Insulin-producing cells and their regulation in physiology and behavior of Drosophila

z2012-009 476..488Insulin-producing cells and their regulation in physiology and behavior of Drosophila1
Dick R. Nässel
Abstract: Insulin-like peptide signaling regulates development, growth, reproduction, metabolism, stress resistance, and life span in a wide spectrum of animals. Not only the peptides, but also their tyrosine kinase receptors and the downstream sig- naling pathways are conserved over evolution. This review summarizes roles of insulin-like peptides (DILPs) in physiology and behavior of Drosophila melanogaster Meigen, 1830. Seven DILPs (DILP1–7) and one receptor (dInR) have been identi- fied in Drosophila. These DILPs display cell and stage specific expression patterns. In the adult, DILP2, 3, and 5 are ex- pressed in insulin-producing cells (IPCs) among the median neurosecretory cells of the brain, DILP7 in 20 neurons of the abdominal ganglion, and DILP6 in the fat body. The DILPs of the IPCs regulate starvation resistance, responses to oxidative and temperature stress, and carbohydrate and lipid metabolism. Furthermore, the IPCs seem to regulate feeding, locomotor activity, sleep and ethanol sensitivity, but the mechanisms are not elucidated. Insulin also alters the sensitivity in the olfac- tory system that affects food search behavior, and regulates peptidergic neurons that control aspects of feeding behavior. Fi- nally, the control of insulin production and release by humoral and neuronal factors is discussed. This includes a fat body derived factor and the neurotransmitters GABA, serotonin, octopamine, and two neuropeptides.
Key words: fruit fly, Drosophila melanogaster, insulin signaling, insulin-like peptide, neuropeptides, neurotransmitters.
Résumé : La signalisation des peptides de type insuline régit la croissance, la reproduction, le métabolisme, la résistance au stress et la durée de vie chez un large éventail d’animaux. Non seulement les peptides, mais aussi leurs récepteurs de tyro- sine kinase et les voies de signalisation inférieures sont conservés au cours de l’évolution. Cette rétrospective résume les rôles des peptides de type insuline (DILP) dans la physiologie et le comportement de Drosophila melanogaster Meigen, 1830. On a identifié sept DILP (DILP1–7) et un récepteur (dlnR) chez Drosophila. Ces DILP possèdent des patrons d’expression spécifiques à la cellule et au stade. Chez l’adulte, DILP2, 3 et 5 sont exprimés dans les cellules productrices d’insuline (IPC) parmi les cellules neurosécrétrices médianes du cerveau, DILP7 dans 20 neurones du ganglion abdominal et DILP6 dans le corps gras. Les DILP des IPC contrôlent la résistance à l’inanition, la réaction aux stress oxydatifs et ther- miques et le métabolisme des hydrates de carbone et des lipides. De plus, les IPC semblent réguler l’alimentation, l’activité locomotrice, le sommeil et la sensibilité à l’éthanol, mais les mécanismes n’ont pas été élucidés. L’insuline modifie aussi la sensibilité du système olfactif qui affecte le comportement de recherche de nourriture et régule les neurones peptidergiques qui contrôlent certains aspects du comportement alimentaire. Enfin, le contrôle de la production d’insuline et de sa libération par les facteurs humoraux et neuronaux fait l’objet d’une discussion qui traite, en particulier, d’un facteur dérivé du corps gras et des neurotransmetteurs GABA, sérotonine, octopamine et deux neuropeptides.
Motsclés : mouche du vinaigre, Drosophila melanogaster, signalisation de l’insuline, peptide de type insuline, neuropeptides, neurotransmetteurs.
