InteractiveFly: GeneBrief

Gonadotropin-releasing hormone receptor: Biological Overview | References


Gene name - Gonadotropin-releasing hormone receptor

Synonyms - Adipokinetic hormone receptor (Akhr)

Cytological map position - 27A1-27A1

Function - receptor

Keywords - fat body, lypolysis, Adipokinetic hormone signaling pathway, transmitter of lipolytic AKH signaling, functional analog of the mammalian glucagon receptor

Symbol - GRHR

FlyBase ID: FBgn0025595

Genetic map position - 2L:6,711,282..6,716,183 [-]

Classification - GPCR

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Palu, R. A. S., Praggastis, S. A. and Thummel, C. S. (2017). Parental obesity leads to metabolic changes in the F2 generation in Drosophila. Mol Metab 6(7): 631-639. PubMed ID: 28702320
Summary:
A significant portion of the heritable risk for complex metabolic disorders cannot be attributed to classic Mendelian genetic factors. At least some of this missing heritability is thought to be due to the epigenetic influence of parental and grandparental metabolic state on offspring health. Previous work suggests that this transgenerational phenomenon is evolutionarily conserved in Drosophila. These studies, however, have all depended on dietary paradigms to alter parental metabolic state, which can have inconsistent heritable effects on the metabolism of offspring. This study use AKHR null alleles to induce obesity in the parental generation and then score both metabolic parameters and genome-wide transcriptional responses in AKHR heterozygote F1 progeny and genetically wild-type F2 progeny. Unexpectedly, elevated glycogen levels and changes in gene expression were observed in AKHR heterozygotes due to haploinsufficiency at this locus. It was also shown that genetic manipulation of parental metabolism using AKHR mutations results in significant physiological changes in F2 wild-type offspring of the grandpaternal/maternal lineage. These results demonstrate that genetic manipulation of parental metabolism in Drosophila can have an effect on the health of F2 progeny, providing a non-dietary paradigm to better understand the mechanisms behind the transgenerational inheritance of metabolic state.
BIOLOGICAL OVERVIEW

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).

A glucagon-like endocrine pathway in Drosophila modulates both lipid and carbohydrate homeostasis

The regulation of energy homeostasis is fundamental to all organisms. The Drosophila fat body serves as a repository for both triglycerides and glycogen, combining the energy storage functions of mammalian adipose and hepatic tissues, respectively. This study shows that mutation of the Drosophila adipokinetic hormone receptor (AKHR), a functional analog of the mammalian glucagon receptor, leads to abnormal accumulation of both lipid and carbohydrate. As a consequence of their obese phenotypes, AKHR mutants are markedly starvation resistant. AKHR is expressed in the fat body, and, intriguingly, in a subset of gustatory neurons that mediate sweet taste. Genetic rescue experiments establish that the metabolic phenotypes arise exclusively from the fat body AKHR expression. Behavioral experiments demonstrate that AKHR mutants are neither sedentary nor hyperphagic, suggesting the metabolic abnormalities derive from a genetic propensity to retain energy stores. Taken together, these results indicate that a single endocrine pathway contributes to both lipid and carbohydrate catabolism in the Drosophila fat body (Bharucha, 2008).

The Drosophila fat body serves as a major depot for storage of carbohydrates and lipids. The AKH pathways serves as a critical determinant of both glycogen and triglyceride homeostasis. Interestingly, Akhr mutants are starvation resistant, retaining the ability to mobilize their lipids stores. Thus, it appears that the AKH pathways acts as a generalized catabolic signal, mobilizing both lipid and carbohydrate energy stores. Interestingly, this work suggests that the obese phenotypes of Akhr mutants do not result from increased food intake. In fact, Akhr mutants appear to ingest less when previously challenged with starvation. It is therefore proposed that the obese phenotypes result from a genetic propensity to retain energy stores rather than by increased food ingestion. Akhr mutants do not have any gross defects in locomotor activity (as measured by DAMS), suggesting that the greater energy reserves of mutant flies do not result from decreased energy expenditure in locomotor behavior (Bharucha, 2008).

