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Ion transport peptide: Biological Overview | References

Animals need to continuously adjust their water metabolism to the internal and external conditions. Homeostasis of body fluids thus requires tight regulation of water intake and excretion, and a balance between ingestion of water and solid food. This study investigated how these processes are coordinated in Drosophila melanogaster. The first thirst-promoting and anti-diuretic hormone of Drosophila was identified, encoded by the gene Ion transport peptide (ITP). This endocrine regulator belongs to the CHH (crustacean hyperglycemic hormone) family of peptide hormones. Using genetic gain- and loss-of-function experiments, this study showed that ITP signaling acts analogous to the human vasopressin and renin-angiotensin systems; expression of ITP is elevated by dehydration of the fly, and the peptide increases thirst while repressing excretion, promoting thus conservation of water resources. ITP responds to both osmotic and desiccation stress, and dysregulation of ITP signaling compromises the fly's ability to cope with these stressors. In addition to the regulation of thirst and excretion, ITP also suppresses food intake. Altogether, this work identifies ITP as an important endocrine regulator of thirst and excretion, which integrates water homeostasis with feeding of Drosophila (Galikova, 2018).

Maintenance of homeostasis is based on ingestion and metabolism of water and nutrients in a manner that reflects the internal needs of the animal, but the precise regulatory mechanisms are incompletely understood. Despite the strong evolutionary conservation of the main pathways underlying energy homeostasis, there is a considerable diversity in the strategies involved in the maintenance of water balance. In insects, this variability arises mainly from the diversity of their habitats and life history strategies. For example, some blood-sucking insects are able to ingest a blood meal that exceeds their body volume up to twelve-fold; their feeding is hence coupled to massive post-prandial diuresis of the excessive water and ions. However, in most of the non-blood sucking terrestrial insects, water conservation is more important than water secretion. Studies on water balance in insects have historically focused mainly on the hormonal regulation of water excretion. These studies investigated the correlations between the hormone titers and diuresis, and analyzed the effects of injections or in vitro applications of the tested compounds. These works contributed to a better understanding of water regulation at the level of fluid secretion by the Malpighian tubules and water reabsorption in the hindgut. Development of genetic tools for Drosophila has allowed analysis of diuretic hormones by direct genetic manipulations. However, no anti-diuretic hormone has been identified in Drosophila until now (Galikova, 2018).

Drosophila is under laboratory conditions raised on media that provide both nutrients and water, and flies therefore do not regulate food and water intake independently. Nevertheless, insects, including Drosophila, can sense water (Cameron, 2010; Chen, 2010) and exhibit hygrotactic behavior (Ji, 2015; Enjin, 2016). If given the opportunity, flies differentiate between food and water sources, and are able to seek and drink free water, or ingest media rich in water but devoid of nutrients. Recently, a small group of neurons were identified in the Drosophila brain that antagonistically regulate thirst and hunger (Jourjine, 2016). These neurons sense osmolarity cell-autonomously with the cation channel Nanchung, and internal nutrients indirectly via Adipokinetic hormone signaling (Jourjine, 2016). Although several hormones have been shown to regulate feeding and satiety, no endocrine regulator of thirst has been identified in Drosophila so far (Galikova, 2018).

The mechanisms that orchestrate water sensing, water-seeking behavior and conservation of water remain unclear. It is hypothesized that these processes are likely coordinated by endocrine signaling. Physiological roles of Drosophila hormones are mostly well characterized; one of the few exceptions is Ion transport peptide (ITP), which belongs to the family of crustacean hyperglycemic hormones (CHH). CHHs promote water reuptake and hence, act as an anti-diuretic hormones in crustaceans. The locust homolog of ITP promotes water reabsorption by acting on chloride channels in the hindgut. Drosophila has a single ITP gene that gives rise to an amidated ITP hormone and to two longer forms called ITP-like peptides (Dircksen, 2009; Dircksen, 2008). The functions of Drosophila ITP have not been investigated so far, except for a study that has shown a role of ITP in modulation of evening activity by the circadian clock circuitry (Hermann-Luibl, 2014). The findings from the crustacean (Chung, 1999) and locust (Phillips, 1996) members of the CHH family suggest that Drosophila ITP might be involved in the regulation of water balance as well. This study tested this hypothesis by investigating the effects of gain- and loss-of-function of ITP on key aspects of water homeostasis, such as body water content, desiccation and osmotic stress resistance, food and water intake, and excretion. This work identified master regulatory roles of ITP in water homeostasis of Drosophila; ITP levels increase under desiccation stress and protect the fly from water loss by increasing thirst, reducing excretion rate, and promoting ingestion of water instead of food. Altogether, this work identifies the first anti-diuretic and drinking-promoting hormone in Drosophila, which also coordinates water balance with feeding behavior (Galikova, 2018).

