InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila

The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. This study shows that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon alpha, which signals via its neuronal receptor DLgr2/Rickets. Bursicon alpha/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon alpha/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, this work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis (Scopelliti, 2018).

Maintaining systemic energy homeostasis is crucial for the physiology of all living organisms. A balanced equilibrium between anabolism and catabolism involves tightly coordinated signaling networks and the communication between multiple organs. Excess nutrients are stored in the liver and adipose tissue as glycogen and lipids, respectively. In times of high energy demand or low nutrient availability, nutrients are mobilized from storage tissues. Understanding how organs communicate to maintain systemic energy homeostasis is of critical importance, as its failure can result in severe metabolic disorders with life-threatening consequences (Scopelliti, 2018).

The intestine is a key endocrine tissue and central regulator of systemic energy homeostasis. Enteroendocrine (ee) cells secrete multiple hormones in response to the nutritional status of the organism and orchestrate systemic metabolic adaptation across tissues. Recent work reveals greater than expected diversity, plasticity, and sensing functions of ee cells. Nevertheless, how ee cells respond to different environmental challenges and how they coordinate systemic responses is unclear. A better understanding of ee cell biology will directly impact understanding of intestinal physiopathology, the regulation of systemic metabolism, and metabolic disorders (Scopelliti, 2018).

Functional studies on inter-organ communication are often challenging in mammalian systems, due to their complex genetics and physiology. The adult Drosophila midgut has emerged as an invaluable model system to address key aspects of systemic physiology, host-pathogen interactions, stem cell biology and metabolism, among other things. As in its mammalian counterpart, the Drosophila adult intestinal epithelium displays multiple subtypes of ee cells with largely unknown functions. Recent work has demonstrated nutrient-sensing roles of ee cells (Scopelliti, 2018 and references therein).

The role of Bursicon/DLgr2 signaling has long been restricted to insect development, where the heterodimeric form of the hormone Bursicon, made by α and β subunits, is produced by a subset of neurons within the CNS during the late pupal stage and released systemically to activate its receptor DLgr2 in peripheral tissues to drive post-molting sclerotization of the cuticle and wing expansion. A recent study demonstrated a post-developmental activity for the α subunit of Bursicon (Bursα), which is produced by a subpopulation of ee cells in the posterior midgut, where it paracrinally activates DLgr2 in the visceral muscle (VM) to maintain homeostatic intestinal stem cell (ISC) quiescence (Scopelliti, 2014; Scopelliti, 2016; Scopelliti, 2018).

This study reports an unprecedented systemic role for Bursα regulating adult energy homeostasis. This work identifies a novel gut/fat body axis, where ee cells orchestrate organismal metabolic homeostasis. Bursα is systemically secreted by ee cells in response to nutrient availability and acts through DLgr2+ neurons to repress adipokinetic hormone (AKH)/AKH receptor (AKHR) signaling within the fat body/adipose tissue to restrict the use of energy stores. Impairment of systemic Bursα/DLgr2 signaling results in exacerbated oxidative metabolism, strong lipodystrophy, and organismal hypersensitivity to nutrient deprivation. This work reveals a central role for ee cells in sensing organismal nutritional status and maintaining systemic metabolic homeostasis through coordination of an intestinal/neuronal/adipose tissue-signaling network (Scopelliti, 2018).

This study shows that ee cells secrete Bursα in the presence of plentiful nutrients, while caloric deprivation reduces its systemic release and consequently results in hormone accumulation within ee cells. Interestingly, it was observed that conditions leading to the latter scenario are accompanied by reduced bursα transcription. The reasons underlying the inverse correlation between midgut bursα mRNA and protein levels are unclear and may represent part of a negative feedback mechanism for ultimate control of further protein production. A similar phenomenon is described during the regulation of the secretion of other endocrine hormones, such as DILPs (Scopelliti, 2018).

The results show that Bursα within ee cells is preferably regulated in response to dietary sugars. This is further supported by the function of Glut1 as at least one of the transmembrane sugar transporters connecting nutrient availability to Bursα signaling. Glut1 is the closest homolog of the mammalian regulator of ee incretin secretion SLC2A2, and it has been shown to positively regulate the secretion of peptide hormones in flies (Park, 2014). Whether Glut1 is a central sensor of dietary sugars and hormone secretion by ee cells remains to be addressed. However, it is likely that, in the face of challenges, such as starvation, multiple mechanisms of nutrient sensing and transport converge to allow a robust organismal adaptation to stressful environmental conditions (Scopelliti, 2018).

Reduction of systemic Bursα/DLgr2 signaling induces a complex metabolic phenotype, characterized by lipodystrophy and hypoglycemia, which is accompanied by hyperphagia. These phenotypes are not due to poor nutrient absorption or uptake by tissues or impaired synthesis of energy stores but are rather a consequence of increased catabolism. This is supported by a higher rate of glucose-derived 13C incorporation into TCA cycle intermediates, accompanied by increased mitochondrial respiration and body-heat production (Scopelliti, 2018).

While glucose tracing experiments help explain the hypoglycemic phenotype of Bursα/DLgr2-compromised animals even in the context of hyperphagia, they do not directly address the reduction in fat body triacylglycerides (TAGs). The latter would require 13C6-palmitate tracing for assessment of the rate of lipid oxidation and incorporation into the TCA cycle. This was precluded by overall poor uptake of 13C6-palmitate into adult animals even after prolonged periods of feeding. However, the depletion of fat body TAG stores in the presence of normal de novo lipid synthesis in Bursα/DLgr2-impaired animals strongly suggests that at least part of the increased rate of O₂ consumption in those animals results from increased lipid breakdown via mitochondrial fatty acid oxidation. Consistently, increased O₂ consumption rates and the thermogenic phenotype of Bursα/DLgr2-deficient animals are attenuated upon reduction of AKH/AKHR signaling. Finally, the functional role of Hormone-sensitive lipase (dHSL) in the fat body further supports the regulation of lipid breakdown by AKH/AKHR signaling as at least one of the key aspects mediating the role of Bursα/DLgr2 signaling in adult metabolic homeostasis (Scopelliti, 2018).