[Traduit par la Rédaction]
Introduction
Insulin-like peptides regulate development, growth, repro- duction, metabolism, as well as stress resistance, aging, and life span both in invertebrates and vertebrates (Kenyon et al. 1993; Brogiolo et al. 2001; Broughton et al. 2005; Géminard
et al. 2006; Wu and Brown 2006; Baker and Thummel 2007; Giannakou and Partridge 2007; Fontana et al. 2010; Grönke et al. 2010; Teleman 2010; Antonova et al. 2012). Although insulin-like peptides, including peptides resembling insulin- like growth factors, have been identified in all invertebrate genomes sequenced so far (see Roller et al. 2008; Veenstra
Received 17 October 2011. Accepted 17 January 2012. Published at www.nrcresearchpress.com/cjz on 4 April 2012.
D.R. Nässel. Department of Zoology, Stockholm University, SE-10691 Stockholm, Sweden.
E-mail for correspondence: [email protected]. 1This review is part of a virtual symposium on recent advances in understanding a variety of complex regulatory processes in insect physiology and endocrinology, including development, metabolism, cold hardiness, food intake and digestion, and diuresis, through the use of omics technologies in the postgenomic era.
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Can. J. Zool. 90: 476–488 (2012) doi:10.1139/Z2012-009 Published by NRC Research Press
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2010; Dircksen et al. 2011; Antonova et al. 2012), their func- tional roles have been investigated primarily in the nematode Caenorhabditis elegans Maupas, 1900, the fruit fly Droso- phila melanogaster Meigen, 1830, and to some extent in mosquitos like Aedes aegypti (Linnaeus in Hasselquist, 1762) and in the silkworm (Bombyx mori (L., 1758)), as well as in the mollusks California seahare (Aplysia californ- ica J.G. Cooper, 1863) and pond snail (Lymnaea stagnalis (L., 1758)) (Kenyon et al. 1993; Kondo et al. 1996; Kimura et al. 1997; Floyd et al. 1999; Riehle and Brown 1999; Riehle et al. 2006; Brown et al. 2008; Okamoto et al. 2009a, 2011; Kenyon 2010). Much of the work on invertebrate insu- lin-like peptides has dealt with their roles in development, growth, and reproduction. These roles will be mostly ignored here and the focus will be on insulin-like peptide signaling in Drosophila in regulation of physiology and behavior, includ- ing metabolic homeostasis, locomotion, feeding, stress re- sponses, and life span. The first identifications of insect insulin-like peptides
(ILPs) were made in B. mori (Nagasawa et al. 1986; Adachi et al. 1989; Kondo et al. 1996) and the migratory locust (Lo- custa migratoria (L., 1758)) (Lagueux et al. 1990; Hetru et al. 1991). There are amazingly 38 ILPs, or bombyxins, in Bombyx (Kondo et al. 1996), whereas there is only 1 ILP known in the locust (Badisco et al. 2008). In dipteran insects, a first suggestion of the presence of ILPs was based on de- tection of insulin-like immunoreactive material in head ex- tract and in immunolabeled median neurosecretory cells (MNCs) of the blow fly Calliphora vomitoria (L., 1758) (Duve and Thorpe 1979; Duve et al. 1979). The genes en- coding the Drosophila DILPs were identified and the cellular expression of transcripts and peptides revealed by various techniques as late as 2001 (Brogiolo et al. 2001; Cao and Brown 2001; Vanden Broeck 2001). The only known Droso- phila insulin receptor (dInR) was, however, identified earlier and shown to be ubiquitously expressed (Garofalo and Rosen 1988; Fernandez et al. 1995). For more detailed data on insu- lin-like peptides and insulin signaling in different inverte- brates refer to Claeys et al. (2002), Wu and Brown (2006), Dircksen et al. (2011), and Antonova et al. (2012). In Drosophila, seven insulin-like peptides (DILP1–7) and
