Fatty acid tryptamide from cacao elongates Drosophila melanogaster lifespan with sirtuin-dependent heat shock protein expression.

Life span is increasing in developed countries as Japan, and an aging society is becoming a problem. In fact, healthy lifespan is not extended, and it is desired to extend it by functional food. Cacao (Theobroma cacao) contains various active components and is considered a preventative agent against metabolic disease. In addition, it has long been thought that regular cacao intake extends a healthy lifespan. However, there is no direct evidence for this belief. The purpose of this study is to identify the cacao component that elongate the lifespan of D. melanogaster as a model organism and to elucidate its functional mechanism. The activation of sirtuins, a family of NAD+-dependent deacetylases, has been reported to extend the lifespans of various organisms. Heat shock factor 1 is known to be deacetylated by reaction with sirtuins, thereby inducing gene expression of various heat shock proteins by heat stress and effectively extending the lifespan of organisms. Therefore, we evaluated whether components in cacao activate sirtuins and extend the lifespan of D. melanogaster. In the process, we discovered the fatty acid tryptamide as a lifespan-elongating component of cacao. Therefore, we investigated whether the fatty acid tryptamide from cacao upregulates the genes of heat shock proteins. As a result, it was confirmed that the gene expression of multiple heat shock proteins was significantly increased. This suggests that fatty acid tryptamide may activate sirtuins, increase gene expression of heat shock proteins, and elongate the lifespan of D. melanogaster.

Loss of CNDP causes a shorter lifespan and higher sensitivity to oxidative stress in Drosophila melanogaster.

Increasing oxidative stress seems to be the result of an imbalance between free radical production and antioxidant defenses. During the course of aging, oxidative stress causes tissue/cellular damage, which is implicated in numerous age-related diseases. Carnosinase (CN or CNDP) is dipeptidase, which is associated with carnosine and/or glutathione (GSH) metabolism, those are the most abundant naturally occurring endogenous dipeptide and tripeptides with antioxidant and free radical scavenger properties. In the present study, we generated Drosophila cndp (dcndp) mutant flies using the CRISPR/Cas9 system to study the roles of dcndp in vivo. We demonstrate that dcndp mutant flies exhibit shorter lifespan and increased sensitivity to paraquat or hydrogen peroxide (H2O2) induced oxidative stress. These results suggest that dcndp maintains homeostatic conditions, protecting cells and tissues against the harmful effects of oxidative stress in the course of aging.

Alkylresorcinols activate SIRT1 and delay ageing in Drosophila melanogaster.

Sirtuins are enzymes that catalyze NAD+ dependent protein deacetylation. The natural polyphenolic compound resveratrol received renewed interest when recent findings implicated resveratrol as a potent SIRT1 activator capable of mimicking the effects of calorie restriction. However, resveratrol directly interacts with fluorophore-containing peptide substrates. It was demonstrated that the SIRT1 activation of resveratrol is affected by the amino acid composition of the substrate. Resveratrol did increase the enzyme activity in cases in which hydrophobic amino acids are at the +1 position to the acetylated lysine in the substrate. Alkylresorcinols (ARs) are compounds that belong to the family of phenolic lipids, and they are found in numerous biological species. Here we show that the natural activators ARs increased the Vmax of recombinant SIRT1 for NAD+ and peptide substrate, and that ARs decreased acetylated histone in human monocyte cells by stimulating SIRT1-dependent deacetylation of substrates. ARs also extended the lifespan of Drosophila melanogaster, which was shown to be dependent on functional Sir2. Our results demonstrated that ARs are natural catalytic activators for sirtuin.

The Combined Effects of Genetic Variation in the CNDP1 and CNDP2 Genes and Dietary Carbohydrate and Carotene Intake on Obesity Risk.

BACKGROUND/AIMS: It is possible that carnosinase (CNDP1) and cellular nonspecific dipeptidase (CNDP2) have important roles in protecting cells and tissues against the damage of oxidative stress. Oxidative stress and subsequent inflammation are key factors in the development of common chronic metabolic diseases, such as obesity. We aimed to investigate the combined effects of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate and carotene intake on obesity risk. METHODS: A total of 1,059 Japanese men were randomly selected from participants who visited a medical center for routine medical checkups. We analyzed the relationships between the genotypes of 4 single-nucleotide polymorphisms (SNPs) (rs12605520, rs7244647, rs4891558, and rs17089368) in the CNDP1/CNDP2 locus and body mass index or prevalence of obesity/overweight taking into account dietary carbohydrate and carotene intake. RESULTS: We found that 2 SNPs (rs7244647 in CNDP1 and rs4891558 in CNDP2) were associated with obesity risk. In addition, these associations were observed only in the group with high carbohydrate and low carotene intake but not in the group with low carbohydrate and high carotene intake. CONCLUSIONS: Our findings indicate that the combination of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate/carotene intake modulate obesity risk.

Autocrine regulation of ecdysone synthesis by ß3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis.