Previous work revealed that ee Bursα is required to maintain homeostatic ISC quiescence in the adult Drosophila midgut; that is, in the midgut of unchallenged and well-fed animals (Scopelliti, 2014, Scopelliti, 2016). Such a role of Bursα is mediated by local or short-range signaling through DLgr2 expressed within the midgut VM (Scopelliti, 2014). This study demonstrates a systemic role of Bursα that does not involve VM-derived DLgr2 but rather signals through its neuronal receptor. In that regard, the paracrine and endocrine functions of Bursα/DLgr2 are uncoupled. However, the regulation of ee-derived Bursα by nutrients is likely to affect local as well as systemic Bursα/DLgr2 signaling. Retention of Bursα within ee as observed in conditions of starvation may impair the hormone's signaling into the VM, which, in principle, would lead to ISC hyperproliferation (Scopelliti, 2014). In fact, under full nutrient conditions, genetic manipulations impairing systemic Bursα signaling, such as ee Glut1 knockdown or osbp overexpression, lead to ISC hyperproliferation comparable with that observed upon bursα knockdown (Scopelliti, 2014). This represents an apparent conundrum, as ISC proliferation is not the expected scenario in the context of starvation. However, starvation completely overcomes ISC proliferation in Bursα-impaired midguts. This is consistent with recent evidence showing that restrictive nutrient conditions, such as the absence of dietary methionine or its derivative S-adenosyl methionine, impair ISC proliferation in the adult fly midgut, even in the presence of activated mitogenic signaling pathways (Obata, 2018). Altogether, these data support a scenario in which starvation, while preventing systemic and local Bursα/DLgr2 signaling, would not result in induction of ISC proliferation as a side effect (Scopelliti, 2018).

Drosophila DLgr2 is the ortholog of mammalian LGR4, -5, and -6 with closer homology to LGR4. While LGR5 and 6 are stem cell markers in several tissues, such as small intestine and skin, LGR4 depicts broader expression patterns and physiological functions. LGR4, -5, and -6 are best known to enhance canonical Wnt signaling through binding to R-spondins. However, several lines of evidence support a more promiscuous binding affinity for LGR4, which can act as a canonical G-protein coupled receptor inducing iCa2+ and cyclic AMP signaling (Scopelliti, 2018).

Interestingly, an activating variant of LGR4 (A750T) is linked to obesity in humans, while the nonsense mutation c.376C>T (p.R126X) is associated with reduced body weight. Recent reports show that LGR4 homozygous mutant (LGR4m/m) mice display reduced adiposity and are resistant to diet- or leptin-induced obesity. These phenotypes appear to derive from increased energy expenditure through white-to-brown fat conversion and are independent of Wnt signaling. The tissue and molecular mechanisms mediating this metabolic role of LGR4 remain unclear. Therefore, the current paradigm may lead to a better understanding of LGR4's contribution to metabolic homeostasis and disease. Importantly, the results highlight the intestine and ee cells in particular as central orchestrators of metabolic homeostasis and potential targets for the treatment of metabolic dysfunctions (Scopelliti, 2018).

Bursicon is an insect-specific hormone. Therefore, direct mammalian translation of the signaling system presented in this study is unlikely. However, given the clear parallels between the metabolic functions of DLgr2 and LGR4, analysis of enteroendocrine cell-secreted factors in mammalian systems may reveal new and unexpected ligands for LGR4 (Scopelliti, 2018).

Drosophila insulin-like peptide dilp1 increases lifespan and glucagon-like Akh expression epistatic to dilp2

The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. This study finds that dilp1 is highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Dilp1 and dilp2 single and double mutants were used to describe interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1-dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 - dilp2 double mutants. dilp1 and dilp2 interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. Repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro-longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. The data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro-longevity insulin-like peptide (Post, 2018).

Based on mutational analyses of the insulin receptor (daf-2, InR) and its associated adaptor proteins and signaling elements, numerous studies in C. elegans and Drosophila established that decreased insulin/IGF signaling (IIS) extends lifespan. Studies on how reduced IIS in Drosophila systemically slows aging also reveal systems of feedback where repressed IIS in peripheral tissue decreases DILP2 production in brain insulin-producing cells (IPC), which may then reinforce a stable state of longevity assurance. This study finds that expression of dilp1 is required for loss of dilp2 to extend longevity. This novel observation contrasts with conventional interpretations where reduced insulin ligand is required to slow aging: Elevated dilp1 is associated with longevity in dilp2 mutants, and transgene expression of dilp1 increases longevity (Post, 2018).

dilp1 and dilp2 are encoded in tandem, likely having arisen from a duplication event. Perhaps as a result, some aspects of dilp1 and dilp2 are regulated in common: Both are expressed in IPCs, are regulated by sNPF, and have strongly correlated responses to dietary composition. Nonetheless, the paralogs are differentially expressed throughout development. While dilp2 is expressed in larvae, dilp1 expression is elevated in the pupal stage when dilp2 expression is minimal. In reproductive adults, dilp1 expression decreases substantially after eclosion and dilp2 expression increases (Post, 2018).

Furthermore, DILP1 production is associated with adult reproductive diapause. IIS regulates adult reproductive diapause in Drosophila, a somatic state that prolongs survival during inclement seasons. DILP1 may stimulate these diapause pro-longevity pathways, while expression in nondiapause adults is sufficient to extend survival even in optimal environments (Post, 2018).

The current data suggest a hypothesis whereby dilp1 extends longevity in part through induction of adipokinetic hormone (AKH), which is also increased during reproductive diapause and acts as a functional homolog of mammalian glucagon. Critically, AKH secretion has been shown to increase Drosophila lifespan and to induce triacylglycerides and free fatty acid catabolism. Here, it is noted that dilp1 mutants were more sensitive to starvation than wild-type and dilp2 mutants, as might occur if DILP1 and AKH help mobilize nutrients during fasting and diapause. Mammalian insulin and glucagon inversely regulate glucose storage and glycogen breakdown, while insulin decreases glucagon mRNA expression. It is proposed that DILP2 in Drosophila indirectly regulates AKH by repressing dilp1 expression, while DILP1 otherwise induces AKH (Post, 2018).

A further connection between dilp1 and diapause involves juvenile hormone (JH). In many insects, adult reproductive diapause and its accompanied longevity are maintained by the absence of JH. Furthermore, ablation of JH-producing cells in adult Drosophila is sufficient to extend lifespan, and JH is greatly reduced in long-lived Drosophila insulin receptor mutants. In each case, exogenous treatment of long-lived flies with a JH analog (methoprene) restores survival to the level of wild-type or nondiapause controls. JH is a terpenoid hormone that interacts with a transcriptional complex consisting of Met (methoprene tolerant), Taimen, and Kruppel homolog 1 (Kr-h1). As well, JH induces expression of kr-h1 mRNA, and this serves as a reliable proxy for functionally active JH. This study finds that dilp2 mutants have reduced kr-h1 mRNA, while the titer of this message is similar to that of wild-type in dilp1 - dilp2 double mutants. DILP1 may normally repress JH activity, as would occur in diapause when DILP1 is highly expressed. Such JH repression may contribute to longevity assurance during diapause as well as in dilp2 mutant flies maintained in laboratory conditions (Post, 2018).

