InteractiveFly: GeneBrief

Gonadotropin-releasing hormone receptor: Biological Overview | References

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, CG11188^A1332 and AKHR^G6244, which are located close to and within the AKHR gene, respectively. CG11188^A1332 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 AKHR^G6244, 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 AKHR^rev, 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 (AKHR^rev bmm^rev) 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 Akhr^null and Akhr^rev 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 Akhr^null and Akhr^rev 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).

Galphaq, Ggamma1 and Plc21C control Drosophila body fat storage

Adaptive mobilization of body fat is essential for energy homeostasis in animals. In insects, the adipokinetic hormone (Akh) systemically controls body fat mobilization. Biochemical evidence supports that Akh signals via a G protein-coupled receptor (GPCR) called Akh receptor (AkhR) using cyclic-AMP (cAMP) and Ca(2+) second messengers to induce storage lipid release from fat body cells. Recently, genetic evidence has been provided that the intracellular calcium [iCa(2+)] level in fat storage cells controls adiposity in Drosophila. However, little is known about the genes which mediate Akh signalling downstream of the AkhR to regulate changes in iCa(2+). This study used thermogenetics to provide in vivo evidence that the GPCR signal transducers G protein alpha q subunit (Galphaq), G protein gamma1 (Ggamma1) and Phospholipase C at 21C (Plc21C) control cellular and organismal fat storage in Drosophila. Transgenic modulation of Galphaq, Ggamma1 and Plc21C affected the iCa(2+) of fat body cells and the expression profile of the lipid metabolism effector genes midway and brummer resulting in severely obese or lean flies. Moreover, functional impairment of Galphaq, Ggamma1 and Plc21C antagonised Akh-induced fat depletion. This study characterizes Galphaq, Ggamma1 and Plc21C as anti-obesity genes and supports the model that Akh employs the Galphaq/Ggamma1/Plc21C module of iCa(2+) control to regulate lipid mobilization in adult Drosophila (Baumbach, 2014).

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).

High-fat diet enhances starvation-induced hyperactivity via sensitizing hunger-sensing neurons in Drosophila

The function of the central nervous system to regulate food intake can be disrupted by sustained metabolic challenges such as high-fat diet (HFD), which may contribute to various metabolic disorders. Previous work has shown that a group of octopaminergic (OA) neurons mediated starvation-induced hyperactivity, an important aspect of food-seeking behavior. This study found that HFD specifically enhances this behavior. Mechanistically, HFD increases the excitability of these OA neurons to a hunger hormone named adipokinetic hormone (AKH), via increasing the accumulation of AKH receptor (AKHR) in these neurons. Upon HFD, excess dietary lipids are transported by a lipoprotein LTP to enter these OA(+)AKHR(+) neurons via the cognate receptor LpR1, which in turn suppresses autophagy-dependent degradation of AKHR. Taken together, this study has uncovered a mechanism that links HFD, neuronal autophagy, and starvation-induced hyperactivity, providing insight in the reshaping of neural circuitry under metabolic challenges and the progression of metabolic diseases (Huang, 2020).

Obesity and obesity-associated metabolic disorders such as type 2 diabetes and cardiovascular diseases have become a global epidemic. Chronic over-nutrition, especially excessive intake of dietary lipids, is one of the leading causes of these metabolic disturbances. Accumulating evidence has shown that HFD imposes adverse effects on the physiology and metabolism of liver, skeletal muscle, the adipose tissue, and the nervous system. It is therefore of importance to understand the mechanisms underlying HFD-induced changes in different organs and cell types, which will offer critical insight into the diagnosis and treatment of obesity and other metabolic diseases (Huang, 2020).

