What controls release of ipCs from cns neurons and glial cells?

In feeding stages of Drosophila a key trigger of DILP release from IPCs appears to be intake of nutrition. The post-feeding increase in circulating glucose and amino acid levels is sensed by the fat body and as a consequence signals are released into the circulation and reach the brain and the IPCs. This was first studied in larvae where nutrient sensing occurs in the fat body and a fat body-secreted factor activates DILP release (Geminard et al., 2009). The fat body nutritional sensor is the amino acid transporter slimfast, that acts on the TOR (target of rapamycin) pathway (Colombani et al., 2003). In the adult fly a similar mechanism has been proposed, and the fat body-derived factor identified as a leptin-like molecule, unpaired 2 (Upd2), that indirectly activates the IPCs by lifting a tonic inhibition from specific GABAergic neurons (Rajan and Perrimon, 2012). This will be dealt with in more detail below. The IPCs do not seem to display clear cell-autonomous nutrient sensitivity, in contrast to the larval secretory cells producing the glucagon-like adipokinetic hormone (AKH) (Kim and Rulifson, 2004). Some evidence, however, exists that the adult IPCs have autonomous glucose sensing by means of ATP-sensitive potassium (K^ATP) channels similar to mammalian pancreatic beta cells (Kreneisz et al., 2010), but no coupling to regulation of DILP release was made. Another membrane-associated channel has been implicated in adult IPCs in regulation of DILP signaling. This is the calcium-activated potassium channel Slowpoke (SLO) that is known to be regulated by a binding partner, the SLO-binding protein (SLOB) (Sheldon et al., 2011). Both SLO and SLOB are expressed by the IPCs, and when their expression is diminished in these cells, Dilp3 transcript level decreases and energy is stored, and DILP signaling is changed. It was proposed that SLO and SLOB modulate action potential duration in the IPCs and thus have a possible role in release of DILPs (Sheldon et al., 2011).

A novel nutrient sensor was reveled recently: the fructose sensitive gustatory receptor Gr43a (Miyamoto et al., 2012). In addition to its expression in sensory cells of the proboscis, Gr43a was found in four pairs of neurons in the region of lateral neurosecretory cells of the brain. The Gr43a expressing brain neurons can sense circulating fructose and were shown to regulate food intake in a satiety-dependent manner (Miyamoto et al., 2012). Possibly the Gr43a-expressing neurons interact with neurons in the lateral neurosecretory cell group that in turn affect feeding circuits or maybe insulin signaling via IPCs.

In mammals release of insulin is modulated by several hormones and neurotransmitters, as well as feed-back from circulating insulin (Adeghate et al., 2001; Aspinwall et al., 1999; Dong et al., 2006; Drucker et al., 1987; Sonoda et al., 2008). Similarly, the Drosophila IPCs appear to be regulated by neurons producing neuropeptides, monoamines and GABA and receive DILP feedback. Roles of short neuropeptide F (sNPF), corazonin Drosophila tachykinin, GABA, serotonin and octopamine in IPC regulation are summarized in the following.

A role of sNPF has been proposed in regulation of feeding, growth and insulin production (Lee et al., 2008b; Lee et al., 2004). Those studies did not clarify which sNPF expressing neurons that are responsible for the regulation of IPCs since they were based on more global knockdown of sNPF and interference with the sNPFR1 in the IPCs. In the larva there are two candidate systems of sNPF neurons: (1) the two DP neurons of the first abdominal ganglion that express DILP7 and sNPF (Miguel-Aliaga et al., 2008; Nässel et al., 2008), and (2) a set of lateral neurons (DLPs; among the lateral neurosecretory cells) that send processes to the presumed dendrite region of the IPCs and to the corpora cardiaca region of the ring gland where the IPC axons terminate (Nässel et al., 2008). In the adult fly the sNPF expressing DLPs coexpress the neuropeptide corazonin and their axons project to the IPC branches in pars intercerebralis, tritocerebrum and corpora cardiaca and could act on the IPCs (Kapan et al., 2012). Indeed it was shown by targeted RNA interference that both sNPF and corazonin from the DLPs stimulate the IPCs and thereby affect carbohydrate and lipid levels (Kapan et al., 2012). After sNPF knockdown in DLPs the transcript levels of Dilp2 and 5 decrease, whereas corazonin-RNAi in the same cells does not affect Dilp transcription. Thus the two peptides co-released from DLPs act in different ways on the IPCs, and corazonin additionally appears to be released into the hemolymph to act on the fat body (Kapan et al., 2012)(unpublished results). The DLPs have been shown to express receptors to allatostatin A and diuretic hormones 31 and 44 (Johnson et al., 2005; Veenstra, 2009). The former two peptides are produced by endocrine cells of the midgut (Veenstra et al., 2008) and may possibly be released into the circulation at feeding to target the DLPs. If this is the case, then the midgut endocrines may act as nutrient sensors or monitor gut distension at feeding. It can also be noted here that the fructose sensing Gr43a-expressing neurons in the area of LNCs might be in a position to act on the DLPs (Miyamoto et al., 2012).

The next neurotransmitter to be proposed in the regulation of IPCs was serotonin. Serotonergic neurons were found to express Drosophila NS3, a nucleostemin family GTPase (Kaplan et al., 2008). NS3 manipulations affect growth in Drosophila via insulin signaling (Kaplan et al., 2008). When an ns3 mutant was rescued by expressing ns3 in serotonergic neurons the growth defects were rescued. These authors suggested that NS3 acts in serotonergic neurons in regulation of insulin signaling and thereby control of organismal growth (Kaplan et al., 2008). The direct connection between serotonergic neurons and IPCs was, however, not revealed in the study. We therefore screened for serotonin receptors on IPCs and found only evidence for 5-HT_1A being expressed in these cells (Luo et al., 2012).