Does DILP1 act as an insulin receptor agonist or inhibitor? Inhibitory DILP1 could directly interact with the insulin receptor to suppress IIS, potentially even in the presence of other insulin peptides. Such action could induce programs for longevity assurance that are associated with activated FOXO. Alternatively, DILP1 may act as a typical insulin receptor agonist that induces autophosphorylation and represses FOXO. In this case, to extend lifespan, DILP1 should stimulate cellular responses distinct from those produced by other insulin peptides such as DILP2 or DILP5. Through a third potential mechanism, DILP1 may interact with binding proteins such as IMPL2 or dALS to indirectly inhibit IIS output. These distinctions may be resolvednin a future study using synthetic DILP1 applied to cells in culture (Post, 2018).

A precedent exists from C. elegans where some insulin-like peptides are thought to function as antagonists. In genetic analyses, ins-23 and ins-18 stimulate larval diapause and longevity, while ins-1 promotes Dauer formation during development and longevity in adulthood. Moreover, C. elegans ins-6 acts through DAF-2 to suppress ins-7 expression in neuronal circuits to affect olfactory learning, where ins-7 expression inhibits DAF-2 signaling. These studies propose that additional amino acid residues of specific insulin peptides contribute to their distinct functions, and notably, the B-chain of DILP1 has an extended N-terminus relative to other DILP sequences (Post, 2018).

While dFOXO and DAF-16 are intimately associated with how reduced IIS regulates aging in Drosophila and C. elegans, in the current work, the behavior of FOXO does not correspond with how longevity is controlled epistatically by dilp1 and dilp2. Mutation of dilp2 did not impact FOXO activity, as measured by expression of target genes InR and 4eBP, and interactions with dilp1 did not modify this result. Some precedence suggests only a limited role for dfoxo as the mediator of reduced IIS in aging, as dfoxo only partially rescues longevity benefits of chico mutants, revealing that IIS extends lifespan through some FOXO-independent pathways. On the other hand, dilp1 expression from a transgene in the dilp1-2 double mutant background did induce FOXO targets. Differences among these results might arise if whole animal analysis of dFOXO targets obscures its role when IIS regulates aging through actions in specific tissues. In this vein, this study found that dilp2 controls thorax ERK signaling but not AKT, suggesting that dilp2 mutants may activate muscle-specific ERK/MAPK anti-aging programs (Post, 2018).

Dilp1 and dilp2 redundantly regulate glycogen levels and blood sugar, while these dilp loci interact synergistically to modulate dilp5 expression and starvation sensitivity. In contrast, dilp1 and dilp2 interact in a classic epistatic fashion to modulate longevity and AKH. Such distinct types of genetic interactions may reflect unique ways DILP1 and DILP2 stimulate different outcomes from their common tyrosine kinase insulin-like receptor, along with outcomes based on cell-specific responses. Understanding how and what is stimulated by DILP1 in the absence of dilp2 will likely reveal critical outputs that specify longevity assurance (Post, 2018).

Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila

Obesity is a progressive, chronic disease, which can be caused by long-term miscommunication between organs. It remains challenging to understand how chronic dysfunction in a particular tissue remotely impairs other organs to eventually imbalance organismal energy homeostasis. This paper introduces RNAi Pulse Induction (RiPI) mediated by short hairpin RNA (shRiPI) or double-stranded RNA (dsRiPI) to generate chronic, organ-specific gene knockdown in the adult Drosophila fat tissue. Organ-restricted RiPI targeting Stromal interaction molecule (Stim), an essential factor of store-operated calcium entry (SOCE), results in progressive fat accumulation in fly adipose tissue. Chronic SOCE-dependent adipose tissue dysfunction manifests in considerable changes of the fat cell transcriptome profile, and in resistance to the glucagon-like Adipokinetic hormone (Akh) signaling. Remotely, the adipose tissue dysfunction promotes hyperphagia likely via increased secretion of Akh from the neuroendocrine system. Collectively, this study presents a novel in vivo paradigm in the fly, which is widely applicable to model and functionally analyze inter-organ communication processes in chronic diseases (Xu, 2019).

This study presents in vivo evidence for chronic targeted gene knockdown following RiPI in the adult Drosophila fat body. A short pulse induction of shRNA targeting the AkhR gene generates persisting siRNAs, which causes significant down-regulation of AkhR for at least 10 days. Persistence of RNAi has been associated with RNA-dependent RNA polymerase (RdRP)-mediated siRNA amplification in C. elegans and in human cells. In Drosophila, however, it remains controversial whether the genome encodes a functional RdRP. Therefore, slow degradation of the transgene-derived siRNAs might confer the chronic gene knockdown. In fact, RNAi effector double-stranded siRNAs (21nt and 24nt) are more stable than the 18nt double-stranded RNAs in the human cytosolic extract. Moreover, in human HEK293T cells, the anti-sense strand of siRNA is more resistant to intracellular nucleases compared to the sense strand of the siRNA duplex, which is likely due to the incorporation of anti-sense siRNAs into the activated RNA induced silencing complex (RISC). Therefore, the involvement of RISC might allow the slow degradation of siRNAs in adult Drosophila fat body cells. The slow decline of the siRNA level is apparently sufficient to chronically knockdown the endogenous gene expression of AkhR and Stim, which causes progressive body fat increase. This mode of action is further supported by the fact that pulsed overexpression of RNAi resistant Stim-mRNA only transiently rescues the fat content increase due to Stim-TRiPI. Consistently, long-term gene silencing (at least 11 days) is also observed in adult flies after injection of low concentrations of dsRNAs. Similarly, in an EGFP-transgenic mouse model, the inhibition of the reporter expression lasts as long as two months after siRNA injection. In summary, this study shows here that in vivo RiPI generates long-lasting RNAi, which allows chronic knockdown of target genes in a tissue-specific manner. It is proposed that RiPI is a versatile tool to study causative relationships and temporal sequences in inter-organ communication processes (Xu, 2019).

Using RiPI, this study has established a Drosophila obesity model based on chronic, adipose tissue-directed knockdown of Stim, which shares remarkable similarity to characteristics of human obesity. First, the visibly enlarged abdomen of the obese flies corresponds to increased waist circumference, which gains importance as meaningful parameter to assess android adiposity. Similarly, body fat accumulation causes significant weight gain, another readout to quantify obesity in rodents and humans. Second, the excessive fat accumulation correlates with climbing deficits of the obese flies, with physical fitness reduction being another hallmark of human adiposity. Moreover, obese Stim-TRiPI flies have reduced life span, which is reminiscent of the higher mortality rates in human obesity patients. Third, this study demonstrates that early-onset hyperphagia drives the positive energy balance in Stim-TRiPI flies. Consistently, increased food intake is the major driver of human obesity. Hyperphagia is linked to increased dietary glucose conversion into storage fat in obese Stim-TRiPI flies. Notably, increased food intake and elevated glucose conversion into storage lipids has also been reported after silencing obesity blocking neurons in the fly central brain (Al-Anzi, 2009). With hyperphagia being an important contributor, obesity development in Stim-RiPI flies is not monocausal. It is noteworthy that the rise in fat storage in Stim-DRiPI substantially exceeds the food intake increase. Moreover, matching the food intake of Stim-TRiPI On and Off flies still results in body fat accumulation. Importantly, there is a significantly reduced metabolic rate of Stim-DRiPI flies. Finally, the observed hyperglycemia at day 10, physical fitness reduction at day 24 and shortened life span of Stim-TRiPI On flies are associated with obesity development, similar to type 2 diabetes (T2D), exercise intolerance and mortality, which are also highly correlated with human obesity. In summary, chronic knockdown of Stim in the adult fat body causes fly obesity by a number of physiological factors culminating in organismal energy imbalance similar to mammalian adiposity (Xu, 2019).