Other lipolytic mechanisms (independent of the AKH pathway) must exist in Drosophila that enable Akhr mutants to utilize their triglyceride stores and affect their starvation resistance. Recently, the AKH and brummer lipase pathways were shown to be two major pathways regulating lipolysis in Drosophila (Gronke, 2007), but that sutd concluded that AKHR does not affect carbohydrate homeostasis. This study, in striking contrast, demonstrates that AKHR affects both total body carbohydrate and lipid content. In the fed state, the percentage differences in glycogen content between Akhrnull and Akhrrev flies were not as pronounced as the differences in lipid content, perhaps accounting for this discrepancy. However, this study shows that differences in glycogen content between Akhrnull and Akhrrev flies are more readily apparent after 24 h of starvation. Genetic rescue experiments provide further support for the effect of Akhr expression on carbohydrate homeostasis. Because Akhr mutants (and brummer mutants) retain their ability to access their glycogen stores, it is predicted that additional pathways exist that regulate carbohydrate homeostasis (Bharucha, 2008).

The selective expression of Akhr in gustatory neurons that mediate attractive taste raises the interesting possibility that the AKH pathway coordinates a fly's response to hunger in two ways: (1) by mobilizing internal energy stores by its action on the fat body, and (2) increasing food intake by its action on attractive-gustatory neurons. Starved Akhr mutants display decreased food intake when re-introduced to food. However, genetic rescue experiments (using flies of the same genotype as those used for rescue of metabolic phenotypes) did not allow this altered behavior to be definitively attributed to loss of AKHR function. Therefore, the possibility that the observed feeding behavior results from a background effect cannot be rigorously excluded. Nonetheless, it is intriguing to speculate that activation of AKHR in the gustatory system promotes food intake in the hungry fly. Further work will be needed to delineate the role of gustatory Akhr expression in the context of an emerging picture of the Drosophila neuronal feeding circuit (Bharucha, 2008).

Genes that modulate the retention of fuel molecules can provide an adaptive survival benefit during periods of decreased food availability. The results are consistent with the idea that specific genetic mutations in Drosophila can serve to prolong long-term survival when flies are challenged with food deprivation. There is evidence that selective pressures can be used to increase the triglyceride content of flies both in nature and in the laboratory. For example, naturally occurring mutants of the adipose gene have higher triglyceride stores and are starvation resistant (Hader, 2003). In addition, flies with higher triglyceride stores can be generated by selecting for starvation-resistant phenotypes over several generations. Overall, more work is needed to understand better how specific genetic mechanisms contribute to the adaptation of Drosophila to specific ecological niches differing in food availability (Bharucha, 2008).

Over the approximately 600 million years of evolution that separate humans from flies from common urbilaterial ancestors, mammals have evolved discrete liver and adipose tissues that have energy storage functions performed jointly by the Drosophila fat body. Thus, AKHR expression in the fat body is uniquely poised to control mobilization of both carbohydrates and lipids. Mammals may require a more elaborate array of endocrine signals that coordinate carbohydrate and lipid homeostasis during periods of food deprivation. For example, specific genetic manipulation of the mammalian glucagon pathway is rendered difficult by the complex structure of the preproglucagon gene. Although murine glucagon receptor knockouts have abnormal carbohydrate metabolism, no obese phenotypes have been observed. Significantly, these results are confounded by upregulation of other hormone pathways. Thus, Drosophila offers a genetically tractable model organism to dissect pathways involved with energy mobilization (Bharucha, 2008).

It is anticipated that further study of the AKHR pathway will provide a better understanding of the downstream signaling components regulating glycogenolysis and lipolysis that are conserved between flies and mammals. In addition, the power of forward genetic screens in the Drosophila may uncover other determinants of energy homeostasis that have relevance to the study of human disorders of lipid and carbohydrate metabolism, such as obesity and diabetes (Bharucha, 2008).

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).

Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila

Starvation induces sustained increase in locomotion, which facilitates food localization and acquisition and hence composes an important aspect of food-seeking behavior. This study investigated how nutritional states modulate starvation-induced hyperactivity in adult Drosophila. The receptor of adipokinetic hormone (AKHR), the insect analog of glucagon, is required for starvation-induced hyperactivity. AKHR is expressed in a small group of octopaminergic neurons in the brain. Silencing AKHR+ neurons and blocking octopamine signaling in these neurons eliminates starvation-induced hyperactivity, whereas activation of these neurons accelerates the onset of hyperactivity upon starvation. Neither AKHR nor AKHR+ neurons are involved in increased food consumption upon starvation, suggesting that starvation-induced hyperactivity and food consumption are independently regulated. Single cell analysis of AKHR+ neurons identified the co-expression of Drosophila insulin-like receptor (dInR), which imposes suppressive effect on starvation-induced hyperactivity. Therefore, insulin and glucagon signaling exert opposite effects on starvation-induced hyperactivity via a common neural target in Drosophila (Yu, 2016).

Food seeking and food consumption are essential for the acquisition of food sources, and hence survival, growth, and reproduction of animal species. Starvation influences food-seeking behavior via both modulating the perception of food cues as well as enhancing flies' locomotor activity. Accumulated evidence has suggested that starvation modulates the activity of ORNs via multiple neural and hormonal cues, which in turn facilitates odor driven food search and food consumption. Similarly, starvation also modulates the perception of food taste via the relative sensitivity of appetitive sweet-sensing and aversive bitter-sensing GRNs,which may in turn increase the attractiveness of food taste. However, how starvation increases the locomotor activity of flies remains largely uncharacterized (Yu, 2016).

Consistent with previous reports, this study has shown that starved fruit flies exhibit sustained increase in their locomotor activity, which can be suppressed by food consumption induced by both nutritive and non-nutritive food cues. The present study has shown that a small group of neurons located in the subesophageal zone (SEZ) region of the fly brain are both necessary and sufficient for starvation induced hyperactivity. These neurons sense the changes in flies' internal nutritional states by directly responding to two sets of hormones, AKH and DILPs, and modulate locomotor activity in response. Single cell analysis has identified that these AKHR+dInR+ neurons are octopaminergic, which offers an entry point to trace the downstream neural circuitry that regulates starvation-induced hyperactivity. For example, there are seven candidate octopamine receptors in fruit flies and it would be of interest to investigate whether any of these receptors and the receptor-expressing neurons are involved in locomotor regulation upon starvation (Yu, 2016).

AKH and DILPs are two sets of functionally counteracting hormones in fruit flies. As its mammalian analog glucagon, the reduction in circulating sugars induces the release of AKH, which in turn mobilizes fat storage and provides energy supply for flies. In contrast, DILPs, the insect analog of mammalian insulin, function as satiety hormones. Dietary nutrient induces the release of DILPs into the hemolymph, which in turn promotes protein synthesis, body growth, and other anabolic processes. This study has shown that these two hormonal signaling systems exert opposite effects on starvation-induced hyperactivity via a small group of AKHR+InR+ octopaminergic neurons. These results suggest that these AKHR+dInR+ neurons can integrate the inputs from the two hormonal signaling systems representing hunger and satiety at the same time, and modulate flies' locomotor activity. This elegant yet concise design allows these neurons to be responsive to rapid changes in the internal nutritional states as well as food availability. Furthermore, it is possible that besides hunger and satiety, other physiological states such as wakefulness, stress, and emotions also influence flies' locomotor activity. Notably, single cell analysis has shown that these AKHR+dInR+ neurons also sparsely express other neuropeptide receptors, suggesting that at least small portions of these neurons may also receive input from other neuropeptidergic systems (Yu, 2016).

