 in bees, which may improve the regulation of cell division and recycling. A poor regulation of these processes is one of the features of human senescence (25).
Regucalcin (RC, senescence marker protein 30) is a calcium-binding protein that regulates calmodulin ac-tivity. It inhibits tyrosine kinases, protein phosphatases, NO synthase, and, after translocation to the nucleus, it regulates nuclear functions (43, 44). It also causes apoptosis of cancerous cells and suppresses signaling in the TNF-a and TGF-B1 pathways (29). Regucalcin protects fruit flies against environmental stress induced by desiccation (34). Therefore the increased expression of AM03777 in bees that consumed PB+PG in our studies (Tab. 1C) may offer them protection against environmental stress and helps maintain homeostasis (cf 1, 28). PB+PG-supplemented diet also increased the expression of genes encoding zinc-binding proteins (CG5880-PA and CG9761-PA) in our bees (Tab. 1C). CG9761-PA is believed to have the activity of human neprilysin (30). It plays an important part in the pro-teolysis of enkephalin and atrial natriuretic factor (41). High activity of zinc carriers positively influences apian immunity (28). The vitellogenin level depends on hemolymphatic zinc concentration (28), and vitello-genin is a key protein both for the apian anti-oxidative barrier and for senescence control (1).
We found (Tab. 1D) that the gene with the most pro-nounced increase in expression was AM06320, corre-sponding to Y4C6B.6 in C. elegans. The human analog protein product, acid-beta-glucosidase, is an enzyme which plays an important role in lysosomal organiza-tion and biogenesis (11). Another over-expressed gene, AM09997, codes for the putative conserved oligo-meric Golgi complex component 6, which in humans is required for a normal Golgi function. A defective function of the Golgi system and lysosomes leads to accelerated aging and neurodegenerative diseases, including Alzheimer’s disease (23). There are also reports describing a reduced longevity of insects with defective lysosomal degradation and recycling (18).
The suppression of immune defense is one of the risk factors of aging (1, 28). Results of this study reveal a substantial increase in the expression of the gene encoding hymenoptaecin (AM10109), which is an im-portant peptide for the apian antimicrobial defense, in response to PB+PG-supplemented diet (in comparison to the control; Tab. 1E). Paleolog et al. (31) showed that PG-supplemented larval diet altered the activity of the bee-cuticle proteolytic defense system, increasing cuticle protein concentrations at the same time. The protein concentrations usually diminish with age (28).
TCA is a major metabolic process in which glucose is utilized to generate ATP. Life extension resulting
from dietary restrictions depends on the activity of TCA (24, 37). The TCA aconitase activity is decreased by 44% in old houseflies as compared to young flies. The up-regulation of TCA increases neuronal glucose metabolism and improves antioxidant defenses. Genes involved in the regulation of metabolism play an im-portant role in bee-caste determination and, therefore, in the longevity of honeybees (9), as well as in that of
Drosophila (20). Hence, the up-regulation of TCA by
PB+PG, as shown in our study (Tab. 1I), may extend the lifespan of bees.
The debilitating effects of aging are associated with the accumulation of ROS (1, 28, 35). In our experi-ment (Tab. 1J), the Glutathione System exhibited up- -regulation in the bees fed with the addition of PB+PG. This pathway plays an important role in the protection against ROS (28).
The major RJ proteins (MRJP) are encoded by nine genes (8). In this study (Tab. 1F), PB+PG feeding increased the expression of Mrjp2 and Mrjp6. PB, PG and PN affect DNA methylation (4, 32). Kucharski et al. (22) and Kamakura (19) showed that DNA meth-ylation plays an important part in the developmental decision leading from larvae to workers or queens, and therefore, in the lifespan pathways. Only the bee larvae that consume RJ for their entire life become queens, and we have identified PB in RJ (17 mg/g; HPLC, Mass Spec; Burzyński Clinic Lab). Therefore the longevity of bees, which results from the link between diet, aging, and epigenetic mechanisms (13, 45), may be influenced by PB+PG consumption.
We can conclude that the most affected genes in this study (Tab. 1) were those encoding proteins involved in energy metabolism, cell-cycle, signaling pathways, apoptosis, detoxification, and anti-ROS defense, as well as antibacterial defense – that is, the genes involved in the lifespan pathways (28). In Drosophila (20), PB also up-regulated genes that were involved in the lifespan pathways, particularly genes connected with chromatin remodeling and detoxification (including ROS), and changed the expression of genes involved in metabolism (including glutathione S transferase) and cell signaling. However, the same pathways were affected differently in our research and in that by Kang et al. (20). Thus, the complex senescence processes appear to be species specific. On the other hand, our results are not fully comparable with those of Kang et al. (20), since different genes involved in the same pathways were analyzed in these studies.
Our cage experiments revealed an increase in the lifespan of bees that consumed PB+PG at the larval stage (Fig. 1). Therefore, PB+PG consumed at the ini-tial larval stages, may change the expression of genes controlling the lifespan of adult worker bees. This long-lasting effect is visible even 3-7 weeks after the termination of PB+PG feeding. This might be expected, considering that most of the developmental decisions
in honeybees are made during the 4 initial days of the larval stages (22). This experiment also showed that a PB+PG concentration of 15 mg/ml was not toxic for larvae. On the other hand, the oral administration of PG+PB at a concentration of 15 mg/ml was associated with intestinal toxicity, which consequently shortened the lifespan of adult workers. Such a concentration might be harmless when the ingredients are applied in a different way. A negligible toxicity of PB/PG was revealed in mammals (16, 25, 32), whereas Kang et al. (20) found that there is a narrow range of PB con-centration (10 mM) in Drosophila food that is effec-tive but not toxic. In our preliminary assays, PB+PB concentrations that were increased from 1 to 10 mg/ ml neither decreased the locomotor abilities of worker bees nor were harmful to them. When the concentra-tions ranged from 15 to 60 mg/ml, toxicity increased and locomotor abilities decreased. This suggests that harmful effects of PB/PG are different in honeybees, fruit flies, and mammals. The toxicity mechanisms of PB/PG in adult worker bees require further studies.
Conclusions
A diet supplemented with PB+PG increases the ex-pression of many genes that play an important role in the development and life extension of honeybees. Up- -regulation was observed in the pathways of chroma-tin remodeling, Hedgehog, TCA, glutathione system, cell division and endocytic recycling, intercellular signaling, lysosome and Golgi complex, antimicro-bial defense, and royal jelly proteins family. Down- -regulation was observed in the Notch pathway. These results facilitate the use of A. mellifera as a model for gerontological research.
The bees that consumed PB+PG at the larval stage, lived longer. Thus, a long-term, probably epigenetic, effect was revealed. The PB+PG concentrations com-monly used in humans were harmful for bees, probably because of intestinal toxicity. This requires further studies.
Acknowledgments
The authors wish to thank Dr. Gene E. Robinson, as well as Dr. Thomas Newman and Dr. Amro Zayed.
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Corresponding author: Aneta Strachecka PhD, University of Life Science, Akademicka 13, 20-950 Lublin, Poland; e-mail: aneta.strachecka@up.lublin.pl
