InteractiveFly: GeneBrief

Adipokinetic hormone: Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References


Gene name - Adipokinetic hormone

Synonyms - dAkh

Cytological map position - 64A7--8

Function - ligand

Keywords - hormones, regulation of blood sugar and lipid, regulation of starvation-induced foraging behavior

Symbol - Akh

FlyBase ID: FBgn0004552

Genetic map position -

Classification - adipokinetic hormone family

Cellular location - secreted



NCBI links: Precomputed BLAST | Entrez Gene | UniGene

Recent literature
Gáliková, M., Diesner, M., Klepsatel, P., Hehlert, P., Xu, Y., Bickmeyer, I., Predel, R. and Kühnlein, R.P. (2015). Energy homeostasis control in Drosophila Adipokinetic hormone mutants. Genetics [Epub ahead of print]. PubMed ID: 26275422
Summary:
Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas Adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based in AKH injections and correlative studies in a broad range of insect species have established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone - termed Adipokinetic hormone precursor related peptide (APRP) - is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. This study employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, the study provides evidence for a negative auto-regulatory loop, in Akh gene regulation.

Galikova, M., Diesner, M., Klepsatel, P., Hehlert, P., Xu, Y., Bickmeyer, I., Predel, R. and Kuhnlein, R. P. (2015). Energy homeostasis control in Drosophila adipokinetic hormone mutants. Genetics [Epub ahead of print]. PubMed ID: 26275422
Summary:
Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas Adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based in AKH injections and correlative studies in a broad range of insect species established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone - termed Adipokinetic hormone precursor related peptide (APRP) - is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. This study employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, evidence is provided for a negative auto-regulatory loop, in Akh gene regulation.

Zemanova, M., Staskova, T. and Kodrik, D. (2016). Role of adipokinetic hormone and adenosine in the anti-stress response in Drosophila melanogaster. J Insect Physiol 91-92: 39-47. PubMed ID: 27374982
Summary:
The role of adipokinetic hormone (AKH) and adenosine in the anti-stress response was studied in Drosophila melanogaster larvae and adults carrying a mutation in the Akh gene (Akh1), the adenosine receptor gene (AdoR1), or in both of these genes (Akh1 AdoR1 double mutant). Stress was induced by starvation or by the addition of an oxidative stressor paraquat (PQ) to food. Mortality tests revealed that the AAkh1 mutant was the most resistant to starvation, while the AdoR1 mutant was the most sensitive. Conversely, the Akh1 AdoR1 double mutant was more sensitive to PQ toxicity than either of the single mutants. Administration of PQ significantly increased the Drome-AKH level in w1118 and AdoR1 larvae; however, this was not accompanied by a simultaneous increase in Akh gene expression. In contrast, PQ significantly increased the expression of the glutathione S-transferase D1 (GstD1) gene. The presence of both a functional adenosine receptor and AKH seem to be important for the proper control of GstD1 gene expression under oxidative stress, however, the latter appears to play more dominant role. On the other hand, differences in glutathione S-transferase (GST) activity among the strains, and between untreated and PQ-treated groups were minimal. In addition, the glutathione level was significantly lower in all untreated AKH- or AdoR-deficient mutant flies as compared with the untreated control w1118 flies and further declined following treatment with PQ. All oxidative stress characteristics modified by mutations in Akh gene were restored or even improved by 'rescue' mutation in flies which ectopically express Akh. Thus, the results demonstrate the important roles of AKH and adenosine in the anti-stress response elicited by PQ in a Drosophila model, and provide the first evidence for the involvement of adenosine in the anti-oxidative stress response in insects.
Laranjeira, A., Schulz, J. and Dotti, C. G. (2016). Genes related to fatty acid beta-oxidation play a role in the functional decline of the Drosophila brain with age. PLoS One 11: e0161143. PubMed ID: 27518101
Summary:
In living organisms, ageing is widely considered to be the result of a multifaceted process consisting of the progressive accumulation of damage over time, having implications both in terms of function and survival. The study of ageing presents several challenges, from the different mechanisms implicated to the great diversity of systems affected over time. The current study set out to identify genes involved in the functional decline of the brain with age and study its relevance in a tissue dependent manner using Drosophila melanogaster as a model system. The age-dependent upregulation is reported of genes involved in the metabolic process of fatty acid beta-oxidation in the nervous tissue of female wild-type flies. Downregulation of CG10814, dHNF4 and lipid mobilizing genes bmm and dAkh rescues the functional decline of the brain with age, both at the cellular and behaviour level, while over-expression worsens performance. The data proposes the occurrence of a metabolic alteration in the fly brain with age, whereby the process of beta-oxidation of fatty acids experiences a genetic gain-of-function. This event proved to be one of the main causes contributing to the functional decline of the brain with age.
Song, W., Cheng, D., Hong, S., Sappe, B., Hu, Y., Wei, N., Zhu, C., O'Connor, M. B., Pissios, P. and Perrimon, N. (2017). Midgut-derived Activin regulates glucagon-like action in the fat body and glycemic control. Cell Metab 25(2): 386-399. PubMed ID: 28178568
Summary:
While high-caloric diet impairs insulin response to cause hyperglycemia, whether and how counter-regulatory hormones are modulated by high-caloric diet is largely unknown. This study found that enhanced response of Drosophila adipokinetic hormone (AKH, the glucagon homolog) in the fat body is essential for hyperglycemia associated with a chronic high-sugar diet. The activin type I receptor Baboon (Babo) autonomously increases AKH signaling without affecting insulin signaling in the fat body via, at least, increase of Akh receptor (AkhR) expression. Further, it was demonstrated that Activin-β (Acβ), an activin ligand predominantly produced in the enteroendocrine cells (EEs) of the midgut, is upregulated by chronic high-sugar diet and signals through Babo to promote AKH action in the fat body, leading to hyperglycemia. Importantly, activin signaling in mouse primary hepatocytes also increases glucagon response and glucagon-induced glucose production, indicating a conserved role for activin in enhancing AKH/glucagon signaling and glycemic control.
Solari, P., Rivelli, N., De Rose, F., Picciau, L., Murru, L., Stoffolano, J. G., Jr. and Liscia, A. (2017). Opposite effects of 5-HT/AKH and octopamine on the crop contractions in adult Drosophila melanogaster: Evidence of a double brain-gut serotonergic circuitry. PLoS One 12(3): e0174172. PubMed ID: 28334024
Summary:
In adult Drosophila melanogaster, the type of sugar - either present within the crop lumen or in the bathing solution of the crop - has no effect on crop muscle contraction. What is important, however, is the volume within the crop lumen. Electrophysiological recordings demonstrated that exogenous applications of serotonin on crop muscles increases both the amplitude and the frequency of crop contraction rate, while adipokinetic hormone mainly enhances the crop contraction frequency. Conversely, octopamine virtually silenced the overall crop activity. The present study reports an analysis of serotonin effects along the gut-brain axis in adult D. melanogaster. Injection of serotonin into the brain between the interocellar area shows that brain applications of serotonin decrease the frequency of crop activity. Based on these results, it is proposed that there are two different, opposite pathways for crop motility control governed by serotonin: excitatory when added in the abdomen (i.e., directly bathing the crop) and inhibitory when supplied within the brain (i.e., by injection). The results point to a double brain-gut serotonergic circuitry suggesting that not only the brain can affect gut functions, but the gut can also affect the central nervous system.
Zhao, X. and Karpac, J. (2017). Muscle directs diurnal energy homeostasis through a Myokine-dependent hormone module in Drosophila. Curr Biol 27(13): 1941-1955 e1946. PubMed ID: 28669758
Summary:
Inter-tissue communication is critical to control organismal energy homeostasis in response to temporal changes in feeding and activity or external challenges. Muscle is emerging as a key mediator of this homeostatic control through consumption of lipids, carbohydrates, and amino acids, as well as governing systemic signaling networks. However, it remains less clear how energy substrate usage tissues, such as muscle, communicate with energy substrate storage tissues in order to adapt with diurnal changes in energy supply and demand. Using Drosophila, this study shows that muscle plays a crucial physiological role in promoting systemic synthesis and accumulation of lipids in fat storage tissues, which subsequently impacts diurnal changes in circulating lipid levels. The data reveal that the metabolic transcription factor Foxo governs expression of the cytokine unpaired 2 (Upd2) in skeletal muscle, which acts as a myokine to control glucagon-like adipokinetic hormone (AKH) secretion from specialized neuroendocrine cells. Circulating AKH levels in turn regulate lipid homeostasis in fat body/adipose and the intestine. The data also reveal that this novel myokine-dependent hormone module is critical to maintain diurnal rhythms in circulating lipids. This tissue crosstalk provides a putative mechanism that allows muscle to integrate autonomous energy demand with systemic energy storage and turnover. Together, these findings reveal a diurnal inter-tissue signaling network between muscle and fat storage tissues that constitutes an ancestral mechanism governing systemic energy homeostasis.

BIOLOGICAL OVERVIEW

Adipokinetic hormones (AKHs) are metabolic neuropeptides, mediating mobilization of energy substrates from the fat body in many insects. In delving into the roles of the Drosophila Adipokinetic hormone-like (dAkh) gene, its developmental expression patterns were examined and the physiological functions of the AKH-producing neurons were investigated using animals devoid of AKH neurons and ones with ectopically expressing dAkh. The dAkh gene is expressed exclusively in the corpora cardiaca (a portion of the ring gland) from late embryonic to adult stages. Projections emanating from the AKH neurons indicate that AKH has multiple target tissues as follows: the prothoracic gland and aorta in the larva and the crop and brain in the adult. Studies using transgenic manipulations of the dAkh gene have demonstrated that AKH induces both hypertrehalosemia and hyperlipemia. Starved wild-type flies display prolonged hyperactivity prior to death; this novel behavioral pattern is associated with food-searching activities in response to starvation. In contrast, flies devoid of AKH neurons not only lack this type of hyperactivity, but also display strong resistance to starvation-induced death. From these findings, another role for AKH in the regulation of starvation-induced foraging behavior is proposed (Lee, 2004).

Homeostatic regulation of blood sugar levels is a fundamental physiological process in both vertebrates and invertebrates. Failure to do so causes serious health problems such as diabetes in humans. In mammals, two important endocrine hormones, glucagon and insulin, are key physiological effectors that regulate blood glucose levels. These peptide hormones are synthesized by the endocrine glands in the pancreas and released into the bloodstream in response to internal changes in sugar levels. In target tissues, such as the liver, these pancreatic hormones activate opposing metabolic pathways (e.g., glycogen breakdown by glucagon and glycogen synthesis by insulin), thereby maintaining steady-state glucose levels (Lee, 2004).

Fundamental endocrine regulations of homeostatic blood sugar levels are also conserved in insects. For instance, an insulin-related peptide, bombyxin, lowers hemolymph sugar concentrations in a dose-dependent manner in the silkworm Bombyx mori, and transgenic ablation of dilp-producing neurons results in the elevation of total blood sugar (Lee, 2004 and references therein).

Insects also produce peptide hormones that act as functional homologs of vertebrate glucagons (Van Der Horst, 2001). Injection of the peptides into cockroaches elevates levels of hemolymph trehalose, a nonreducing disaccharide that is one of the major blood sugar molecules in insects. Thus the glucagon-like peptide in insects is referred to as hypertrehalosemic hormone (HTH). However, injection of this peptide into locusts elicits both carbohydrate and lipid mobilization from the fat body, leading to the alternative name adipokinetic hormone (AKH). These peptide hormones form the largest neuropeptide family in arthropods, including >30 isoforms identified in >80 species encompassing all major insect phyla and several crustacean species (Lee, 2004 and references therein).

Like other neuropeptides, AKHs are multifunctional. Other known physiological effects observed for this substance include cardioacceleration in cockroaches and migration of tegumentary and retinal distal pigments in crustaceans. AKH also induces transcription of the cytochrome P450 gene in the fat body of cockroaches, and expression of a gene encoding fatty acid binding protein in the flight muscle of locusts. In addition, AKH peptides have excitatory effects on motor neurons in moths, and enhance amplitudes of the electroretinogram in the crayfish (Lee, 2004 and references therein).

Despite the physiological studies just described, biological functions of the AKH-encoding gene are unknown, in part due to the lack of genetic variants involving this substance. Drosophila AKH peptide and its encoding gene sequences have been reported (Schaffer, 1990; Noyes, 1995). To gain insight into in vivo roles of AKH in Drosophila, anatomical details of AKH-expressing (AKHergic) neurons were examined in various developmental stages. Targeted ablation to obtain AKH-cell-deficient (AKH-CD) flies and ectopic dAkh expression were carried out, followed by analyses of physiological and behavioral phenotypes resulting from these transgenic manipulations. The results show that AKH functions as a metabolic stimulator causing both hypertrehalosemia and hyperlipemia. These data also suggest that AKH is involved in the regulation of starvation-induced locomotor activities, and such roles are likely to be associated with AKH's metabolic roles to maximize the likelihood of the fly's survival when foods are scarce (Lee, 2004).

These studies have explored developmental regulation of Drosophila Akh gene expression, its essential roles in energy metabolism, and function associated with starvation-induced feeding behavior. Unlike in other insect species, larval CC of Drosophila and other cyclorraphous dipterans are fused to other endocrine glands, forming a ring-like structure called the ring gland. Using dAkh as a marker gene for the CC, detailed neuro-anatomical aspects of the CC in Drosophila were described. (1) Most (if not all) of the CC cells are AKH-positive; therefore, characteristics of AKHergic neurons represent overall morphology of the CC at least in larvae. There are ~7 AKHergic cells in each larval CC lobe and 13 such cells in the entire adult CC. The latter count (of adult AKHergic cells) agrees with an electron microscopic observation, which estimated ~12 intrinsic cells in the CC of Drosophila adults. (2) The adult CC also form bilobed structure (analogous to the larval version of this organ). The lobes are closely associated with each other, so that they often appear to be a single mass of tissue. (3) Larval AKHergic neurons send projections into the aorta, where AKH is likely to be released into the circulatory system to reach distantly located target tissues (e.g., fat body). In addition, projections were found invading the prothoracic gland, which is the source of a molting hormone ecdysteroid. Thus, it is tempting to speculate that AKH has a role in metamorphosing processes. However, since AKH cell deficient larvae and pupae molt in a normal fashion, the neurological roles of the projections just described are unknown. (4) Adult AKHergic neurons project to the brain and the crop. These potential targets are likely to be associated with metabolism/feeding-related roles of AKH (Lee, 2004).

