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Regulation of insulin producing cells, stress responses and metabolism in Drosophila

Doctoral dissertation 2012 Neval Kapan Department of Zoology Stockholm University 106 91 Stockholm ABSTRACT In Drosophila, neuropeptides have regulatory roles in development, growth, metabolism and reproduction. This study focused on GABA and the neuropeptides Drosophila tachykinin (DTK), short neuropeptide F (sNPF), adipokinetic hormone (AKH), corazonin (CRZ) and Drosophila insulin-like peptides (DILPs) as possible regulators of metabolic stress responses and homeostasis. We showed that metabotropic GABAB receptors (GBRs) are expressed on brain insulin producing cells (IPCs), suggesting an inhibitory regulation of these cells by GABA. Knockdown of GBR on IPCs shortened lifespan and stress resistance, altered carbohydrate and lipid metabolism at stress (paper I). We showed that three different neuropeptides; DTK, sNPF and ITP, are co-expressed in five pairs of adult neurosecretory cells (paper II). ITP-knock down was not studied yet, but sNPF- and DTK-knock down flies showed decreased stress resistance at desiccation and starvation and decreased water levels at desiccation, suggesting that these peptides are involved in water homeostasis during stress conditions. sNPF was previously shown to affect feeding, growth and DILP expression via the IPCs, but it was not known which sNPF-expressing neurons are responsible for these actions. We could identify a specific set of bilateral neurons (DLPs) that co-express sNPF and corazonin that target the IPCs. We showed that these peptides co-released from DLPs regulate DILP transcription and probably release in the adult Drosophila brain and thus have roles in regulation of stress resistance and metabolism (paper III). AKH signaling was previously shown to affect hemolymph carbohydrate levels and lipid stores in Drosophila. Insulin (DILP) signaling and AKH signaling are suggested to have opposing effects on lipid and sugar metabolism in Drosophila. We studied the possible functional relationship between these two systems; do they mutually regulate each other? Our results suggest action of DILPs via the Insulin Receptor on the IPCs and the AKH producing cells, but we could not provide evidence for AKH action on IPCs or AKH cells (paper IV).

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TABLE OF CONTENTS List of papers 3 1. Introduction 4

1.1. Peptide Hormones in Regulation of Feeding, Metabolism and Stress 5 1.1.1. Insulin-like Peptides (ILPs) 6 1.1.2. Short Neuropeptide F (sNPF)-like Peptides and Head Peptides 7 1.1.3. Tachykinin Related Peptides (TRPs) 8 1.1.4. Ion Transport Peptide (ITP) 9 1.1.5. Corazonin (Crz) 9 1.1.6. Adipokinetic hormone (AKH) 10

2. Aims of the Thesis 11 3. Materials and Methods 12

3.1. GAL4/UAS Method 12 3.2. Functional Assays 15 3.3. Other Assays 15

4. Results 16 4.1. Paper I 16 4.2. Paper II 17 4.3. Paper III 18 4.4. Paper IV 20

5. Conclusions and Future Perspectives 20 6. References 22 7. Acknowledgements 31

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LIST OF PAPERS This thesis is based on the following papers and will be referred to by their Roman numerals. _____________________________________________________________________

I. Enell LE*., Kapan N*., Söderberg JAE., Kahsai L., Nässel DR., (2010). Insulin signaling, lifespan and stress resistance are modulated by metabotropic GABA receptors on insulin producing cells in the brain of Drosophila. PLoS ONE. Dec 30;5(12):e15780. PMID: 21209905. * Authors contributed equally.

II. Kahsai L., Kapan N., Dircksen H., Winther Å.M.E., Nässel DR., (2010). Metabolic stress responses in Drosophila are modulated by brain neurosecretory cells that produce multiple neuropeptides. PLoS One. Jul 8;5(7):e11480. PMID: 20628603.

III. Kapan N., Lushchak OV., Luo J., Nässel DR., (2012). Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin. Cellular and Molecular Life Sciences. July 2012, DOI: 10.1007/s00018-012-1097-z.

IV. Kapan N., Lushchak OV., Nässel DR., (2012). A search for reciprocal signaling between insulin and adipokinetic hormone producing cells in Drosophila. (Manuscript).

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1. INTRODUCTION Insect neuropeptides and peptide hormones are produced in less than 1% of the cells of the central nervous system, yet they are involved in regulation of fundamental physiological processes, such as growth, development, osmotic and metabolic homeostasis, reproduction, control of muscle activity and various behaviors (Gäde et al., 1997; Nässel and Winther, 2010). Therefore it is essential to examine the insect neuroendocrine system to understand regulation of these physiological processes.

Invertebrate endocrine systems consist of neurosecretory cells and classical endocrine glands (e.g. corpora cardiaca, corpora allata and prothoracic glands). Neurosecretory cells are present in all ganglia of the central nervous system and partly in the visceral nervous system. Due to their nervous and endocrine properties, neurosecretory cells can act directly on distant target cells or release hormones into the circulation via their axons terminating in storage and release sites (neurohaemal organs).

The corpora cardiaca (CC) are a pair of glandular lobes and neurohemal organs located on either side of the aorta, behind the brain. CC store and release neurohormones into the circulation, where they act at distant targets. In the CC intrinsic neurosecretory cells produce peptides called adipokinetic hormones (AKHs).

The corpora allata (CA) is located adjacent to the corpora cardiaca and intrinsic cells synthesize and release juvenile hormones that have major roles in metamorphosis, growth and development. The activity of the CA is known to be regulated by a variety of factors, including several different classes of peptide hormones (Weaver and Audsley, 2010). Many neuropeptides are shown to be present in nerves projecting from the brain into the corpora cardiaca-corpora allata complex, such as insulin-like peptides, tachykinins, FLRFamides and crustacean cardioactive peptide (CCAP) (Veelaert et al., 1998; Predel et al., 2004b; Wegener et al., 2006; 2010).

Brain lateral neurosecretory cells that produce neuropeptides including prothoracicotropic hormone, short neuropeptide F and corazonin, median neurosecretory cells that produce for instance insulin-like peptides and diuretic hormones are part of the insect endocrine system and have release sites in the CC/CA.

The publication of genome sequences of several insects (including the fruit fly Drosophila melanogaster, the honey bee Apis mellifera, the mosquito Anopheles gambiae) and nematode worms (e.g. Caenorhabditis elegans, Caenorhabditis briggsae) provided masses of genomic data on a number of peptides and their G protein-coupled receptors (GPCR) and other genes encoding proteins vital to hormonal and neuronal signaling. These findings resulted in an increasing number of studies using molecular genetics approaches for physiological analysis of neuropeptides that are known to regulate vital physiological events in insects. Knocking-down or ablating certain Drosophila genes encoding neuropeptide precursors or their receptors that are involved in various physiological processes and analyzing the resulting behavior and physiology helps us to understand the neuropeptide functions.