With the colonization of dry land and evolution of terrestrial life, conservation, rather than elimination of water became the main challenge for the maintenance of water homeostasis. Despite the differences in the organization of the endocrine systems, the main principles of fluid homeostasis are the same in vertebrates and invertebrates; these include thirst, compensation for the feeding-induced increase in osmolarity by water intake, and water re-absorption by the excretory systems. In humans, water homeostasis is regulated primarily by an osmostat located in the hypothalamus. This osmostat increases water levels by triggering thirst, and reduces the water loss by inducing release of the anti-diuretic hormone vasopressin. In addition to the regulation by osmolarity, thirst is also induced by the changes in the blood volume both via vasopressin and the renin-angiotensin system. Even though thirst and water retention are physiologically coupled, their regulation occurs independently. This study shows that these regulations are simplified in Drosophila, where the same hormone promotes thirst, reduces appetite, and increases water storage. Thus, ITP acts as a functional analog of both vasopressin and renin-angiotensin. Interestingly, like the vasopressin and renin-angiotensin system, also ITP is regulated by body water content (Galikova, 2018).

Over-expression of ITP increases water content by 4.5%, whereas RNAi dehydrates the fly by 3.3%. The physiological consequences of such mild changes of water levels are not known in Drosophila, but for comparison, in human patients, loss of as little as 2% water significantly impairs cognitive abilities, and liquid overload and hypervolemia represent harmful conditions as well (Galikova, 2018).

The current findings show that knockdown of ITP leads to increased water excretion similar to human disorders caused by defective water re-absorbance in kidney, such as diabetes insipidus. Conversely, ITP over-expression results in increased water retention reminiscent of the human syndrome of inappropriate anti-diuretic hormone secretion (SIADH). ITP manipulations may thus become useful tools to induce and study pathologies associated with these human disorders in Drosophila (Galikova, 2018).

ITP is the first identified hormone that regulates drinking in Drosophila. Thus, it acts as a functional analog of the renin-angiotensin system of mammals. Similar to the renin-angiotensin system, ITP is most likely activated by hypovolemia. The neural circuits that control drinking and are regulated by ITP, however, remain to be investigated. Neurons that repress drinking in Drosophila have already been identified in the suboesophageal zone (Jourjine, 2016). These neurons are regulated cell autonomously by an ion channel that senses osmolarity (Jourjine, 2016). ITP-knockdown flies do not have the drive to drink despite their state of dehydration, whereas ITP over-expressing flies drink despite their excessive water content. Thus, unlike the Nanchung-expressing repressors of drinking (Jourjine, 2016), the ITP-regulated neurons are not regulated by the volume of body water, but rather by ITP itself (Galikova, 2018).

In insects, primary urine is produced by the Malpighian tubules that are functional analogs of mammalian kidneys. Water enters the lumen of these tubules by passive diffusion along the ionic gradient maintained by the vacuolar V-H+-ATPase. The function of the Malpighian tubules is hormonally regulated by diuretic hormones, which in Drosophila include products of the genes capa (Terhzaz, 2015; Davies, 2013), DH31 (Coast, 2001), DH44 and leucokinin (Cannell, 2016). Urine then enters the hindgut, where it mixes with the gut contents. Importantly, considerable parts of the water and ions are subsequently re-absorbed in the ileum and rectum. This study shows that ITP reduces excretion of water by reducing the defection rate. Thus, it is likely that Drosophila ITP promotes water reabsorption in the hindgut similar to its homologs in the desert locust Schistocerca gregaria or in the European green crab Carcinus maenas. It is noteworthy that ITP-expressing neurons in the abdominal ganglia innervate Drosophila hindgut, suggesting that in addition to the hormonal regulation, the hindgut may also be regulated by ITP in a paracrine fashion. In crabs and in the red flour beetle Tribolium castaneum¸ CHH- or ITP-producing endocrine cells, respectively, have even been detected in gut epithelia. Thus, whether produced in the neurosecretory cells or in the endocrine cells of the gut, the actions of CHHs and ITPs on the hindgut appear to be evolutionarily conserved (Galikova, 2018).