The central nervous system plays a critical role in regulating energy intake and expenditure. In rodent models, neurons located in the arcuate nucleus of the hypothalamus, particularly neurons expressing Neuropeptide Y (NPY) and Agouti-Related Neuropeptide (AgRP) or those expressing Pro-opiomelanocortin (POMC), are important behavioral and metabolic regulators. These neurons detect various neural and hormonal cues such as circulating glucose and fatty acids, leptin, and ghrelin, and modulate energy intake and expenditure accordingly. Upon the reduction of the internal energy state, NPY/AgRP neurons are activated and exert a robust orexigenic effect. Genetic ablation of NPY/AgRP neurons in neonatal mice completely abolishes food consumption whereas acute activation of these neurons significantly enhances food consumption. NPY/AgRP neurons also antagonize the function of POMC neurons that plays a suppressive role on food consumption. Taken together, these two groups of neurons, among other neuronal populations, work in synergy to ensure a refined balance between energy intake and expenditure, and hence organismal metabolism (Huang, 2020).

In spite of their critical roles, the function of the nervous system to accurately regulate appetite and metabolism may be disrupted by sustained metabolic stress, resulting in eating disorders and various metabolic diseases such as obesity and type 2 diabetes. Several lines of evidence have begun to reveal the underlying neural mechanisms. For example, HFD increases the intrinsic excitability of orexigenic NPY/AgRP neurons, induces leptin resistance, and enhances their inhibitory innervations with anorexigenic POMC neurons, altogether resulting in hypersensitivity to starvation and increased food consumption. Interestingly, besides HFD, other metabolic challenges, including maternal HFD, alcohol consumption, as well as aging, also disrupt normal food intake via affecting the excitability and/or innervation of NPY/AgRP neurons. All these interventions may contribute to the onset and progression of metabolic disorders (Huang, 2020).

Before the actual food consumption, food-seeking behavior is a critical yet largely overlooked behavioral component for the localization and occupation of desirable food sources. Food-seeking behavior has been characterized in rodent models, primarily by the elevation of locomotor activity and increased food approach of starved animals. It has been reported that NPY/AgRP neurons also play a role in food-seeking behavior. However, to ensure adequate food intake, food seeking and food consumption are temporally and spatially separated and even reciprocally inhibited. It remains largely unclear how the neural circuitry of food seeking and food consumption segregated and independently regulated in rodent models. Furthermore, it remains unknown whether HFD also affects food seeking, and if so whether its effects on both food seeking and food consumption share common mechanisms or not. To fully understand the intervention of energy homeostasis by sustained metabolic stress, it is necessary to dissect the neural circuitry underlying food seeking and examine whether and how it is affected by HFD (Huang, 2020).

Fruit flies Drosophila melanogaster share fundamental analogy to vertebrate counterparts on the regulation of energy homeostasis and organismal metabolism despite that they diverged several hundred million years ago. Therefore, it offers a good model to characterize food-seeking behavior in depth and provides insight into the regulation of energy intake and the pathogenesis of metabolic disorders in more complex organisms such as rodents and human (Huang, 2020).

Previous work showed that fruit flies exhibited robust starvation-induced hyperactivity that was directed towards the localization and acquisition of food sources, therefore resembling an important aspect of food-seeking behavior upon starvation (Yang, 2015). A small subset of OA neurons in the fly brain were identified that specifically regulated starvation-induced hyperactivity (Yu, 2016). Analogous to mammalian systems, a number of neural and hormonal cues are involved in the systemic control of nutrient metabolism and food intake in fruit flies. Among them, Neuropeptide F (NPF), short NPF (sNPF), Leucokinin, and Allatostatin A (AstA), have been shown to regulate food consumption, all of which have known mammalian homologs that regulate food intake. In particular, starvation-induced hyperactivity is regulated by two classes of neuroendocrine cells (Yu, 2016). One is functionally analogous to pancreatic α cells and produce AKH upon starvation, whereas the other produces Drosophila insulin-like peptides (DILPs), resembling the function of pancreatic β cells. These two classes of Drosophila hormones exert antagonistic functions on starvation-induced hyperactivity via the same group of OA neurons in the fly brain (Huang, 2020).