Most small peptide hormones are known to bind to GPCRs which have seven transmembrane domains. GPCRs constitute one of the largest superfamilies of proteins, and are ubiquitous throughout the Metazoa. Studies on the Drosophila genome so far have revealed the presence of more than 45 GPCRs that are likely to

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have peptides as their ligands. In Drosophila, these peptide receptors can be grouped into two families of GPCR; rhodopsin-like (Family A) and the secretin class (Family B). Many of these GPCRs have common ancestors with the mammalian peptide GPCRs in several cases, suggesting a highly conserved peptidergic signaling mechanism through evolution. 1.1 Peptide Hormones in Regulation of Feeding, Metabolism and Stress Control of food intake and energy expenditure is very complex and imbalances can cause diabetes, obesity and other serious health problems. In mammals hunger, satiety and metabolism is under complex regulation of various peptide hormones. These hormones signal in an interaction between the brain, endocrine organs, the intestine and adipose tissues. Caenorhabditis elegans and Drosophila melanogaster are two of the invertebrate model organisms that have been used most in analysis of peptidergic control of feeding and metabolism, as well as metabolic stress and longevity (Broughton et al., 2005; Baker and Thummel, 2007). Interestingly, in Drosophila many of the peptide hormones and their receptors involved in feeding and metabolism appear to be related to those in mammals. Among these are peptides which are very similar to insulin, glucagon, cholecystokinin, neuropeptide Y, that all play different roles in feeding, growth and metabolism. These neuropeptides may act at different levels of the nervous system and peripheral targets and we are interested in peptidergic links between the higher brain centers and behavioral outputs that regulate stress resistance and metabolism. Of these hormones, insulin-like peptides (DILP-2, 3 and 5), short Neuropeptide F (sNPF), a tachykinin-related peptide (DTK), corazonin (CRZ) and adipokinetic hormone (AKH) were primarily investigated in this study.

The insulin signaling pathway is conserved in all multicellular organisms (Garofalo, 2002; Wu and Brown, 2006; Teleman, 2010) and has regulatory roles on diverse physiological functions such as growth, development, reproduction, metabolic homeostasis, and lifespan (Brogiolo et al., 2001; Ikeya et al., 2002; Broughton et al., 2005). Ablation of Drosophila insulin-like peptide-producing cells (IPCs) in the brain resulted in increased lipid and carbohydrate levels, reduced fecundity, as well as an increase of lifespan and increased resistance to oxidative stress and starvation (Broughton et al., 2005). This clearly suggests a role for the Drosophila DILPs in regulation of major aspects of survival of the organism. Thus there is a need for further study of regulation of the IPCs and links between metabolic status and insulin release. So far there are no clear studies on the factors regulating the IPCs in Drosophila and Caenorhabditis elegans. In larval Drosophila nutritional sensing takes place in the fat body and this tissue signals to the IPCs (Colombani et al., 2003, Scott et al., 2004, Geminard et al., 2009) whereas in the adult fly glucose sensing is present in IPCs where ATP-sensitive potassium (KATP) channels are utilized (Fridell et al., 2009; Kreneisz et al., 2010).

Recent studies indicate that the IPCs in Drosophila brain may be additionally regulated by sNPF via its GPCR and GABA (α-aminobutyric acid) via metabotopic GABAB receptors (GABABR) (Lee et al., 2004, 2008; Paper I). A role has been indicated for GABA and the GABABR in regulation of insulin release in the pancreas of mammals (Bonaventura et al., 2008) and a recent study in this thesis has suggested a role of GABA in regulation of IPCs in Drosophila (Paper I). GABA is the major inhibitory neurotransmitter in most animals. In Drosophila, GABA is mainly utilized as a neurotransmitter by interneurons in the central nervous system. There are two types of GABA receptors: an ion channel (GABAA-R) and a GPCR (GABAB-

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R) for fast and slow transmission, respectively. GABAB-R immunolabeling showed that the receptor is mostly expressed in the optic lobes, mushroom body calyces and antennal lobes among the brain neuropils (Enell et al., 2007) and is expressed on IPCs in the brain (Paper I).

Another peptide hormone that was shown to have a regulatory role in the metabolic homeostasis is the functional analog of mammalian glucagon: adipokinetic hormone (AKH) (Kim and Rulifson, 2004; Lee and Park, 2004).

1.1.1. Insulin-like Peptides (ILPs) Insulin-like peptides (ILPs) are key hormones, not only in carbohydrate and lipid metabolism, growth and reproduction of Drosophila, but also in regulation of stress responses and life span. Diminished insulin signaling increases stress resistance and extends life span, at the cost of growth and fertility. Extensive studies have been made on insulin signaling in Drosophila, especially downstream of the insulin receptor (Brogiolo et al., 2001; Ikeya et al., 2002; Broughton et al., 2005; Zhang et al., 2000; Wu et al., 2005; Yu et al., 2009). However, the factors regulating production and release of insulin in insects still remain unclear. Also neuronal pathways that may regulate IPCs are unknown in Drosophila and other insects.

The insulin superfamily consists of vertebrate insulins, mammalian relaxin, insulin-like growth factors (IGF 1 and 2) and insulin-like peptides in invertebrates. In Drosophila there are 8 different ILPs (DILP-1 to 8) which are produced by neurons or neurosecretory cells of the CNS or by cells in other tissues such as fat body, midgut and renal tubules (REFs). The DILPs act in neuroendocrine signaling and play roles in the regulation of carbohydrate and lipid metabolism, growth and reproduction (Brogiolo et al., 2001; Grönke et al., 2010; Colombani et al., 2012; Garelli et al., 2012). In Drosophila, there are eight genes encoding these peptides (Brogiolo et al., 2001; Hewes and Taghert, 2001; Vanden Broeck, 2001; Colombani et al., 2012). The DILPs (DILP 2, 3 and 5) resemble mammalian insulins based on amino acid sequences, domain structures and spacing of cysteines, DILP 6 resembles insulin-like growth factors (IGFs) and DILP 7 resembles relaxin (Brogiolo et al., 2001; Miguel-Aliaga et al., 2008; Slaidina et al., 2009). DILP 8 is a protein of the insulin and relaxin type which was shown to coordinate Drosophila tissue growth by delaying the onset of metamorphosis (Colombani et al., 2012; Garelli et al., 2012; Hariharan 2012).

DILP 2, 3 and 5 are found in median neurosecretory cells of the protocerebrum, termed insulin producing cells, IPCs (Brogiolo et al., 2001; Broughton et al., 2005; Rulifson et al., 2002). In Drosophila, DILP 7 was shown to be expressed in neurons of the abdominal ganglia in third instar larvae and adults (Brogiolo et al., 2001; Miguel-Aliaga et al., 2008; Yang et al., 2008). DILP 6 was shown to be produced by fat body cells (Slaidina et al., 2009), DILP 8 was recently shown to be produced by imaginal discs (Colombani et al., 2012; Garelli et al., 2012), however DILP 1 and 4 expression has still not been shown.

The only insulin receptor (InR; CG18402), that has been identified so far in Drosophila and other insects (Fernandez et al., 1995; Brogiolo et al., 2001; Broughton and Partridge, 2009) share structural and functional similarities with the insulin tyrosine kinase receptors of vertebrates and other invertebrates (Fernandez et al., 1995; Brogiolo et al., 2001; Garofalo, 2002; Piper et al., 2008).