a single insulin receptor (dInR) have been identified (Bro- giolo et al. 2001; Grönke et al. 2010). These DILPs are pro- duced in a cell and developmental stage specific manner (Brogiolo et al. 2001; Rulifson et al. 2002; Okamoto et al. 2009b, 2011; Slaidina et al. 2009; Grönke et al. 2010). In the adult fly, three of the DILPs (DILP2, 3, and 5) are pro- duced by a set of MNCs in the brain of Drosophila (Fig. 1) and are thought to be released into the circulation from axon terminals in neurohemal areas in the corpora cardiaca, ante- rior aorta, and foregut (Brogiolo et al. 2001; Cao and Brown 2001; Rulifson et al. 2002). DILP5 was also detected in prin- cipal cells of the renal (Malpighian) tubules (Söderberg et al. 2011) (Fig. 1); DILP3 was detected in midgut muscle cells (Veenstra 2010); DILP6 was produced in the fat body (Oka- moto et al. 2009b; Slaidina et al. 2009); and DILP7 was ex- pressed in 20 neurons of the abdominal ganglia (Miguel- Aliaga et al. 2008; Yang et al. 2008) (Fig. 1). The cellular origin of the others, DILP1 and 4, is not clear in the adult fly (see Lee et al. 2008; Broughton and Partridge 2009). Studies of insulin signaling in Drosophila have to a large
extent focused on the action of DILPs on fat body (or ova- ries) and subsequent effects on growth, metabolism, fecund- ity, and stress responses (reviewed in Tatar et al. 2003; Géminard et al. 2006; Baker and Thummel 2007; Giannakou and Partridge 2007; Teleman 2010). Overall, we know more about the DILPs expressed by the IPCs of the brain (DILP2, 3, and 5), although recently some work has been performed on DILP6 and 7 (see Yang et al. 2008; Okamoto et al. 2009b; Slaidina et al. 2009; Cognigni et al. 2011). Ablation of the IPCs by Dilp2-Gal4-driven apoptosis
(UAS-reaper) leads to retarded growth, extended lifesspan, increased levels of carbohydrate in the circulation, increased storage of carbohydrate and lipids, and increased resistance to various stresses (Rulifson et al. 2002; Broughton et al. 2005). Also simultaneous mutations in the three genes encod- ing DILP2, 3, and 5 produce these phenotypes, in addition to defects in growth and fertility (Zhang et al. 2009; Grönke et al. 2010). Thus, DILP2, 3, and 5 display pleiotropic func- tions and it has been shown by mutations of the single genes that the individual DILPs display redundant functions (Grönke et al. 2010). In spite of their partial redundancy, the production of the three DILPs co-localized in the IPCs can be individually regulated transcriptionally (Ikeya et al. 2002; Hwangbo et al. 2004; Karpac et al. 2009; Broughton et al. 2010; Birse et al. 2011). DILP2 is the one most highly ex- pressed in IPCs and if the other DILPs are deleted, ectopic expression of DILP2 is sufficient to rescue many of the DILP functions (Rulifson et al. 2002; Broughton et al. 2008; Grönke et al. 2010). Interestingly, some basic questions remain unsolved in
both larval and adult Drosophila. One is how production and especially release of brain-derived DILPs are regulated in daily life. What are the regulators of DILP release for in- stance during stress? Another basic question is whether the single known dInR mediates the action of the multiple DILPs in a selective manner. Do the individual DILPs activate the dInR differentially? Furthermore, it may be that interference with IPCs produces some phenotypes that are due to other factors released from these cells, or actions of DILPs on tar- gets other than the fat body. So, the question is how diverse are the targets of the brain IPCs and the signaling pathways downstream of the dInR? This review summarizes data on signaling from the IPCs in the Drosophila brain, including regulation of production and release of DILPs from these cells and physiological and behavioral roles of brain-derived DILPs. Although the main focus is on insulin signaling in the adult fly, several examples are given where larvae were studied.