It has been well documented that members of the AKH family play a pivotal role in the stimulation of intermediary metabolism in the fat body of various insects (Van Der Horst, 2001). For instance, in locusts, AKH-mediated lipid and carbohydrate mobilizations from the fat body provide energy substrates for the flight muscles. In the horse fly (Tabanus atratus), injection of AKH causes hyperlipemia but not hypertrehalosemia (Jaffe, 1989), and in the blow fly (Phormia terraenovae), it causes hypertrehalosemia, but not the other (Gade, 1990). By comparison, genetic data show that AKH induces both hyperlipemia and hypertrehalosemia in Drosophila. Perhaps the fruit flies may need (as do locusts) a combination of carbohydrates and lipids as energy sources for a variety of energy-requiring conditions such as starvation, flight, and other locomotor activities (Lee, 2004).

Insect AKH is apparently a functional homolog of vertebrate glucagon. Recently, Drosophila insulin-like peptide (dilp) has been shown to produce a physiological activity opposite to AKH with respect to carbohydrate metabolism (Rulifson, 2002). These studies combined with results from this study suggest that hormonal regulatory mechanisms for homeostatic carbohydrate metabolism are conserved between Drosophila and vertebrates. Of interest, nerve fibers from the dilp neurons project to the AKHergic neurons, implicating intercellular interactions between these cell types. If in fact this is true, it will be interesting to determine whether these peptidergic neurons regulate each other, so that only one type of peptide is dominantly produced under a certain physiological circumstance. Exploiting cellular and molecular mechanisms involved in sensing hemolymph sugar titers is another avenue of inquiry prompted by the results presented (Lee, 2004).

Although AKH-mediated carbohydrate metabolism in the fat body is the principal cause of hyperglycemia in some insects, studies done in hymenopteran insects have proposed another mechanism of hyperglycemia caused by this peptide. Lorenz (1999) reported that workers of bumblebees, honeybees, and Vespula vulgaris store most carbohydrates in the crop and essentially lack fat body storage for carbohydrates. Despite this, injection of AKH into well-fed animals (whose crops were presumably full) still elicited significant hyperglycemia, whereas no such effect was found in the animals with empty crops (Lorenz, 2001). The results suggest that the crop is a principal carbohydrate storage organ in certain insects and that AKH induces hyperglycemia perhaps by stimulating crop-emptying activity. In line with this, potential innervation of the crop by AKHergic neurons indicates that the crop could be another source of AKH-dependent hyperglycemia in Drosophila. AKH may modulate crop muscle contractions, squeezing out sugar-containing fluid into the midgut from which sugar molecules are transported into the hemocoel through the gut epithelium (Lee, 2004).

When foods are abundant, wild-type flies show robust daily activity-rest rhythms that are governed by a circadian pacemaker system. However, the clock system fails to control normal rhythmicity when animals are stressed by adverse environmental conditions. Prolonged hyperactivities displayed by starved wild-type flies prior to death could be a desperate attempt to acquire food that would be the key to their survival. Food is not always available in nature; thus, this kind of accentuated locomotion, regardless of the time of day, might be an important behavioral component for the survival of hungry animals. This theory is supported by evidence that food availability is an important environmental factor that controls animals' circadian behavior (reviewed in Stephan, 2002; Lee, 2004).

Intuitively, persistent hyperactive behavior may augment the likelihood of starvation-induced death, since this would facilitate rapid consumption of energy resources. Conversely, suppression of such behavior may help animals to survive longer during periods of starvation. This is what is observed in AKH cell deficient flies, which not only lacked hyperactive locomotion, but also survive ~24 hr longer than wild type under starvation condition. Assuming that average life spans for humans and flies, under normal living conditions, are 70 years and 45 days, respectively, 24 hr of fly life is equivalent to ~570 days of that in humans. By comparison, timings of starvation-induced death of AKH ectopic expression flies did not deviate from those of wild type, perhaps because AKH ectopic expression flies displayed wild-type-like hyperactivity patterns. From these data, it is speculated that prolonged hyperactive locomotion is causally associated with starvation-induced lethality (Lee, 2004).

On the basis of these findings, it is proposed that AKH acts in two ways to regulate separate phenotypes in Drosophila; in one way, AKH stimulates intermediary metabolism in the fat body, leading to hypertrehalosemia and hyperlipemia. In the other way, AKH may carry out a central function involving hyperactive behavior in response to starvation. Apparently the central brain controls the fly's locomotor activities, because lack of pacemaker neurons or 'behavioral output factor' (PDF peptide) normally possessed by such cells disrupts circadian activity rhythms. The fact that no motor neurons in the brain are responsible for locomotion implies the presence of a complex neural network that controls the fly's general locomotion. AKHergic neuronal projections entering the brain may be a part of the network. Evidence from studies in other insects supports the central role of AKH for locomotion; for instance, injection of AKH into the mesothoracic neuropile elicits marked motor response in a moth (Milde, 1995). Nevertheless, central functions of AKH seem to be complementary to its hormonal roles, since AKH-mediated prolonged hyperactivities (central role) are likely to be supported by AKH-dependent fat body metabolism (hormonal role). Therefore, such multidirectional AKH functions maximize the fly's best chances for survival particularly when the food source is limited (Lee, 2004).

To understand AKH functions in Drosophila, AKH-cell-deficient (AKH-CD) flies were obtained by expressing a preapoptotic gene, reaper (rpr), in the AKHergic neurons. Ectopic expression of rpr in various peptidergic neurons has been successfully employed to trigger the apoptosis of these neurons. The AKH-CD flies were generated by crossing dAkh-gal4 flies to a UAS-rpr or a double transgenic UAS-rpr:lacZ strain, and their progeny were examined histologically to confirm the absence of the AKHergic neurons. The UAS-rpr:lacZ line was useful particularly for checking ablation status, since the presence or absence of target neurons could be easily judged by simple X-gal staining. Indeed, no ß-gal activity could be detected in the CC of the dAkh-gal4/UAS-rpr:lacZ larvae and adults, which therefore indicates a complete loss of dAkh neurons. This was further confirmed by the lack of AKH immunoreactivities. The rpr-mediated cell death was rescued partially by coexpression of the anti-apoptotic protein p35, since fewer numbers of X-gal-stained cells or less intensive AKH immunosignals were observed in the CC of p35-rescued animals compared with such stainings in the control animals (Lee, 2004).

The AKHergic neurons apparently do not play a vital role, since AKH-CD animals developed in an ostensibly normal manner. No noticeable defects in growth, metamorphosis, eclosion, and longevity were observed. Adult AKH-CD flies also showed normal reproductive capabilities and courtship behavior. The results suggest that AKH functions are not essential for overall development and reproduction at normal growth conditions (Lee, 2004).

Trehalose is a disaccharide composed of two glucose molecules and is the principal blood sugar in insects. Physiological studies in other insects have shown that AKHs elevate hemolymph trehalose titers at the expense of glycogen storage in the fat body (e.g., Park, 1995). This prompted an examination of whether AKH also plays a role in the regulation of carbohydrate metabolism in Drosophila (Lee, 2004).

Hemolymph trehalose levels in AKH-CD larvae were a mere 7%-26% of normal, whereas the glucose levels were unaffected. Moreover, the trehalose titers in p35-rescued larvae were intermediate between controls and AKH-CD, thus revealing a positive correlation between the levels of dAkh expression and hemolymph trehalose concentrations. The results suggest that the AKH neurons produce a hypertrehalosemic factor essential for normal carbohydrate metabolism (Lee, 2004).

Despite the results, it was still uncertain whether subnormal trehalose levels observed in AKH-CD are due to the lack of AKH or other coexisting hypertrehalosemic factor(s). Thus, the effects of overexpression and misexpression of the dAkh gene on trehalose titers were examined. If AKH is the principal effector for hypertrehalosemia, then increasing AKH production in such transgenically modified animals should elevate trehalose concentrations in the hemolymph (Lee, 2004).

Overexpression of dAkh in the native neurons was accomplished by crossing dAkh-gal4 flies to a UAS-dAkh; however, the overexpression did not alter the trehalose levels. This is perhaps because dAkh expression levels are not proportional to the amounts of AKH peptide released; thus, circulating AKH levels in UAS-dAkh/+; dAkh-gal4/+ animals may approximate those in wild type. In support of this, there is no coupling between release and biosynthesis of AKH peptides in locust CC (Harthoorn, 2001). The lack of phenotypic effect by overexpression of a given neuropeptide gene in its usual location is not unprecedented. For instance, overexpression of the Pdf gene (which encodes a principal circadian clock output factor) in pacemaker neurons that normally contain PDF peptides does not affect circadian rhythmicity (Lee, 2004).

As an alternative tactic, dAkh was misexpressed in the fat body, using a fat body-specific GAL4 driver (r4-gal4) that directs strong and constitutive expression of a reporter gene in the fat body in a sex-nonspecific manner from late embryo to adult stages. It was reasoned that expression of AKH in its target tissue could be the most effective way of activating AKH-dependent metabolism. Since adipose tissues are an important endocrine organ, producing several bioactive peptides in mammals and growth factors in flies, it was speculated that AKH precursors encoded by dAkh in the fat body undergo appropriate processing, thereby producing functional AKH peptides (Lee, 2004).

Ectopic expression of dAkh (AKH-EE) in the fat body was accomplished by crossing the r4-gal4 to a UAS-dAkh. Overall developmental processes were not interfered with by the misexpression of dAkh. Production of AKH peptides in the fat body was verified by AKH immunofluorescence. Although wild-type fat bodies do not produce AKH, the peptides bound to fat body receptors could misguide any interpretation of the origin of AKH immunosignals. To avoid this, the fat body from AKH-CD larvae was employed as control tissue. AKH-CD fat bodies gave rise to background signals originating from endogenous autofluorogenic materials in this tissue. By comparison, immunofluorescent signals detected in AKH-EE fat bodies were considerably greater (~1.5-fold) than those in AKH-CD, thus verifying that AKH is indeed overproduced by this type of transgenic modification (Lee, 2004).

Next, attempts were made to determine whether hemolymph trehalose levels are altered in AKH-EE. Consistent with AKH's suggested role as a hypertrehalosemic effector, significant elevation of trehalose levels (~34% above normal) was observed in AKH-EE larvae. Such hypertrehalosemic response to AKH-EE is unlikely due to an ectopic overexpression artifact, since the trehalose titers were unchanged by ectopic expression of another neuropeptide Pdf gene in the fat body (Lee, 2004).

Hemolymph trehalose titers are nicely correlated with the levels of dAkh expression affected by various transgenic modifications. The data thus strongly suggest that AKH plays a major role in the regulation of carbohydrate metabolism in Drosophila. However, there must be AKH-independent pathways for this type of physiological reaction, since detectable amounts of trehalose are still present in animals devoid of AKHergic neurons (Lee, 2004).

Another well-documented physiological AKH function is to mobilize lipid storage from the fat body via lipase activation; the resulting metabolites serve as energy substrates in locusts for long-term flight. The fat body of Drosophila also stores large amounts of lipids, which are consumed rapidly upon starvation. However, it is unknown whether lipid metabolism is regulated by AKH in Drosophila. To address this question, lipid droplets stored in the fat cells were visually examined in well-fed AKH-EE and control larvae by using the Sudan Black staining method. The lipid droplets were substantially smaller and fewer in the AKH-EE than in the controls. Consistent with the results, a quantitative assay also revealed a significant decrease of triglyceride content -- the main storage form of lipids in the fat body -- in AKH-EE (Lee, 2004).

Reduction of endogenous triglyceride levels in AKH-EE fat bodies could be a consequence of either subnormal synthesis or supernormal degradation (hydrolysis) of the triglycerides. If the latter is the case, one can expect an increase of metabolites derived from the hydrolysis of triglycerides (i.e., free fatty acids and glycerol) in the serum of AKH-EE. In accordance with the prediction, hemolymph glycerol concentrations were significantly higher in AKH-EE than in controls, thus supporting that reduction of triglyceride contents in the AKH-EE fat body is due to an enhanced lipolytic response to AKH (Lee, 2004).

If AKH is the sole effector for the hydrolysis of triglycerides, then complete suppression of lipolysis in AKH-CD would increase triglyceride storage in AKH-CD fat body. The data, however, showed that fat body triglyceride contents in AKH-CD were comparable to those of controls. The results indicate that lipid metabolism occurs normally in the absence of AKH, thus foretelling the existence of alternative lipolytic pathways that are independent of AKH (Lee, 2004).

Since animals have to survive on nutrients stored in the body when foods are not available, slower catabolism of such limited resources would help them to survive longer. In this context, AKH-CD flies are expected to live longer than wild type, since the foregoing results indicate that catabolic activities are appreciably attenuated in AKH-CD. To test the hypothesis, mortalities of AKH-CD and control flies, when supplied only with water, were monitored (Lee, 2004).

Remarkably, AKH-CD flies survived for at least 24 hr longer than wild type or any other genetic controls. Resistance to starvation-induced death was consistently observed for all dAkh-gal4 transgenic lines regardless of gender. Of importance, the survival rate of p35-rescued flies was intermediate between those of controls and AKH-CD. This nicely correlates with dAkh expression levels in the p35-rescued flies that are also intermediate between normal and fully ablated. The data suggest that degrees of resistance to the starvation-induced death are most likely AKH-dose dependent (Lee, 2004).

Extended longevity of AKH-CD flies under starvation may be due to their abnormal feeding habits (for instance, more frequent feeding or a larger amount of food intake per meal) in response to the reduction of blood sugar levels, resulting in a larger amount of nutrients taken in by AKH-CD flies than by wild types. If so, then young flies have less time to feed than the older flies do, thereby storing relatively low energy reserves. As a consequence, young AKH-CD flies could be more sensitive to starvation than older AKH-CD flies. This hypothesis was tested by assessing the phenotype of very young flies (the majority of whom were younger than 30 hr after eclosion). Survival rates displayed by young AKH-CD flies were similar to those of older flies, suggesting that the feeding anomaly may not be an influential factor for the phenotype exhibited by Drosophila ablated of their AKH cells (Lee, 2004).

Recent studies show that locomotor activities of honeybees and wasps are unable to be sustained in the absence of available energy substrates. Such studies led to a proposition that subnormal levels of energy substrates observed in AKH-CD may affect motility of these animals. To test this hypothesis, the flies' circadian locomotor activities were monitored using an infrared emitter-detector system (Lee, 2004).

First daily locomotor activities were measured of wild-type and AKH-CD flies fed on 4% sucrose-agar medium. Under 12-hr:12-hr LD conditions, wild type showed typical bimodal activity peaks, one at dawn and the other at dusk; in subsequent DD conditions, robust circadian rhythmicity was sustained. Quite similar rhythmic activity patterns were observed in AKH-CD flies, suggesting that normal functions of AKH are not involved in clock-controlled locomotor activity rhythms (Lee, 2004).