This study highlights the critical roles played by Stim in interaction with Akh/AkhR signaling and insulin signaling in the fly fat body tissue. Reduced expression of Mdh1 and Gprk2 suggests impaired Akh/AkhR signaling in the fat body of Stim-TRiPI flies. Mammalian MDH1 has been linked to glycolysis in cells with mitochondrial dysfunction. Obese Stim-TRiPI On flies display normal glycogen storage and mobilization during starvation. Similar findings are also observed in AkhA, AkhAP, and AkhR1 mutant fly larvae and adult flies, albeit their capability to mobilize glycogen is weakly impaired. A possible explanation is that flies employ corazonin, a starvation-responsive pathway complementary to Akh, to utilize glycogen. In addition to storage glycogen, the reduced expression of genes involved in lipolysis predicts an impairment of starvation-induced storage lipid mobilization. Indeed, obese Stim-TRiPI flies display an abnormal lipid mobilization profile under starvation and die with residual fat resources. Similarly, impaired lipid mobilization is also observed in flies with loss-of-function mutation in the TAG lipase gene bmm or in flies lacking either InsP3R or AkhR. Consistently, loss-of-function of STIM1/2 in mammalian cells, also impairs lipolysis via down-regulation of cAMP. Moreover, decreased catecholamine-stimulated lipolysis has been identified in human obese individuals. Collectively, these results show that fat body tissue of obese Stim-RiPI On flies is resistant in response to Akh signaling, which drives the obesity development (Xu, 2019).

Moreover, this study supports the possibility to model T2D in adult flies. Obese Stim-TRiPI flies show reduced expression of the glucose clearance gene Hex-C, whose mammalian homolog was also suppressed in T2D patients. Besides, evidence is provided to support that obese Stim-TRiPI flies have hyperglycemia, impairment of insulin signaling in fat body tissue, and larger lipid droplets. Similar features were also described in fly larvae reared on high sugar diet, which resemble mammalian insulin resistance. Regarding unchanged circulating dIlp-2 level in obese Stim-DRiPI flies, insulin-like peptide secretion might be interfered by the knockdown of Stim in the insulin producing cells of Stim-DRiPI flies mediated by ubiquitous driver daGS, more investigation on circulating insulin levels of obese Stim-DRiPI flies by specific driver needs to be done in future. Interestingly, the indicators of insulin signaling impairment mentioned above occur at later stage of Stim-RiPI obesity development, and accordingly are possibly the consequence of Stim-TRiPI On mediated-fat gain, which also supports the concept that obesity compromises insulin signaling (Xu, 2019).

Apart from the specific role of the fat body in storage lipid handling and glucose clearance, this study shows that chronic knockdown of Stim in this organ remotely promotes Akh secretion from the fly CC neuroendocrine cells, which leads to hyperphagia. The RNAseq and gene expression analysis indicate a list of genes encoding candidate hormone or secreted proteins. Among them, CCHa2, daw, and Lst have been shown to function as hormones to regulate insulin-like peptide secretion. In addition, CCHa2, daw and Lst are also regulated by Akh overexpression in opposite direction. Whether differential expression of these genes mentioned above mediate the (mis)communication between the fat body and the CC cells is currently unknown. Nevertheless, the communication between the fat body and the CC cells is essential for the food intake increase as well as further obesity development induced by long-term knockdown of Stim. Interestingly, a study provided evidence that muscle tissue in flies communicates with the CC cells to control Akh secretion via the myokine Unpaired2 (Upd2). Upd2 had been previously shown to act as adipokine, which signals the fed state from the fat body. Unlike mammalian leptin, Upd2 remotely acts on insulin-producing cells in the central brain to regulate insulin secretion but not food intake. Recently, Akh mRNA expression was shown to be regulated by a gut-neuronal relay via midgut-secreted peptide Buriscon α in response to nutrients. Given the fact that the transcription of Akh is unaffected in Stim-RiPI On flies, identification of the adipokine, which regulates the Akh release directly or indirectly to affect food intake in the Stim-RiPI fly obesity model requires future research efforts (Xu, 2019).

In conclusion, this work introduces RNAi Pulse Induction as a novel in vivo paradigm for chronic, tissue-specific gene interference. RiPI makes essential genes accessible to long-term functional analysis in the adult fly, as exemplified here by establishing a Drosophila obesity model caused by chronic knockdown of Stim in the adult fat body. Moreover, this study reveals, that the fat body integrates the tissue-autonomous and the systemic branches of Akh signalling: by regulation of lipid mobilization via SOCE in the fat body, and possibly by remote-control of Akh secretion from the CC cells. Recently, the evolutionarily conserved role of SOCE in controlling energy metabolism has attracted the interest of mammalian studies. While Akh is structurally not conserved to humans, there is a growing number of remotely-controlled orexigenic peptide hormones in mammals with asprosin being one of the latest additions. Collectively, these findings in the fly add further evidence to the existence of conserved regulatory principles in animal energy homeostasis control emanating from SOCE signalling in fat storage tissues (Xu, 2019).

A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. This study has identified a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (Dilp2) and the other extends to Adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and Dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. It is proposed that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of Dilp2 and suppressing the release of AKH when haemolymph glucose levels are high (Oh, 2019).

Glucose-sensing neurons respond to glucose or its metabolites, which act as signalling cues to regulate their neuronal activity. According to the glucostatic hypothesis proposed in 1953, feeding and related behaviours are regulated by neurons in the brain that sense changes in glucose levels in the blood. Despite the discovery of glucose-sensing neurons in the hypothalamus through electrophysiological methods more than ten years later, the physiological role of these neurons remained unclear until recently, when a population of glucose-excited neurons in the Drosophila brain were determined to function as an internal nutrient sensor to mediate the animal's consumption of sugar (Dus, 2015). A large number of glucose-sensing neurons appear to be present in animals; it is speculated that these neurons mediate physiological functions that are critical for the wellbeing of the animal, including glucose homeostasis. This study reports the identification of a pair of glucose-excited neurons in the Drosophila brain that maintain glucose homeostasis by coordinating the activity of the two key hormones involved in the process: insulin and glucagon (Oh, 2019).