Based on these findings, this study sought to examine whether HFD disrupted the regulation of starvation-induced hyperactivity in fruit flies and aimed to investigate the underlying mechanism. The present study found that HFD-fed flies became significantly more sensitive to starvation and exhibited starvation-induced hyperactivity earlier and stronger than flies fed with normal diet (ND). Meanwhile, HFD did not alter flies' food consumption, suggesting that starvation-induced hyperactivity and food consumption are independently affected by HFD. Several days of HFD treatment did not alter the production of important hormonal cues like AKH and DILPs, but rather increased the sensitivity of the OA neurons that regulated starvation-induced hyperactivity to the hunger hormone AKH. In these OA neurons, constitutive autophagy maintained the homeostasis of AKHR protein, which determined their sensitivity to AKH and hence starvation. HFD feeding suppressed neuronal autophagy via AMPK-TOR signaling and in turn increased the level of AKHR in these OA neurons. Consistently, eliminating autophagy in these neurons mimicked the effect of HFD on starvation-induced hyperactivity whereas promoting autophagy inhibited the induction of hyperactivity by starvation. Furthermore, this study also showed that a specific lipoprotein LTP and its cognate receptor LpR1 likely mediated the effect of HFD on the neuronal autophagy of OA neurons and hence its effect on starvation-induced hyperactivity. Taken together, this study uncovered a novel mechanism that linked HFD, AMPK-TOR signaling, neuronal autophagy, and starvation-induced hyperactivity, shedding crucial light on the reshaping of neural circuitry under metabolic stress and the progression of metabolic diseases (Huang, 2020).

There is accumulating evidence that notes the effect of HFD on food consumption from insects to human, which results in obesity and obesity-associated metabolic diseases. But the effect of HFD on another critical food intake related behavior, food seeking, remains largely uncharacterized. Conceptually, food-seeking behavior in the fruit fly is composed of two behavioral components, increased sensitivity to food cues, and enhanced exploratory locomotion, which altogether facilitates the localization and acquisition of desirable food sources. Previous work has shown that starvation promotes starvation-induced hyperactivity, the exploratory component of food-seeking behavior, via a small group of OA neurons in the fly brain. These hunger-sensing OA neurons sample the metabolic status by detecting two groups of functionally antagonistic hormones, AKH and DILPs, and promote starvation-induced hyperactivity (Yu, 2016; Huang, 2020).

This study has demonstrated that this behavior is compromised by metabolic challenges. After a few days of HFD feeding, flies became behaviorally hypersensitive to starvation and as a result their starvation-induced hyperactivity was greatly enhanced, despite that their food intake and expenditure were not affected. These results suggest that HFD feeding may specifically modulate the activity of the neural circuitry underlying starvation-induced hyperactivity and offers an opportunity to further elucidate the cellular and circuitry mechanisms underlying behavioral abnormalities upon metabolic challenges (Huang, 2020).

As an insect counterpart of mammalian glucagon, AKH acts as a hunger signal to activate its cognate receptor AKHR expressed in the fat body and subsequently triggers lipid mobilization and energy allocation. In the fly brain, a small number of OA neurons also express AKHR. These neurons have been shown to be responsive to starvation and modulate various behaviors including food seeking and drinking (Jourjine, 2016; Yu, 2016). In that sense, these OA+AKHR+ neurons are functionally analogous to mammalian NPY/AgRP neurons in the hypothalamus, which also senses organismal metabolic states and regulates specific food intake behaviors. This study found that OA+AKHR+ neurons exhibited higher AKHR protein accumulation and became hypersensitive to AKH after HFD feeding. Notably, HFD feeding in mammals also increases the excitability of NPY/AgRP neurons, which contributes to the hypersensitivity to starvation and increased food consumption (Vernia, 2016). Thus, HFD may exert a conserved effect in the regulation of neuronal excitability and food intake related behaviors in both fruit flies and mammals (Huang, 2020).