These studies were extended to detect any differences in locomotor activities between starved and fed wild-type flies or between wild-type and AKH-CD in the absence of food. In doing so, flies were provided with water only in a form of agarose block. Under this assay condition, the nonfeeding wild-type flies were persistently active at Zeitgeber times (in LD cycles) while feeding flies were normally quiescent. Most of the starved flies died after the onset of accentuated locomotion. Although the durations and amplitudes of such hyperactivities varied individually, this type of behavioral pattern was observed in >90% of hungry wild-type flies and other genetic controls, UAS-rpr/+, and dAkh-gal4/+. The hunger-driven prolonged hyperactivity may reflect avid (even desperate) search for food (Lee, 2004).

Intriguingly, the majority of AKH-CD flies did not show pronounced starvation-induced hyperactivity, suggesting a role for AKH in the regulation of this novel phenotype. Lack of hyperactivity in starved AKH-CD flies is unlikely due to their general weakness, since they are as robust as wild type when food is ample. Instead, this could be a consequence of lower levels of energy substrates in the hemolymph of AKH-CD. If this is true, then higher levels of energy substrates in the hemolymph of AKH-EE may cause them to be excessively hyperactive. However, starvation-dependent activity patterns of AKH-EE were not much different from those of the control, indicating that the fat body's metabolic activity may not be a causative factor for the accentuated locomotive behavior. Perhaps neural inputs from the AKH neurons play a role in the starvation-induced behavioral change (Lee, 2004).

Dual lipolytic control of body fat storage and mobilization in Drosophila

Energy homeostasis is a fundamental property of animal life, providing a genetically fixed balance between fat storage and mobilization. The importance of body fat regulation is emphasized by dysfunctions resulting in obesity and lipodystrophy in humans. Packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, are conserved between mammals and insects. A Drosophila mutant was generated lacking the receptor (AKHR; FlyBase name -- Gonadotropin-releasing hormone receptor or GRHR) of the adipokinetic hormone signaling pathway, an insect lipolytic pathway related to ss-adrenergic signaling in mammals. Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR is as important for chronic accumulation and acute mobilization of storage fat as is the Brummer lipase, the homolog of mammalian adipose triglyceride lipase (ATGL). Simultaneous loss of Brummer and AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. These data demonstrate that storage-fat mobilization in the fly is coordinated by two lipocatabolic systems, which are essential to adjust normal body fat content and ensure lifelong fat-storage homeostasis (Grönke, 2007).

Expression studies in a heterologous tissue culture system (Staubli, 2002) and in Xenopus oocytes (Park, 2002) identified AKH-responsive G protein-coupled receptors in Drosophila, such as the one encoded by the AKHR (or CG11325) gene. AKHR is expressed during all ontogenetic stages of the fly (Hauser, 1998). It consists of seven exons, which encode a predicted protein of 443 amino acids. In late embryonic and larval stages, AKHR is expressed in the fat body. This finding is consistent with its predicted role as transmitter of the lipolytic AKH signal in this organ (Grönke, 2007).

In order to examine the effect of AKHR signaling on fat storage and mobilization in vivo, two different P element-insertion mutants were used, CG11188A1332 and AKHRG6244, which are located close to and within the AKHR gene, respectively. CG11188A1332 flies carrying the transposable element integration designated A1332 allow for the transcriptional activation of the adjacent AKHR gene. This ability was used for AKHR gain-of-function studies by overexpression of AKHR in the fat body of flies. Overexpression of AKHR in response to a fat body-specific Gal4 inducer causes dramatic reduction of organismal fat storage. This finding could be recapitulated by fat body-targeted AKHR expression from a cDNA-based upstream activation sequence (UAS)-driven AKHR transgene. These gain-of-function results suggest a critical in vivo role for AKHR in storage-lipid homeostasis of the adult fly (Grönke, 2007).

Flies of strain AKHRG6244, which carry a P element integration in the AKHR untranslated leader region, were used to generate the AKHR deletion mutants AKHR1 and AKHR2, as well as the genetically matched control AKHRrev, which possesses a functionally restored AKHR allele. As exemplified for embryonic and larval stages, AKHR1 mutants lack AKHR transcript. Ad libitum-fed flies without AKHR function are viable, fertile, and have a normal lifespan. However, such flies accumulate lipid storage droplets in the fat body and have 65%-127% more body fat than the controls. These results indicate that AKHR1 mutants develop an obese phenotype. The same result was obtained with AKHR2 and AKHR1/AKHR2 transheterozygous mutant flies, as well as with flies lacking the AKH-producing cells of the neuroendocrine system due to targeted ablation by the cell-directed activity of the proapoptotic gene reaper. Conversely, chronic overexpression of AKH provided by a fat body-targeted AKH transgene of otherwise wild-type flies largely depletes lipid storage droplets and organismal fat stores. However, the obese phenotype of AKHR mutants is unresponsive to AKH, indicating that AKHR is the only receptor transmitting the lipolytic signal induced by AKH in vivo. Collectively, these data demonstrate that AKH-dependent AKHR signaling is critical for the chronic lipid-storage homeostasis in ad libitum-fed flies (Grönke, 2007).

Studies on various insect species helped elucidate several components and mediators of the lipolytic AKH/AKHR signal transduction pathway (for review, see [Van der Horst, 2001). However, the identity of the TAG lipase(s) executing the AKH-induced fat mobilization program remained elusive. Besides the Drosophila homolog of the TG lipase from the tobacco hornworm Manduca sexta (Arrese, 2006), the recently identified Brummer lipase, a homolog of the mammalian ATGL, is a candidate member of the AKH/AKHR pathway. This is based on the striking similarity between the phenotypes of AKHR and bmm mutants. Ad libitum-fed flies lacking either AKHR or bmm activity, store excessive fat. Both mutants show incomplete storage-fat mobilization (Grönke, 2005) and starvation resistance (Grönke, 2005) in response to food deprivation. Starvation resistance of these mutants might be caused by their increased metabolically accessible fat stores and/or changes in their energy expenditure due to locomotor activity reduction as described for flies with impaired AKH signaling (Lee, 2004; Isabel, 2005). Despite the phenotypic similarities of their mutants, however, AKHR and bmm are members of two different fat-mobilization systems in vivo. Several lines of evidence support this conclusion. On one hand, AKH overexpression reduces the excessive TAG storage of bmm mutants, while on the other, bmm-induced fat mobilization can be executed in AKHR mutants. Thus, AKH/AKHR signaling is not a prerequisite for Brummer activity. Moreover, genetic epistasis experiments support this idea that AKHR and bmm belong to different control systems of lipocatabolism in vivo. Double-mutant analysis reveals that the obesity of AKHR and bmm single mutants is additive. Accordingly, AKHR bmm double-mutant flies store about four times as much body fat as control flies and accumulate excessive lipid droplets in their fat body cells (Grönke, 2007).

Thin layer chromatography (TLC) analysis was used to compare the storage-fat composition of AKHR and bmm single mutants with AKHR bmm double-mutant and control flies. Excessive body fat accumulation in AKHR bmm double mutants is on the one hand due to TAG, which is increased compared to AKHR and bmm single-mutant flies. Additionally, an uncharacterized class of TAG (TAGX) appears exclusively in AKHR bmm double mutants. In contrast to TAG, changes in diacylglycerol (DAG) content do not substantially contribute to the differences in body fat content in any of the analyzed genotypes. Taken together a quantitative increase and a qualitative change in the TAG composition account for the extreme obesity in AKHR bmm double-mutant flies (Grönke, 2007).

To address the in vivo response of AKHR bmm double mutants to induced energy-storage mobilization, flies were starved and their survival curve monitored. AKHR bmm double mutants die rapidly after food deprivation. In contrast to the starvation-resistant obese AKHR and bmm single mutants, the double mutants are not capable of mobilizing even part of their excessive fat stores. AKHR bmm double mutants do not, however, suffer from a general block of energy-storage mobilization because they can access and deplete their carbohydrate stores. These data demonstrate that energy homeostasis in AKHR bmm double-mutant flies is imbalanced by a severe and specific lipometabolism defect, which cannot be compensated in vivo (Grönke, 2007).

The nature of Brummer as a TAG lipase and AKHR as a transmitter of lipolytic AKH signaling suggests that the extreme storage-fat accumulation and starvation sensitivity of ad libitum-fed AKHR bmm double mutants is due to severe lipolysis dysfunction. To address this possibility in vitro, lipolysis rate measurements on fly fat body cell lysates and lysate fractions of control flies were performed. Results show that the cytosolic fraction of fat body cells contains the majority of basal and starvation-induced lipolytic activity against TAG, similar to the activity distribution in mammalian adipose tissue. Little basal and induced total TAG cleavage activity localizes to the lipid droplet fraction, whereas the pellet fraction including cellular membranes shows low basal, non-inducible TAG lipolysis. Lipolysis activity against DAG is similarly distributed between fat body cell fractions. However, in accordance with the function of DAG as major transport lipid in Drosophila, DAG lipolysis in fat body cells is not induced in response to starvation (Grönke, 2007).

Based on the lysate fraction analysis of control flies, cytosolic fat body cell extracts were used to assess the basal and starvation-induced lipolytic activity of mutant and control flies on TAG, DAG, and cholesterol oleate substrates. Whereas DAG and cholesterol oleate cleavage activity of fat body cells is comparable between all genotypes and physiological conditions tested, TAG lipolysis varies widely. Compared to control flies, basal TAG lipolysis of AKHR bmm double mutants is reduced by 80% and induced TAG cleavage is completely blocked, consistent with the flies' extreme obesity and their inability to mobilize storage fat. The impairment of basal lipolysis in the double mutants is largely due to the absence of bmm function, because it is also detectable in bmm single-mutant cells, whereas basal lipolysis in AKHR mutants is not reduced. Interestingly, bmm mutants mount a starvation-induced TAG lipolysis response after short-term (6 h), but not after extended (12 h), food deprivation. Conversely, AKHR mutant cells lack an early lipolysis response, but exhibit strong TAG cleavage activity after extended food deprivation. These data suggest that induced storage-fat mobilization in fly adipocytes relies on at least two lipolytic phases: an early, AKH/AKHR-dependent phase and a later, Brummer-dependent phase. Accordingly, it is speculated that the obesity of bmm and AKHR mutant flies is caused by different mechanisms: chronically low basal lipolysis in bmm mutants and, in AKHR mutants, lack of induced lipolysis during short-term starvation periods that is characteristic of organisms with discontinuous feeding behavior. It is acknowledged, however, that in vitro lipolysis assays on artificially emulsified substrates allow only a limited representation of the lipocatabolism in vivo, because lipid droplet-associated proteins modulate the lipolytic response in the insect fat body (Patel, 2005; Patel, 2006) and mammalian tissue. Moreover, excessive fat accumulation in AKHR mutants may be in part due to increased lipogenesis because AKH signaling has been demonstrated (Lee, 1995; Ziegler, 1997; Lorenz, 2001) to repress this process in various insects (Grönke, 2007).

Fat body cells of control flies (AKHRrev bmmrev) exhibit basal TAG lipolysis, which is doubled by starvation-induced lipolysis after 6 h or 12 h of food deprivation. bmm mutant cells have reduced basal lipolysis and lack induced lipolysis after 12 h starvation. AKHR mutant cells lack early (6 h) induced lipolysis, but show strong starvation-induced lipolysis after 12 h food deprivation. AKHR bmm double mutants have reduced basal lipolysis and lack starvation-induced lipolysis altogether (Grönke, 2007).

The finding of the dual lipolytic control in the fly raises the question of whether the two systems involved act independently of each other or whether one system responds to the impairment of the other. Modulation of transcription is an evolutionarily conserved regulatory mechanism of lipases from the ATGL/Brummer family. ATGL is transcriptionally up-regulated in fasting mice, as is bmm transcription in starving flies. Moreover bmm overexpression depletes lipid stores in the fat body of transgenic flies. Accordingly, bmm transcription was analyzed in response to modulation of AKH/AKHR signaling to assess a potential regulatory interaction between the two lipolytic systems. Compared to the moderate starvation-induced up-regulation of bmm in control flies, the gene is hyperstimulated in flies with impaired AKH signaling. As early as 6 h after food deprivation, bmm transcription is up-regulated by a factor of 2.5-3 in flies lacking the AKH-producing neuroendocrine cells (AKH-ZD) or in AKHR mutant flies. Conversely, chronic expression of AKH in the fat body suppresses bmm transcription. Bmm hyperstimulation in AKHR mutants is consistent with a subsequent strong increase of starvation-induced TAG lipolysis observed 12 h after food deprivation. Taken together, these data demonstrate an AKH/AKHR-independent activation mechanism of bmm and suggest the existence of compensatory regulation between bmm and the AKH/AKHR lipolytic systems, the mechanism of which is currently unknown (Grönke, 2007).

The results presented in this study provide in vivo evidence that the fly contains two induced lipolytic systems. One system confers AKH/AKHR-dependent lipolysis, a signaling pathway, which assures rapid fat mobilization by cAMP signaling and PKA activity. Drosophila's second lipolytic system involves the Brummer lipase, which is responsible for most of the basal and part of the induced lipolysis in fly fat body cells, likely via transcriptional regulation. Currently, it is unknown whether Brummer activity is post-translationally modulated by an α/β hydrolase domain-containing protein like the regulation of its mammalian homolog ATGL by CGI-58. Homology searches between mammalian and Drosophila genomes identify the CGI-58-related fly gene CG1882 and the putative Hsl homolog CG11055, providing additional support for the evolutionary conservation of fat-mobilization systems. However, differences in lipid transport physiology (i.e., DAG transport in Drosophila, and FFA in mammals) suggest a different substrate specificity or tissue distribution of fly Hsl compared to its mammalian relative (Grönke, 2007).

Future studies will not only unravel the crosstalk between the two Drosophila lipocatabolic systems, but also disclose the identity of additional genes involved in this process, such as the upstream regulators of bmm. This study substantiates the emerging picture of the evolutionary conservation between insect and mammalian fat-storage regulation and emphasizes the value of Drosophila as a powerful model system for the study of human lipometabolic disorders (Grönke, 2007).

Energy-dependent modulation of glucagon-like signaling in Drosophila via the AMP-activated protein kinase

Adipokinetic Hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. This study reports that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases lifespan during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting AMPK promotes AKH secretion. Reduced secretion was observed in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and ultimately metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks are highly conserved and AMPK plays a prominent role in these networks (Braco, 2012).