In mammals, an increase in osmolarity due to food intake results in postprandial thirst, and conversely, dehydration inhibits feeding when water is not available and this is likely also the case in Drosophila. The current findings of the ITP-driven positive regulation of water intake, concomitant with a negative regulation of feeding likely represents another level of regulation of thirst and hunger, acting in parallel to that of the four drink-repressing neurons in the suboesophageal zone (Galikova, 2018).

Whereas many terrestrial arthropods frequently experience arid conditions, salt stress is not very common in non-blood feeding terrestrial insects. Nevertheless, desiccation and salt stress resistance have been traditional tests in the studies of Drosophila diuretic hormones. RNAi against diuretic hormones increases desiccation resistance, as shown for capa, DH44 and leucokinin genes. However, it remains unclear whether these hormones contribute to the natural response to the desiccation and osmotic stress. For example, desiccation does not change expression of diuretic hormones DH44 and leucokinin. In contrast, ITP seems to be a natural component of the desiccation and osmotic stress responses, since both stressors trigger an increase in ITP expression. The role of ITP in thirst, hunger and excretion suggest that the ITP-regulated changes in behavior and physiology represent natural responses to cope with the reduction of body water. Consistently, knockdown of ITP reduces survival under desiccation and osmotic stress. However, it is unclear why over-expression of ITP reduces resistance to desiccation and osmotic stress. The UAS-GAL4 based manipulations may increase ITP levels far beyond the physiological range, which (although not lethal under standard feeding) might reduce survival under stressful conditions. Given the role of ITP in the ion transport across the hindgut epithelia of locusts, it is tempting to speculate that a similar mechanism exists in Drosophila. In such a scenario, the non-physiological doses of ITP might considerably increase osmolarity of hemolymph. This would be toxic when feeding on a food medium with a high salt content, as well as under desiccation conditions (which further increase osmolarity) (Galikova, 2018).

Although ITP has been known for a long time, its function has remained enigmatic in Drosophila. Pioneering work on its roles in Drosophila physiology suggests that ITP codes for a master regulator of water balance, which also integrates the water homeostasis with energy metabolism. Thus, this study not only shows that this member of the CHH family has an evolutionarily conserved anti-diuretic role in Drosophila as it has in other arthropods, but also reveals novel functions of this peptide family in food and water intake. It remains to be investigated to what extent these roles are conserved in other insect species or even in crustaceans, but the strong evolutionary conservation of the gene structure suggests that this might be the case. It is possible that the fly ITP regulates, in addition to its role in water balance, other processes that are known to be CHH-regulated in crustaceans. For example, the high developmental lethality of ITP RNAi, together with the previously described lethality of ITP mutants imply that Drosophila ITP plays a critical role during development, perhaps analogous to the role of CHHs in crustacean molting (Galikova, 2018).

Although identification of the cellular sources of ITP that are responsible for the functions of this hormone was beyond the scope of this manuscript, the expression pattern of the gene already provides some tempting hints. Previous in situ-hybridizations and immunohistochemistry experiments based on a locust anti-ITP antibody showed that Drosophila ITP is expressed in several neuronal types. Using an antibody specific to Drosophila ITP, this study confirmed that these cells include ipc-1 and ipc-2a neurosecretory neurons in the brain, ipc-3 and ipc-4 interneurons, three pairs of iag cells in the abdominal ganglia, and the LBD neurons in abdominal segments A7 and A8. Although ITP is expressed in several interneurons, the most prominent cells of the brain that express ITP are the neurosecretory protocerebral ipc-1 and the ipc-2a neurons, which send axons towards neurohemal release sites in the corpora cardiaca, corpora allata, and aorta. Experiments based on the Impl2 driver showed that a proper response to desiccation and osmotic stress requires production of ITP in the ipc-1 neurons, ipc-2a neurons, or LBD neurons, or in their combination. The ITP production in these cells becomes nevertheless critical only under desiccation and osmotic stress. In contrast to the global manipulations, ITPi targeted to these neurons is not sufficient to impair water balance under standard conditions. Thus, water content is regulated either via ITP produced by cells outside of the Impl2 expression pattern, or the ITP-producing neurons are redundant in their ability to produce sufficient ITP to maintain water homeostasis under standard conditions. Altogether, additional cell type-specific manipulations are required to differentiate whether thirst, excretion and food intake are regulated by specific neurons, or whether different ITP-producing neurosecretory cells act redundantly to produce sufficient amount of the hormone to regulate physiology of the fly (Galikova, 2018).