In several other invertebrates insulin was shown to play roles in regulation of carbohydrate and lipid metabolism, reproduction, development, growth, metabolism and stress resistance (Jonas et al., 1996; Floyd et al., 1999; Iwami, 2000; Guarente

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and Kenyon, 2000; Brogiolo et al., 2001; Tatar et al., 2001; Baker and Thummel, 2007; Giannakou and Partridge, 2007; Paper I).

To test the functional roles of these insulin peptides in Drosophila they were targeted for genetic knock down in numerous studies. DILP 2, 3 and 5 which are produced by the IPCs in the brain, have been extensively studied. A Dilp 2-knockdown in these cells resulted in an upregulation of DILP 3 and 5 levels (Brogiolo et al., 2001) which suggests a compensatory balance mechanism involving these three peptides (DILP 2, 3 and 5). Ablation of the IPCs (which will eliminate the production of DILP 2, 3 and 5), or inactivation of the insulin receptor (InR) lead to increased starvation resistance, defects in growth, elevated levels of circulating carbohydrates, increased lipid storage and extended life span (Brogiolo et al., 2001; Tatar et al., 2001; Rulifson et al., 2002; Broughton et al., 2005).

DILP 6 was shown to be related to IGF and has a role in growth (Slaidina et al., 2009). DILP 7 was suggested to have a role in egg-laying behavior, feeding and gut function (Yang et al., 2008; Cognigni et al 2012). DILP 8 was recently discovered to coordinates the growth status of tissues with developmental timing (Colombani et al., 2012). However DILP 1 and DILP 4 have no known functions yet. Thus there may be a complex set of control systems for differential release of the different DILPs in different cell systems. 1.1.2. Short Neuropeptide F (sNPF)-like peptides and head peptides Short Neuropeptide F (sNPF) probably has no ancestral relations to the Neuropeptide F (NPF)-like peptide family. On the other hand the NPF-like peptides (also called long NPF) show functional and structural similarities to the vertebrate neuropeptide Y (NPY)-like peptides (Brown et al., 1999; Nässel, 2002; Nässel and Wegener 2011). NPY-like peptides have a tyrosine (Y) amide C-terminus, whereas NPF-like peptides end with phenylalanine (F).

The name NPF (both long and short) for the insect peptides is derived from the methodology used to purify the neuropeptides using an antiserum to the tapeworm long NPF which resulted in isolation of the first sNPFs (Spittaels et al., 1996). The head peptides (also sNPF) were isolated from whole heads of the mosquito Aedes aegypti (Matsumoto et al., 1991). In Drosophila there are different genes that encode long (Brown et al., 1999) and short (Hewes and Taghert, 2001; Vanden Broeck, 2001a) NPF-like peptides. The similar names (sNPF and NPF) are unfortunate and we will not be concerned with the long forms in the following.

The sNPF family of peptides consists of three peptides from the mosquito Aedes aegypti (Aedes head peptides; Matsumoto et al., 1989; Veenstra, 1999), two peptides from the beetle Leptinotarsa decemlineata (Led-NPF-1 and -2; Spittaels et al., 1996), two peptides from midgut of the moth H. zea (Hez-MP-I and II; Huang et al., 1998), four predicted peptides from Drosophila (short NPF 1-4; Hewes and Taghert, 2001; Vanden Broeck, 2001a) and one peptide from the locust Schistocerca gregaria (Scg-NPF; De Loof et al., 2001; Schoofs et al., 2001) and multiple peptides that have been identified in sequenced insect genomes more recently.

A receptor (CG7395; NPFR76F or sNPFR1) that is expressed in the Drosophila CNS was shown to be activated by the Drosophila sNPFs, and displays slight sequence similarities to the vertebrate NPY receptors (Feng et al., 2003; Mertens et al., 2002). sNPF and its receptor are shown to be involved in the regulation of feeding, growth and insulin production in brain neurosecretory cells (Lee et al., 2004, 2008, 2009).

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sNPF is widely expressed in the larval and the adult Drosophila CNS (Lee et al., 2004; Nässel et al., 2008). In the adult CNS a large number of neurons express sNPF (Johard et al., 2008; Nässel et al., 2008). Based on the diverse neuronal expression of the peptide it is quite likely that sNPF function is highly diverse and dependent on which neuron type or circuit is utilizing the peptide (Nässel et al., 2008; Nässel and Winther, 2010).

The longer peptide NPF and its receptor are known to regulate larval feeding behavior (Wu et al., 2003), whereas sNPF is considered to, in addition to modulation of food intake, be involved in many other physiological processes, including olfaction, and locomotion (Root et al. 2011, Kahsai et al, 2010, Carlsson et al., 2010). This is based on the presence of sNPF in numerous neurons of different types throughout the CNS during all developmental stages (Lee et al., 2004; Nässel et al., 2008). In a feeding assay, using transgenic flies, it was shown that either downregulation or overexpression of the snpf gene had effects. Overexpression of the gene promoted food intake, whereas downregulation of sNPF levels suppressed it. The snpf over expression also produced bigger and heavier flies than in the wild-type (Lee et al., 2004). In a later study, production of DILP-2 was found to be upregulated after sNPF overexpression (Lee et al., 2008).

In this study only sNPF was studied due to its previously documented regulatory roles in Drosophila feeding, growth and insulin production (Lee et al., 2004, 2008, 2009). A main focus in this thesis was on the role of sNPF and its receptor in possible regulation of feeding/metabolism, water and ion balance and stress responses of Drosophila. 1.1.3. Tachykinin Related Peptides (TRPs) The invertebrate tachykinin-related peptide (TRP) family consists of various structurally related peptides (Nässel, 1999; Vanden Broeck et al., 1999). These TRPs were first isolated from Locusta migratoria and named locustatachykinins (LomTKs) due to the structural resemblance to the vertebrate tachykinins (Schoofs et al., 1990a; Nässel, 1999; Vanden Broeck et al., 1999).

The tachykinin family includes the mammalian peptides substance P, neurokinin A, neurokinin B, neuropeptide K and neuropeptide γ. The mammalian peptide substance P has roles in diverse physiological behaviors such as; sensory processing, motor control, pituitary hormone release, intestinal motility, vasodilation, depression, anxiety and stress (Otsuka and Yoshioka, 1993; Kramer et al., 1998; Murtra et al., 2000).

TRPs have been identified from several invertebrate species including insects, crustaceans and molluscs (Schoofs et al., 1990a, 1993a,b,c; Nässel, 1999; Vanden Broeck et al., 1999). Structural similarities between the mammalian peptide substance P and locust TRP (LomTK-I) (Nachman et al., 1999) and the fact that vertebrate tachykinins of the substance P subfamily (NK1 receptor agonists) activate invertebrate receptors (Li et al., 1991; Nässel, 1999), suggests that vertebrate tachykinins and invertebrate TRPs are ancestrally related.