To identify neurons that respond to sugar on the basis of its nutritional value, a two-choice assay was used to screen Vienna tiles (VT)-Gal4 Drosophila lines that had been crossed to UAS-Kir2.1, tub-Gal80ts flies (inward-rectifier potassium ion channel allele Kir2.1 with tubulin-temperature-sensitive Gal80) for defects in their ability to select nutritive D-glucose over non-nutritive L-glucose. Two independent Gal4 lines, VT58471 and VT43147-Gal4, were isolated that failed to select D-glucose after periods of starvation and appeared to contain dorsolateral cells that resemble those that are labelled by the corazonin (Crz)-Gal4 line. Flies in which Crz-Gal4-expressing neurons had been inactivated failed to select D-glucose even when starved. These results suggest that the dorsolateral neurons labelled by Crz-Gal4 and two candidate Gal4 lines mediate the behavioural response to sugar (Oh, 2019).

A Crz antibody was used to confirm the identity of the dorsolateral neurons. A previous study demonstrated that a subset of Crz-expressing neurons also express short neuropeptide F (sNPF). Immunolabelling revealed that the dorsolateral neurons expressing Crz indeed express sNPF. On the basis of these findings, these Crz+sNPF+ neurons were named CN neurons. To restrict Gal4 expression to a few cells that include the dorsolateral neurons, T58471-Gal4 was crossed to choline acetyltransferase (ChAT)-Gal80, generating CN-Gal4, which unambiguously labelled a pair of CN neurons when crossed to UAS-mCD8::GFP. Flies in which these dorsolateral neurons were inactivated using CN-Gal4 failed to select D-glucose when starved. Each CN cell body projects an axon that bifurcates to form two major branches. One branch (axon 1) projects to the pars intercerebralis (PI) region of the brain and the other branch (axon 2) projects ventrolaterally towards the corpora cardiaca (CC). An intersectional approach was used to define these projections further, thereby validating that axon 1 innervates the PI and axon 2 projects to the CC. This approach was also used to induce the expression of tetanus toxin (TNT) to silence a pair of CN neurons. These flies failed to choose D-glucose even after starvation when CN neurons were inactivated. This provided further evidence of the contribution of the pair of the dorsolateral CN neurons to glucose-evoked behaviour (Oh, 2019).

Attempts were made to determine whether CN neurons respond to glucose and other sugars. Calcium-imaging studies using ex vivo brain preparations of flies carrying the calcium indicator UAS-GCaMP6s14 and CN-Gal4 revealed that CN neurons were robustly activated by D-glucose with substantial calcium oscillations. CN neurons also responded to D-trehalose and D-fructose, which are found in the haemolymph, but failed to respond to (1) the non-nutritive sugar L-glucose; (2) the non-haemolymph sugar sucrose; and (3) the non-sugar nutrients amino acids. D-Glucose and D-trehalose are key sugars in the haemolymph, although D-trehalose stimulates the activity of CN neurons only after a substantial delay (about 12 min), possibly because it requires additional metabolic steps to be converted to glucose. D-Fructose applied at 20 mM activated CN neurons, although the concentration of D-fructose in the haemolymph is much lower (<2 mM). These findings suggest that the pair of CN neurons responds only to D-glucose under normal physiological conditions (Oh, 2019).

It was next determined whether activation of CN neurons by D-glucose requires glucose metabolism inside the cell. Exposing the brain to D-glucose mixed with 2DG, phlorizin or nimodipine, which inhibits glycolysis, glucose transport or voltage-gated calcium channels, respectively, blunted the glucose-induced stimulation of CN neurons. In the presence of pyruvate (an end product of glycolysis), the CN neurons demonstrated activity similar to that seen in the presence of other haemolymph sugars. Application of the ATP-sensitive potassium channel (KATP)16 blocker glibenclamide resulted in activation of CN neurons (Fig. 2b, c). Furthermore, glucose-induced calcium transients of these neurons were not abrogated by the application of the sodium-channel blocker tetrodotoxin (TTX). Using RNA-mediated interference (RNAi) lines, it was also determined that glucose transporter 1 (Glut1), hexokinase C (Hex-C), a subunit of the KATP channel (SUR1) and the voltage-gated calcium channel are required in CN neurons for the two-choice behaviour. Consistent with the behavioural results, the glucose-induced calcium response of CN neurons requires Glut1, SUR1 and a voltage-gated calcium channel, further supporting the role of the intracellular glucose metabolic pathway in stimulating CN neuronal activity (Oh, 2019).

The calcium-dependent nuclear import of LexA (CaLexA) system was used to measure cellular activity in CN neurons in intact flies; GFP signal driven by the CaLexA system in starved flies was significantly reduced compared to the signal in fed flies, and the signal was restored when starved flies were refed D-glucose. These results suggest that the activity of CN neurons is stimulated by the increase in glucose levels observed under fed conditions. In addition to the altered CaLexA signals, the effect of glucose on the number and intensity of synaptotagmin (Syt)-GPF+ puncta in fed, starved and refed animals. The Syt-GPF+ signals decreased significantly in axon 1 in starved animals and returned to normal levels after the flies were fed with D-glucose. However, this nutrient-dependent plasticity was not observed in Crz-Gal4-labelled axonal processes that did not originate from the dorsolateral CN neurons (Oh, 2019).

Next attempts were made to determine whether CN neurons are coupled with IPCs20 at the synaptic level. A modified GFP reconstitution across synaptic partners (GRASP) method was used, and the GRASP signals were found to be visible around the synapse between CN neurons and insulin-producing cells (IPCs), indicating physical coupling between CN neurons and IPCs (Oh, 2019).

To determine whether the coupling between CN neurons and IPCs is functional, ATP-gated P2X2 purine receptors were used in CN neurons and the calcium indicator GCaMP6s14 in IPCs, and then CN neurons were stimulated using ATP while recording from the IPCs. ATP-induced CN-neuron activity was accompanied by a significant increase in the amplitude of GCaMP signals in the IPCs in fed flies; this effect was reduced in starved flies. This finding supports the hypothesis that the nutrient-dependent synaptic changes observed between CN neurons and IPCs have functional consequences. The CN neurons did not appear to be functionally coupled to glucose-excited diuretic hormone 44 (Dh44) neurons. Furthermore, whether CN neuronal activity is required for Dilp2 secretion from IPCs was tested. A significant reduction in the intensity of Dilp2 immunoreactivity was observed in the IPCs of fed control flies, but not in fed flies in which CN neurons had been inactivated. These results suggest that an excitatory signal from the CN neurons contributes to the secretion of Dilp2 from IPCs in response to increased glucose levels. Using mass spectrometry and dot blot assay, it was further validated that the flies carrying CN-Gal4 and UAS-Kir2.1 had lower Dilp2 levels circulating in the haemolymph than wild-type flies, in contrast to the higher dilp2 levels found in IPCs (Oh, 2019).