Another key step towards understanding the ITP actions is the identification of the hitherto unknown Drosophila ITP receptor. This will facilitate cell- and tissue-specific manipulations to unravel the neural circuit(s) responsible for the roles of ITP in the control of thirst and hunger, and allow more detailed studies of the peripheral roles of ITP in defecation and water excretion (Galikova, 2018).

The ion transport peptide is a new functional clock neuropeptide in the fruit fly Drosophila melanogaster

The clock network of Drosophila melanogaster expresses various neuropeptides, but a function in clock-mediated behavioral control was so far only found for the neuropeptide Pigment dispersing factor (PDF). This paper proposes a role in the control of behavioral rhythms for the Ion transport peptide (ITP), which is expressed in the fifth small ventral lateral neuron, one dorsal lateral neuron, and in only a few nonclock cells in the brain. Immunocytochemical analyses revealed that ITP, like PDF, is most probably released in a rhythmic manner at projection terminals in the dorsal protocerebrum. This rhythm continues under constant dark conditions, indicating that ITP release is clock controlled. ITP expression is reduced in the hypomorph mutant Clk(AR), suggesting that ITP expression is regulated by CLOCK. Using a genetically encoded RNAi construct, ITP was knocked down in the two clock cells, and these flies were found to show reduced evening activity and increased nocturnal activity. Overexpression of ITP with two independent timeless-GAL4 lines completely disrupted behavioral rhythms, but only slightly dampened PER cycling in important pacemaker neurons, suggesting a role for ITP in clock output pathways rather than in the communication within the clock network. Simultaneous knockdown (KD) of ITP and PDF made the flies hyperactive and almost completely arrhythmic under constant conditions. Under light-dark conditions, the double-KD combined the behavioral characteristics of the single-KD flies. In addition, it reduced the flies' sleep. It is concluded that ITP and PDF are the clock's main output signals that cooperate in controlling the flies' activity rhythms (Hermann-Luibl, 2014).

Ion transport peptide splice forms in central and peripheral neurons throughout postembryogenesis of Drosophila melanogaster

Ion transport peptides (ITPs) belong to a large arthropod neuropeptide family including crustacean hyperglycaemic hormones and are antidiuretic hormones in locusts. Because long and short ITP isoforms are generated by alternative splicing from a single gene in locusts and moths, this study investigated whether similarly spliced gene products occur in the nervous system of Drosophila melanogaster throughout postembryogenesis. The itp gene CG13586 was reanalyzed, and three instead of the two previously annotated alternatively spliced mRNAs were found. These give rise to three different neuropeptides, two long C-terminally carboxylated isoforms (DrmITPL1 and DrmITPL2, both 87 amino acids) and one short amidated DrmITP (73 amino acids), which were partially identified biochemically. Immunocytochemistry and in situ hybridization reveal nine larval and 14 adult identified neurons: four pars lateralis neurosecretory neurons, three hindgut-innervating neurons in abdominal ganglia, and a stage-specific number of interneurons and peripheral bipolar neurons. The neurosecretory neurons persist throughout postembryogenesis, form release sites in corpora cardiaca, and invade corpora allata. One type of ITP-expressing interneuron exists only in the larval and prepupal subesophageal ganglia, whereas three types of interneurons in the adult brain arise in late pupae and invade circumscribed neuropils in superior median and lateral brain areas. One peripheral bipolar and putative sensory neuron type occurs in the larval, pupal, and adult preterminal abdominal segments. Although the neurosecretory neurons may release DrmITP and DrmITPL2 into the haemolymph, possible physiological roles of the hindgut-innervating and peripheral neurons as well as the interneurons are yet to be identified (Dircksen, 2008).