In Drosophila the gene Tk (or dtk; CG14734) encodes six TRP isoforms, DTK-1–6 (Siviter et al., 2000). DTK-1–5, have been identified by mass spectrometry in the adult brain and the larval CNS (Baggerman et al., 2005; Winther et al., 2003). The sequence of DTK-6 was proposed recently (Poels et al., 2009).

Two G-protein-coupled receptors (NKD and DTKR) have been cloned from Drosophila that show similarities to mammalian tachykinin receptors (Li et al., 1991; Monnier et al., 1992). NKD receptor (CG6515) is shown to be activated by the DTK6

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only and DTKR (CG7887) is shown to be activated by DTK 1-6 (Birse et al., 2006; Poels et al., 2009).

In adult Drosophila, DTKs are distributed in about 150 interneurons in the brain, with processes in the central complex, optic lobes and the antennal lobe neuropil and there are also endocrine cells of the intestine expressing DTKs (Nässel, 1993b, 1999; Kim et al., 1998; Nässel, 1999 ; Vitzthum and Homberg, 1998; Winther et al., 2003). In the larval Drosophila CNS, DTKs are mainly expressed in interneurons (Winther et al., 2003; Park et al., 2008b).

Several functional roles have been attributed to TRPs based on in vivo and electrophysiological experiments: modulating the activity of locust interneurons, crayfish photoreceptors and motor circuits (Christie et al., 1997; Lundquist and Nässel, 1997; Nusbaum, 2002), playing roles in chemosensory information processing and central control of locomotion (Ignell et al., 2009; Winther et al., 2006; Kahsai et al. 2010). Also roles in regulation of secretion in renal tubules have been demonstrated in locusts and a moth (Johard et al., 2003; Skaer et al., 2002) and in Drosophila the DTKR is expressed in principal cells of the renal tubules and affect local insulin signaling in these cells (Söderberg et al. 2011).

1.1.4. Ion Transport Peptide (ITP) ITP was first identified in the corpora cardiaca of the locust Schistocerca gregaria and from other insects, including Drosophila later on (Audsley et al., 1992; Dai et al., 2007; Dircksen et al., 2008). In locust it was shown to have a role in stimulation of Cl- ion transport in the hindgut (Audsley et al., 1992).

The Drosophila gene that encodes ITP is similar to that of the locust ITPs and has two longer forms: ITPL1 and ITPL2. The ITP peptide has a sequence similarity to those of crustacean hyperglycemic hormones (CHHs) (Dircksen et al., 2001; Keller, 1992; Vanden Broeck, 2001; Dircksen et al., 2009).

The diverse distribution pattern of ITP in different types of neurons and peripheral cells suggests that the peptide has multiple functions. ITP was initially identified as regulating the electrochemical balance in locust hindgut, by stimulating the ileum to transport Cl- ions from lumen to hemolymph (Audsley et al., 1992; Dircksen, 2009). ITP‟s role in Drosophila is not known clearly yet, however a conserved antidiuretic activity is suggested. Also a role in the neuronal circuits of the biological clock has been suggested (Johard et al., 2009).

1.1.5. Corazonin (CRZ) Crz was originally isolated as a cardioactive factor in the cockroach, Periplaneta americana (Veenstra, 1989a). In later studies it has been shown that corazonin has a role in the regulation of the ecdysis behavior in Manduca sexta and is also expressed in presumptive circadian pacemaker neurons in this same insect (Wise et al., 2002; Kim et al., 2004).

In Drosophila, corazonin is expressed in twenty four larval neurons mapping to two different anatomical loci – a cluster of eight pairs along the ventral nerve cord and a set of three pairs of lateral neurosecretory cells (LNCs) with axons terminating in the corpora cardiaca and anterior aorta (Cantera et al., 1994; Siegmund and Korge, 2001; Choi et al., 2005, 2008; Lee et al., 2008).

The neuronal distribution patterns of corazonin may suggest that it is acting as a circulating hormone or as a neuromodulator due to its possible role in regulation of AKH release (Choi et al., 2008). Choi and colleagues showed that ablation of corazonin in LNCs resulted in a slight but statistically significant decrease in

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trehalose levels suggesting that either Crz modulates AKH-cell functions through neuronal pathways or that Crz released from the LNCs modulates trehalose levels by acting as a hormone on either fat cells or AKH cells.

The receptors for corazonin and AKH are ancestrally related to mammalian gonadotropin releasing hormone (GnRH) receptors and are presumptive arthropod homologs of GnRHs (Park et al., 2002; Cazzamali et al., 2002).

Figure 1. Arrangement of neurons and endocrine cells producing adipokinetic hormone (AKH), insulin-like peptides (DILP 2, 3 and 5), short neuropeptide F (sNPF) and corazonin in the brain and ring gland of the third instar larva of Drosophila. (Image is taken from Nässel and Winther, 2010).

1.1.6. Adipokinetic Hormone (AKH) AKH is an octomeric peptide and has a pyroglutamine at its N terminal. This peptide is both synthesized and released from intrinsic endocrine cells of the corpus cardiacum. AKHs constitute a family of related peptides with diverse functions in insects. The most important and well-studied function of AKHs is in the regulation of lipid recruitment from fat body for flight in locusts, increase of hemolymph trehalose levels in several insects, stimulation of heart beat frequency in cockroaches, inhibition of protein synthesis in locust and cricket, inhibition of fatty acid and RNA synthesis in fat body, activation of P4504Cl biosynthesis in locust fat body (Orchard, 1987; Gäde et al., 1997; Goldsworthy et al., 1997; Van der Horst et al., 2001; Nässel, 2002; Nässel and Winther, 2010). AKH is released in response to stimuli from prolonged flight and acts on the fat body which is the equivalent to the vertebrate liver and white adipose tissue (Nässel and Winther, 2010). AKH also acts on flight muscles to cause the mobilization and utilization of diglycerides once hemolymph carbohydrates are exhausted (Lee and Park, 2004; Isabel et al., 2005). The AKHs are thus called the endocrine regulators of energy store mobilization.

Within the arthropods the number and sequences of AKH peptides are quite diverse. There are three different known AKH members (AKH-I to III) which are colocalized in the glandular cells and have different bioactivities in Locusta migratoria (Vroemen et al., 1998) and in Drosophila only a single form of AKH peptide is known. It is suggested that for the migratory locust performing long-distance flights is in part possible because of this neuropeptide multiplicity, with AKH-I being the strongest lipid-mobilizing hormone, AKH-II the most efficient carbohydrate mobilizer and AKH-III being a modulator that provides the animal with energy at rest (Vroemen et al., 1998). Hansen et al. have identified a peptide in Aedes gambiae that has sequence similarities to both AKH and corazonin, thus naming it AKH/corazonin related peptide (Hansen et al., 2010).

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In Drosophila, the AKH receptor shows resemblance to vertebrate gonadotropin-releasing hormone receptors (Hauser et al. 1997; Staubli, 2002). The cellular distribution of the AKH receptor is yet to be studied, however fat body is thought to be the main tissue of AKH receptor expression in most insects (Goldsworthy et al. 1997).