To further clarify the role of CN neurons in mediating glucose-evoked activity in IPCs, CN neurons were inactivated by expressing TNT13, and then the responsiveness of IPCs to glucose was evaluated. The amplitude of calcium signals in IPCs that had been exposed to D-glucose was significantly reduced when the CN neurons were inactivated. Furthermore, it was found that IPCs harbour at least three subpopulations of neurons with distinct responses to glucose or KATP channel blocker. These findings suggest that CN neuronal activity is required for the majority of IPCs to respond to glucose (Oh, 2019).

To determine whether nutrient-dependent plasticity also occurs in axon 2 of the CN neurons, the number and intensity of Syt-GFP+ puncta was monitored before and after feeding flies with D-glucose. A significant reduction was observed in these parameters in starved flies, and a restoration to normal levels was found after refeeding starved flies with D-glucose. This raised the possibility of coupling between CN neurons and AKH-producing cells. Using a modified GRASP method, GRASP fluorescent signals were observed around AKH-producing cells. To determine whether there is any functional connectivity between these cells, the CN neurons were activated while monitoring the activity of AKH-producing cells, and calcium transients found in the AKH-producing cells appeared to decrease during activation of CN neurons (Oh, 2019).

To probe this observation further, the Arclight receptor, which increases fluorescent signals when cells become hyperpolarized, was expressed in AKH-producing cells, and P2X2 receptors were expressed in CN neurons. When the CN neurons were activated using ATP, the Arclight fluorescence intensity in fed flies increased significantly compared with that in starved flies, validating the occurrence of nutrient-dependent changes in the synapses between CN neurons and AKH-producing cells. Notably, when CN neurons were inactivated, the intracellular AKH levels decreased significantly compared with controls. Using mass spectrometry and dot blot assay, significantly higher levels of AKH were expressed in haemolymph of flies carrying CN-Gal4 and UAS-Kir2.1 compared with those in control flies. These findings suggest that CN neuronal activity inhibits the release of AKH from the CC and the increase of AKH levels in haemolymph (Oh, 2019).

Next the identities were investigated of the key neurotransmitters in axon 1 and axon 2 for regulating the functionally opposing synaptic activities. The role of Crz and sNPF was tested in the two-choice behaviour using RNAi lines, and sNPF in CN neurons and sNPF receptor in the postsynaptic IPCs, but not Crz or its receptor, were found to be important. sNPF but not Crz levels in CN neurons were significantly reduced when CN neurons were exposed to D-glucose. Approximately a half of the IPCs that had responded to glucose failed to respond glucose when the dominant-negative allele of sNPF receptor was expressed in IPCs. Furthermore, it was observed that intracellular AKH levels remained high in AKH-producing cells in fed control flies, but declined significantly in fed flies in which the function of sNPF receptor was inhibited in AKH-producing cells (Oh, 2019).

Finally, whether sNPF alters activity of IPCs and/or the CC was determined. The activity of IPCs was significantly stimulated by the application of sNPF26, whereas CC activity was significantly inhibited by sNPF. These functionally opposing effects of sNPF are probably mediated by Gq in IPCs and by Gi/o in AKH-producing cells via the sNPF receptor, which is a G-protein-coupled receptor. Exposing the brain to U73122, a PLC inhibitor that inhibits the Gq pathway, eliminated the glucose-evoked activation of IPCs, but had no effect on sNPF-induced inhibition of AKH-expressing cells. Conversely, exposing the brain to pertussis toxin, a Gi inhibitor, blunted the sNPF-induced inhibition of AKH-producing cells, but had no effect on the glucose-evoked activation of IPCs. These results indicate that axon 1 and axon 2 can have opposing synaptic activities through a mechanism involving the same neurotransmitter and receptor but with distinct downstream factors coupled with opposing outputs (Oh, 2019).

To determine whether CN neuronal activity can alter circulating sugar levels in flies, circulating concentrations of glucose and trehalose in haemolymph were monitored; they were significantly increased in flies in which CN neurons were inactivated compared with controls. This finding illustrates that dysfunctional CN neuronal input to IPCs and AKH-producing cells results in a defect in glucose homeostasis (Oh, 2019).

This study identified and characterized a pair of glucose-sensing neurons in the Drosophila brain that have an essential role in maintaining glucose homeostasis. This was achieved by counterbalancing the activities of Drosophila equivalents of insulin- and glucagon-producing cells. When food consumption leads to a rise in haemolymph sugar levels, CN neurons excite the IPCs through sNPF and its receptor, which appear to be coupled to the Gq signalling cascade to induce the secretion of Dilp2, while suppressing the release of AKH by using the same sNPF receptor, which in this case is coupled with Gi signalling pathway. It is speculated that precise control of these opposing functions is facilitated because the nutrient-dependent plastic changes arise from a single cell (Oh, 2019).

This study demonstrates how the activity of the two key endocrine systems is coordinated in metazoans and that their coordination is under the direct control of glucose-sensing neurons. Such coordination has been proposed to occur in mammals via the sympathetic and parasympathetic nerves that connect the pancreatic islets with glucose-sensing neurons in the hypothalamus and hindbrain. The finding that a large proportion of IPCs respond to glucose through CN neurons in insects raises an intriguing possibility that both direct and indirect mechanisms control endocrine function in mammals. Finally, this work may shed light on the function of glucose-sensing neurons. Further research is needed to understand how these regulatory processes are affected by excessive nutrition and other metabolic disturbances, including obesity (Oh, 2019).

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. 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).

Sex determination gene transformer regulates the male-female difference in Drosophila fat storage via the adipokinetic hormone pathway

Sex differences in whole-body fat storage exist in many species. For example, Drosophila females store more fat than males. Yet, the mechanisms underlying this sex difference in fat storage remain incompletely understood. This study identified a key role for sex determination gene transformer (tra) in regulating the male-female difference in fat storage. Normally, a functional tra protein is present only in females, where it promotes female sexual development. This study shows that loss of tra in females reduced whole-body fat storage, whereas gain of tra in males augmented fat storage. Tra's role in promoting fat storage was largely due to its function in neurons, specifically the Adipokinetic hormone (Akh)-producing cells (APCs). Analysis of Akh pathway regulation revealed a male bias in APC activity and Akh pathway function, where this sex-biased regulation influenced the sex difference in fat storage by limiting triglyceride accumulation in males. Importantly, tra loss in females increased Akh pathway activity, and genetically manipulating the Akh pathway rescued Tra-dependent effects on fat storage. This identifies sex-specific regulation of Akh as one mechanism underlying the male-female difference in whole-body triglyceride levels, and provides important insight into the conserved mechanisms underlying sexual dimorphism in whole-body fat storage (Wat, 2021).