This study reports that the selective reduction of AMPK function in AKH neuroendocrine cells results in a series of behavioral and physiological phenotypes consistent with a loss of function of AKH itself. Specifically, animals have increased lifespan during starvation as do animals lacking the AKH hormone. Furthermore, animals deficient in AKH exhibit a loss of starvation-induced hyperactivity, and the selective loss of AMPK function in these cells leads to a delay in this behavioral response. It is concluded that AMPK is a critical component that regulates AKH secretion via modulation of cell excitability based on the observations that AMPK is not necessary for cell survival or AKH expression, and the results demonstrating reduced secretion and GCaMP fluorescence during starvation challenges. A model is proposed in which AMPK acts as an energy sensor in the AKH cell population to control secretion and ultimately coordinate physiological and behavioral responses to maintain metabolic homeostasis. Processing of the AKH peptide relies on cleavage of the prohormone and subsequent amidation of the N-terminus and these events are required for AKH bioactivity. While the possibility cannot be ruled out that AMPK may be impacting AKH hormone processing, this is considered insufficient to explain the ensemble of phenotypes associated with reduced AMPK function in AKH cells. First, partial phenocopies were observed of AKH cell ablation, whereas in contrast, the loss of processing causes complete loss of function phenotypes (Rhea, 2010). Second, the observation of delayed locomotor responses present in animals with compromised AMPK function suggests at least minimal levels of bioactive AKH as the complete loss of the hormone eliminates this behavioral response. Third, reduced levels of bioactive AKH, which may be caused by reduced AMPK function, are insufficient to explain the changes in AKH cell excitability. While it cannot be completely ruled that another hormone co-expressed in the AKH cell population is responsible for some of the behavioral phenotypes observed, this is considered unlikely: (1) there is an extensive literature establishing the roles of Adipokinetic Hormone in mediating metabolic homeostasis; (2) the observations targeting the AKH hormone with a specific RNAi leads to phenotypes consistent with the AKH cell ablations; (3) results with a deletion of the receptor highly specific for the AKH peptide are also consistent with the behavioral phenotypes from AKH cell ablations. Collectively, these results make a compelling case that it is AKH as opposed to another hormone which is relevant in mediating metabolic homeostasis. Nonetheless, it is noted that even if there were other hormones co- expressed with AKH that are relevant, the actions of AMPK strongly cement this kinase as a critical regulator of AKH cell excitability and by extension, hormonal regulation (Braco, 2012).

How might AMPK be altering AKH cell excitability? The results implicate an acute modulation of channel activity. AMPK has been shown to modulate the biophysical properties of the twin pore K+ (TWIK) channels, and while it is currently unknown if AKH cells express similar channels, it is speculated that AMPK is similarly modulating an unknown channel conductance in AKH cells. There is evidence that AKH cells express the K+ATP channels, based on in situ analysis and that dietary introduction of a specific K+ATP channel antagonist, tolbutamide, leads to behavioral phenotypes consistent with blocking AKH release. AMPK has been shown to regulate the activation of this channel subtype (Yoshida, 2012). Given the energy sensing roles of the K+ATP channel conductance, the contribution of this conductance in the regulation of AKH signaling and whether this intersects with AMPK signaling is currently being tested. It is noted that AMPK deficient AKH cells still respond to sugar changes as directly observed with GCaMP, albeit those responses are diminished and delayed. This may reflect residual wild-type AMPK function or more likely, redundant mechanisms present in AKH cells to regulate AKH secretion. Therefore, it is suspected that AKH release in an AMPK deficient background may result from other signaling processes. In support of that notion, autophagy, which also facilitates increased cellular energy availability, has been shown to occur independent of AMPK activation. Another candidate that may be involved in AMPK- independent regulation of AKH is the activity-regulated cytoskeletal-associated (ARC) gene, which is specifically expressed in AKH cells and mutants in this gene fail to exhibit normal starvation-induced hyperactivity (Braco, 2012).

While the distinct changes in the responses to sugar transitions in explanted AKH cells implicate other AKH cell-autonomous elements, the delay in hyperactive behaviors was also noted in animals with reduced AMPK function. Many different hormones in a variety of insects have been implicated as AKH release factors, including but not limited to tachykinin-like peptides, octopamine, and proctolin. While it is currently unknown if these hormones are operating in a similar fashion in Drosophila, it is speculated that these or other hormonal factors may also be responsible for AKH release in animals with reduced AMPK function in AKH cells. It is also suspected that some of these or other regulatory hormones may operate through AMPK. For example, AMPK has been shown to be a critical component of leptin signaling and a target of FSH modulation in mammals. Which hormones regulate AKH secretion is currently being evaluated and whether hormonal signaling pathways modulate AMPK activity is being assessed (Braco, 2012).

It is noted that the regulation of AKH via AMPK is similar to the regulation of glucagon signaling via AMPK. Specifically, AMPK activity in pancreatic alpha cells is required for elevated calcium levels upon lowered glucose levels, akin to the demonstration of AKH calcium levels requiring AMPK. These similarities suggest that the signaling networks dedicated to maintain metabolic homeostasis are highly conserved across broad phylogenetic distances. These results suggest that the mechanism underlying AMPK regulation of glucagon signaling in mammals may be caused by changes in pancreatic alpha cell excitability (Braco, 2012).

REGULATION

Promoter Structure

To define a regulatory region responsible for CC-specific dAkh expression, three independent fly lines were generated bearing the dAkh-gal4 transgene. The dAkh-gal4 flies were crossed to a UAS-lacZ reporter line and the progeny were processed for X-gal histochemistry. As seen in the in situ hybridization and immunohistochemistry results, ß-galactosidase (ß-gal) activity was detected only in the CC of larvae and adults. Identical expression patterns were obtained from all three dAkh-gal4 lines. Lack of ectopic expression sites directed by this promoter was further confirmed by dAkh-gal4-driven gfp expression in 'live' larvae. The results suggest that cis-acting regulatory elements necessary for CC-specific dAkh expression are present within the 1-kb upstream sequence (Lee, 2004).

Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells

Antagonistic activities of glucagon and insulin control metabolism in mammals, and disruption of this balance underlies diabetes pathogenesis. Insulin-producing cells (IPCs) in the brain of insects such as Drosophila also regulate serum glucose, but it remains unclear whether insulin is the sole hormonal regulator of glucose homeostasis and whether mechanisms of glucose-sensing and response in IPCs resemble those in pancreatic islets. This study shows, by targeted cell ablation, that Drosophila corpora cardiaca (CC) cells of the ring gland are also essential for larval glucose homeostasis. Unlike IPCs, CC cells express Drosophila cognates of sulphonylurea receptor (Sur) and potassium channel (Ir), proteins that comprise ATP-sensitive potassium channels regulating hormone secretion by islets and other mammalian glucose-sensing cells. They also produce adipokinetic hormone, a polypeptide with glucagon-like functions. Glucose regulation by CC cells is impaired by exposure to sulphonylureas, drugs that target the Sur subunit. Furthermore, ubiquitous expression of an akh transgene reverses the effect of CC ablation on serum glucose. Thus, Drosophila CC cells are crucial regulators of glucose homeostasis and they use glucose-sensing and response mechanisms similar to islet cells (Kim, 2004).

Insect corpora cardiaca (CC) are clusters of endocrine cells in the ring gland adjacent to the prothoracic gland and corpus allatum. A principal CC product is adipokinetic hormone (AKH), a polypeptide that mobilizes stored macromolecular energy reserves to sustain energy-consuming activities, such as crawling and flight. AKH is similar to mammalian glucagon; like glucagon in pancreatic islet α-cells, AKH is synthesized as a pre-prohormone, processed, and stored in dense core vesicles. Like mammalian glucagon activity in liver, AKH has been shown to bind a G-protein-coupled transmembrane receptor and to increase lipolysis, glycogenolysis and production of trehalose in the insect fat body, a storage organ for lipid and glycogen (Kim, 2004).

Previous studies of AKH microinjection and ring gland transplantation in locusts and other insects suggest that AKH is sufficient to increase haemolymph glucose concentrations, but have not yet shown a requirement for AKH in glucose homeostasis. To examine phenotypes resulting from CC cell ablation and AKH deficiency, 1,000-base-pair DNA segment derived from sequences immediately 5' of the Drosophila akh gene was used to drive the expression of the transcriptional trans-activator GAL4 in CC cells. The akh-GAL4 construct, when crossed with a UAS-mCD8GFP (membrane-tethered green fluorescent protein, mGFP) reporter line, directed a GFP expression pattern that reflected endogenous akh expression in the ring gland corpora cardiaca of third-instar larvae. Using in situ hybridizations, it was establised that embryonic akh messenger RNA expression initiates in cells of the presumptive CC anlage and that in later larval stages it is maintained only in CC cells. To assess the role of the CC as an endocrine regulator of haemolymph glucose concentrations, akh-GAL4 lines were used to express the cell death factor Reaper in akh-expressing CC cells. This resulted in the ablation of only CC cells at high efficiency: in more than 96% of newly hatched first-instar larvae harbouring akh-GAL4, UAS-Reaper and UAS-mCD8GFP, no mGFP-labelled CC cells were detected. In contrast, mGFP was detected in CC cells within all control larvae at the same stage, and at later stages. In Drosophila , haemolymph glucose is composed of trehalose (a disaccharide of glucose) and monomeric free glucose, and the combined circulating concentration of these (hereafter referred to as total haemolymph glucose) is maintained in a narrow range for a given feeding condition. Ablation of akh-expressing CC cells in larvae raised on dextrose-supplemented medium decreased the mean total haemolymph glucose and trehalose by 50%, an effect similar to that recently reported by others. CC cell deficiency did not result in discernible growth reduction, developmental delay or lethality, phenotypes that arise after the ablation of IPCs in the brain. Thus, like glucagon, AKH is an essential regulator of energy metabolism but might be dispensable for developmental growth control (Kim, 2004).

To test whether AKH activity alone could account for the glucose-regulating action of CC cells, the ability was tested of an akh transgene with ubiquitous expression from a heat shock promoter to reverse the effect of CC ablation. Lower haemolymph glucose concentrations resulting from CC ablation were partly restored by the ubiquitous expression of an akh transgene. Thus, bioactive AKH from the akh transgene might be produced in target tissues, as has been shown for transgene-encoded neuropeptides such as Drosophila insulin. These data indicate that AKH is an essential regulator of haemolymph carbohydrate concentrations in Drosophila . It is suggested that the hyperglycaemic effects of AKH counter-regulate the activity of other systemic hormones such as insulin and that these antagonistic activities might refine the levels of circulating energy to match systemic energy requirements. If so, it is postulated that the negative energy balance accompanying starvation might worsen the hypoglycaemic effects of AKH deficiency. In comparison with starved control larvae, total haemolymph glucose was decreased by 75% in starving larvae after CC cell ablation. Thus, starvation increased the severity of hypoglycaemia in animals lacking CC cells, indicating that AKH might be required for the compensatory mechanisms that maintain circulating glucose during periods of food deprivation in Drosophila larvae (Kim, 2004).

Labelling of CC cell processes with mGFP and an antibody against AKH revealed that AKH-producing cells extend processes that terminate on the heart and on the prothoracic gland compartment of the ring gland. On the surface of the heart, CC cell processes have extensive contact with axons that project from insulin-producing cells from the brain. Labelling of CC cell processes with mGFP and an antibody against AKH revealed localization of AKH within the processes that contact the IPCs, and AKH peptide on the processes contacting the heart. These results indicate that the heart surface is the principal site of AKH release into the circulating haemolymph. Thus, like glucagon-producing cells in mammalian islets and brain, AKH-producing CC cells in the Drosophila ring gland have direct systemic vascular access, consistent with their role as endocrine regulators of metabolism (Kim, 2004).

ATP-sensitive potassium (KATP) channels regulate neuroendocrine cell function in organs such as the mammalian pancreas and brain, and this study examined whether KATP functions regulate CC cell activity. KATP channels are heteromeric protein complexes composed of sulphonylurea receptor (Sur) and inward-rectifying potassium channel (Ir; also called Kir) subunits. An ATP-binding domain in the Ir subunit regulates KATP channel activity, allowing these channels to serve as cellular energy sensors, opening or closing in response to the intracellular ADP/ATP ratio, thus influencing membrane potential and subsequent calcium currents that regulate hormone secretion. Using mRNA in situ hybridization, it was showm that larval CC cells expressed Sur (Nasonkin, 1999) and Ir (Döring, 2002), which have sequence similarity to mammalian Sur1 and Kir6 proteins, respectively. Expression of Sur or Ir was not detected in the larval brain IPCs, another group of cells known to regulate haemolymph glucose. Drosophila Sur has been shown to be sufficient to allow K+ currents that polarize membrane potentials (Nasonkin, 1999). Drosophila Ir was demonstrated to evoke an inwardly rectifying K+ current (Kim, 2004).

Tests were performed to see whether increased haemolymph glucose concentrations might result from excess AKH secretion brought about by sulphonylurea inhibition of the Sur and K+-dependent depolarization of CC cells. Glyburide and tolbutamide are representative members of the two major classes of sulphonylureas. These drugs promote the closure of KATP channels and cellular depolarization, thereby regulating secretion in mammalian neuroendocrine cells. For example, sulphonylureas stimulate glucagon secretion in diabetic patients. Glyburide has previously been shown to inhibit Drosophila Sur-mediated outward K+ currents, resulting in the depolarization of cell potential. Exposure of feeding third-instar larvae to glyburide mixed in yeast paste (standard dextrose medium did not permit drug delivery) produced a 10% increase in mean total haemolymph glucose concentration compared with controls. Exposure of larvae to tolbutamide had a greater effect, producing a 40% increase in mean total haemolymph glucose, and tolbutamide was used in subsequent studies. Average haemolymph glucose concentrations were generally decreased in animals fed with yeast paste compared with animals fed with standard dextrose medium, and this might have accentuated the hyperglycaemic effect of sulphonylureas administered in yeast paste. Moreover, the hypoglycaemic effect induced by CC cell ablation (or hyperpolarization) seemed attenuated in yeast-fed animals, further supporting the hypothesis that requirements for AKH might be altered by manipulating feeding conditions (Kim, 2004).

To test the hypothesis that Sur and Ir function in the CC to regulate haemolymph glucose concentrations in Drosophila , CC cells were ablated in larvae fed with tolbutamide. Ablation of the CC cells using Akh-GAL4 and UAS-Reaper blocked the hyperglycaemic effect of tolbutamide, indicating that CC cells must be present to support the hyperglycaemic action of tolbutamide. To determine whether the hyperglycaemic effect of tolbutamide resulted from Sur and Ir-mediated depolarization of CC cells, membrane potential was hyperpolarized in CC cells, in the presence and absence of tolbutamide. Kir2.1 is a human K+ channel that evokes an outward K+ current, independently of ATP regulation, and has previously been used to impair cellular depolarization in vivo in Drosophila by inducing persistent outward K+ current and a hyperpolarized resting potential. One indication that AKH release by CC cells requires membrane depolarization and might be regulated by K+-channel-dependent membrane potential comes from the observation that, on standard dextrose medium, third-instar larvae expressing Kir2.1 in CC cells had a 23% decrease in mean haemolymph glucose concentration, compared with controls. Expression of the Kir2.1 channel in CC cells prevented the hyperglycaemic effect of tolbutamide, indicating that K+-channel-dependent CC cell depolarization resulted from exposure to sulphonylurea. Together, these pharmacological and genetic data support the view that KATP channel activity in CC cells governs AKH release, thereby controlling concentrations of circulating glucose in Drosophila (Kim, 2004).