AKH-producing and Insulin producing cells (IPCs) are presumed to communicate via their processes in the anterior aorta (Kim and Rulifson, 2004; Lee et al., 2004). Several reports (Kim and Rulifson, 2004; Lee et al., 2004; Baker and Thummel, 2007; Grönke et al., 2007; Buch et al., 2008) have shown evidence for a joint regulation of metabolic homeostasis by AKH and Insulin which is a topic that remains to be further studied in order to better understand the underlying mechanisms.

In a recent report, ablation of corazonin (Crz; see below) expressing cells in protocerebrum resulted in reduced trehalose levels suggesting that Crz may also have a role in modulation of AKH production or release (Lee et al., 2008).

It has been suggested, based on these roles of AKH, that this peptide is the arthropod functional equivalent of mammalian glucagon, however the identification of the AKH receptor suggests that this peptide is evolutionarily related to the Gonadotropin releasing hormones (GnRH) of mammals (Johnson, 2006).

AKH distribution in Drosophila is limited to a small group of glandular cells in the corpus cardiacum. Ablation of these cells resulted in hypotrehalosaemia under starvation conditions and changes in sugar metabolism which in turn led to increased survival (Isabel et al., 2005; Kim et al., 2004; Lee et al., 2004). Thus AKH acts to adjust the effects of starvation and is a major regulatory element in sugar homeostasis (Johnson, 2006).

AKH is also suggested to also have a role in the regulation of locomotor activity. Starved wild-type flies showed prolonged hyperactivity which is attributed to the fly‟s survival response (food-search behavior) to starvation. The AKH-ablated flies were devoid of this type of hyperactivity, which suggests a regulatory role for AKH in starvation-induced foraging behavior (Lee and Park, 2004; Isabel et al., 2005). 2. AIMS OF THE THESIS In Drosophila, insulin signaling has regulatory roles in major physiological functions such as life span, stress resistance, metabolic homeostasis, reproduction and growth. However neuronal pathways that may regulate IPCs as well as chemosensory links between metabolic status and insulin release are still unclear. The primary aim of my study is to understand regulation of peptide hormone release, the neuronal factors and circuits that regulate insulin signalling, metabolism and fly‟s response to nutritional stress conditions. In this thesis I have focused on DTK, sNPF, Crz, AKH neuropeptides and GABA and their role in insulin signalling, stress resistance and metabolism. Thus in general I wanted to study the roles of DTK, sNPF, Crz, AKH and GABA in the physiology of Drosophila. This was mainly performed by utilizing transgenic flies for knockdown or overexpression of the genes involved in DTK, sNPF, Crz, AKH and GABA signaling in specific neurons of interest in the adult Drosophila brain. In paper I, the research primarily focused on the role of GABA in the control of DILP production and release. We could show that GABABR is expressed on the IPCs and wanted to investigate the possible roles of GABABR on insulin signalling. In paper II, downstream actions and peripheral roles of DTK and sNPF were studied. We showed that a set of protocerebral cells in the adult Drosophila brain co-

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expresses three different peptides, namely ITP, DTK and sNPF. Of these three, we focused mainly on the hormonal roles of DTK and sNPF and their role in metabolic stress responses. In paper III, we identified a set of dorsolateral neurosecretory cells that co-express sNPF and Crz peptides and their axon terminations run adjacent to the IPCs. Previous studies showed that sNPF has a role in regulation of food intake, growth and Dilp transcript levels. However, sNPF is abundantly produced in the Drosophila brain and it was not known which set of cells specifically would be responsible for these actions. Crz was shown to have regulatory roles during nutritional stress conditions and since it is co-expressed with sNPF in same set of cells, we asked whether there is a dual regulation of insulin signalling by sNPF and Crz. In paper IV, we asked questions to study the possible relationship between the insulin signalling in the brain and the AKH signalling originating from the CC cells. These two systems in Drosophila are suggested to be reminiscent of the insulin/glucagon secretion in mammals and previous studies suggested a crosstalk between them. Thus, we attempted to manipulate levels of Insulin, AKH, InR and AKH-R in a reciprocal fashion to observe the resulting effects and their possible roles in regulation of metabolism. 3. MATERIALS AND METHODS Many peptide hormones have been identified utilizing traditional isolation and fractionation methods, Maldi-TOF mass spectroscopy or bioinformatic analysis of the genome. Mass spectrometry, in situ hybridization, immunocytochemical methods, as well as Gal4/UAS methods are used to explore the peptidergic signaling systems and the latter also for analysis of roles in behavior.

During this study, in particular Gal4/UAS and immunocytochemical methods and additional functional assays (see below) were employed to better understand the chemical signaling in the brain in regulation of metabolic stress, feeding and growth.

To provide such phenotypes the GAL4-UAS system was applied. By this method we were able to observe effects of the gain-of-function and loss-of-function constructs in different behaviors and physiology. 3.1. Gal4/UAS Method (Papers I, II, III and IV) This method is often used in studies of neuronal signaling. Gal4 is a protein (881 amino acids long) identified in the yeast Saccharomyces cerevisiae as a regulator of genes which is induced by galactose. The Gal4 protein binds to a sequence called Upstream Activating Sequence (UAS) element, analogous to an enhancer element defined in multicellular eukaryotes. The GAL4 system is a bipartite system in which one transgenic line, the driver, expresses GAL4 in a known temporal or spatial pattern and a second transgenic line, the responder, contains a UAS-dependent transgene (U). The responder (gene „U‟) is under the control of UAS sequence. In order to activate the transcription, flies carrying the UAS gene (U), need to be crossed to flies with GAL4 gene which will express in particular cell or tissue, then the resulting progeny will express the gene „U‟ (Duffy, 2002). The gene „U‟ can be selected to encode for proteins that are toxic (tetanus toxin), apoptotic (reaper) or for receptors or neuropeptides, or many other proteins. A very important use of UAS constructs is to have gene „U‟ producing double stranded RNA representing genes of interest. This, so called RNA interference (RNAi) can thus be targeted to knock down a gene of interest. Since it was shown that this system has no deleterious phenotypic

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effects (Brand and Perrimond, 1993), it has since been widely used for targeted gene expression in Drosophila and remains a powerful tool for in vivo genetic research.

