Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. Here, we report the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gainof-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster.Neuropeptides regulate a wide range of animal behavior. In vertebrates, neuropeptide Y (NPY) 1 regulates food consumption, circadian rhythms, anxiety, and other physiological processes (1). NPY, a 36-amino acid neuromodulator, is expressed abundantly in the mammalian brain and controls feeding (2). The NPY injection into the hypothalamus of rat brain resulted in hyperphagia and obesity, whereas the NPY-deficient mouse in the leptin mutant background (NPYϪ/Ϫ; ob/ob) showed the less obese phenotype (3).In invertebrates, neuropeptide F (NPF) peptides share structural similarity with the vertebrate NPY (4). NPF peptides isolated from various invertebrate animals have the conserved (A/L)R(P/L)RF amide sequence at their C-terminal ends (5, 6). From a completely sequenced Drosophila melanogaster genome, short NPF (sNPF; CG13968) and Drosophila NPF (dNPF; CG10342) were found. The dNPF peptide was isolated by the radioimmunoassay. The preprotein is processed to the 36-amino acid peptide containing RVRF amide in the C terminus. dNPF is considered the homolog of the vertebrate NPY. dNPF mRNA and peptide are expressed in the brain and midgut of Drosophila larvae and adults (7). The dNPF neural network in the larval central nervous system (CNS) is changed by the gustatory stimulation of sugar, indicating that dNPF is an integral part of the chemosensory system that regulates eating behavior (8). Drosophila larvae eat continuously and grow in a relatively short period. About 5 days after egg laying, they s...

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. Here we show that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. We show that these effects are mediated through activation of extracellular signal-related kinases (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan.

SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan.

Angiotensin II (Ang II) stimulates norepinephrine transporter (NET) and tyrosine hydroxylase (TH) in the neurons, but the signal transduction mechanism of this neuromodulation is not understood. Treatment of neuronal cultures of hypothalamusbrainstem with Ang II resulted in a time-and dose-dependent activation of Ras, Raf-1, and mitogen-activated protein kinase. This activation was mediated by the interaction of Ang II with the AT 1 receptor subtype and was associated with the redistribution of AT 1 receptor with Ras and Raf-1 on the neuronal membrane. Treatment with antisense oligonucleotide (AON) to mitogen-activated protein kinase decreased mitogen-activated protein kinase immunoreactivity by 70% and attenuated Ang II stimulation of c-fos, NET, and TH mRNA levels. This demonstrates that induction of these genes requires mitogenactivated protein kinase activation by Ang II. In contrast, AON to mitogen-activated protein kinase failed to inhibit Ang II stimulation of plasminogen activator inhibitor-1 mRNA levels. These results suggest that AT 1 receptors are coupled to a Ras-Raf-1 mitogen-activated protein kinase signal transduction pathway that is responsible for stimulation of NET and TH, two neuromodulatory actions of Ang II in the brain.

SUMMARY Mitochondria play central roles in buffering intracellular Ca2+ transients. While basal mitochondrial Ca2+ (Ca2+mito) is needed to maintain organellar physiology, Ca2+mito overload can lead to cell death. How Ca2+mito homeostasis is regulated is not well understood. Here we show that Miro, a known component of the mitochondrial transport machinery, regulates Drosophila neural stem cell (NSC) development through Ca2+mito homeostasis control independent of its role in mitochondrial transport. Miro interacts with Ca2+ transporters at the ER-mitochondria contact site (ERMCS). Its inactivation causes Ca2+mito depletion and metabolic impairment, whereas its overexpression results in Ca2+mito overload, mitochondrial morphology change, and apoptotic response. Both conditions impaired NSC lineage progression. Ca2+mito homeostasis is influenced by Polo-mediated phosphorylation of a conserved residue in Miro, which positively regulates Miro localization to, and the integrity of, ERMCS. Our results elucidate a regulatory mechanism underlying Ca2+mito homeostasis and how its dysregulation may impact NSC metabolism/development and contribute to disease.

The observed positive effects of curcumin on life span and health span in two different D. melanogaster strains demonstrate a potential applicability of curcumin treatment in mammals. The ability of curcumin to mitigate the expression levels of age-associated genes in young flies suggests that the action of curcumin on these genes is a cause, rather than an effect, of its life span-extending effects.

Dietary restriction (DR) extends lifespan of many animals including Drosophila melanogaster. Recent work with flies shows longevity is controlled by the ratio of consumed protein relative to carbohydrates. Since reduced insulin/IGF and TOR signaling increase Drosophila lifespan, these pathways are candidate mediators of DR. This idea, however, has ambiguous experimental support. The Nutritional Geometric Framework, which dissects the impact of nutrient protein relative to carbohydrates, may provide an approach to resolving the roles for these pathways in DR. Nutrient sensing of protein and carbohydrate may occur in the fat body through signals to hypothalamic-like neurons in the fly brain, and thus control secretion of insulin-like peptides that regulate longevity.

The inducible isoform II of nitric-oxide synthase (iNOS) was recently cloned from brain and identified in astroglial cells. Induced nitric oxide biosynthesis occurs in brain cells only if extracellular cerebrospinal fluid contains -arginine. This study demonstrates for the first time that induced iNOS activity is strictly dependent on concomitant induction of an alternatively spliced transcript of the cat-2 gene encoding high affinity -arginine transporter System y+ in cultured rat astrocytes. Inhibition profiles of radiolabeled -arginine and -leucine uptake identified the dominance of Na+-independent transport System y+ serving cationic amino acids, with insignificant activities of Systems y+L, bo,+, or Bo,+. A reverse transcription-polymerase chain reaction/sequencing/cloning strategy was used to identify a single 123-base nucleotide sequence coding the high affinity domain of alternatively spliced CAT-2 (not CAT-2a) in astrocytes activated by lipopolysaccharide/interferon-gamma. Using this sequence as a cDNA probe, it was determined that CAT-2 mRNA, iNOS mRNA, and System y+ activity were concomitantly and strongly induced in astrocytes. Constitutive CAT-1 mRNA was weakly present in neurons and astrocytes, was not inducible in either cell type, and contributed <3% to total System y+ activity. Although astroglial iNOS Km approximately 10 microM L-arginine for intracellular substrate, hyperbolic kinetics of inducible iNOS activity measured as a function of extracellular L-arginine concentration gave Km approximately 50 microM L-arginine with intact cells. The same Km approximately 50 microM was obtained for induced membrane transport System y+ activity. iNOS activity was reduced to zero in the absence of extracellular L-arginine uptake via System y+. These findings expand the current understanding of NO biosynthesis modulation and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane L-arginine transport in brain.