In pancreatic α-cells, hypoglycaemia stimulates increased intracellular calcium concentrations promoting glucagon secretion, whereas hyperglycaemia inhibits these responses. To test whether Drosophila CC cells sense glucose changes and, like pancreatic α-cells, modulate intracellular calcium concentrations, CC cells were mared with fluorescent transgene-encoded calcium sensors ('camgaroos'). The fluorescence intensity of camgaroos increases in response to elevated intracellular calcium concentration, an effect used previously to measure cytoplasmic calcium transients in depolarized Drosophila neurons. Elevation of cytoplasmic calcium concentration after CC cell depolarization stimulates AKH secretion; thus, in these experiments elevated intracellular calcium concentration in CC cells was used as an indicator of AKH secretion. Fluorescence of camgaroo-2 (cg-2) in cultured CC cells increased as extracellular trehalose or glucose concentration decreased. Direct CC cell depolarization with increased extracellular potassium concentration similarly led to increased cg-2 fluorescence. In contrast, fluorescence in cg-2-labelled CC cells decreased as extracellular trehalose concentration increased. These results corroborate previous studies of locust CC cells showing that decreases in extracellular trehalose or glucose concentration stimulated AKH secretion. Drosophila CC cells express the enzyme trehalase, raising the possibility that the sensing of trehalose by CC cells involves the hydrolysis of trehalose to glucose, a view also supported by similar effects of trehalose and glucose in in vitro studies. Thus, hypoglycaemic sensing in CC cells leads to increased concentrations of the intracellular second messenger calcium, a signal for subsequent regulated exocytosis of AKH -- a mechanism similar to those regulating glucagon secretion by mammalian pancreatic α-cells (Kim, 2004).

Thus, there are remarkable parallels in endocrine cell functions that ensure the supply of circulating glucose in Diptera and in mammals. On the basis of these parallels, it is speculated that insect CC cells and mammalian neuroendocrine cells that regulate metabolism might have arisen from an ancestral energy-sensing cell. If so, it is further speculated that pancreatic islet cells, including β-cells, might have evolved from an ancient α-cell. Similarly to pancreatic islets, insect CC cells might delaminate from embryonic epithelial cells that give rise to both gut and neuroendocrine structures. Thus, common mechanisms might regulate the development of CC and pancreatic islet cells. Understanding CC cell development could therefore accelerate the discovery of cell-replacement therapies for type 1 diabetes mellitus. This Drosophila model might also serve to elucidate the mechanisms that control stimulus-secretion coupling in CC cells, and hence the biology of hypoglycaemia. Moreover, the sensitivity of CC cells to drugs commonly prescribed for disorders such as type 2 diabetes indicates that Drosophila might provide a model system for the discovery of pharmacological agents to treat human endocrine diseases (Kim, 2004).

Ablation of AKH-producing neuroendocrine cells decreases trehalose levels and induces behavioral changes in Drosophila

Adipokinetic hormone (AKH) is a metabolic neuropeptide principally known for its mobilization of energy substrates, notably lipid and trehalose during energy-requiring activities, such as flight and locomotion. Drosophila AKH cells localization in corpora cardiaca, as in other insects species, was confirmed by immunoreactivity and by a genetic approach using UAS/GAL4 system. To assess AKH general physiological rules, AKH endocrine cells were ablated by specifically driving the expression of apoptosis transgenes in AKH cells. Trehalose levels were decreased in larvae and starved adults, when the stimulation by AKH of the production of trehalose from fat body glycogen is no longer possible. Moreover, these adults without AKH-cells become progressively hypoactive. Finally, under starvation conditions, those hypoactive AKH-knockout cells flies survived about 50% longer than control wild-type flies, suggesting that the slower rate at which AKH-ablated flies mobilize their energy resources, extends their survival (Isabel, 2004).

The sleeping beauty: How reproductive diapause affects hormone signaling, Metabolism, immune response and somatic maintenance in Drosophila melanogaster

Some organisms can adapt to seasonal and other environmental challenges by entering a state of dormancy, diapause. Thus, insects exposed to decreased temperature and short photoperiod enter a state of arrested development, lowered metabolism, and increased stress resistance. Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation. Diapause was induced in D. melanogaster and effects were monitored over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity. During diapause food intake diminishes drastically, but circulating and stored carbohydrates and lipids are elevated. Gene transcripts of glucagon- and insulin-like peptides increase, and expression of several target genes of these peptides also change. Four key genes in innate immunity can be induced by infection in diapausing flies, and two of these, Drosomycin and Cecropin A1, are upregulated by diapause independently of infection. Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium. Many phenotypes induced by diapause are reversed after one week of recovery from diapause conditions. Furthermore, mutant flies lacking specific insulin-like peptides (dilp5 and dilp2-3) display increased diapause incidence. This study provides a first comprehensive characterization of reproductive diapause in D. melanogaster, and evidence that glucagon- and insulin-like signaling are among the key regulators of the altered physiology during this dormancy (Kubrak, 2014: 25393614).

The neuropeptide Allatostatin A regulates metabolism and feeding decisions in Drosophila

Coordinating metabolism and feeding is important to avoid obesity and metabolic diseases, yet the underlying mechanisms, balancing nutrient intake and metabolic expenditure, are poorly understood. Several mechanisms controlling these processes are conserved in Drosophila, where homeostasis and energy mobilization are regulated by the glucagon-related adipokinetic hormone (AKH) and the Drosophila insulin-like peptides (DILPs). This study provides evidence that the Drosophila neuropeptide Allatostatin A (AstA) regulates AKH and DILP signaling. The AstA receptor gene, Dar-2, is expressed in both the insulin and AKH producing cells. Silencing of Dar-2 in these cells results in changes in gene expression and physiology associated with reduced DILP and AKH signaling and animals lacking AstA accumulate high lipid levels. This suggests that AstA is regulating the balance between DILP and AKH, believed to be important for the maintenance of nutrient homeostasis in response to changing ratios of dietary sugar and protein. Furthermore, AstA and Dar-2 are regulated differentially by dietary carbohydrates and protein and AstA-neuronal activity modulates feeding choices between these types of nutrients. These results suggest that AstA is involved in assigning value to these nutrients to coordinate metabolic and feeding decisions, responses that are important to balance food intake according to metabolic needs (Hentze, 2015).

Imbalance between the amount and type of nutrients consumed and metabolized can cause obesity. It is therefore important to understand how animals maintain energy balancing, which is determined by mechanisms that guide feeding decisions according to the internal nutritional status. The fruit fly Drosophila melanogaster has become an important model for studies of feeding and metabolism, as the regulation of metabolic homeostasis is conserved from flies to mammals. In Drosophila, hormones similar to insulin and glucagon regulate metabolic programs and nutrient homeostasis. Adipokinetic hormone (AKH) is an important metabolic hormone and considered functionally related to human glucagon and a key regulator of sugar homeostasis. The release of AKH promotes mobilization of stored energy from the fat body, the equivalent of the mammalian liver and adipose tissues. Neuroendocrine cells in the corpus cardiacum (CC) express and release AKH3 that binds to the AKH receptor (AKHR), a G-protein coupled receptor (GPCR) expressed mainly in the fat body, and promotes mobilization of stored sugar and fat. Insulin and glucagon have opposing effects important to maintain balanced blood glucose levels. The Drosophila genome contains 7 genes coding for insulin-like peptides (DILPs), called dilp1-7, which are homologous to the mammalian insulin and insulin-like growth factors (IGFs). The seven DILPs are believed to act through one ortholog of the human insulin receptor that activates conserved intracellular signaling pathways. The DILPs are important regulators of metabolism, sugar homeostasis and cell growth. DILP2, 3 and 5 are produced in 14 neurosecretory cells in the brain; the insulin producing cells (IPCs). Genetic ablation of the IPCs results in a diabetic phenotype, increased lifespan and reduced growth. Because of the growth promoting effects, the activity of the DILPs is tightly linked to dietary amino acid concentrations (Hentze, 2015).

Although metabolism has been extensively studied, the mechanisms that coordinate metabolism and feeding decisions to maintain energy balancing are poorly understood. Neuropeptides are major regulators of behavior and metabolism in mammals and insects making them obvious candidates to coordinate these processes. Peptides with a FGL-amide carboxy terminus, called type A allatostatins, have previously been related to feeding and foraging behavior. Four Drosophila Allatostatin A (AstA) peptides have been identified that are ligands for two GPCRs, the Drosophila Allatostatin A receptors DAR-1 and DAR-2. AstA peptides were originally identified as inhibitors of juvenile hormone (JH) synthesis from the corpora allata (CA) of the cockroach Diploptera punctata. However, recently it was shown that AstA does not regulate JH in Drosophila. Moreover, DAR-1 and DAR-2 are homologs of the mammalian galanin receptors, known to be involved in both feeding behavior and metabolic regulation (Hentze, 2015).

The function of AstA in Drosophila was examined in an effort to determine whether it is involved in the neuroendocrine mechanisms coupling feeding behavior to metabolic pathways that manage energy supplies. The data suggest that AstA is a modulator of AKH and DILP signaling. Dar-2 is expressed in both the IPCs and the AKH producing cells (APCs) of the CC. Silencing of AstA receptor gene Dar-2 in the APCs or IPCs resulted in changes in expression of genes associated with reduced AKH or DILP signaling, respectively. Moreover, loss of AstA is associated with increased fat body lipid levels, resembling the phenotype of mutants in the DILP and AKH pathways. The connection between nutrients and AstA signaling was also investigated, and AstA and Dar-2 were found to be regulated differently in response to dietary carbohydrates and protein, and activation of AstA-neurons was found to increase the preference for a protein rich diet, while AstA loss enhances sugar consumption. The results suggest that AstA is a key coordinator of metabolism and feeding behavior (Hentze, 2015).

In order to adjust energy homeostasis to different environmental conditions, feeding-related behavior needs to be coordinated with nutrient sensing and metabolism. The current data suggest that AstA is a modulator of AKH and DILP signaling that control metabolism and nutrient storage, but also affects feeding decisions. The positive effect of AstA on AKH signaling indicated by these observations is supported by the recent finding that expression of a presumably constitutive active mu opioid receptor, a mammalian GPCR which is also closely related to DAR-2, stimulates AKH release from the APCs in Drosophila. Moreover, AstA-type peptides have also been shown to stimulate AKH release in Locusta migratoria. AKH is primarily regulated at the level of secretion to allow a rapid response to metabolic needs. Considering that only a minor effect of Dar-2 silencing in the APCs on Akh transcription was detected, it is likely that AstA primarily acts at the level of AKH release in Drosophila (Hentze, 2015).

The data suggest regulation of both the DILPs and AKH by AstA indicating a close coupling between the activity of these two hormones. Consistent with this notion, the results also indicate a feedback relationship between the IPCs and APCs. The IPCs have processes that contact the corpora cardiaca (CC) cells of the ring gland and it is possible that DILPs released from these affect AKH release. The current findings are supported by a previous study that identifies a tight association between DILPs and AKH secretion in Drosophila. Furthermore, it was recently found that AKH regulates DILP3 release from the IPCs, and that sugar promotes DILP3 release, while DILP2 release is amino acid dependent. Interestingly, the data, which suggest that AstA is involved in the cross-talk between DILPs and AKH related specifically to sugar and protein, also indicate that AstA has a strong influence on dilp3 expression. Why is the relationship between the DILPs and AKH so tight? Even though insulin-like peptides reduce hemolymph sugar, they also reduce the content of stored glycogen and lipids, like AKH. Consistent with this, both AKH and the DILPs stimulate expression of tobi, which encodes a glycosidase believed to be involved in glycogen breakdown. However, since AKH and the DILPs have opposing effect on hemolymph sugar levels, a balance between these hormones is presumably required to maintain homeostasis. It is likely that different sources of AstA affect these two hormones, since the IPCs are located in the brain in proximity of AstA-positive neurites, while AstA-positive processes do not innervate the CC. Thus, it is likely that neuronal-derived AstA affects DILP secretion from the IPCs, while circulating AstA, which may be released from the endocrine cells of the gut, may be the source of AstA that acts on the APCs to regulate AKH. AstA regulation of DILP and AKH release may therefore not occur simultaneously and could also depend on the type of nutrient ingested, or be sequential. Since the data suggest feedback regulation between AKH and DILP, the overall outcome of simultaneous AstA induced activation of both cell types will not necessarily be a strong and equal increase in both hormones in the hemolymph. It is possible that AstA is involved in metabolic balancing, adjusting the ratio between AKH and DILPs in response to different dietary conditions. In mammals, glucagon and insulin are secreted simultaneously when the animal feeds on a protein-rich diet, to prevent hypoglycemia and promote cellular protein synthesis, since insulin is strongly induced after ingestion of amino acids. A similar mechanism has been proposed to explain the relationship between DILPs and AKH in Drosophila. The balance between DILP and AKH therefore may be important for resource allocation into growth and reproduction (Hentze, 2015).

Several differences in the expression of genes involved in energy mobilization were observed between males and females, which possibly reflects sex-specific strategies for energy mobilization and allocation of resources towards reproduction. Interestingly, 4EBP expression was significantly decreased in females with reduced AKH signaling, but upregulated in males. This suggests that in females AKH has a strong negative influence on DILP signaling that is not present in males. Why does the interaction between AKH and DILPs differ between sexes? An interesting possibility is that this sexually dimorphic interaction is related to the different preferences and requirements for sugar and protein in males and females. Males generally have a higher preference for sugar compared to females that prefer more dietary protein and show strong correlation between amino acid uptake, insulin and reproduction. In both mammals and Drosophila the balance between insulin and glucagon/AKH is important for nutrient homeostasis in response to high-protein versus high-sugar diets. This balance ensures that insulin promotes protein synthesis in response to dietary amino acids, while maintaining sugar levels stable, a function possibly important in females to allocate the high consumption of amino acids into reproduction. Thus, the sex-specific interplay between DILPs and AKH likely reflects difference in the metabolic wiring of males and females that underlie the sexually dimorphic reproductive requirements for dietary sugar and protein (Hentze, 2015).