Organization of the insulin-producing cells in Drosophila In Drosophila, DILP2, 3, and 5 are expressed in 12–16
brain IPCs (Fig. 1) embedded in a cluster of median neuro- secretory cells in the pars intercerebralis (PI). The three DILPs are actually co-localized in each of these IPCs (Ikeya et al. 2002; Geminard et al. 2009). The morphology of these IPCs has been described from immunolabeling with various DILP antisera and by use of Dilp2-Gal4-driven GFP (Bro- giolo et al. 2001; Cao and Brown 2001; Ikeya et al. 2002; Rulifson et al. 2002; Cognigni et al. 2011). It appears that
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these IPCs all share the same anatomy with cell bodies lo- cated in the PI, two sets of branches in the PI, another set in tritocerebrum, and axons terminating with varicosities in the corpora cardiaca, anterior aorta, proventriculus, and crop (Figs. 1, 2). It is not clear whether the branches in the PI and tritocerebrum are dendritic or perhaps a mix between dendrites and release sites. Antisera to DILPs label the IPC branches in the PI, as well as in the tritocerebrum, suggesting storage and perhaps release sites of the peptides within the brain. It is possible therefore that DILPs are released both into the circulation from neurohemal release sites and in a paracrine fashion from branches within the brain. In some papers, Dilp2-Gal4-expressing neurons have been
described also in the larval and adult abdominal and thoracic ganglia (Kaplan et al. 2008; Agrawal et al. 2010) and these cells can indeed be seen in at least two different Dilp2-Gal4 drivers (unpublished observations). The expression of DILP2 peptide or Dilp2 transcript in these cells has not yet been confirmed in contrast to the brain IPCs. An older paper using antiserum to mammalian insulin described immunolabeling in neurons of the larval abdominal ganglia (Gorczyca et al. 1993); these neurons, however, do not resemble the ones de- tected by the Dilp2-Gal4. The DILP7-producing neurons localized in the abdominal
ganglia are of at least two types (Fig. 1). Eight neurons (dMP2) in abdominal neuromeres A6-9 are efferent with ax- ons that terminate on the hindgut. One pair of interneurons (DP) in A1 arborize in the abdominal ganglion and with ax- ons reaching the brain (Miguel-Aliaga et al. 2008). In the third-instar larva, the DP neurons send axons with varicose terminations to the protocerebral branches of the IPCs (Miguel-Aliaga et al. 2008; Nässel et al. 2008). Curiously the DPs, but not the other DILP7-expressing neurons, co- express short neuropeptide F (sNPF), and weak Cha-Gal4 ex-
pression, indicating a cholinergic phenotype (Nässel et al. 2008). It was claimed that also in the adult the DPs impinge on the IPCs, but in the subesophageal ganglion (Cognigni et al. 2011). In the larva it is possible that the DPs modulate the IPCs with DILP7 and sNPF (and possibly acetylcholine). It seems likely that the DILP7-producing neurons release the peptide in a paracrine fashion within the CNS and at the hindgut structures (Miguel-Aliaga et al. 2008; Cognigni et al. 2011). A role of DILP7 has also been detected in repro- ductive behavior in female egg laying (Yang et al. 2008). The integration of the brain IPCs into the neuroendocrine
system of the brain and neurons involved in control of the IPCs is dealt with in later sections. However, it should be noted that the IPCs are a subpopulation of the MNCs and other MNCs produce distinct neurohormones. In the Droso- phila larva, these MNCs express the peptides myosuppressin and diuretic hormone, and the IPCs may co-express sulfaki- nins (Park et al. 2008).
Insulin functions in Drosophila DILPs may affect many aspects of growth and differentia-
tion of the nervous system (and other tissues) during devel- opmental stages. Therefore, it is not always clear to what extent the phenotypes in adult flies that result from genetic manipulations of insulin signaling are derived from (i) altered neuronal function or wiring as a result of developmental ef- fects (including compensatory changes in morphology and physiology) or (ii) caused by alteration of bona fide DILP signaling in the adult fly in an otherwise unaltered organism. Only rarely have conditional interference (e.g., gene switch and Gal80ts constructs) been utilized to ensure that the ob- served effects are not contaminated by effects of altered or compensated development. A summary of DILP functions is shown in Fig. 2.
