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).
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).
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).
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).
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. 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).
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