Starved animals exhibited increased locomotion and food consumption, the transition of which relies on the detection of food cues. But whether these two behaviors are interdependently or independently regulated remains unclear. This study has shown that these two behaviors are dissociable from each other in fruit flies. On the one hand, although AKHR+ neurons exert robust modulatory effect on starvation-induced hyperactivity, these neurons are neither necessary nor sufficient for starvation-induced food consumption. On the other hand, the regulation of food consumption is independent of starvation-induced hyperactivity as well. Previous studies have shown that a small subset of GABAergic neurons in the fly brain regulates food consumption but exerts no effect on 10 starvation-induced hyperactivity (Pool, 2014). In addition, several neuropeptides are known to regulate food consumption, such as Hugin, NPF, sNPF, Leucokinin, and AstA. However this study found in an RNAi screen that the receptors of these neuropeptides were not involved in the regulation of starvation-induced hyperactivity. Taken together, it is likely that starvation-induced hyperactivity and food consumption are independently regulated by different sets of hormonal cues, and that AKHR+ neurons are only involved in the former but not the latter. These results may shed light on the regulation of food intake in mammals, especially whether starvation-induced hyperactivity and food consumption are also independently regulated by different sets of hormones and distinct neural circuitry in mammals (Yu, 2016).

The evolution and nomenclature of GnRH-type and corazonin-type neuropeptide signaling systems

Gonadotropin-releasing hormone (GnRH) was first discovered in mammals on account of its effect in triggering pituitary release of gonadotropins and the importance of this discovery was recognized forty years ago in the award of the 1977 Nobel Prize for Physiology or Medicine. Investigation of the evolution of GnRH revealed that GnRH-type signaling systems occur throughout the chordates, including agnathans (e.g. lampreys) and urochordates (e.g., sea squirts). Furthermore, the discovery that adipokinetic hormone (AKH) is the ligand for a GnRH-type receptor in the arthropod Drosophila melanogaster provided evidence of the antiquity of GnRH-type signaling. However, the occurrence of other AKH-like peptides in arthropods, which include corazonin and AKH/corazonin-related peptide (ACP), has complicated efforts to reconstruct the evolutionary history of this family of related neuropeptides. Genome/transcriptome sequencing has revealed that both GnRH-type receptors and corazonin-type receptors occur in lophotrochozoan protostomes (annelids, mollusks) and in deuterostomian invertebrates (cephalochordates, hemichordates, echinoderms). Furthermore, peptides that act as ligands for GnRH-type and corazonin-type receptors have been identified in mollusks. However, what has been lacking is experimental evidence that distinct GnRH-type and corazonin-type peptide-receptor signaling pathways occur in deuterostomes. Two neuropeptides that act as ligands for either a GnRH-type receptor or a corazonin-type receptor have been identified in an echinoderm species - the common European starfish Asterias rubens. Discovery of distinct GnRH-type and corazonin-type signaling pathways in this deuterostomian invertebrate has demonstrated for the first time that the evolutionarily origin of these paralogous systems can be traced to the common ancestor of protostomes and deuterostomes. Furthermore, lineage-specific losses of corazonin signaling (in vertebrates, urochordates and nematodes) and duplication of the GnRH signaling system in arthropods (giving rise to the AKH and ACP signaling systems) and quadruplication of the GnRH signaling system in vertebrates (followed by lineage-specific losses or duplications) accounts for the phylogenetic distribution of GnRH/corazonin-type peptide-receptor pathways in extant animals. A standardized nomenclature for GnRH/corazonin-type neuropeptides is proposed wherein peptides are either named 'GnRH' or 'corazonin', with the exception of the paralogous GnRH-type peptides that have arisen by gene duplication in the arthropod lineage and which are referred to as 'AKH' (or red pigment concentrating hormone, 'RCPH', in crustaceans) and 'ACP' (Zandawala, 2017).

Molecular identification of the insect adipokinetic hormone receptors

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)?


Functions of Gonadotropin-releasing hormone receptor orthologs in other species

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).


REFERENCES

Search PubMed for articles about Drosophila Akhr

Arrese, E. L., Patel, R. T., Soulages, J. L. (2006). The main triglyceride-lipase from the insect fat body is an active phospholipase A1: Identification and characterization. J. Lipid Res. 47: 2656-2667. PubMed ID: 17005997

Bharucha, K. N., Tarr, P., Zipursky, S. L. (2008). A glucagon-like endocrine pathway in Drosophila modulates both lipid and carbohydrate homeostasis. J. Exp. Biol. 211(19): 3103-3110. Full text of article

