The insulin͞insulin-like growth factor-like signaling pathway, present in all multicellular organisms, regulates diverse functions including growth, development, fecundity, metabolic homeostasis, and lifespan. In flies, ligands of the insulin͞insulin-like growth factor-like signaling pathway, the Drosophila insulin-like peptides, regulate growth and hemolymph carbohydrate homeostasis during development and are expressed in a stage-and tissue-specific manner. Here, we show that ablation of Drosophila insulin-like peptide-producing median neurosecretory cells in the brain leads to increased fasting glucose levels in the hemolymph of adults similar to that found in diabetic mammals. They also exhibit increased storage of lipid and carbohydrate, reduced fecundity, and reduced tolerance of heat and cold. However, the ablated flies show an extension of median and maximal lifespan and increased resistance to oxidative stress and starvation.

Dietary restriction (DR) extends life span in many organisms, through unknown mechanisms that may or may not be evolutionarily conserved. Because different laboratories use different diets and techniques for implementing DR, the outcomes may not be strictly comparable. This complicates intra- and interspecific comparisons of the mechanisms of DR and is therefore central to the use of model organisms to research this topic. Drosophila melanogaster is an important model for the study of DR, but the nutritional content of its diet is typically poorly defined. We have compared fly diets composed of different yeasts for their effect on life span and fecundity. We found that only one diet was appropriate for DR experiments, indicating that much of the published work on fly "DR" may have included adverse effects of food composition. We propose procedures to ensure that diets are suitable for the study of DR in Drosophila.

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The insulin/IGF-like signalling (IIS) pathway has diverse functions in all multicellular organisms, including determination of lifespan. The seven insulin-like peptides (DILPs) in Drosophila are expressed in a stage- and tissue-specific manner. Partial ablation of the median neurosecretory cells (mNSCs) in the brain, which produce three DILPs, extends lifespan, reduces fecundity, alters lipid and carbohydrate metabolism and increases oxidative stress resistance. To determine if reduced expression of DILPs is causal in these effects, and to investigate possible functional diversification and redundancy between DILPs, we used RNA interference to lower specifically the transcript and protein levels of dilp2, the most highly expressed of the mNSC-derived DILPs. We found that DILP2 was limiting only for the increased whole-body trehalose content associated with mNSC-ablation. We observed a compensatory increase in dilp3 and 5 mRNA upon dilp2 knock down. By manipulation of dfoxo and dInR, we showed that the increase in dilp3 is regulated via autocrine insulin signaling in the mNSCs. Our study demonstrates that, despite the correlation between reduced dilp2 mRNA levels and lifespan-extension often observed, DILP2 reduction is not sufficient to extend lifespan. Nor is the increased trehalose storage associated with reduced IIS sufficient to extend lifespan. To understand the normal regulation of expression of the dilps and any functional diversification between them will require independent control of the expression of different dilps.

SummaryDietary restriction extends lifespan in diverse organisms, but the gene regulatory mechanisms and tissues mediating the increased survival are still unclear. Studies in worms and flies have revealed a number of candidate mechanisms, including the target of rapamycin and insulin ⁄ IGF-like signalling (IIS) pathways and suggested a specific role for the nervous system in mediating the response. A pair of sensory neurons in Caenorhabditis elegans has been found to specifically mediate DR lifespan extension, but a neuronal focus in the Drosophila nervous system has not yet been identified. We have previously shown that reducing IIS via the partial ablation of median neurosecretory cells in the Drosophila adult brain, which produce three of the seven fly insulin-like peptides, extends lifespan. Here, we show that these cells are required to mediate the response of lifespan to full feeding in a yeast dilution DR regime and that they appear to do so by mechanisms that involve both altered IIS and other endocrine effects. We also present evidence of an interaction between these mNSCs, nutrition and sleep, further emphasising the functional homology between the DILP-producing neurosecretory cells in the Drosophila brain and the hypothalamus of mammals in their roles as integration sites of many inputs for the control of lifespan and behaviour.

BackgroundOutcomes of lifespan studies in model organisms are particularly susceptible to variations in technical procedures. This is especially true of dietary restriction, which is implemented in many different ways among laboratories.Principal FindingsIn this study, we have examined the effect of laboratory stock maintenance, genotype differences and microbial infection on the ability of dietary restriction (DR) to extend life in the fruit fly Drosophila melanogaster. None of these factors block the DR effect.ConclusionsThese data lend support to the idea that nutrient restriction genuinely extends lifespan in flies, and that any mechanistic discoveries made with this model are of potential relevance to the determinants of lifespan in other organisms.

Dietary restriction (DR) is a moderate reduction in food intake, without malnutrition, that extends the healthy life span in many organisms, including Drosophila (1). Although fruit flies have many practical advantages for studying DR, various technical complexities can have large effects on experimental outcomes (1).For DR, it is important that the basic food conditions are optimal. This ensures that increased longevity due to food restriction prolongs a healthy life span rather than returning sick animals to normal health by limiting access to a nutritionally inappropriate diet. We have systematically optimized our conditions for Drosophila DR, eliminated several non-nutritional explanations including water imbalance, and recommend a Brewer's-yeast-based diet (2, 3). Ja et al. (4) report contradictory data. Here, we present additional data in support of our conclusions and point out a flaw in the data concerning the Brewer's yeast diet presented by Ja et al.First, we developed a system that effectively hydrates flies under salt stress (Table 1, experiment 1). Adding 8 g·L −1 NaCl to our standard food [1.0 SYBrewer's (2)] shortened median life span by 24%. Adding to each vial a 200-μl pipette tip filled with water (1% agar) restored normal life span. The rescue was not due to the tip itself because an identical tip filled with dry cotton wool had no effect. Furthermore, addition of a tip containing wet cotton wool also restored normal life span. This was reproducible when the food was made from a different yeast [SYBaker's (2)] with salt added (Table 1, experiment 2). Thus, our technique is effective in delivering water to salt-stressed flies and in rescuing the associated shortening of the life span.Next, we established that the life-span change associated with DR was not rescued by water addition [ Table 1, experiment 3; replicate of our published data (2)].These data demonstrate that DR in Drosophila under our conditions is not due to rescue of hydration stress.These results directly contradict data from an unreplicated experiment by Ja et al. using conditions ostensibly replicating ours, which reported that life-span extension by DR was eliminated by the addition of water, concluding that hydration stress explains DR (figure 2, G-I, in ref. 4). There are two problems with this conclusion. First, Ja et al. used more sugar (100 g·L −1 ) in their concentrated medium (CM) than did we, and this higher concentration causes a significant reduction in egg laying compared with the level we use (50 g·L −1 ) (2), perhaps because of water stress. [Furthermore, fly feeding behavior can be dramatically reduced as sugar concentration increases in this range, which may explain why Ja et al. observed lowered feeding in their CM ( figure 1F in ref. 4) and we do not.] Second, the data in figure 2, G-I, from Ja et al. (4) demonstrate that water addition shortened DR life span to the level of the CM (Fig. 1). Thus the "rescue" of the DR effect by water addition could also be explained by water shortening the life span of DR fli...