This study used the fruit fly Drosophila melanogaster to improve the knowledge of the mechanisms underlying the male-female difference in whole-body triglyceride levels. The presence of a functional tra protein in females, which directs many aspects of female sexual development, promotes whole-body fat storage. Tra's ability to promote fat storage arises largely due to its function in neurons, where the APCs were identified as one neuronal population in which tra function influences whole-body triglyceride levels. Examination of Akh/AkhR mRNA levels and APC activity revealed several differences between the sexes, where these differences lead to higher Akh pathway activity in males than in females. Genetic manipulation of APCs and Akh pathway activity suggest a model in which the sex bias in Akh pathway activity contributes to the male-female difference in fat storage by limiting whole-body triglyceride storage in males. Importantly, this study showed that tra function influences Akh pathway activity, and that Akh acts genetically downstream of tra in regulating whole-body triglyceride levels. This reveals a previously unrecognized genetic and physiological mechanism that contributes to the sex difference in fat storage (Wat, 2021).

One key finding from this study was the identification of sex determination gene tra as an upstream regulator of the male-female difference in fat storage. In females, a functional Tra protein promotes fat storage, whereas lack of tra in males leads to reduced fat storage. While an extensive body of literature has demonstrated important roles for tra in regulating neural circuits, behavior, abdominal pigmentation, and gonad development, uncovering a role for tra in regulating fat storage significantly extends understanding of how sex differences in metabolism arise. Given that sex differences exist in other aspects of metabolism (e.g., oxygen consumption), this new insight suggests that more work will be needed to determine whether tra contributes to sexual dimorphism in additional metabolic traits. Indeed, one study showed that tra influences the sex difference in adaptation to hydrogen peroxide stress. Beyond metabolism, tra also regulates multiple aspects of development and physiology such as intestinal stem cell proliferation, carbohydrate metabolism, body size, phenotypic plasticity, and lifespan responses to dietary restriction. Because some, but not all, of these studies identify a cell type in which tra function influences these diverse phenotypes, future studies will need to determine which cell types and tissues require tra expression to establish a female metabolic and physiological state. Indeed, recent single-cell analyses reveal widespread gene expression differences in shared cell types between the sexes (Wat, 2021).

Identifying neurons as the anatomical focus of Tra's effects on fat storage was another key finding from this study. While many sexually dimorphic neural circuits related to behavior and reproduction have been identified, less is known about sex differences in neurons that regulate physiology and metabolism. Indeed, while many studies have identified neurons that regulate fat metabolism, these studies were conducted in single- or mixed-sex populations. Because male-female differences in neuron number, morphology, and connectivity have all been described across the brain and ventral nerve cord, a detailed analysis of neuronal populations that influence metabolism will be needed in both sexes to understand how neurons contribute to the sex-specific regulation of metabolism and physiology. Indeed, while identification of a role for APC sexual identity in regulating the male-female difference in fat storage represents a significant step forward in understanding how sex differences in neurons influence metabolic traits, more knowledge is needed of how tra regulates sexual dimorphism in this critical neuronal subset. For example, while this study showed that females normally have lower Akh mRNA levels and APC activity, it remains unclear how the presence of tra regulates these distinct traits. tra may regulate Akh mRNA levels via known target genes fruitless (fru) and doublesex (dsx) , or alternatively through a fru- and dsx-independent pathway. To influence the sex difference in APC activity and Akh release, tra may regulate factors such as ATP-sensitive potassium (KATP) channels and 5' adenosine monophosphate-activated protein kinase (AMPK)-dependent signaling, both of which are known to modulate APC activity. Future studies will therefore need to investigate Tra-dependent changes to KATP channel expression and function in APCs, and characterize Tra's effects on ATP levels and AMPK signaling within APCs (Wat, 2021).

Additional ways to learn more about the sex-specific regulation of fat storage by the APCs will include examining how sexual identity influences physical connections between the APCs and other neurons, and monitoring APC responses to circulating hormones. For example, there are physical connections between Corazonin- and Neuropeptide F (NPF)-positive (CN) neurons and APCs in adult male flies, and between the APCs and a bursicon-α-responsive subset of DLgr2 neurons in females. These connections inhibit APC activity: CN neurons inhibit APC activity in response to high hemolymph sugar levels, whereas binding of bursicon-α to DLgr2 neurons inhibits APC activity in nutrient-rich conditions. Future studies will therefore need to determine whether these physical connections exist in both sexes. Further, it will be important to identify male-female differences in circulating factors that regulate the APCs. While gut-derived Allatostatin C (AstC) was recently shown to bind its receptor on the APCs to trigger Akh release, loss of AstC affects fat metabolism and starvation resistance only in females. This suggests sex differences in AstC-dependent regulation of fat metabolism may exist (Wat, 2021).

Given that gut-derived NPF binds to its receptor on the APCs to inhibit Akh release, that skeletal muscle-derived unpaired 2 (upd2) regulates hemolymph Akh levels, and that circulating peptides such as Allatostatin A (AstA), Drosophila insulin-like peptides (Dilps), and activin ligands influence Akh pathway activity, it is clear that a systematic survey of circulating factors that modulate Akh production, release, and Akh pathway activity in each sex will be needed to fully understand the sex-specific regulation of fat storage. Another important point to address in future studies will be confirming results from previous studies that the fat body is the main anatomical focus of Akh-dependent regulation of fat storage. Given that the sex-biased effects of triglyceride lipase bmm arise from a male-female difference in the cell type-specific requirements for bmm function, it will be important to determine which cell types mediate Akh's effects on fat storage in each sex. This line of enquiry will also clarify the underlying processes that support increased fat storage in females. At present, it remains unclear whether the higher whole-body fat storage in females is caused by lower fat breakdown, increased lipogenesis, or both. Given that Akh pathway activity plays a role in regulating both lipolysis and lipogenesis in Drosophila and other insects, it will be important to identify the cellular mechanism underlying Akh's effects on the sex difference in fat storage (Wat, 2021).