UAS-RNAi and UAS-reaper constructs help to knock-down and ablate cells of interest respectively and UAS-GFP construct helps to visualize those cells using immunocytochemistry. Table 1. GAL4 strains that are used in this thesis

Gal4 line Description Reference Paper

Akh (II) Adipokinetic

hormone Lee and Park.,

2004 IV

Akh (III) Adipokinetic

hormone Lee and Park.,

2004 III, IV

Akh-R Adipokinetic

hormone receptor Bharucha et al.,

2008 IV

c929 Large peptidergic

neurons, endocrine cells

Hewes et al., 2003 II

Crz Corazonin Choi et al., 2008 III, IV

Dilp2 Insulin-like peptide

2 Wu et al., 2005 I, II, III, IV

Dilp3 Insulin-like peptide

3 Buch et al., 2008 I

GABABR2 Metabotropic

GABA receptor Root et al., 2008 I

Gad1 GABAergic

neurons Ng et al., 2002 I

Gad2b GABAergic

neurons Mehren and Griffith, 2006

I

Kurs6 Corpora

cardiaca/ring gland cells

Siegm and Korge, 2001

II

MB247 Intrinsic neurons of

the mushroom body

Aso et al., 2009 I

OK107 Mushroom body

and neurosecretory cells

Lee et al., 1999 I

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Rdl GABAAR subunit Kolodziejczyk et

al., 2008 I

sNPF Short neuropeptide

F Nässel et al., 2008 III

sNPFR1 Short neuropeptide

F receptor 1 Kahsai et al., 2012 III

Table 2. UAS strains that are used in this thesis

UAS line Description Reference Paper

Akh-R-RNAi Adipokinetic

hormone receptor Park et al., 2002 IV

Crz-RNAi Corazonin Veenstra, 1994 III

Crz-R-RNAi Corazonin receptor Park et al., 2002 III, IV

Dilp2 Insulin-like peptide

2 Wu et al., 2005 I

dOrk1 Drosophila open

rectifier K+ channel Nitabach et al.,

2002 III

Dtk-RNAi Drosophila tachykinin

Winther et al., 2006 II

GABABR2-RNAi Metabotropic

GABA receptor Root et al., 2008 I

InRCA Insulin receptor

constitutively active Wu et al., 2005 IV

InRDN Insulin receptor

dominant negative Wu et al., 2005 IV

InR-RNAi Insulin receptor Söderberg et al.,

2011 IV

Irk3-RNAi Inwardly rectifying

K+ channel Dietzl et al., 2007 I

mcd8-Gfp Green fluorescent

protein I, II, III, IV

Rdl-RNAi Ionotropic GABA receptor subunit

Liu et al., 2009 I

s65t-Gfp Green fluorescent

protein I, II, III, IV

sNPF Short neuropeptide

F Lee et al., 2004 III

sNPFc00448 Short neuropeptide

F Lee et al., 2008 III

sNPF-RNAi Short neuropeptide

F Lee et al., 2004 II, III

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3.2. Functional Assays All crossed experimental and parental flies were kept in a 25 °C incubator, with 12h light:12h dark cycle and controlled humidity. 3-6 day old male and female flies were tested. All experiments were done at least in triplicates, with 3 independent crosses. Survival at Starvation (Papers I, II, III and IV) In this assay we tested the stress resistance by starving flies. Flies received no food, but only water, while they were kept individually in 0.5% aqueous agarose filled glass vials (papers I, II and III) or in 50 ml vials in groups of 15 flies (paper IV). We monitored the number of dead flies over 12 hour periods. Survival at Desiccation (Papers I and II) Desiccation assays were in principal similar to the starvation assay, only with harsher conditions. The stress resistance of the flies were monitored under a no food, no water regime (dry starvation) and the number of dead flies were recorded around the clock. Longevity Assay (Paper I) In this assay the flies received fresh food every second day and number of dead flies was recorded every day, until the last fly had perished. Locomotor Activity Assay (Paper II) Locomotor activity of the flies was monitored at starvation by placing them individually in 5 mm diameter glass tubes filled with aqueous agarose. These tubes were placed in the Trikinetics activity monitor which recorded the locomotor activity of each fly by counting the number of crossings of an infrared beam in the middle of the tube every 15 minutes. 3.3. Other Assays Growth Rate Measurement (Paper I) Larvae, pupae and adult flies (male and female) were weighed on a microbalance in groups of 5 individuals after being anesthetized. Length of late pupae was measured with the help of a microscopic scale. Lipid Measurement (Papers I and II) Whole body lipid content of fed (0 hour) and starved (12 hours and 24 hours) flies was measured via ether extraction. Flies were weighed to obtain their wet weight and their dry weight was obtained after leaving them for 24h in 65°C. Finally flies were left in diethyl ether for 24h to obtain their lean dry weight. Lipid content was measured by subtracting the lean dry weight from the dry weight. Trehalose (Carbohydrate) Measurement (Paper I) Whole body carbohydrate content of fed (0 hour) and starved (5 hours and 12 hours) flies was measured in a set of steps described in Isabel et al., 2005. Carbohydrate Measurements (Papers III and IV) Concentrations of circulating glucose and trehalose together with whole body glycogen and trehalose of fed flies were measured via enzymatic reactions utilizing

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commercially available kits. Decapitated flies were centrifugated to collect hemolymph which was used to measure circulating glucose and trehalose, and wholebody to measure glycogen and stored trehalose (body trehalose). Lipid (Triacylglyceride-TAG) Measurements (Papers III and IV) Whole body lipid content of fed and starved (24 hours) flies was measured via enzymatic reaction. Flies were subjected to a set of steps including centrifugation and homogenization of the whole body to obtain supernatants for enzymatic reaction with a commercially available triacylglyceride kit. The results are shown as per cent change after starvation. Water Loss Measurement (Paper II) Fed (0 hour) and desiccated (16 hours – no food and no water condition) flies were weighed in groups of 5, at consecutive time points to obtain wet, dry and dead weights. The whole body water content was then calculated. Quantitative real-time PCR (qPCR) (Paper III) Amounts of Dilp2, 3 and 5 RNA were measured in Drosophila heads by qPCR as described in detail in paper III. 4. RESULTS 4.1. Paper I There are eight insulin-like peptides in Drosophila (DILP-1 to 8) of which three (DILP-2, 3 and 5) are produced by median neurosecretory cells in the brain. Ablation of these insulin producing cells (IPCs) in the Drosophila brain leads to increased stress resistance, increased lipid and carbohydrate levels, retarded growth and increased lifespan (Rulifson et al., 2002; Brougton et al., 2005).

The insulin signaling pathway is important in the regulation of physiological responses to nutritional stress, reproduction, growth and lifespan (Géminard et al., 2006; Baker and Thummel, 2007). The fat body, an insect organ that has the endocrine and storage functions of the vertebrate liver, has been shown to act as a nutrient sensor and to regulate organismal growth via acting on insulin signaling in response to changes in nutrient levels (Colombani et al., 2003). However less is known, so far, about the chemical and hormonal pathways regulating the insulin release mechanisms from the IPCs.

We investigated the possible regulatory role of GABA on IPCs. The metabotropic GABAB receptors (GBRs) are expressed on brain IPCs, whereas the ionotropic GABAA receptor subunit RDL was not expressed. This suggests that GABA has a role in inhibitory regulation of these IPCs via metabotropic receptors.

Initially we visualized the GABA receptor expression on IPCs by immunocytochemistry and GFP driven by GAL4 to localize the GABABR2 subunit of the GABAB receptor. Using an antiserum to DILP2, we could show that most of DILP immunolabeled cells also co-expressed GABAB R2-GFP.

By utilizing the Gal4/UAS method we knocked down the expression of GBRs in IPCs by RNAi (GBRi) and monitored the effects on life span, stress resistance and metabolism. To test the effect of GBR on longevity, we monitored normally fed flies (Dilp2-Gal4>UAS-GBRi) and observed that GBR-knock down flies had a significantly reduced lifespan compared to controls, although not drastic. This suggests that

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diminished levels of inhibitory GABA signaling led to more insulin release resulting in shorter lifespan.