In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). Here, we show that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, ß3-octopamine receptor (Octß3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octß3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulates ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. We propose that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant.

Tea polyphenols ameliorate fat storage induced by high-fat diet in Drosophila melanogaster.

BACKGROUND: Polyphenols in tea are considered beneficial to human health. However, many such claims of their bioactivity still require in vitro and in vivo evidence. RESULTS: Using Drosophila melanogaster as a model multicellular organism, we assess the fat accumulation-suppressing effects of theaflavin (TF), a tea polyphenol; epitheaflagallin (ETG), which has an unknown function; and epigallocatechin gallate (EGCg), a prominent component of green tea. Dietary TF reduced the malondialdehyde accumulation related to a high-fat diet in adult flies. Other physiological and genetic responses induced by the high-fat diet, such as lipid accumulation in the fat body and expression of lipid metabolism-related genes, were ameliorated by the addition of TF, ETG, and EGCg, in some cases approaching respective levels without high-fat diet exposure. Continuous ingestion of the three polyphenols resulted in a shortened lifespan. CONCLUSION: We provide evidence in Drosophila that tea polyphenols have a fat accumulation-suppressing effect that has received recent attention. We also suggest that tea polyphenols can provide different desirable biological activities depending on their composition and the presence or absence of other chemical components.

Effect of a dipeptide-enriched diet in an adult Drosophila melanogaster laboratory strain.

Here we present free amino acid profiles for Drosophila melanogaster adults. Imidazol dipeptides anserine and carnosine, which are abundant in mammalian muscle tissue, are not present in Drosophila. Dipeptide-enriched food altered the amino acid balance, suggesting that the free amino acid content is nutrition-dependent and probably mediated by dipeptides.

A heteroallelic Drosophila insulin-like receptor mutant and its use in validating physiological activities of food constituents.

Here we report an additional Drosophila transheterozygote InR(GS15311)/InR(GS50346) carrying two different P-element-inducible alleles of insulin-like receptor gene (InR). InR(GS15311)/InR(GS50346) flies exhibit the following phenotypes previously reported in InR and insulin/IGF-1 signaling (IIS) pathway-related gene mutants: small bodies, developmental delay, shortened lifespan, and increased fasting resistance. All of these characteristics are shared among flies carrying mutated genes implicated in the pathway. This heteroallelic combination exhibited fertility but resulted in male semilethality, while females were viable and grew into adults. Furthermore, an experimental model employing the InR(GS15311)/InR(GS50346) strain confirmed negligible involvement of royal jelly in IIS. Thus, the heteroallelic InR mutant, discovered in this study, will serve as a good model for multiple purposes: investigating the IIS mechanisms; identifying and validating the ingredients that prevent type II diabetes; and screening of food constituents associated with IIS.

The binding of multiple nuclear receptors to a single regulatory region is important for the proper expression of EDG84A in Drosophila melanogaster.

Nuclear receptor transcription factor family members share target sequence similarity; however, little is known about how these factors exert their specific regulatory control. Here, we examine the mechanism regulating the expression of the Drosophila EDG84A gene, a target gene of the orphan nuclear receptor ßFTZ-F1, as a model to study the cooperative behavior among nuclear receptors. We show that the three nuclear receptors ßFTZ-F1, DHR3, and DHR39 bind to a common element in the EDG84A promoter. The expression level of the EDG84A promoter-lacZ reporter genes in DHR39-induced and mutant animals, respectively, suggests that DHR39 works as a repressor. The activity of a reporter gene carrying a mutation preventing DHR3 binding was reduced in ftz-f1 mutants and rescued by the induced expression of ßFTZ-F1, suggesting that DHR3 and ßFTZ-F1 activate the EDG84A gene in a redundant manner. A reporter gene carrying a mutation that abolishes DHR39 and FTZ-F1 binding was prematurely expressed, and the expression level of the reporter gene carrying a mutation preventing DHR3 binding was reduced. These findings suggest that the temporal expression of this gene is mainly controlled by ßFTZ-F1 but that the binding of DHR3 is also important. Comparison of the binding site sequence among Drosophila species suggests that DHR3 binding ability was gained after the melanogaster subgroup evolved, and this ability may contribute to the robust expression of this gene. These results show the complicated regulatory mechanisms utilized by multiple nuclear receptors to properly regulate the expression of their target gene through a single target site.

Freeze-dried royal jelly maintains its developmental and physiological bioactivity in Drosophila melanogaster.

Royal jelly (RJ), a honeybee-derived product, has been found to possess developmental and physiological bioactivity in the fruit fly, Drosophila melanogaster, but little is known about the in vivo bioactivity of freeze-dried RJ (FDRJ) powder, which is another form of RJ processed for human use. To address this, we used Drosophila as a model animal to examine the effects of FDRJ in multicellular organisms. When flies were reared on food supplemented with FDRJ, the developmental time from larva to adult was shortened, the adult male lifespan was prolonged, and female fecundity was increased without any significant morphological alterations. Moreover, the expression of dilp5, an insulin-like peptide, its receptor InR, and the nutrient sensing molecule TOR, the target of rapamycin, was significantly increased in FDRJ-fed female flies as compared with ones reared on standard and on protein-enriched food. These findings suggest that like RJ, FDRJ maintains its bioactivity even after processing from RJ, what is expected to have bioactivity for multicellular organisms, including humans.