Autophagy, a lysosomal degradative process that maintains cellular homeostasis, is critical for energy homeostasis. Upon cellular starvation, autophagy generates additional energy supply by breaking down macromolecules and subcellular organelles. At the organismal level, autophagy also contributes to the regulation of food intake and hence organismal energy homeostasis. For example, fasting induces autophagy in NPY/AgRP neurons via fatty acid uptake and promotes AgRP expression, which in turn enhances food intake (Kaushik, 2011). In line with these results, eliminating autophagy in NPY/AgRP neurons reduces food intake and hence body weight and fat deposits (Kaushik, 2011). Conversely, loss of autophagy in POMC neurons displays increased food intake and adiposity (Coupe, 2012). Consistently, in the current study, fruit flies neuronal autophagy was critical for the function of OA+AKHR+ neurons to sense hunger and regulate starvation-induced hyperactivity (Huang, 2020).

Accumulating evidence suggests that HFD suppresses autophagy in different peripheral tissue types such as liver, skeletal muscle, and the adipose tissue. Similarly, HFD suppresses autophagy in the hypothalamus, whereas blocking hypothalamic autophagy, particularly in POMC neurons, exacerbates HFD induced obesity. This study showed that HFD suppressed neuronal autophagy in OA+AKHR+ neurons and enhanced AKHR accumulation in these neurons. As a result, OA+AKHR+ neurons became hypersensitive to starvation and promoted starvation-induced hyperactivity. It will be of interest to examine whether HFD also reduces autophagy and increases the accumulation of specific membrane receptors in mammalian NPY/AgRP neurons (Huang, 2020).

This study also sought to examine the cellular mechanism that linked HFD feeding to the reduction of autophagy. HFD feeding activated TOR signaling. TOR, a highly conserved serine-threonine kinase, controls numerous anabolic cellular processes. Yhis study found that TOR signaling was tightly associated with the activity of AKHR+ neurons and the behavioral responses upon HFD feeding. Genetic enhancement of TOR activity in AKHR+ neurons increased AKHR protein accumulation, the sensitivity of these neurons to AKH, and hence starvation-induced hyperactivity, all of which mimicked the effect of HFD feeding. Inhibiting TOR activity exerted an opposite effect. In addition, the effect of HFD on TOR signaling was found to be mediated by AMPK signaling. These results altogether suggest that AMPK-TOR signaling in AKHR+ neurons plays an important role in maintaining the homeostasis of these neurons and determining the responsiveness to HFD feeding. Similarly, rodent studies have shown that manipulating AMPK-TOR signaling results in the dysfunction of NPY/AgRP neurons as well as POMC neurons, which leads to abnormal food consumption and adiposity. It will be of interest to examine whether HFD also modulates AMPK-TOR signaling in these specific hypothalamic neurons (Huang, 2020).

This study next sought to understand how AKHR+ neurons detected HFD, or more specifically, excess lipid ingested by the flies. As an essential nutrient and important energy reserve, dietary lipids were transported via their carrier proteins, named lipoproteins, in the circulation system and regulated multiple cellular signaling pathways. Proteomic analysis helped identify one lipoprotein LTP that was enriched in flies' hemolymph after HFD feeding. Single-cell RNAseq of AKHR+ neurons identified a number of lipoprotein receptors, especially LpR1, highly expressed in these neurons. Therefore, it is proposed that AKHR+ neurons might sense HFD feeding via LTP-LpR1 signaling. Evidently, it was found that eliminating LpR1 in AKHR+ neurons could protect flies from HFD, reducing AKHR accumulation and abolishing the effect of HFD to enhance starvation-induced hyperactivity. Conversely, eliminating LpR1 in the fat body, the major lipid reservoir of flies, created diet-independent hyperlipidemia and mimicked the effect of HFD feeding on flies' starvation-induced hyperactivity. Taken together, a working model is proposed that upon HFD feeding, excess dietary lipids are transported by LTP in the hemolymph, which interacts with its cognate receptor LpR1 in OA+AKHR+ neurons. As a result, these neurons undergo a number of cellular signaling processes and eventually become hypersensitive to starvation (Huang, 2020).

To summarize, the present study establishes a link between an unhealthy diet and abnormalities of food intake related behaviors in a model organism. The underlying mechanism was also deciphered, involving intracellular AMPK-TOR signaling, reduced neuronal autophagy, accumulation of a specific hormone receptor, and increased excitability of a small group of hunger-sensing neurons. This study will shed crucial light on the pathological changes in the central nervous system upon metabolic challenges. Given that the central control of metabolism and food intake related behaviors are highly conserved across different species, it will be of importance to further examine whether similar mechanisms also mediate the effect of HFD feeding on food intake and metabolic diseases in mammals (Huang, 2020).