Next we investigated the effects of GBR-knock down on survival at starvation and desiccation. Diminished insulin signaling is reported to elevate the metabolic stress resistance (Rulifson et al., 2002; Broughton et al., 2005). Two GAL4 lines (Dilp2-Gal4 and Dilp3-Gal4) which exclusively target DILP2 and DILP3 cells (= IPCs) were crossed with UAS-GBRi flies to knock down the expression of GBRs in those specific cells. Both crosses showed diminished survival at starvation compared to controls. The Dilp2-Gal4 driver demonstrated a stronger phenotype than the Dilp3-Gal4 driver. The Dilp2-Gal4>UAS-GBRi cross was also tested at desiccation which resulted in decreased resistance compared to its controls. The Dilp2-Gal4 line was also crossed to a second UAS-GBRi line and tested at starvation and desiccation which resulted in same phenotype as the first UAS-GBRi line. A third Gal4 (OK107-Gal4) line which includes most IPCs producing DILPs was tested at starvation and desiccation showing a strong reduction in survival. These three Gal4 drivers (Dilp2, Dilp3 and OK107) crossed to UAS-GBRi were also investigated to observe the effect on growth during development. Weight of late larvae and adult flies and length of pupae were monitored. GBR-knockdown did not result in any significant effect on growth, suggesting that the diminished GBR levels on IPCs do not affect growth. We also tested the effects of over expression of DILP2 in IPCs at starvation which resulted in strongly diminished resistance, as in the GBR-knock down flies supporting that increased DILP signaling shortens lifespan and that the GBR normally inhibits DILP release from the IPCs.

Activation of GABAB receptors (GBRs) results in generation of slow inhibitory postsynaptic potentials through the stimulation of G-protein-coupled inwardly rectifying K+ channels, GIRKs, which in turn induces a hyperpolarization (Mezler et al., 2001; Mitrovic et al., 2003). Assuming that GBRs have a downstream GIRK in IPCs, we knocked down a GIRK channel subunit: Irk3 in DILP2 cells. This led to strongly decreased survival at starvation compared to its controls, phenocopying the GBR-knock down flies.

In many Drosophila studies it has been shown that insulin-like peptides have a regulatory role in carbohydrate and lipid storage (Broughton et al., 2005; Baker and Thummel, 2007; Zhang et al., 2009). We investigated the whole body trehalose and lipid levels in normally fed and starved Dilp2-Gal4>UAS-GBRi flies at different time points. In knock-down flies, trehalose levels gradually but significantly diminished by time compared to the controls, whereas lipid levels drastically dropped after the first 12 hours of starvation. This might be due to the faster lipid mobilization in response to metabolic stress in GBR-knock down flies. 4.2. Paper II The peptide sNPF and its receptor are suggested to have modulatory roles in food intake, growth and insulin production (Schoofs et al., 2001; Lee et al., 2004, 2008; Nässel et al., 2008; Nässel and Winther 2010). Tachykinin-related peptides have been shown to have modulatory roles in AKH release in locusts (Nässel et al., 1995) and stimulation of secretion in Malphigian tubules of moths and locusts (Skaer et al., 2002; Johard et al., 2003, reviewed in Nässel 2002). ITP was initially identified as regulating the electrochemical balance and ion transport in locust hindgut (Audsley et al., 1992; Dircksen, 2009).

We identified three neuropeptides (sNPF, DTK and ITP) colocalized in five pairs of large protocerebral neurosecretory cells in two neuron clusters (ipc-1 and ipc-

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2) in the adult Drosophila brain. Both clusters have axonal terminations in neurohemal release sites including the corpora cardiaca which is also the release site for AKH and DILPs. DILPs and AKH have demonstrated roles in carbohydrate metabolism under starvation and metabolic stress conditions (Rulifson et al., 2002; Lee and Park, 2004; Isabel et al., 2005).

Based on previous findings and our current immunocytochemical findings we wanted to investigate the functional roles of these neurons that co-express three different neuropeptides. Thus we tested whether these neuropeptides were involved in metabolic stress response and in maintaining the metabolic and water homeostasis. For this purpose we employed two different enhancer trap Gal4 lines (C929 and Kurs 6) that are both expressed in the ipc-1 and ipc-2a neurons. The Gal4/UAS method was employed either by utilizing the GFP expression to visualize the cell expression or by utilizing RNA interference (RNAi) to knock down a peptide gene of interest and thereafter to observe the outcomes under stress conditions.

In the larval stage, only four pairs of large ITP immunoreactive neurosecretory cells are visualized (Dircksen et al., 2008) which do not co-express sNPF, DTK nor c929. We show that these peptides along with the transcription factor DIMMED (DIMM) can only be visualized in the ipc-1 and 2a neurons at the adult stage.

By targeting these neuronal clusters by UAS-dtk-RNAi and UAS-snpf-RNAi fly lines, we knocked down the DTK and sNPF expression levels in those cell groups. In these flies we monitored the stress resistance at starvation and desiccation. Peptide-knock down flies showed diminished life span when compared to the control flies under both stress conditions. The same flies were subjected to a water retention assay at desiccation. This experiment showed higher water loss in DTK-knock down flies compared to the controls, when exposed to desiccation.

In summary, this study shows that three diverse neuropeptides are colocalized in two groups of large neurosecretory cells, and that these directly or indirectly regulate the homeostasis at metabolic stress conditions especially during desiccation in the adult Drosophila brain. 4.3. Paper III In paper III, we have identified a bilateral set of peptidergic neurons (DLPs) in the lateral brain, that coexpress sNPF and Crz neuropeptides which play important roles in regulation of activity in the brain IPCs of Drosophila. This is the first time for demonstrating a specific group of sNPF expressing cells, that regulate insulin signaling although general sNPF-knockdown has been shown to do so (Lee et al., 2008b; Lee et al., 2004). We could also provide further evidence for Crz in regulation of stress responses previously proposed in different studies (Boerjan et al., 2010; Veenstra, 2009; Zhao et al., 2010).

Diminished levels of Crz and sNPF in the DLPs resulted in similar starvation resistance and metabolic phenotypes, leading to increased starvation resistance and increased levels of carbohydrates and lipid. Our data suggests that Crz and sNPF regulate stress resistance and metabolism by acting on the IPCs to increase production and release of DILPs. We could confirm the presence of sNPFR1 on adult IPCs but we could not verify the distribution of CrzR on the same cells. However when we knocked down CrzR in IPCs, we obtained same phenotypes in stress resistance and metabolic assays as in peptide knock downs. This strongly suggests that the receptor is expressed in these cells.