Interestingly, AstA expression showed a general increase after feeding with a stronger transcriptional response of both AstA and Dar-2 to the carbohydrate rich diet compared to the protein rich diet. AstA may therefore be important for coordinating carbohydrate and protein dependent metabolic programs. The strong response to carbohydrates indicates that AstA may be involved in signaling related to carbohydrate feeding, although increased transcription may not necessarily result in elevated release of the mature AstA peptide. Nonetheless, the data indicate that feeding regulates AstA-signaling and that the response is influenced by the food composition. Consistent with the notion that AstA is involved in different responses to dietary carbohydrate and protein, this study found that flies with increased AstA neuronal activity increase their protein preference on the expense of their natural preference for sucrose. The AstA regulated circuitry may therefore be important for guiding the decision to feed on protein or sugar, a decision influenced by metabolic needs. The AstA neurons have projections that may contact the Gr5a sugar sensing neurons and AstA>NaChBac flies with increased activity of the AstA neurons display reduced feeding and responsiveness to sucrose under starvation. Thus, the increased preference for dietary protein in AstA>NaChBac flies observed in this study may be caused by reduced sucrose responsiveness. If AstA signaling is high after feeding on carbohydrates as indicated by the data showing increased expression of AstA and Dar-2, then an increase in AstA signaling might mimic carbohydrate satiety. In line with this view, the data show that animals lacking AstA enhance their intake of dietary sugar. AstA signaling may therefore increase the animals preference for essential amino acids, as suggested by a recent study indicating that amino acid depleted flies increased their taste sensitivity for amino acids, even when they were replete with glucose. Based on the current data, it is therefore proposed that AstA plays a central role in a circuitry important for encoding nutritional value related to these distinct nutrients and the regulation of feeding decisions and metabolic programs. Excess dietary sugar is associated with obesity, and this study found that flies lacking AstA enhance intake of sugar and have increased lipid storage droplets in their fat bodies, like animals lacking AKH or its receptor. Thus, the data implicate AstA in regulation of appetite and food intake related to sugar, which is relevant for understanding obesity (Hentze, 2015).

This study suggests that AstA affects metabolism through its action on two key players, the DILPs and AKH. AstA expression is induced by feeding, but exhibits a differential nutritional response to dietary sugar and protein and influence metabolic programs and feeding choices associated with the intake of these nutrients. Interestingly, the homolog of AstA, galanin, regulates both feeding and metabolism in mammals and in Caenorhabditis elegans loss of the Allatostatin/galanin-like receptor npr9 affects foraging behavior and nutrient storage. Altogether the data suggest that AstA is part of a conserved mechanism involved in coordinating nutrient sensing, feeding decisions and metabolism to ensure adequate intake of amino acids and sugar to maintain nutrient homeostasis under different feeding conditions (Hentze, 2015).

Protein Interactions

The insect adipokinetic hormones (AKHs) are a large family of peptide hormones that are involved in the mobilization of sugar and lipids from the insect fat body during energy-requiring activities such as flight and locomotion, but that also contribute to hemolymph sugar homeostasis. The first insect AKH receptors, namely those from the fruitfly Drosophila melanogaster and the silkworm Bombyx mori, have been identified (see Gonadotropin-releasing hormone receptor or GRHR). These results represent a breakthrough for insect molecular endocrinology, because it will lead to the cloning of all AKH receptors from all model insects used in AKH research, and, therefore, to a better understanding of AKH heterogeneity and actions. Interestingly, the insect AKH receptors are structurally and evolutionarily related to the gonadotropin-releasing hormone receptors from vertebrates (Staubli, 2002).

A Drosophila G protein-coupled receptor has been cloned that is structurally and evolutionarily related to the three known mammalian glycoprotein hormone (gonadotropin and thyroxin stimulating-hormone) receptors. To find additional possible Drosophila glycoprotein hormone receptors, a screen was performed, using the BLAST algorithm, of the Drosophila Genome Project database with each of the seven transmembrane helices of the first Drosophila glycoprotein hormone receptor, which resulted in the cloning of a Drosophila G protein-coupled receptor that was structurally related to the vertebrate gonadotropin-releasing-hormone (GnRH) receptors (36% amino acid residue identity with the catfish and 31% with the rat GnRH receptor). One intron in the Drosophila receptor gene occurred at the same position and had the same intron phasing as one intron in the rat GnRH receptor gene, showing that the two receptors were not only structurally related, but also evolutionarily related (Staubli, 2002).

The Drosophila GnRH receptor-related (GnRHR) receptor is an orphan receptor, and its ligand is unknown, although it is expected to be related to one of the vertebrate GnRH peptides. To find the cognate Drosophila GnRHR receptor ligand, the receptor was stably expressed in CHO cells that were also stably expressing the alpha subunit of the 'promiscuous' human G protein, G16, and one cell line (CHO/G16/PCG.6) was cloned expressing the receptor most abundantly. Two days before the assay, these cells were transiently transfected with a vector containing DNA coding for aequorin, and 3 h before the assay coelenterazine was added to the cell culture medium. Activation of the Drosophila GnRHR receptor in these pretreated cells would result in a Ca2+-induced bioluminescence response, which could easily be measured and quantified (Staubli, 2002).

The Drosophila GnHR receptor is mostly expressed in third-instar larvae. An aqueous extract was made from 400 g of third-instar larvae (about 4 × 105 animals) and whether the extract contained the GnRHR receptor ligand was investigated, by using the bioluminescence response of the above-mentioned transformed CHO cells as a bioassay. This, indeed, turned out to be the case, which enabled the purification of the ligand by HPLC. After seven HPLC purification steps, the natural ligand was purified to apparent homogeneity, i.e., a single peak of the expected form (Staubli, 2002).

The structure of the purified ligand was determined by CID experiments using an electrospray mass spectrometer. The CID spectrum showed that the structure of the purified ligand was identical to that of a previously isolated, identified, and cloned Drosophila peptide, Drm-AKH. Because the mass spectra suggested this structure, Drm-AKH was synthesized and compared the CID spectra from the natural ligand and synthetic Drm-AKH. This comparison showed that the two spectra were identical, confirming the proposed sequence of the Drosophila GnRHR receptor ligand (Staubli, 2002).

Synthetic Drm-AKH was also tested on the transformed (CHO/G16/PCG.6) cells, showing that Drm-AKH gives a clear bioluminescence response indistinguishable from that of the natural ligand. Dose-response curves showed that the bioluminescence responses induced by synthetic Drm-AKH have an EC50 of 8 × 10-10 M. Synthetic AKHs from other insect species also induced a bioluminescence response in the transformed cells, but with much less potency (e.g., hypertrehalosaemic hormone from the moth H. zea, Hez-HrTH; EC50, 2 × 10-8 M). Other neuropeptides, e.g., the Drosophila A-type allatostatins, did not activate the receptor. Even Drosophila corazonin, which has some structural features in common with the insect AKHs, did not give a response in the transformed CHO cells (Staubli, 2002).

The above data, thus, clearly show that the cognate ligand of the Drosophila GnRHR receptor is Drm-AKH. These findings illustrate that it is dangerous to put names on orphan receptors based on structural and evolutionary relationships alone ('annotations' -- they might, of course, be very useful in other contexts). Furthermore, the data represent a breakthrough for decades of work by other insect scientists to find or characterize insect adipokinetic hormone receptors. These results will now make it possible to clone all AKH receptors from all insects, and, because insect AKHs are structurally closely related to the red-pigment-concentrating hormone from crustaceans (AKH injected into crustaceans induces pigment concentration in chromatophores and red-pigment-concentrating hormone injected into insects induces lipid mobilization), it will now also be possible to clone the crustacean red-pigment-concentrating hormone receptors (Staubli, 2002).

From some insects it is known that they produce two or more different types of AKH, and it can be expected that these species have two or more different AKH receptors. The present paper identified one Drosophila AKH receptor, but the Drosophila Genome Project database contains the sequence of a second G protein-coupled receptor (CG10698) that is closely related to the first Drosophila AKH receptor (now called Drm-AKH receptor-1) both with respect to amino acid sequence and gene structure. This receptor, therefore, is most likely to be a second Drm-AKH receptor, suggesting that many or perhaps all insect species have two or more AKH receptors (Staubli, 2002).

To illustrate the opportunities that these present findings offer, an AKH receptor was cloned from another model insect, the silkworm B. mori (which belongs to a different insect order, the Lepidoptera, or moths and butterflies). This cloning was done by aligning the sequence of the Drm-AKH receptor-1 with that of the probable Drm-AKH receptor-2 (CG10698) and by using primers against their conserved regions, in conjunction with PCR and 3'/5'-RACE. The primary structure of the cloned Bombyx receptor shows that it has 48% identical amino acid residues (68% conserved residues) in common with the Drm-AKH receptor-1. Furthermore, two potential glycosylation sites occur at the same positions within the two receptors (Staubli, 2002).

The B. mori AKH (Bom-AKH) receptor was expressed in CHO/G16 cells; it was found to be activated by low concentrations of a moth AKH peptide, the H. zea hypertrehalosaemic hormone (Hez-HrTH; EC50, 3 × 10-10 M). Hez-HrTH has not been isolated from Bombyx so far, but another AKH peptide has been purified from this silkworm, which turned out to be identical to Mas-AKH, an AKH peptide originally isolated from the moth M. sexta. Mas-AKH also activated the Bom-AKH receptor, but with a lower affinity than Hez-HrTH (EC50, 8 × 10-9 M). These results suggest that Bombyx has a second intrinsic AKH that is more related to Hez-HrTH than to Mas-AKH and that the Bom-AKH receptor is the high-affinity receptor for this second Bombyx AKH peptide. Drm-AKH did also activate the Bombyx receptor, but with a much lower potency than Hez-HrTH (EC50, 2 × 10-8 M), whereas other insect AKHs, such as the locust peptide Schistocerca-AKH-II, were less effective. Corazonin did not stimulate the receptor, nor did other insect peptides that were unrelated to AKH, or PBS alone. All of these data show that the Bombyx receptor is an AKH receptor that reacts to Bombyx and other moth AKHs with high affinity (Staubli, 2002).

The insect AKH receptors are structurally and evolutionarily related to the GnRH receptors from mammals. It is often true that evolutionarily related G protein-coupled receptors in different animal groups might have exchanged their ligands, but that their basic functional properties have roughly remained unchanged. The allatostatin receptors from insects, for example, are structurally clearly related to the somatostatin, galanin, and opioid receptors from mammals. Both the insect and the mammalian receptors are generally inhibitory receptors, a function that, thus, has been conserved, but their ligands are different in structure. Another example is that of the oxytocin/vasopressin receptor family, where the ligands have remained relatively similar during evolution (five of nine residues and a disulfide ring structure have been conserved). These receptors have been cloned from mammals (there exists one oxytocin and three vasopressin receptors in humans), lower vertebrates, and invertebrates and they are structurally and evolutionarily clearly related to each other (both within a mammalian species and across the different animal classes and phyla). The mammalian oxytocin receptors are often involved in various aspects of reproduction (estrous cycle length, partner bond, sexual behavior, birth, milk ejection during lactation, and offspring care). Similar functions of these receptors can be found in other vertebrates and even in invertebrates, such as snails. The involvement of the oxytocin/vasopressin receptors with reproductive processes, has thus been conserved during a very long period of animal evolution. The obvious question that might be raised, therefore, is in how far insect AKH and mammalian GnRH receptors are functionally related. Does sugar and fat mobilization have something to do with sex and reproduction (Staubli, 2002)?

G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous 'orphan' GPCRs have become available for functional analysis. This study analyzes two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were constructed and tested. The insect PRXa peptides are evolutionarily related to the vertebrate peptide neuromedin U (NMU), or are related to arginine vasopressin (AVP), both of which have PRXa motifs. Seven Drosophila GPCRs related to receptors for NMU and AVP were cloned and expressed in Xenopus oocytes for functional analysis. Four Drosophila GPCRs in the NMU group (CG11475, CG8795, CG9918, CG8784) are activated by insect PRXa pyrokinins, (FXPRXamide), Cap2b-like peptides (FPRXamide), or ecdysis triggering hormones (PRXamide). Three Drosophila GPCRs in the vasopressin receptor group respond to crustacean cardioactive peptide (Cardioacceleratory peptide), corazonin, or adipokinetic hormone (AKH), none of which are PRXa peptides. These findings support a theory of coevolution for NMU and Drosophila PRXa peptides and their respective receptors (Park, 2002).

Examination of the three Drosophila GPCRs homologous to the AVP receptor yielded serendipitous findings. CG6111, orthologous to the vasopressin receptor, is activated by CCAP and AKH. CG10698 and CG11325 are activated by corazonin and AKH, respectively. The EC50 values for receptors in the AVP group are consistently lower than those observed in the NMU PRXa group (Park, 2002).

It is surprising that CG6111, an orthologous gene of AVP receptor, is activated by CCAP and AKH, but not by AVP. The presence of an insect vasopressin-like peptide was reported in locust, but searches of the Drosophila genome sequence to locate a candidate AVP-like peptide sequence were unsuccessful. CCAP and AVP both are C-terminally amidated, disulfide bridged peptides, but share no significant sequence similarity. Current data set favors assignment of CG6111 as an authentic CCAP receptor because of ligand cross-reactivity within this group of GPCRs. It seems reasonable to have residual functional cross-activity within recent evolutionary diverged GPCRs. Further work is needed to verify whether CG6111 is an authentic CCAP receptor or is a receptor for unidentified Drosophila AVP-like peptide cross-reacting to the CCAP (Park, 2002).

CG10698 is activated by corazonin with an EC50 of 1 nM. Similarly, CG11325, previously cloned by its homology to GNRHR, is activated by AKH with an EC50 of 0.3 nM. These evolutionarily related GPCRs, activated by structurally similar signaling peptides, reveal a clear case of receptor-ligand coevolution (Park, 2002).

CCAP, corazonin, and AKH have overlapping biological functions, and thus it is not unexpected that their receptors would fall into an evolutionarily related group. CCAP was initially identified by its cardioacceleratory action on the heart of the shore crab and in the tobacco hawkmoth, Manduca. The primary structure of this peptide appears to be strictly conserved across the arthropods. Additional functions of CCAP include myotropic actions, induction of AKH release in corpora cardiaca of locust, and induction of ecdysis behaviors. Corazonin is known for its cardioactive function in cockroach and pigment modulation in locust. AKH and related peptides, grouped with red pigment concentrating hormone of crustacea are cardioacceleratory and have metabolic functions such as lipid and carbohydrate mobilization (Park, 2002).