Targeted knock-down of the 5-HT_1A receptor in IPCs produced phenotypes that suggest an effect on insulin signaling (Luo et al., 2012). Diminished receptor levels decreased survival of flies at starvation and altered lipid levels, but the responses to temperature stress were the opposite of what was expected for a presumed action of an inhibitory 5-HT_1A receptor. The 5-HT_1A knockdown flies displayed decreased survival at 39 °C and required longer to recover from cold coma at 0°C (Luo et al., 2012). This suggested that the receptor normally has a stimulatory effect on insulin signaling, since an earlier study had shown that decreased insulin signaling by means of IPC ablation led to decreased tolerance to heat and cold treatment (Broughton et al., 2005). We speculate that the 5-HT_1A receptor is indeed inhibitory and inhibits adenylate cyclase (AC) and protein kinase A (PKA) and subsequently inactivates cAMP response element binding protein (CREB) [see (Nichols and Nichols, 2008)]. Activated CREB is known to inhibit insulin signaling (Walkiewicz and Stern, 2009), and therefore inhibition of AC, PKA and CREB would stimulate insulin signaling. The adverse effect of 5-HT_1A knockdown on survival at starvation and on lipid levels could be caused by an increase in locomotor activity as a response to starvation (Lee and Park, 2004), where more energy is consumed and thus overrides the insulin-mediated effect on starvation resistance.

The inhibitory neurotransmitter GABA acts via ionotropic or metabotropic receptors, but only the metabotropic GABA_B receptor (GBR) was detected on the Drosophila IPCs (Enell et al., 2010). Targeted knockdown of the GBR in IPCs resulted in phenotypes indicating that its role is to inhibit production and/or release of DILPs. Flies with diminished GBR lived longer than controls, displayed increased starvation resistance and altered carbohydrate and lipid metabolism (Enell et al., 2010). The ionotropic GABA_A receptor subunit RDL was not found on the IPCs, although it is otherwise very widespread. This study indicated that GABA inhibits the production and/or release of DILPs in the IPCs via its metabotropic receptor, but it is was not shown what triggers GABA signaling to the IPCs. The regulation of GABAergic neurons acting on the IPCs was, however, shown in a later study. It was found that the leptin-like Upd2 (related to type 1 cytokinins) is released from the fat body after feeding and acts on its receptor Dome that is expressed on GABAergic neurons adjacent to the IPCs (Rajan and Perrimon, 2012). Upd2 activates JAK/STAT signaling in the Dome-expressing GABAergic neurons and thereby lifts the tonic inhibition of the IPCs and allows DILP release. Thus a nutrient-triggered signal from the fat body acts indirectly via GABAergic neurons to induce systemic DILP signaling. This model requires that Upd2 passes through the blood-brain barrier to be able to act on GABAergic neurons in the brain.

Another modulatory control of IPCs is by means of products of the Drosophila tachykinin (DTK) precursor gene (Dtk or Tk). Six DTK peptides have been identified, five of which are expressed in the CNS (Siviter et al., 2000; Winther et al., 2003). We found that one of the two known DTK receptors, DTKR, is expressed on IPCs and that knockdown of this receptor affects the IPCs (Birse et al., 2011). Diminishment of DTKR expression on IPCs results in increased lifespan at starvation, and a more rapid decrease of whole body trehalose, but has no effect on lipid levels. These results suggest that also DTKR inhibits insulin signaling from the IPCs. An important finding in this context was that DTKR knockdown in IPCs affects Dilp transcript levels in brains of fed and starved flies. It could be shown that only Dilp2 and Dilp3, but not Dilp5 transcripts were affected by receptor knockdown (Birse et al., 2011). After DTKR-RNAi the Dilp2 and 3 transcripts both increased in fed flies, whereas after 24h starvation the Dilp3 transcript decreased and Dilp2 increased. This suggests that the DTKR activation induces transcriptional effects that are differential for the three Dilp genes. Similar individual regulation of Dilp transcription in the brain IPCs has been shown in multiple other experiments, for instance after nutritional restriction, manipulations of JNK signaling or SLO/SLOB expression in IPCs and NS3 in serotonergic neurons and by manipulation of sNPF in specific neurons (Broughton et al., 2008; Broughton et al., 2010; Kapan et al., 2012; Kaplan et al., 2008; Karpac et al., 2009; Sheldon et al., 2011). We will return to the possible role of differential transcriptional control of Dilps in the concluding section.

A further neurotransmitter/neuromodulator that has been suggested to modulate IPC activity is octopamine (Crocker et al., 2010). It was shown that the IPCs express one of the octopamine receptors, OAMB and knockdown of this receptor altered sleep-wake patterns in the flies. Stimulation of the IPCs with octopamine increased cAMP in these neurons and the wake-promoting effect of octopamine appears to be dependent on PKA activation in the IPCs (Crocker et al., 2010). These authors, however, did not provide any evidence that octopaminergic activation of the IPCs affects systemic insulin signaling.

Finally there is indirect evidence that various DILPs may provide feedback onto the brain IPCs. DILP7 .. DILP6 (Bai et al., 2012). DILP3 (Grönke et al., 2010). Relationship between DILPs (IPCs) and AKH (CC)… see (Buch et al., 2008).