Peroxisomal ERK mediates Akh/glucagon action and glycemic control

The enhanced response of glucagon and its Drosophila homolog, Adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. This study found that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. Mammalian glucagon-induced hepatic ERK peroxisomal translocation was also observed in diabetic subjects. Therefore, these results conclude that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control (Li, 2023).

Insulin and glucagon play evolutionarily conserved roles in maintaining stable circulating carbohydrate levels in response to nutritional cues in both vertebrates and invertebrates. The glucagon promotes the release of carbohydrates into circulation, while insulin enhances storages of carbohydrates from circulation. In addition to the well-established impairment of insulin response, enhanced response of glucagon under chronic high-caloric feeding also results in hyperglycemia, a common problem for patients with diabetes (Li, 2023).

Drosophila has emerged as an important model organism to study metabolic hormones and glycemic control.Drosophila adipokinetic hormone (Akh) is equivalent to mammalian glucagon to elevate glycemic levels through glycogenolysis and gluconeogenesis two highly spatialized metabolic processes. For example, glycogen breakdown has been reported to occur in the autophagosome and lysosome to release glucose while conversion of amino acids into pyruvate or other metabolites in the peroxisome and TCA cycle in the mitochondria provides substrates to support gluconeogenesis (Li, 2023).

Many metabolic enzymes that catalyze these processes have been characterized as well. Previous studies indicated that, similar to glucagon, Akh signals to its G-coupled receptor Akh receptor (AkhR) and activates Ca2+/calcium/calmodulin-dependent protein kinase II (CaMKII) and cAMP/PKA/CREB cascades and their downstream regulators to control carbohydrate metabolism in the fat body. However, beside the linear molecular cascades, the spatial modulation of intracellular Akh signaling at the organelle level associated with these signaling pathways is still largely unknown (Li, 2023).

The extracellular signal-regulated kinase (ERK) cascade is a highly conserved mitogen-activated protein kinase (MAPK) pathway that modulates various biological processes, including proliferation, differentiation, and stress responses, in both vertebrates and invertebrates. Recent studies have shown that ERK executes distinct and even opposing outcomes depending on the physiological stimulus. In addition to the well-known temporal regulation and binding competition, the spatial regulation of the ERK cascade has recently been shown to be essential for activation of downstream targets at certain organelles to execute specific physiological functions. For instance, growth factors promote ERK translocation to nuclear and plasma membrane to induce proliferative and migratory programs, respectively (Li, 2023).

Endosomal ERK targeting is triggered by certain G protein-coupled receptors (GPCRs; β2-adrenergic receptor and angiotensin II type 1 receptor) and receptor tyrosine kinases (RTKs) to regulate receptor turnover. Mitochondrial ERK translocation is mediated by unknown stress signaling to modulate mitochondrial activity and cell apoptosis (Li, 2023).

Studies in dividing cells have also indicated that the ERK cascade regulates other cellular activities via translocating to Golgi and the autophagosome, as well as cytoskeletal elements. In addition to these proliferation-associated activities, investigating spatial ERK regulation in other compartments or organelles in non-dividing cells would be insightful for understanding the diverse effects ERK, such as maintenance of metabolic homeostasis. In vivo RNAi screening was performed to identify ERK in the fat body as an important regulator of Akh-induced peroxisomal carbohydrate metabolism and hyperglycemia. Interestingly, by labeling different subcellular organelles, Akh was found to promote ERK translocation to the peroxisome, for the first time, via Ca2+/CaMKII signaling, to enhance carbohydrate production and cause hyperglycemia. Finally, the conserved carbohydrate regulation was validated by the glucagon-peroxisomal ERK axis in both obese mice and patients (Li, 2023).