Structure-activity studies of Drosophila adipokinetic hormone (AKH) by a cellular expression system of dipteran AKH receptors

Structure-activity studies for the adipokinetic hormone receptor of insects were for the first time performed in a cellular expression system. A series of single amino acid replacement analogues for the endogenous adipokinetic hormone of Drosophila melanogaster [pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH(2)] were screened for activity with a bioluminescence cellular assay, expressing the G-protein coupled receptor. For this series of peptide analogues, one amino acid of the N-terminal tetrapeptide was successively replaced by alanine, while those of the C-terminal tetrapeptide were successively substituted by glycine; other modifications included the blocked N- and C-termini that were replaced by an acetylated alanine and a hydroxyl group, respectively. The analogue series was tested on the AKH receptors of two dipteran species, D. melanogaster and Anopheles gambiae. The blocked termini of the AKH peptide probably play a minor role in receptor interaction and activation, but are considered functionally important elements to protect the peptide against exopeptidases. In contrast, the amino acids at positions 2, 3, 4 and 5 from the N-terminus all seem to be crucial for receptor activation. This can be explained by the potential presence of a β-strand in this part of the peptide that interacts with the receptor. The inferred β-strand is probably followed by a β-turn in which the amino acids at positions 5-8 are involved. In this β-turn, the residues at positions 6 and 8 seem to be essential, as their substitutions induce only a very low degree of receptor activation. Replacement of Asp(7), by contrast, does not influence receptor activation at all. This implies that its side chain is folded inside the β-turn so that no interaction with the receptor occurs (Caers, 2012).

DEVELOPMENTAL BIOLOGY

In situ hybridization identifies sites of Drosophila AKH synthesis towards the base of the third larval instar ring gland. Like other RPCH (red pigment concentrating hormone)/AKH family peptides, DAKH can act as a cardioaccelerator at least in prepupae. Peptide levels measured in wildtype and mutant flies possessing one or three copies of the DAKH gene suggest that the amount of neuropeptide per fly is tightly regulated (Noyes, 1995).

The larval ring gland is an important endocrine organ in the cyclorraphous Diptera, consisting of the corpus allatum (CA), prothoracic gland, and corpora cardiaca (CC). The dAkh gene was cloned and shown to produce mRNA exclusively in the CC of third-instar larvae (Noyes, 1995); however, dAkh-expressing tissue types beyond this juvenile stage have not been examined (Lee, 2004).

Prior to the examination of dAkh expression patterns in adult flies, whole-mount in situ hybridizations was performed on third-instar larval tissues to validate a new antisense dAkh riboprobe. In agreement with previous results, the probe produced specific signals exclusively in the CC. Using this probe, the assay was extended to adult tissues. During metamorphosis, the ring gland migrates posteriorly and finally attaches to the esophagus, just anterior to the cardia (or proventriculus) in the adult thorax. Strong and unique in situ hybridization signals were detected in a tiny structure located at this position. The results suggest that the CC-specific dAkh expression pattern remains unchanged during metamorphosis (Lee, 2004).

To determine whether AKH peptides are actually synthesized in the CC cells, whole-mount immunohistochemistry was performed using anti-AKH antibodies. Consistent with dAkh mRNA expression patterns, AKH-immunoreactive signals were limited to the CC of both larvae and adults, suggesting that intrinsic neurosecretory cells in the CC actively produce AKH peptides during both juvenile and adult stages. Essentially identical expression patterns obtained by both techniques also verify the specificity of the antibody to the AKH peptides. From the results, it is concluded that the CC is the only tissue type expressing the dAkh gene in Drosophila melanogaster (Lee, 2004).

Using the Gal4-UAS system to drive lacZ in the pattern of normal Akh expression (dAkh-gal4/UAS-lacZ), the earliest developmental stage of dAkh expression was determined. The ß-gal activity was at first faint in a paired structure in approximately stage-14 embryos and then became stronger in older embryos. CC-specific expression was also observed in first-instar larvae; however, projections from the CC neurons were undetectable, suggesting that the dAkh neurons in first-instar larvae have not yet been fully differentiated. Nevertheless, the overall results suggest that normal dAkh gene functions might be necessary from late embryonic stages onward (Lee, 2004).

Little is known about neuro-anatomical details of the intrinsic neurosecretory cells in the CC of Drosophila. Since dAkh gene products could serve as a useful marker for such cells, characteristics of these cells were further examined in great detail, using various transgenic manipulations and histochemical assays. (1) In determining the number of dAkh-expressing cells, dAkh-gal4 flies were crossed to a UAS-NZ reporter to express ß-gal in the nuclei of dAkh cells. As a result, ~7 cells per each lobe of larval CC were identified. For adult CC, the total number of dAkh cells was counted from a whole CC structure instead of per each lobe, since the boundary between lobes was not clearly recognizable, thus hampering precise counting. This yielded an average of 13 cells per CC, ranging from 11 to 16. Since the counts in an adult CC are comparable to those observed in an entire larval CC, dAkh cells might be present persistently during metamorphosis (Lee, 2004).

To determine the population of dAkh cells in the CC, somata of dAkh neurons were simultaneously marked by dAkh promoter-driven gfp expression and nuclei of entire CC cells were marked DAPI staining. A majority of the DAPI-positive cells expressed gfp, suggesting that dAkh cells represent most of the CC cells (Lee, 2004).

Stainings mediated by anti-AKH antibody and X-gal histochemistry were examined at higher resolution to construct a fine neural mapping involving the AKHergic neurons. In larvae, two potential targets were detected innervated by AKHergic neurons, one of which is the prothoracic gland located immediately adjacent to the CC and known to produce a molting hormone ecdysteroid. The AKHergic neurons sent two or three projections to this structure. The other target is the aorta (or dorsal vessel) that is closely associated with the CC. Extensive AKH-immunoreactive varicosities observed on the aorta indicate that AKHs are released into the circulatory system (Lee, 2004).

Although it is not as clear as in larval CC, adult CC also form a bi-lobed structure and the dAkh neurons are present in both lobes. Processes stemming from the anterior side of the lobes were traced proximate to the esophagus foramen where they are likely to enter the protocerebrum. A pair of long processes arising from the posterior side reached the crop duct at which the crop begins its expansion. In some insects, such as honeybees and blow flies, the crop stores liquid foods (e.g., nectar or soluble nutrients), and its volume is highly variable depending on the amounts of liquid deposit. AKH-homologous peptides have been proposed to cause regurgitation of nectars from the crop in some wasp species to increase hemolymph trehalose titers. In this regard, the findings of AKH nerve terminals at the crop duct support the idea that AKH may control the crop volume in Drosophila. In addition to the brain and the crop, a process whose target could not be identified was occasionally observed. Nonetheless, this implies additional physiological functions attributed to AKH in adult flies (Lee, 2004).


Functions of Adipokinetic hormone orthologs in other species

AKH in non-insect arthropods

The role of the crustacean octapeptide red pigment concentrating hormone (RPCH) in the control of crayfish retinal activity was explored. RPCH injection into intact animals resulted, after a latency of 10-30 min, in a dose-dependent enhancement of electroretinogram (ERG) amplitude lasting 60-120 min. RPCH was able to potentiate ERG amplitude in both light-adapted and dark-adapted animals. Following light-adaptation, responsiveness to RPCH was five times higher than following dark-adaptation. In conjunction with ERG enhancement, in light-adapted animals, RPCH injection elicited a dose-dependent retraction of distal retinal pigment, but did not affect proximal retinal pigment position. The effects of RPCH were blocked by a polyclonal antibody raised against a tyrosinated form of RPCH (A-tyr-RPCH). The antibody was also capable of partially blocking the nocturnal phase of the circadian rhythm of ERG amplitude and the darkness-induced retraction of distal retinal pigment. These results suggest that RPCH acts both on the retinal photoreceptors and on the distal pigment cells, playing a physiological role as a mediator of the effects induced by darkness and by the nocturnal phase of the circadian rhythm (Garfias, 1995).

The octapeptide red pigment-concentrating hormone is capable of eliciting the aggregation of intracellular pigment granules in distal retinal pigment cells of isolated retinas of the crayfish Procambarus clarkii (Girard). The final level and the time course of pigment aggregation are dose dependent within a range of 10-10 mol l-1 to 10-4 mol l-1. The effect of red pigment-concentrating hormone is prevented by previous incubation with an anti-red pigment-concentrating hormone antibody; however, application of the antibody after the onset of the red pigment-concentrating hormone effect, does not prevent its full development. A similar effect to that elicited by red pigment-concentrating hormone is induced by the calcium ionophores ionomycin and A-23187. Red pigment-concentrating hormone evokes entry of 45Ca2+ to retinal cells. However, the red pigment-concentrating hormone-induced pigment aggregation persists in the presence of the calcium channel blocker verapamil and in Ca2+-free solutions. Caffeine and thapsigargin, known to release calcium from intracellular stores, elicit distal pigment aggregation, while ryanodine and dantrolene, blockers of intracellular calcium release, as well as the intracellular calcium chelator bapta-AM suppress the effect of red pigment-concentrating hormone. These results suggest that red pigment-concentrating hormone elicits distal retinal pigment aggregation by increasing intracellular calcium concentration, acting via a dual mechanism: (1) promoting calcium entry, and (2) releasing intracellular calcium (Porras, 2001).

Adipokinetic hormones and their G protein-coupled receptors emerged in Lophotrochozoa

Most multicellular animals belong to two evolutionary lineages, the Proto- and Deuterostomia, which diverged 640-760 million years (MYR) ago. Neuropeptide signaling is abundant in animals belonging to both lineages, but it is often unclear whether there exist evolutionary relationships between the neuropeptide systems used by proto- or deuterostomes. An exception, however, are members of the gonadotropin-releasing hormone (GnRH) receptor superfamily, which occur in both evolutionary lineages, where GnRHs are the ligands in Deuterostomia and GnRH-like peptides, adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP) are the ligands in Protostomia. AKH is a well-studied insect neuropeptide that mobilizes lipids and carbohydrates from the insect fat body during flight. This paper shows that AKH is not only widespread in insects, but also in other Ecdysozoa and in Lophotrochozoa. Furthermore, two G protein-coupled receptors (GPCRs) from the oyster Crassostrea gigas (Mollusca) that are activated by low nanomolar concentrations of oyster AKH (pQVSFSTNWGSamide) were cloned and deorphanized . The discovery of functional AKH receptors in molluscs is especially significant, because it traces the emergence of AKH signaling back to about 550 MYR ago and brings closer a more complete understanding of the evolutionary origins of the GnRH receptor superfamily (Li, 2016).

Physiological effects of AKH in hormones

The primary structures of two neuropeptides, Tabanus atratus adipokinetic hormone (Taa-AKH) and Tabanus atratus hypotrehalosemic hormone (Taa-HoTH), from the corpora cardiaca of horse flies (Diptera: Tabanidae) have been determined. Amino acid sequences of Taa-AKH (less than Glu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-NH2) and Taa-HoTH (less than Glu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-Gly-Tyr-NH2) (where less than Glu = pyroglutamic acid) were determined by automated gas-phase Edman degradation of the peptides deblocked by pyroglutamate aminopeptidase and by fast atom bombardment mass spectrometry. The hormones were synthesized, and the natural and synthetic peptides exhibited identical chromatographic, spectroscopic, and biological properties. When assayed in adult face fly males, Taa-AKH and Taa-HoTH demonstrated hyperlipemic activity, in addition, Taa-HoTH also demonstrated a significant hypotrehalosemic activity (Jaffee, 1989).

A hypertrehalosaemic neuropeptide from the corpora cardiaca of the blowfly Phormia terraenovae has been isolated by reversed-phase h.p.l.c., and its primary structure was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The octapeptide has the sequence pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 and is clearly defined as a novel member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of peptides. It is the first charged member of this family to be found. The synthetic peptide causes an increase in the haemolymph carbohydrate concentration in a dose-dependent fashion in blowflies and therefore is named 'Phormia terraenovae hypertrehalosaemic hormone' (Pht-HrTH). In addition, receptors in the fat-body of the American cockroach (Periplaneta americana) recognize the peptide, resulting in carbohydrate elevation in the blood. However, fat-body receptors of the migratory locust (Locusta migratoria) do not recognize this charged molecule, and thus no lipid mobilization is observed in this species (Gade, 1990).

The peptide hormone which controls activation of fat body glycogen phosphorylase in starving larvae of Manduca sexta was isolated from larval corpora cardiaca and sequenced by FAB tandem mass spectrometry. It was found to be identical with Manduca AKH. This, together with earlier observations, demonstrates that in M. sexta AKH controls glycogen phosphorylase activation in starving larvae while in adults it controls lipid mobilization during flight. Larval corpora cardiaca contain about 10 times less AKH than the corpora cardiaca of adults. The corpora cardiaca of M. sexta appear to contain only one AKH (Ziegler, 1990).

Hypertrehalosemic hormone (a carbohydrate-mobilizing neuroendocrine decapeptide) and starvation markedly increases levels of a cockroach (Blaberus discoidalis) fat body cytochrome P450 message. The gene represented by the cloned P450 cDNA has been named CYP4C1 (cytochrome P450 family 4, subfamily C, gene 1), a newly identified member of the ubiquitous cytochrome P450 monooxygenase gene superfamily. Blaberus CYP4C1 (511 amino acids, Mr = 58,485) has a hydrophobic NH2 terminus and a sequence near the COOH terminus that is homologous to the cysteine-containing heme-binding region definitive of cytochromes P450. The cockroach sequence is 32%-36% identical to mammalian family 4A and 4B enzymes. It contains a 13-residue sequence characteristic of family 4 but not other P450s. This study suggests that CYP4C1 is hormonally regulated in association with energy substrate mobilization and supports the idea that family 4 is an old and widespread gene family (Bradfield, 1991).

A simple preparation designed to screen and compare the central action of putative neuroactive agents in the moth Manduca sexta is described. This approach combines microinjections into the central nervous system with myograms recorded from a pair of spontaneously active mesothoracic muscles. Pressure injection of either octopamine or Manduca adipokinetic hormone (M-AKH) into the mesothoracic neuropile increases the monitored motor activity. Under the conditions used, the excitatory effects of M-AKH exceed those of the potent neuromodulator octopamine. This suggests that M-AKH plays a role in the central nervous system in addition to its known metabolic functions and supports recent evidence that neuropeptides in insects can be multifunctional (Milde, 1995).

