Molecular Evolution and Functional Characterization of Drosophila Insulin-Like Peptides

Abstract

Multicellular animals match costly activities, such as growth and reproduction, to the environment through nutrient-sensing pathways. The insulin/IGF signaling (IIS) pathway plays key roles in growth, metabolism, stress resistance, reproduction, and longevity in diverse organisms including mammals. Invertebrate genomes often contain multiple genes encoding insulin-like ligands, including seven Drosophila insulin-like peptides (DILPs). We investigated the evolution, diversification, redundancy, and functions of the DILPs, combining evolutionary analysis, based on the completed genome sequences of 12 Drosophila species, and functional analysis, based on newly-generated knock-out mutations for all 7 dilp genes in D. melanogaster. Diversification of the 7 DILPs preceded diversification of Drosophila species, with stable gene diversification and family membership, suggesting stabilising selection for gene function. Gene knock-outs demonstrated both synergy and compensation of expression between different DILPs, notably with DILP3 required for normal expression of DILPs 2 and 5 in brain neurosecretory cells and expression of DILP6 in the fat body compensating for loss of brain DILPs. Loss of DILP2 increased lifespan and loss of DILP6 reduced growth, while loss of DILP7 did not affect fertility, contrary to its proposed role as a Drosophila relaxin. Importantly, loss of DILPs produced in the brain greatly extended lifespan but only in the presence of the endosymbiontic bacterium Wolbachia, demonstrating a specific interaction between IIS and Wolbachia in lifespan regulation. Furthermore, loss of brain DILPs blocked the responses of lifespan and fecundity to dietary restriction (DR) and the DR response of these mutants suggests that IIS extends lifespan through mechanisms that both overlap with those of DR and through additional mechanisms that are independent of those at work in DR. Evolutionary conservation has thus been accompanied by synergy, redundancy, and functional differentiation between DILPs, and these features may themselves be of evolutionary advantage.

Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes

“…4C). We confirmed these dilp6 loss-of-function phenotypes using the loss-of-function allele dilp6 41 (29) (Fig. 4D).…”

“…The role of oenocytes in metabolic adaptation resembles the function of the mammalian liver, as has been predicted from studies in larvae (14). The various insulin-like peptides of flies influence growth and stress responses in different contexts, providing a complex, spatiotemporally defined regulatory system to sustain local cell growth, proliferation, cell survival, and metabolism (10,11,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). So far, the molecular codes that ensure specificity in this signaling network remain unclear.…”

“…In Drosophila, IIS activity is stimulated by a number of different insulin-like peptides (ILPs) that are independently regulated and can act in endocrine as well as local, paracrine fashion. A major source of dILPs are the median neurosecretory cells (mNSCs, also known as insulin-producing cells, IPCs) in the pars intercerebralis of the head, but expression of individual dILPs has also been reported in the gut, the ovary, glial cells, fat body, and abnormally growing imaginal discs (10,11,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Whereas secretion of dILP2, -3, and -5 from mNSCs is regulated by nutritional cues, transcriptional regulation of these peptides depends on the stimulus (9,21).…”

Energy Homeostasis Control in Drosophila Adipokinetic Hormone Mutants

“…Taken together, in contrast to the dramatic effects of insulin signaling deficiency (Grönke et al 2010), absence of AKH does not cause gross abnormalities in development or reproduction.…”

“…We noticed slightly, but significantly reduced hatchability in Akh A . However, this effect was not caused by the deficiency in the AKH signaling itself, as the AkhR mutant and AKH plus APRP double mutants had normal hatchability and larval to adult survival (Figure 3, A and B).Taken together, in contrast to the dramatic effects of insulin signaling deficiency (Grönke et al 2010), absence of AKH does not cause gross abnormalities in development or reproduction.AKH signaling and APRP are dispensable for mobilization of lipids and glycogen during Drosophila development Development encompasses nonfeeding periods like embryogenesis, moltings, and metamorphosis, when the organism relies exclusively on stored energy reserves. To address the fuel utilization during Drosophila metamorphosis, and to test a possible involvement of AKH in this process, we followed the glycogen and lipid changes in Drosophila from the wandering stage of the third instar larvae (3L, end of the feeding) through pupation (P0), termination of the pupal development (P13-P15), until the posteclosion values in the immature adults (within 10 hr after eclosion) (Figure 4, A and B).…”