Fig. 1. Insulin-producing cells (IPCs) in the fruit fly Drosophila nervous system and gut. A set of IPCs in pars intercerebralis of the brain send axons to the tritocerebrum (adjacent to the subesophageal ganglion, SEG), to the corpora cardiaca (CC) with associated aorta, proven- triculus (PV), and the crop. Likely release sites for circulating Drosophila insulin-like peptides (DILPs) are in CC, aorta, PV, and crop. These IPCs produce DILP2, 3, and 5. The branches in protocerebrum (near cell bodies) and tritocerebrum of the brain could be dendritic and (or) further release sites. A second set of cells (aIPCs) is found in the abdominal ganglion. These produce DILP7 and supply axon terminations to the hindgut including the rectal papillae (RP), and in females to reproductive organs (not shown here). At least two of the aIPCs send axons to the SEG. It is not clear whether their branches in the SEG (open circles) are dendrites or release sites. Additionally the principal cells of the renal tubules (RT) produce DILP5. This DILP may act locally in the tubules. DILP6 is produced in fat body cells in the head and abdomen (not shown). This figure is partly based on data from Cognigni et al. (2011).
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Insulin-like peptides and stress resistance All animals experience stress at some points of their life
cycle or even in their daily life. Stress is induced by environ- mental challenges to the well being of the animal and pro- duce imbalances in their homeostasis. Commonly, the organism responds to stress by adaptive behavioral or physio- logical adjustments (Selye 1950; Karpac et al. 2009; Zhao et al. 2010). Stress can be induced by environmental factors like temperature, lack of nutrition or water, and infection, or by factors producing reactive oxygen species (oxidative stress). Several papers have demonstrated a role for insulin signaling in regulation of responses to stresses such as extreme temper- atures, starvation, desiccation, oxidative stress, and infection. Ablation of the brain IPCs lead to a reduced tolerance of
extreme temperatures, but an increased resistance to oxidative stress and starvation (Broughton et al. 2005). In these experi- ments, the flies with diminished DILP production were tested for time to heat knockdown at 39 °C and recovery from im- mobilization at 0 °C, and median and maximal life span at starvation or feeding of paraquat that induces oxidative stress. It is not clear why the flies display opposite reactions to tem-
perature stress on one hand and starvation and oxidative stress on the other, but apparently insulin signaling may tar- get different pathways depending on type of stress. Maybe the difference is due to the fact that oxidative stress is an im- portant factor in the slower aging process, whereas tempera- ture induces more acute stresses. It has been shown that Jun-N-terminal Kinase (JNK) sig-
naling in IPCs (and fat body) is part of a mechanism regulat- ing responses to stress and thereby reallocating resources from metabolic processes to ones required for defense against stress and repair of damage (Wang et al. 2005; Karpac et al. 2009). Karpac et al. (2009) found that repression of insulin signaling via JNK in IPCs is important in responses to stress, but also represses growth during development. Other manip- ulations of insulin signaling, like knockdown of neurotrans- mitter receptors in IPCs, have also been shown to affect stress responses (Enell et al. 2010; Luo et al. 2011; Söder- berg et al. 2011), and will be discussed in a later section.
Insulin-like peptides and sleep-wakefulness Fruit flies display a state closely resembling sleep (Shaw et
Fig. 2. The brain insulin-producing cells (IPCs) produce Drosophila insulin-like peptides (DILP2, 3, and 5) and may act on many targets. The IPCs have their cell bodies in the pars intercerebralis where also their likely dendrites are located (arrow). Another set of arborizations of the IPCs can be seen in the tritocerebrum (Trito) ventral to the antennal lobes (AL). The axons destined for the corpora cardiaca, aorta, and foregut structures are not shown here (they exit…