Blaberus hypertrehalosemic hormone (Bld-HTH)-dependent glycogen phosphorylase activation was investigated using in vitro fat bodies from the cockroach, Blaberus discoidalis. Resting levels of active phosphorylase were decreased by the presence of trehalose and glucose. Phosphorylase activation was dose-responsive to Bld-HTH and increased ca. 3-fold over a range of 0.02 to 2 nM Bld-HTH. Maximum phosphorylase activation required only 5-min exposure to Bld-HTH; reversion to the inactive state began within 15 min after Bld-HTH removal and was completed by 60 min. Octopamine also activated phosphorylase but required 10(3)-fold higher concentrations than did Bld-HTH. Concentrations of Bld-HTH and octopamine that increased active phosphorylase did not elevate fat body cAMP levels, although a high concentration of octopamine increased tissue cAMP levels. cAMP did not increase phosphorylase activity, but Ca2+ was important for both Bld-HTH- and octopamine-dependent phosphorylase activation (Park, 1995).

Signaling mechanisms for Blaberus discoidalis hypertrehalosemic hormone (Bld-HrTH)-dependent glycogen phosphorylase activation were investigated in vitro using fat body of the tropical cockroach, B. discoidalis. Brief treatment of fat bodies with Bld-HrTH in the absence of extracellular Ca2+ showed a significant activation of phosphorylase. Although extracellular Ca2+ was required for a full activation of phosphorylase by Bld-HrTH, stimulation in the absence of extracellular Ca2+ suggested that intracellular Ca2+ was also involved in Bld-HrTH signal transduction. Thapsigargin and thimerosal mobilize Ca2+ from intracellular stores by different mechanisms, and both chemicals stimulated phosphorylase activities as effectively as a maximum dose of Bld-HrTH. Bld-HrTH likely induces intracellular Ca2+ release followed by extracellular Ca2+ entry across the plasma membrane. Inositol-1,4,5-trisphosphate (InsP3) levels were greatly increased by Bld HrTH in a time- and dose-dependent manner, suggesting that InsP3 might be a Ca(2+)-mobilizing intracellular second messenger for Bld-HrTH (Park, 1996).

The pathway for the adipokinetic hormone-stimulated synthesis of sn-1,2-diacylglycerols in the adult Manduca sexta fat body was studied. Adult fat body lipids were labeled by feeding 5th instar larvae either with labelled oleic acid or glycerol and after 32 days insects at the adult stage were used. This long-term prelabeling led to labeled fat body acylglycerols in which triacylglycerols comprised the main radioactive lipid component (95.5%), regardless of the radiolabeled compound used. Because the distribution of radioactivity among the lipid classes was very close to the mass distribution of the fat body lipid subspecies, it was concluded that homogeneous labeling of fat body lipids was obtained. After adipokinetic hormone treatment, an accumulation of radioactivity in the sn-1,2-diacylglycerol fraction was the only significant change found in the distribution of radioactivity among fat body lipids. The size of diacylglycerol pool increased 280% 60 min after adipokinetic hormone stimulation, whereas the fatty acid, monoacylglycerol and phosphatidic acid pool sizes remained constant. These results support the hypothesis that adipokinetic hormone-stimulated synthesis of sn-1,2-diacylglycerol in the fat body involves stereospecific hydrolysis of the triacylglycerol stores (Arrese, 1997).

The corpora cardiaca (CC) of the Italian race (including also the africanised variety) of the honeybee (Apis mellifera ligustica) contain approximately 3 pmol of a hypertrehalosaemic peptide. This peptide is identical in structure to the adipokinetic hormone (AKH) found in Manduca sexta, Mas-AKH. The CC of the dark European race of the honeybee (Apis mellifera carnica) contain no detectable Mas-AKH or any other adipokinetic/hypertrehalosaemic peptide. This is the first report of the occurrence of this peptide in a non-lepidopteran insect and of an intraspecific variation with regards to the presence or absence of a hypertrehalosaemic peptide in the CC of an insect. Extracts of A. m. ligustica CC elicit a strong adipokinetic/hypertrehalosaemic response when injected into crickets and cockroaches but extracts of A. m. carnica CC elicit no such responses when injected into crickets, cockroaches and butterflies. A weak hypertrehalosaemic response to injected Mas-AKH was observed in winter bees of both races, but there was no response in spring/summer bees. However, if a seasonal difference exists, it is at best minimal. Honeybees always have access to a more than adequate supply of high energy food in the form of nectar or honey stored in the hive. Thus, though A. m. ligustica CC contain a hypertrehalosaemic peptide, there is neither a glycogen-mobilising function of this hormone nor an adequate glycogen store in their fat body for its effective utilisation (Lorenz, 1999).

Stereospecific hydrolysis of stored triacylglycerol by a phosphorylatable triacylglycerol-lipase is the pathway for the adipokinetic hormone-stimulated synthesis of sn -1, 2-diacylglycerol in insect fat body. The current series of experiments were designed to determine whether cAMP and/or calcium are involved in the signal transduction pathway for adipokinetic hormone in the fat body. After adipokinetic hormone treatment, cAMP-dependent protein kinase activity in the fat body rapidly increased and reached a maximum after 20 min, suggesting that adipokinetic hormone causes an increase in cAMP. Forskolin, an adenylate cyclase activator, induced up to a 97% increase in the secretion of diacylglycerol from the fat body. 8Br-cAMP (a membrane-permeable analog of cAMP) produced a 40% increase in the hemolymph diacylglycerol content. Treatment with cholera toxin, which also stimulates adenylate cyclase, induced up to a 145% increase in diacylglycerol production. Chelation of extracellular calcium produced up to 70% inhibition of the adipokinetic hormone-dependent mobilization of lipids. Calcium-mobilizing agents, ionomycin and thapsigargin, greatly stimulated DG production by up to 130%. Finally, adipokinetic hormone caused a rapid increase of calcium uptake into the fat body. These findings indicate that the action of adipokinetic hormone in mobilizing lipids from the insect fat body involves both cAMP and calcium as intracellular messengers (Arrese, 1999).

Four locustatachykinins (LomTK I-IV) were identified in about equal amounts in extracts of corpora cardiaca of locusts, using reverse-phase high-performance liquid chromatography and radioimmunoassay with synthetic LomTK I-IV as standards. Brain extracts also contained the four isoforms in roughly equimolar concentrations. Retrograde tracing of the nervi corporis cardiaci II (NCC II) in vitro with Lucifer yellow in combination with LomTK immunocytochemistry revealed that about half of the secretomotor neurons in the lateral part of the protocerebrum projecting into the glandular lobe of the corpora cardiaca (CCG) contain LomTK-immunoreactive material. Since the four LomTKs are present in the CCG, these four or five neurons in each hemisphere are likely to contain colocalized LomTK I-IV. The role of two of the LomTKs in the regulation of the release of adipokinetic hormones (AKHs) from the adipokinetic cells in the CCG in the locust was investigated. Experiments performed in vitro showed that LomTK I and II induced release of AKH in a dose-dependent manner. These peptides also rapidly and transiently elevated the cyclic AMP-content of the CCG. The peak level of cyclic AMP occurred about 45 seconds after stimulation with LomTK. These results support the proposal that LomTKs are involved in controlling the release of the adipokinetic hormones and suggest that all LomTK isoforms may participate in this cyclic AMP-mediated event (Nassel, 1999).

Feeding effects on hypertrehalosemic hormone (HTH) transcript levels in corpora cardiaca (CC) of adult females of the cockroach, Blaberus discoidalis were measured using dot blot hybridization. HTH transcript levels were nearly doubled in CC from females withheld from food and water for ten days compared to CC from fed females. The increase in HTH-mRNA was a response to starvation, not dehydration, and reversed within 2 days after exposure to food. HTH-mRNA was elevated in CC from fed insects that had their recurrent nerve severed, but low fecal output by insects with severed nerves indicated that feeding and digestion were impaired. Thus, the increased HTH synthesis likely resulted from starvation rather than disruption of neural regulation. CC from starved females that were refed with either solutions or agar that contained glucose did not show down-regulation of HTH-mRNA. Likewise, injections of glucose or trehalose did not suppress HTH-mRNA levels in CC of starving insects. Down-regulation of the starvation-related increase in HTH-mRNA appears to be a response to consumption of a complex of nutrients and not to increased carbohydrates or mechanical aspects of feeding (Keeley, 1998).

Adipokinetic hormone (AKH)-producing cells in the corpus cardiacum of the insect Locusta migratoria represent a neuroendocrine system containing large quantities of stored secretory peptides. The question whether the release of AKHs from these cells induces a concomitant enhancement of their biosynthesis has been addressed. The effects of hormone release in vivo (by flight activity) and in vitro (using crustacean cardioactive peptide, locustamyoinhibiting peptide, and activation of protein kinase A and C) on the biosynthetic activity for AKHs were measured. The intracellular levels of prepro-AKH mRNAs, the intracellular levels of pro-AKHs, and the rate of synthesis of (pro-)AKHs were used as parameters for biosynthetic activity. The effectiveness of in vitro treatment was assessed from the amounts of AKHs released. Neither flight activity as the natural stimulus for AKH release, nor in vitro treatment with the regulatory peptides or signal transduction activators appeared to affect the biosynthetic activity for AKHs. This points to an absence of coupling between release and biosynthesis of AKHs. The strategy of the AKH-producing cells to cope with variations in secretory stimulation seems to rely on a pool of secretory material that is readily releasable and continuously replenished by a process of steady biosynthesis (Hawthoorn, 2001).

A new hypertrehalosaemic peptide [Tea-HrTH; pQLNFSTGWGG-NH(2)] was isolated from the corpora cardiaca (CC) of the sawfly Tenthredo arcuata. The hypertrehalosaemic peptides found in the CC of five Bombus species and the paper wasp Polistes fuscata were identical to the adipokinetic hormone II of the desert locust, Schistocerca gregaria (Scg-AKH-II). The hypertrehalosaemic peptides found in the yellowjacket Vespula vulgaris and the hornet Vespa crabro were identical to the adipokinetic hormone of the cricket, Gryllus bimaculatus (Grb-AKH). All species examined had a large storage crop which, when filled with honey, held up to one-third of their total body weight. Overwintering queens of P. fuscata had large stores of carbohydrates and lipids in the abdomen, and were able to survive months of fasting. Workers of Bombus hortorum (bumble-bee), Apis mellifera (honey-bee) and V. vulgaris had little or no fat body. These species could fly as long as sugar was present in their crops, but they stopped flying as the carbohydrates in the crop disappeared. There was no significant increase in the haemolymph carbohydrate titres after injections of CC extracts or corresponding synthetic peptides into workers of B. hortorum or into males and females of T. arcuata. There was a moderate increase in haemolymph carbohydrate titres when these peptides were injected into overwintering queens of P. fuscata and into workers of V. crabro, both with significant amounts of fat body. However, well-fed V. vulgaris workers, with very little fat body, also responded to their own hypertrehalosaemic peptide (Lorenz, 2001).

This report examines three aspects of adipokinetic hormone (AKH) involvement in migratory flight behavior in the grasshopper, Melanoplus sanguinipes. The titer of hemolymph AKH I during long-duration tethered flight was examined using radioimmunoassay (RIA) after narrow bore RP-HPLC. The hemolymph fraction containing AKH I was assayed using commercially available anti-Tyr1-AKH I serum. Titer determinations of hemolymph AKH were done at rest and after various periods of flight. The amount of AKH I released from the corpora cardiaca during flight was estimated. When resting levels of AKH I and II in corpora cardiaca (CC) of migrants and non-migrants were examined with HPLC, no significant differences in AKH levels were detected between non-migrants, animals that had flown for 1 h to identify them as migrants, and animals that had flown to exhaustion (i.e., voluntary cessation). CC levels of both AKH I and II were less in this species than in locusts. When the lipid mobilization in response to AKH I and II was compared in migrants (animals that had self-identified as migrants in a 1-h tethered flight test) and non-migrants (animals that would not perform a 1-h flight in a tethered flight test), the adipokinetic response to AKH I was greater in migrants than in non-migrants, possibly indicating differences in level of sensitivity or number of receptors in the target tissues. AKH II had little effect on hemolymph lipid levels in either flight group, and may not play a significant role in lipid mobilization in this species (Min, 2004).

Adipokinetic hormone-induced mobilization of fat body triglyceride stores in Manduca sexta: Role of TG-lipase and lipid droplets

Triglycerides (TG) stores build up in the insect fat body as lipid droplets at times of excess of food. The mobilization of fat body triglyceride (TG) is stimulated by adipokinetic hormones (AKH). The action of AKH involves a rapid activation of cAMP-dependent protein kinase (PKA). Recent in vitro studies have shown that PKA phosphorylates and activates the TG-lipase substrate, the lipid droplets. Conversely, purified TG-lipase from Manduca sexta fat body is phosphorylated by PKA in vitro but is not activated. This study was directed to learn whether or not AKH promotes a change in the state of phosphorylation of the lipase in vivo, and what are the relative contributions of cytosol and lipid droplets to the overall increase of lipolysis triggered by AKH. TG-lipase activity of fat body cytosols isolated from control and AKH-treated insects was determined against the native substrate, in vivo [3H]-TG radiolabeled lipid droplets, obtained from control and AKH-treated insects. The lipase activity of the system composed of AKH-cytosol and AKH-lipid droplets was 3.1-fold higher than that determined with control cytosol and lipid droplets. Evaluation of the role of AKH-induced changes in the lipid droplets on lipolysis showed that changes in the lipid droplets are responsible for 70% of the lipolytic response to AKH. The remaining 30% appears to be due to AKH-dependent changes in the cytosol. However, the phosphorylation level of the TG-lipase was unchanged by AKH, indicating that phosphorylation of the TG-lipase plays no role in the activation of lipolysis induced by AKH (Patel, 2006).


REFERENCES

Search PubMed for articles about Drosophila Adipokinetic hormone

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Arrese, E. L., et al. (1999). Calcium and cAMP are second messengers in the adipokinetic hormone-induced lipolysis of triacylglycerols in Manduca sexta fat body. J. Lipid Res. 40(3): 556-64. 10064744

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Gade, G., Wilps, H. and Kellner, R. (1990). Isolation and structure of a novel charged member of the red-pigment-concentrating hormone-adipokinetic hormone family of peptides isolated from the corpora cardiaca of the blowfly Phormia terraenovae (Diptera). Biochem. J. 269(2): 309-13. 2386478

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Lee, G. and Park, J. H. (2004). Hemolymph sugar homeostasis and starvation-induced hyperactivity affected by genetic manipulations of the Adipokinetic hormone-encoding gene in Drosophila melanogaster. Genetics 167: 311-323. 15166157

Li, S., Hauser, F., Skadborg, S. K., Nielsen, S. V., Kirketerp-Moller, N. and Grimmelikhuijzen, C. J. (2016). Adipokinetic hormones and their G protein-coupled receptors emerged in Lophotrochozoa. Sci Rep 6: 32789. PubMed ID: 27628442

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Biological Overview

date revised: 10 October 2017

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