Since the spatial regulation of Akh/glucagon response and glycemic control at the organelle level in unknown, this study combined studies in fly and mammals to demonstrate the essential roles of peroxisomal ERK translocation and peroxisomal carbohydrate metabolism in Akh/glucagon signaling and diet-induced hyperglycemia across species. It was further revealed that the Ca2+/CaMKII cascade, but not the cAMP/PKA pathway, contributes to ERK translocation onto peroxisome (Li, 2023).

Akh has been shown to elevate glycemic levels through glycogenolysis and gluconeogenesis, two metabolic processes that occur in specialized subcellular compartments. For instance, glycogen is broken down in the autophagosome and lysosome to release glucose. Circulating amino acids are delivered into the peroxisome to convert into pyruvate or other metabolites that can be used for gluconeogenesis (Li, 2023).

The TCA cycle in mitochondria also provides substrates for glucose synthesis. This study found that Akh predominantly translocases pERK to the peroxisomes to impact carbohydrate metabolism regarding glycemic control. It is the first evidence for ERK peroxisomal translocation, as opposed to the well-known subcellular localization of ERK. These results thus provide novel insights into spatial ERK regulation of carbohydrate metabolism and Akh-induced hyperglycemia (Li, 2023).

Even though this study failed to find autophagosomal or lysosomal ERK translocation by Akh, a small portion of pERK translocates to mitochondria. Consistent with the notion that conversion of oxaloacetate into phosphoenolpyruvate (PEP) in mitochondria supports gluconeogenesis, the results revealed that Akh increased phosphoenolpyruvate (PEP) release into circulating in an ERK-dependent manner, suggesting that mitochondrial ERK translocation might also participate in Akh-associated carbohydrate metabolism. It would be very insightful to investigate how ERK translocation to individual organelles, such as peroxisome, mitochondria, ER, and Golgi, collectively modulates metabolic homeostasis in future study (Li, 2023).

How ERK enhances peroxisome activity with respect to carbohydrate synthesis is an interesting question to address. The most convincing evidence in this study included that APEX2 (an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics) assays in S2R+ cells revealed ERK interaction with a few peroxisomal proteins, including CG1640 (GPT1/2), Got1, and Mdh1, which catalyze the conversion of amino acids to gluconeogenic substrates, under Akh stimulation. It is possible that ERK directly activates these metabolic enzymes in peroxisome to promote gluconeogenesis. Second, it was observed that Akh stimulation increases peroxisomal translocation of both pERK and GFP.SKL in S2R+ cells, indicating a correlation between ERK activation and import of peroxisomal proteins. A recent study interestingly uncovered that mammalian ERK1/2 phosphorylates Pex14, which regulates overall import of peroxisomal proteins, consistently suggesting that ERK might control general peroxisomal protein import to affect gluconeogenesis. Finally, Akh enhances ERK interaction with a few anti-oxidant proteins such as Sod2, Cat, Lon, and Prx5. Because redox balance is critical for peroxisomal integrity and functions, it is also possible that ERK impacts these anti-oxidant regulators to maintain peroxisome activity (Li, 2023).

To exploit the mechanisms how Akh promotes peroxisomal ERK translocation, the major regulators of Akh signaling, including the cAMP/PKA and Ca2+/CaMKII cascades, were examined. Interestingly, only the Ca2+/CaMKII cascade robustly enhances peroxisomal ERK translocation. Because no I peroxisomal targeting signal(s) (PTS) were found in ERK protein, it was speculated that certain binding proteins facilitate ERK translocation downstream of CaMKII signaling. However, the only two candidates that met all three criteria-(1) had a CaMKII phosphorylation site, (2) had PTS1 at the C terminus, and (3) increased binding to ERK by Akh-from the MS dataset were Mfe2 and Mtpa, two established proteins residing in peroxisome in the absence of Akh treatment. This suggested the involvement of non-canonical peroxisomal import. After performing RNAi screening of candidates with both CaMKII phosphorylation site(s) and increased binding to ERK by Akh, it was so far found that knockdown of at least Faf significantly diminished Akh-induced peroxisomal ERK translocation and hyperglycemia. Future validation using more comprehensive tools would be helpful to elucidate detailed mechanisms (Li, 2023).

