Bioproduction of n-3 polyunsaturated fatty acids by nematode fatty acid desaturases and elongase in Drosophila melanogaster.

n-3 polyunsaturated fatty acids (n-3 PUFAs), including α-linolenic acid and eicosapentaenoic acid (EPA), are essential nutrients for vertebrates including humans. Vertebrates are n-3 PUFA-auxotrophic; hence, dietary intake of n-3 PUFAs is required for their normal physiology and development. Although fish meal and oil have been utilized as primary sources of n-3 PUFAs by humans and aquaculture, these traditional n-3 PUFA sources are expected to be exhausted because of the increasing consumption requirements of humans. Hence, it is necessary to establish alternative n-3 PUFA sources to reduce the gap between the supply and demand of n-3 PUFAs. Here, we investigated whether insects, which are considered as a novel source of essential nutrients, could store n-3 PUFAs by the forced expression of n-3 PUFA biosynthetic enzymes. We utilized Drosophila as an insect model to generate transgenic strains expressing Caenorhabditis elegans PUFA biosynthetic enzymes and examined their effects on the proportion of fatty acids. The ubiquitous expression of methyl-end desaturase FAT-1 prominently enhanced the proportions of α-linolenic acid, indicating that FAT-1 is useful for metabolic engineering to fortify α-linolenic acid in insect. Furthermore, the ubiquitous expression of nematode front-end desaturases (FAT-3 and FAT-4), PUFA elongase (ELO-1), and FAT-1 led to EPA bioproduction. Hence, nematode PUFA biosynthetic genes may serve as powerful genetic tools for enhancing the proportion of EPA in insects. This study represents the first step toward the establishment of n-3 PUFA-producing insects.

Chaperonin TRiC/CCT supports mitotic exit and entry into endocycle in Drosophila.

Endocycle is a commonly observed cell cycle variant through which cells undergo repeated rounds of genome DNA replication without mitosis. Endocycling cells arise from mitotic cells through a switch of the cell cycle mode, called the mitotic-to-endocycle switch (MES), to initiate cell growth and terminal differentiation. However, the underlying regulatory mechanisms of MES remain unclear. Here we used the Drosophila steroidogenic organ, called the prothoracic gland (PG), to study regulatory mechanisms of MES, which is critical for the PG to upregulate biosynthesis of the steroid hormone ecdysone. We demonstrate that PG cells undergo MES through downregulation of mitotic cyclins, which is mediated by Fizzy-related (Fzr). Moreover, we performed a RNAi screen to further elucidate the regulatory mechanisms of MES, and identified the evolutionarily conserved chaperonin TCP-1 ring complex (TRiC) as a novel regulator of MES. Knockdown of TRiC subunits in the PG caused a prolonged mitotic period, probably due to impaired nuclear translocation of Fzr, which also caused loss of ecdysteroidogenic activity. These results indicate that TRiC supports proper MES and endocycle progression by regulating Fzr folding. We propose that TRiC-mediated protein quality control is a conserved mechanism supporting MES and endocycling, as well as subsequent terminal differentiation.

The presence of odd-chain fatty acids in Drosophila phospholipids.

Most fatty acids in phospholipids and other lipid species carry an even number of carbon atoms. Also odd-chain fatty acids (OCFAs), such as C15:0 and C17:0, are widespread throughout the living organism. However, the qualitative and quantitative profiles of OCFAs-containing lipids in living organisms remain unclear. Here, we show that OCFAs are present in Drosophila phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and that their level increases in accordance with progression of growth. Furthermore, we found that food-derived propionic acid/propanoic acid (C3:0) is utilized for production of OCFA-containing PC and PE. This study provides the basis for understanding in vivo function of OCFA-containing phospholipids in development and lipid homeostasis.

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.

Polymorphism in microRNA-binding site in HNF1B influences the susceptibility of type 2 diabetes mellitus: a population based case-control study.

BACKGROUND: Recent genome-wide association studies (GWAS) have identified many SNPs associated with type 2 diabetes mellitus (T2DM). However, the functional roles for most of the SNPs have not been elucidated. MicroRNAs (miRNAs) are key regulators of gene expression involved in the development and progression of various diseases including T2DM. In this study, we investigated whether commonly occurring SNPs modulate miRNA-directed regulation of gene expression, and whether such SNPs in miRNA-binding sites are associated with the susceptibility for T2DM. METHODS: Genotypes of eleven 3' untranslated region (UTR) SNPs of seven susceptibility genes for T2DM were determined in 353 T2DM patients and 448 control subjects. In addition, the interactions of miRNAs with the 3'UTR in the hepatocyte nuclear factor 1ß (HNF1B) gene were investigated using luciferase reporter assays. RESULTS: One 3'UTR SNP (rs2229295) in the HNF1B gene was significantly associated with T2DM, and the frequency of an A allele (rs2229295) in T2DM patients was decreased compared with that in controls. Luciferase reporter assays showed that the SNP (rs2229295) altered the binding of two miRNAs (hsa-miR-214-5p and hsa-miR-550a-5p). CONCLUSIONS: We have detected the interactions of hsa-miR-214-5p/hsa-miR-550a-5p and the 3'UTR SNP of the HNF1B gene by in vitro luciferase reporter assays, and propose that the binding of such miRNAs regulates the expression of the HNF1B gene and the susceptibility of T2DM.