Caers, J., et al. (2012). Structure-activity studies of Drosophila adipokinetic hormone (AKH) by a cellular expression system of dipteran AKH receptors. Gen. Comp. Endocrinol. 177(3): 332-7. PubMed ID: 22569168

Grönke S, Mildner A, Fellert S, Tennagels N, Petry S, et al. (2005) Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila. Cell Metab. 1: 323-330. PubMed ID: 16054079

Grönke, S., Müller, G., Hirsch, J., Fellert, S., Andreou, A., Haase, T., Jäckle, H. and Kühnlein, R. P. (2007). Dual lipolytic control of body fat storage and mobilization in Drosophila. PLoS Biol. 5(6): e137. PubMed ID: 17488184

Hader, T., Muller, S., Aguilera, M., Eulenberg, K. G., Steuernagel, A., Ciossek, T., Kuhnlein, R. P., Lemaire, L., Fritsch, R., Dohrmann, C. et al. (2003). Control of triglyceride storage by a WD40/TPR-domain protein. EMBO Rep. 4: 511-6. PubMed ID: 12717455

Hauser, F., Sondergaard, L. and Grimmelikhuijzen, C. J. (1998). Molecular cloning, genomic organization and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to gonadotropin-releasing hormone receptors for vertebrates. Biochem. Biophys. Res. Commun. 249: 822-828. PubMed ID: 9731220

Isabel, G., Martin, J. R., Chidami, S., Veenstra, J. A. and Rosay, P. (2005). AKH-producing neuroendocrine cell ablation decreases trehalose and induces behavioral changes in Drosophila. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288: R531-538. PubMed ID: 15374818

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

Lee, M. J. and Goldsworthy, G. J. (1995). The preparation and use of dispersed cells from fat body of Locusta migratoria in a filtration plate assay for adipokinetic peptides. Anal. Biochem. 228: 155-161. PubMed ID: 8572272

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

Lorenz, M. W. (2001). Synthesis of lipids in the fat body of Gryllus bimaculatus: Age-dependency and regulation by adipokinetic hormone. Arch. Insect Biochem. Physiol. 47: 198-214. PubMed ID: 11462224

Park, Y., Kim, Y. J. and Adams, M. E. (2002). Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution. Proc. Natl. Acad. Sci. 99: 11423-11428. 12177421

Patel, R. T., Soulages, J. L., Hariharasundaram, B. and Arrese, E. L. (2005). Activation of the lipid droplet controls the rate of lipolysis of triglycerides in the insect fat body. J. Biol. Chem. 280: 22624-22631. PubMed ID: 15829485

Patel, R. T., Soulages, J. L. and Arrese, E. L. (2006). Adipokinetic hormone-induced mobilization of fat body triglyceride stores in Manduca sexta: Role of TG-lipase and lipid droplets. Arch Insect Biochem Physiol 63: 73-81. PubMed ID: 16983668

Pool, A. H., Kvello, P., Mann, K., Cheung, S. K., Gordon, M. D., Wang, L. and Scott, K. (2014). Four GABAergic interneurons impose feeding restraint in Drosophila. Neuron 83: 164-177. PubMed ID: 24991960

Staubli, F., et al. (2002). Molecular identification of the insect adipokinetic hormone receptors. Proc. Natl. Acad. Sci. 99(6): 3446-51. 11904407

Van der Horst, D. J., Van Marrewijk, W. J. and Diederen, J. H. (2001). Adipokinetic hormones of insect: release, signal transduction, and responses. Int. Rev. Cytol. 211: 179-240. PubMed ID: 11597004

Yu, Y., Huang, R., Ye, J., Zhang, V., Wu, C., Cheng, G., Jia, J. and Wang, L. (2016). Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila. Elife 5: e15693. PubMed ID: 27612383

Zandawala, M., Tian, S. and Elphick, M. R. (2017). The evolution and nomenclature of GnRH-type and corazonin-type neuropeptide signaling systems. Gen Comp Endocrinol. PubMed ID: 28622978

Ziegler, R. (1997). Lipid synthesis by ovaries and fat body of Aedes aegypti (Diptera: Culicidae). Eur. J. Entomol. 94: 385-391


Biological Overview

date revised: 10 April 2017

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.