Systemic pharmaceutical inhibition of ERK has been established to improve high-caloric-diet-induced hyperglycemia in mammals. Despite the evidence that ERK modulates Cdk5/PPARγ signaling and lipolytic programs in the adipose tissues to cause insulin resistance and impair glucose uptake, the molecular mechanisms of how ERK perturbs hepatic glucose production are not well understood. Previous studies have revealed that activation of hepatic ERK signaling causes hyperglycemia and suggested that this could be caused by cytosolic FOXO1 retention and impaired lipid oxidation, as well as feedback regulation of PKA activity (Li, 2023).

However, the direct evidence of how ERK perturbs hepatic glucose production, especially in a spatial fashion, is still missing. This study uncovered that mouse glucagon enhances peroxisomal ERK translocation in the liver and promotes hepatic glucose production in an ERK-dependent manner. The clinical observations in obese patients further indicated the positive correlation between hepatic peroxisomal ERK translocation, circulating glucagon levels, and hyperglycemia. These results collectively demonstrate the conserved roles of the Akh/glucagon-peroxisomal ERK axis in glycemic control from Drosophila to human and provide novel therapeutic opportunities targeting peroxisomal ERK in diabetes treatment (Li, 2023).

While it was noticed that ERK knockdown in the larval fat body suppresses Akh-induced conversion of amino acids into carbohydrates and reduces hyperglycemia, it remains uncertain whether this regulation relies on peroxisomal ERK translocation. Conversely, the associations observed in obese mice and patients imply that hepatic peroxisomal ERK translocation could contribute to hyperglycemia. Nevertheless, additional validation is required to substantiate this hypothesis (Li, 2023).

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)?

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).

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

Baumbach, J., Xu, Y., Hehlert, P. and Kuhnlein, R. P. (2014). Galphaq, Ggamma1 and Plc21C control Drosophila body fat storage. J Genet Genomics 41(5): 283-292. PubMed ID: 24894355

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

Coupe, B., Ishii, Y., Dietrich, M. O., Komatsu, M., Horvath, T. L. and Bouret, S. G. (2012). Loss of autophagy in pro-opiomelanocortin neurons perturbs axon growth and causes metabolic dysregulation. Cell Metab 15(2): 247-255. PubMed ID: 22285542

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

Huang, R., Song, T., Su, H., Lai, Z., Qin, W., Tian, Y., Dong, X. and Wang, L. (2020). High-fat diet enhances starvation-induced hyperactivity via sensitizing hunger-sensing neurons in Drosophila. Elife 9. PubMed ID: 32324135

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

Jourjine, N., Mullaney, B. C., Mann, K. and Scott, K. (2016). Coupled sensing of hunger and thirst signals balances sugar and water consumption. Cell 166(4): 855-866. PubMed ID: 27477513

Kaushik, S., Rodriguez-Navarro, J. A., Arias, E., Kiffin, R., Sahu, S., Schwartz, G. J., Cuervo, A. M. and Singh, R. (2011). Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance. Cell Metab 14(2): 173-183. PubMed ID: 21803288

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, J., Dang, P., Li, Z., Zhao, T., Cheng, D., Pan, D., Yuan, Y., Song, W. (2023). Peroxisomal ERK mediates Akh/glucagon action and glycemic control. Cell Rep, 42(10):113200 PubMed ID: 37796662

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

Vernia, S., Edwards, Y. J., Han, M. S., Cavanagh-Kyros, J., Barrett, T., Kim, J. K. and Davis, R. J. (2016). An alternative splicing program promotes adipose tissue thermogenesis. Elife 5. PubMed ID: 27635635

Yang, Z., Yu, Y., Zhang, V., Tian, Y., Qi, W. and Wang, L. (2015). Octopamine mediates starvation-induced hyperactivity in adult Drosophila. Proc Natl Acad Sci U S A 112(16): 5219-5224. PubMed ID: 25848004

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

date revised: 10 September 2024

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