Search PubMed for articles about Drosophila Ion transport peptide

Cameron, P., Hiroi, M., Ngai, J. and Scott, K. (2010). The molecular basis for water taste in Drosophila. Nature 465(7294): 91-95. PubMed ID: 20364123

Cannell, E., Dornan, A. J., Halberg, K. A., Terhzaz, S., Dow, J. A. T. and Davies, S. A. (2016). The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster. Peptides 80: 96-107. PubMed ID: 26896569

Chen, Z., Wang, Q. and Wang, Z. (2010). The amiloride-sensitive epithelial Na+ channel PPK28 is essential for drosophila gustatory water reception. J Neurosci 30(18): 6247-6252. PubMed ID: 20445050

Chung, J. S., Dircksen, H. and Webster, S. G. (1999). A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas. Proc Natl Acad Sci U S A 96(23): 13103-13107. PubMed ID: 10557280

Coast, G. M., Webster, S. G., Schegg, K. M., Tobe, S. S. and Schooley, D. A. (2001). The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 204(Pt 10): 1795-1804. PubMed ID: 11316500

Davies, S. A., Cabrero, P., Povsic, M., Johnston, N. R., Terhzaz, S. and Dow, J. A. (2013). Signaling by Drosophila capa neuropeptides. Gen Comp Endocrinol 188: 60-66. PubMed ID: 23557645

Dircksen, H. (2009). Insect ion transport peptides are derived from alternatively spliced genes and differentially expressed in the central and peripheral nervous system. J Exp Biol 212(Pt 3): 401-412. PubMed ID: 19151215

Dircksen, H., Tesfai, L. K., Albus, C. and Nassel, D. R. (2008). Ion transport peptide splice forms in central and peripheral neurons throughout postembryogenesis of Drosophila melanogaster. J Comp Neurol 509(1): 23-41. PubMed ID: 18418898

Dircksen, H., Tesfai, L. K., Albus, C. and Nassel, D. R. (2008). Ion transport peptide splice forms in central and peripheral neurons throughout postembryogenesis of Drosophila melanogaster. J Comp Neurol 509(1): 23-41. PubMed ID: 18418898

Enjin, A., Zaharieva, E. E., Frank, D. D., Mansourian, S., Suh, G. S., Gallio, M. and Stensmyr, M. C. (2016). Humidity Sensing in Drosophila. Curr Biol 26(10): 1352-1358. PubMed ID: 27161501

Galikova, M., Dircksen, H. and Nassel, D. R. (2018). The thirsty fly: Ion transport peptide (ITP) is a novel endocrine regulator of water homeostasis in Drosophila. PLoS Genet 14(8): e1007618. PubMed ID: 30138334

Hermann-Luibl, C., Yoshii, T., Senthilan, P. R., Dircksen, H. and Helfrich-Forster, C. (2014). The ion transport peptide is a new functional clock neuropeptide in the fruit fly Drosophila melanogaster. J Neurosci 34(29): 9522-9536. PubMed ID: 25031396

Ji, F. and Zhu, Y. (2015). A novel assay reveals hygrotactic behavior in Drosophila. PLoS One 10(3): e0119162. PubMed ID: 25738801

Jourjine, N., Mullaney, B. C., Mann, K. and Scott, K. (2016). Coupled Sensing of Hunger and Thirst Signals Balances Sugar and Water Consumption. Cell 166(4): 855-866. PubMed ID: 27477513

Phillips, J. E., Wiens, C., Audsley, N., Jeffs, L., Bilgen, T. and Meredith, J. (1996). Nature and control of chloride transport in insect absorptive epithelia. J Exp Zool 275(4): 292-299. PubMed ID: 8759926

Terhzaz, S., Teets, N. M., Cabrero, P., Henderson, L., Ritchie, M. G., Nachman, R. J., Dow, J. A., Denlinger, D. L. and Davies, S. A. (2015). Insect capa neuropeptides impact desiccation and cold tolerance. Proc Natl Acad Sci U S A 112(9): 2882-2887. PubMed ID: 25730885

date revised: 26 November 2018

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