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In Drosophila DLPs also express receptors for the diuretic hormones DH31, DH44 and the allatostatin A receptor DAR-2 which is mainly expressed in crop, midgut and hindgut in both larvae and adults (Johnson et al., 2005; Veenstra, 2009b; Chintapalli et al., 2007). The DHs are known to stimulate epithelial fluid transport by acting on the principal cells of the renal tubules (Cabrero et al., 2002). Allatostatin A was shown to stimulate the release of amylase and protease enzymes in cockroaches (Fuse et al., 1999). Assumably, DHs and AstA are components of a signaling pathway which modulate the secretion of Crz from the DLPs into the hemolymph based on nutritional signals. We next expressed CrzR-RNAi in the fat body cells by utilizing Pumpless-Gal4 (ppl-Gal4). Here we observed increased stress resistance in the CrzR knockdown flies (Fig 2a). We could also show the expression of pumpless in the adult fatbody for the first time (Figure 2b, c). These results strongly suggest the presence of the CrzR in the fat body cells.

Figure 2. a Knockdown of the corazonin receptor in the fatbody cells increases resistance to starvation in flies. Knockdown of the corazonin receptor by CrzR-RNAi targeted to fat body cells (pplG4 > CrzR-RNAi) extends the life span in starving flies compared to controls (p

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4.4. Paper IV DILPS released from brain IPCs and AKH released from the corpora cardiaca (CC) cells are known to be crucial regulators of carbohydrate and lipid levels in Drosophila (Brogiolo et al., 2001; Lee et al., 2004; Isabel et al., 2005; Bharucha et al., 2008; Buch et al., 2008; Broughton and Partridge, 2009). We investigated the two cell types (IPCs and CC-cells) for expression of dInR and AKH-R by targeted RNAi. One question was whether CC-cells and IPCs signal to each other reciprocally and another question was whether those cells express auto-receptors.

By knocking down the InR on IPCs and CC-cells and by testing these flies for their resistance at starvation, we could indicate that insulins act on the IPCs and CC cells via the InR. We have also observed moderately increased carbohydrate levels when we knocked down InR levels on CC cells. This is suggestive of DILPs and the InR inhibiting AKH signaling from the CC cells. However we could not demonstrate the expression of the InR on theIPCs or CC cells immunocytochemically.

Although we could also show that diminished InR levels on IPCs lead to increased starvation resistance, these same genotypes, gave variable results when tested for their carbohydrate and TAG levels. We can suggest that the InR normally acts stimulatory on IPCs and is part of a positive DILP feedback system, possibly an autocrine feedback utilizing DILP3 released from the IPCs, as suggested by Grönke et al., 2010.

In summary, in this study we could show some evidence for InR activity in IPCs and AKH-producing CC-cells, but we could not demonstrate the action of AKH receptor on IPCs or on CC cells. 5. CONCLUSIONS AND FUTURE PERSPECTIVES In Paper I, we investigated the regulatory role of GABA, the GABAB receptor and a downstream element (K+-channel) in the activity of IPCs in the adult Drosophila brain. In our study we showed that brain median neurosecretory cells that produce insulin-like peptides express the metabotropic GABAB receptors (GBRs) and in fact these GBRs are involved in inhibitory regulation of insulin signaling in the Drosophila brain. In line with this finding, we also detected an increase in DILP production, however we did not observe any effects of GBR-knock down on growth. This latter contradicting result might be because GABA‟s inhibitory regulation role via GBRs on IPCs is more pronounced at metabolic stress conditions. Indeed, we observed a stronger reduction in lifespan in GBR-knock down flies under stress.

In Paper II, we showed that three different neuropeptides: DTK, sNPF and ITP colocalize in five pairs of large protocerebral neurosecretory cells in the adult Drosophila brain. Individual knock-down of sNPF and DTK resulted in decreased stress resistance at starvation and desiccation and increased water loss at desiccation, which suggests a role for these peptides in antidiuresis and metabolic stress responses. The locomotor activity of the DTK-knock down flies was not altered at starvation in comparison to the control flies, which suggests that the foraging behaviour is not affected by diminished DTK signaling in ipc-1 and ipc-2a cell clusters.

In light of previous research and based on our findings we can presume that ITP, DTK and sNPF work in conjunction to maintain the water/ion balance in order to increase survival at stress conditions. Neither the hormonal roles of sNPF and DTK nor the receptor distribution sites of ITP, DTK and sNPF are clearly known yet, thus it

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would be interesting to investigate the targets of the neurons producing these peptides. Our data does not support a direct link between the ipc1 and 2a cells and the IPCs or AKH producing cells. Possibly other sNPF expressing neurons (maybe other LNCs) could target AKH- or insulin-producing cells.

Indeed, in paper III we investigated a set of dorsolateral neurons (DLPs), co-expressing sNPF and Crz that target the IPCs. We showed that knocking down peptide levels in DLPs, increased stress resistance. This finding seems to contradict the result of paper II. However the DLPs and ipc1 and 2 neurons probably use sNPF to act on different targets; the DLPs target IPCs and stimulate insulin signaling whereas ipc neurons may act hormonally on peripheral targets such as the hindgut. This shows that the same neuropeptide (sNPF) originating from different cell clusters may act differently and affect metabolism antagonistic to one another.

In paper III we show that diminished levels sNPF and Crz peptides, as well as CrzR in DLPs result in increased stress resistance and metabolic component levels. We could confirm the previously demonstrated presence of sNPFR1 in IPCs, but not the presence of markers for CrzR. Corazonin expressing cells also express diuretic hormone DH31, DH44 receptors and the allatostatin A receptor DAR-2 (Johnson et al., 2005; Veenstra, 2009b; Chintapalli et al., 2007). It is suggested that DHs and AstA are components of a signaling pathway that modulates the secretion of Crz from the DLPs to cope with nutritional stress.

In paper IV, we searched for a possible feedback mechanism between Insulin signaling and AKH signaling systems in Drosophila. Previous studies suggest a crosstalk between IPCs and the AKH producing CC cells, therefore we attempted to manipulate those set of cells and their respective receptors in search of a reciprocal signaling system between those cell types. We showed that there is a communication between the IPCs and the CC cells via the InR but we could not find evidence for AKH-R action on the IPCs.

It seems we still know relatively little about regulation of the insulin signaling pathway and its many players due to its complexity. It is of great interest to further study the peptidergic and hormonal links that are involved in regulation of production and release of DILPs, to better understand the many physiological processes under DILP regulation. By utilizing molecular genetics, immunocytochemical and functional assays we studied Drosophila metabolic stress resistance and homeostasis mechanisms which are under complex regulation of neuropeptides and neurotransmitters which may act in conjunction as well as individually. In summary, we mainly studied the possible roles of DTK, sNPF, Crz and AKH on metabolic/osmotic stress responses and GABA acting on ILP production in the brain IPCs during regulation of homeostasis at metabolic stress. Our results suggest that DTK, sNPF and Crz have regulatory roles in the homeostatis during metabolic stress conditions. We also showed the first evidence for a direct neuronal inhibitory control of IPC signaling in Drosophila by GABA via its metabotropic receptor, and identified for the first time a specific set of bilateral sNPF expressing neurons that stimulate DILP production/release.

For the future it would be interesting to identify further neurons and downstream elements that regulate AKH and DILP release. Also the study of hormonal corazonin action on the CrzR in the fat body would help us to better understand the regulation of metabolism in the fly and how the fly copes with stress.

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