The bioconversion of dietary α-linolenic acid to eicosapentaenoic acid in Bombyx mori.

n-3 Long-chain polyunsaturated fatty acids (n-3 LC-PUFAs), including eicosapentaenoic acid (EPA), are essential multifunctional nutrients in animals. Microorganisms such as microalgae are known to be n-3 LC-PUFA producers in aquatic environments. Various aquatic invertebrates, including Harpacticoida copepods, and a few terrestrial invertebrates, such as the nematode Caenorhabditis elegans, possess n-3 LC-PUFA biosynthetic enzymes. However, the capacity for n-3 LC-PUFA biosynthesis and the underlying molecular mechanisms in terrestrial insects are largely unclear. In this study, we investigated the fatty acid biosynthetic pathway in the silkworm Bombyx mori and found that EPA was present in silkworms throughout their development. Stable isotope tracing revealed that dietary α-linolenic acid (ALA) was metabolized to EPA in silkworm larvae. These results indicated that silkworms synthesize EPA from ALA. Given that EPA is enriched in the central nervous system, we propose that EPA confers optimal neuronal functions, similar to docosahexaenoic acid, in the mammalian nervous system.

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.

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.

The combined effects of genetic variation in the SIRT1 gene and dietary intake of n-3 and n-6 polyunsaturated fatty acids on serum LDL-C and HDL-C levels: a population based study.

BACKGROUND: Dyslipidemia due to high total cholesterol, LDL-cholesterol, triglycerides, or low HDL-cholesterol is an important risk factor for coronary heart disease (CHD). Both SIRT1 and PUFAs can influence the expression of genes for nuclear receptors and transcription factors related to lipid metabolism such as LXRα, LXRß, PPARα, SREBP-1c. METHODS: A total of 707 Japanese males and 723 females were randomly selected from the participants who visited a medical center for routine medical check-ups. We analyzed the combined effects of the genotype/haplotype of the SIRT1 gene and dietary n-6/n-3 PUFA intake ratio on the determination of serum lipid levels. RESULTS: We found that the SIRT1 gene marked with haplotype 2 was associated with decreased serum LDL-cholesterol and increased HDL-cholesterol levels. In addition, the associations between the SIRT1 haplotype 2 and decreased LDL-C and increased HDL-C levels were only observed in the low n-6/n-3 PUFA intake ratio group, but not in the high n-6/n-3 PUFA intake ratio group. CONCLUSIONS: Our findings indicate that the combination of genetic variation in the SIRT1 gene and dietary n-6 and/or n-3 PUFA intake influence the determination of inter-individual variations of serum levels of LDL-C and HDL-C.

The combined effect of the T2DM susceptibility genes is an important risk factor for T2DM in non-obese Japanese: a population based case-control study.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a complex endocrine and metabolic disorder. Recently, several genome-wide association studies (GWAS) have identified many novel susceptibility loci for T2DM, and indicated that there are common genetic causes contributing to the susceptibility to T2DM in multiple populations worldwide. In addition, clinical and epidemiological studies have indicated that obesity is a major risk factor for T2DM. However, the prevalence of obesity varies among the various ethnic groups. We aimed to determine the combined effects of these susceptibility loci and obesity/overweight for development of T2DM in the Japanese. METHODS: Single nucleotide polymorphisms (SNPs) in or near 17 susceptibility loci for T2DM, identified through GWAS in Caucasian and Asian populations, were genotyped in 333 cases with T2DM and 417 control subjects. RESULTS: We confirmed that the cumulative number of risk alleles based on 17 susceptibility loci for T2DM was an important risk factor in the development of T2DM in Japanese population (P<0.0001), although the effect of each risk allele was relatively small. In addition, the significant association between an increased number of risk alleles and an increased risk of T2DM was observed in the non-obese group (P<0.0001 for trend), but not in the obese/overweight group (P=0.88 for trend). CONCLUSIONS: Our findings indicate that there is an etiological heterogeneity of T2DM between obese/overweight and non-obese subjects.