Involvement of Prx3, a Drosophila ortholog of the thiol-dependent peroxidase PRDX3, in age-dependent oxidative stress resistance.

Peroxiredoxins (Prxs) are a family of multifunctional antioxidant thioredoxin-dependent peroxidases. We used Drosophila melanogaster to examine the function of Prx3, the Drosophila homolog of human PRDX3. The oxidative stress response in adult Drosophila is age-dependent. RNAiinduced Prx3 knockdown in adult flies did not change their phenotype in normal conditions, but they had a shorter survival than the Prx3(+) controls in the presence of H2O2. The expression levels of the Prx3 were reduced by aging. These results suggest that Prx3 plays an important role in the oxidative stress response and is involved in the age-dependent competence of the oxidative stress response.

Expression of ß-adrenergic-like octopamine receptors during Drosophila development.

An invertebrate biogenic amine, octopamine, plays diverse roles in multiple physiological processes (e.g. neurotransmitter, neuromodulator, and circulating neurohormone). Octopamine is thought to function by binding to G-protein-coupled receptors. In Drosophila, three ß-adrenergic-like octopamine receptors (Octß1R, Octß2R, and Octß3R) have been identified. We investigated the expression of three OctßR genes in embryos, larvae, and adults. These OctßRs showed distinct expression patterns in the central nervous system (CNS) throughout development, and Octß3R expression was evident in an endocrine organ, the ring gland, in larvae. In larvae, Octß1R, Octß2R, and Octß3R were expressed in salivary glands and imaginal discs, Octß2R and Octß3R in midgut, and Octß3R in gonads. In adult, besides in the CNS, each OctßR was strongly expressed in ovary and testis. Our findings provide a basis for understanding the mechanisms by which OctßRs mediate multiple diverse octopaminergic functions during development.

Gene duplication of endothelin 3 is closely correlated with the hyperpigmentation of the internal organs (Fibromelanosis) in silky chickens.

During early development in vertebrates, pluripotent cells are generated from the neural crest and migrate according to their presumptive fate. In birds and mammals, one of the progeny cells, melanoblasts, generally migrate through a dorsolateral route of the trunk region and differentiate to melanocytes. However, Silky is an exceptional chicken in which numerous melanoblasts travel via a ventral pathway and disperse into internal organs. Finally, these ectopic melanocytes induce heavy dermal and visceral melanization known as Fibromelanosis (Fm). To identify the genetic basis of this phenotype, we confirmed the mode of inheritance of Fm as autosomal dominant and then performed linkage analysis with microsatellite markers and sequence-tagged site markers. Using 85 backcross progeny from crossing Black Minorca chickens (BM-C) with F(1) individuals between White Silky (WS) and BM-C Fm was located on 10.2-11.7 Mb of chicken chromosome 20. In addition, we noticed a DNA marker that all Silky chickens and the F(1) individuals showed heterozygous genotyping patterns, suggesting gene duplication in the Fm region. By quantitative real-time PCR assay, Silky line-specific gene duplication was detected as an ~130-kb interval. It contained five genes including endothelin 3 (EDN3), which encoded a potent mitogen for melanoblasts/melanocytes. EDN3 with another three of these duplicated genes in Silky chickens expressed almost twofold of those in BM-C. Present results strongly suggest that the increase of the expression levels resulting from the gene duplication in the Fm region is the trigger of hypermelanization in internal organs of Silky chickens.

Anterior epidermis-specific expression of the cuticle gene EDG84A is controlled by many cis-regulatory elements in Drosophila melanogaster.

During insect metamorphosis, a pulse of ecdysteroids induces many different morphological changes depending on different parts of the body. In Drosophila, although a number of transcription factors are expressed in a stage-specific manner in response to an ecdysteroid pulse, little is known on the regulatory mechanism for space-specific gene expression during metamorphosis. The EDG84A gene encoding pupal cuticle protein is one of the targets of ecdysteroid-inducible transcription factor betaFTZ-F1 and is expressed only in anterior epidermis of the body during mid- to late prepupal period, whereas betaFTZ-F1 is expressed in almost all tissues. To address the regulatory mechanism of the tissue-specific expression of the EDG84A gene, we established transgenic fly lines which carry various upstream regions of the gene fused to the LacZ gene and examined the expression pattern of the reporter gene. Results of the transgenic fly reporter assays showed that the space-specific expression is controlled by at least four positive and two negative elements within a 263-bp region near the transcription start site, and at least three of them showed space-specific effects to the anterior body trunk. These results suggest that both high expression level and differential expression are achieved through many cis-regulatory elements.