Beyond fat metabolism, it will be important to extend understanding of how sex-specific Akh regulation affects additional Akh-regulated phenotypes. Given that Akh affects fertility and fecundity, future studies will need to determine whether these phenotypes are due to Akh-dependent regulation of fat metabolism, or due to direct effects of Akh on gonads. Similarly, while Akh has been linked with the regulation of lifespan, carbohydrate metabolism, starvation resistance, locomotion, immune responses, cardiac function, and oxidative stress responses, most studies were performed in mixed- or single-sex populations. Additional work is therefore needed to determine how changes to Akh pathway function affect physiology, carbohydrate levels, development, and life history in each sex. Importantly, the lessons learned may also extend to other species. Akh signalling is highly conserved across invertebrates, and is functionally similar to the mammalian β-adrenergic and glucagon systems. Because sex-specific regulation of both glucagon and the β-adrenergic systems have been described in mammalian models and in humans, detailed studies on sex-specific Akh regulation and function in flies may provide vital clues into the mechanisms underlying male-female differences in physiology and metabolism in other animals (Wat, 2021).

A gut-derived hormone suppresses sugar appetite and regulates food choice in Drosophila

Animals must adapt their dietary choices to meet their nutritional needs. How these needs are detected and translated into nutrient-specific appetites that drive food-choice behaviours is poorly understood. This study shows that enteroendocrine cells of the adult female Drosophila midgut sense nutrients and in response release neuropeptide F (NPF), which is an ortholog of mammalian neuropeptide Y-family gut-brain hormones. Gut-derived NPF acts on glucagon-like adipokinetic hormone (AKH) signalling to induce sugar satiety and increase consumption of protein-rich food, and on adipose tissue to promote storage of ingested nutrients. Suppression of NPF-mediated gut signalling leads to overconsumption of dietary sugar while simultaneously decreasing intake of protein-rich yeast. Furthermore, gut-derived NPF has a female-specific function in promoting consumption of protein-containing food in mated females. Together, these findings suggest that gut NPF-to-AKH signalling modulates specific appetites and regulates food choice to ensure homeostatic consumption of nutrients, providing insight into the hormonal mechanisms that underlie nutrient-specific hungers (Malita, 2022).

To maintain nutritional homeostasis, animals need to match their ingestion of specific nutrients to their needs. This is achieved by modulating appetite towards the specific nutrients needed. A number of factors, including gut hormones, that regulate food consumption have been identified in both flies and mammals, and reports have also described central brain mechanisms that induce ingestion of protein food in response to amino-acid deprivation, that sense amino acids and promote food consumption and that reject food lacking essential amino acids. However, little is known about the hormonal mechanisms that regulate nutrient-specific appetite, and gut hormones that regulate selective food intake are completely unknown. The current findings indicate that, in mated female Drosophila, gut-derived NPF is a selective driver of sugar satiety and protein consumption, providing a basis for understanding these mechanisms. Hormone-based therapies that inhibit appetite offer promising new directions for weight-loss treatment. For example, Fibroblast growth factor 21 (FGF21) is a liver-derived hormone that promotes protein consumption, and it is emerging as a promising target for metabolic disorders. Uncovering appetite-regulatory hormones such as gut-derived NPF that specifically inhibit sugar consumption while promoting the intake of protein-rich foods could provide effective new weight-management strategies by promoting healthier food choices (Malita, 2022).

The SLC2-family sugar transporter Sut2 is the closest Drosophila homologue of human SLC2A7 (GLUT7), a transporter expressed mainly in the intestine whose function is poorly defined. In flies, GLUT1 is important for Bursicon secretion from the EECs, and Sut1, another SLC2-family sugar transporter protein, was recently shown to be involved in midgut NPF release in virgin females. The current results implicate Sut2 in the release of NPF from EECs in mated females and thus link it to the mechanism by which NPF-mediated gut signalling controls feeding decisions. This indicates that both Sut1 and Sut2 sugar transporters are involved in glucose-stimulated NPF secretion from the gut. In mammals, several mechanisms also regulate glucose-stimulated GLP-1 secretion from intestinal endocrine cells, which involves sodium-glucose cotransporter 1 (SGLT1), the glucose transporter GLUT2 and sweet taste receptors. Targeting of these intestinal glucose-sensing mechanisms therefore has become a focus of weight-management therapies because of its potential in regulating appetite and incretin effects. Future studies should investigate whether GLUT7, like its Drosophila homologue Sut2, affects appetite-regulatory mechanisms in the mammalian gut (Malita, 2022).

NPF is orthologous with the mammalian NPY family of gut-brain peptides, including peptide YY (PYY), pancreatic polypeptide and NPY itself, that regulate food-seeking behaviours and metabolism. Like mammalian NPY-family hormones, Drosophila NPF is expressed in both the nervous system and the gut. While NPY is abundant in the nervous system and, like brain NPF, promotes food intake, PYY is mainly produced by endocrine cells of the gut as a satiety factor. Gut-expressed PYY is homologous to NPY, and both act through specific G-protein coupled receptors, called NPY receptors (NPYRs), that are orthologous with Drosophila NPFR. Thus, in mammals, multiple NPY-family peptides from different tissues sources exert their functions on target organs through several related NPYRs, while in Drosophila, these functions may be regulated through the single peptide-receptor pair of NPF and NPFR (Malita, 2022).

The results indicate that gut-derived Drosophila NPF fulfills the function of mammalian PYY. PYY is produced by the endocrine L-cells of the gut, which, like the EECs of Drosophila, produce a context-dependent combination of multiple hormones49. The physiological role of PYY in feeding regulation has been difficult to clarify, but it is believed to act through different NPYRs on tissues including the hypothalamus and the pancreatic islets to suppress appetite. These findings show that, in flies, NPF injection strongly reduces the intake of sugar-containing food and promotes the ingestion of protein-rich food. In humans, PYY infusion also been shown to strongly reduce food intake. Although the satiety function of human PYY has made it a prime therapeutic target for potential weight management, it is not clear whether PYY regulates nutrient-specific appetite, which would be important from a therapeutic perspective. These results indicate that Drosophila gut NPF, perhaps filling the role of mammalian gut PYY, acts to mediate sugar-specific satiety, illustrating a key hormonal mechanism that underlies selective hunger by which animals adjust their intake of specific nutrients (Malita, 2022).

Feeding decisions are based on internal state and exhibit sexual dimorphism. In Drosophila, males and females differ in their preference for and intake of dietary sugar and protein6. The current findings define a complex interorgan communication system through which mating influences food choices in females. Midgut NPF was found to be involved in mediating SP-induced postmating responses in females, inhibiting sugar appetite and promoting the ingestion of protein-rich yeast food, and it was further shown that AKH is required for mediating the effects of NPF. When mated females consume dietary carbohydrates, NPF is released from the EECs and inhibits the AKH axis by directly suppressing AKH release from the AKH-producing cells (APCs) as well as by inhibiting the release of midgut AstC, a factor that stimulates AKH secretion. Furthermore, NPF acts directly on the fat body through NPFR to inhibit energy mobilization, thereby antagonizing AKH-mediated signalling in the adipose tissue. Likewise, mammalian NPY-family peptides also regulate metabolism by direct actions on adipose tissue via NPYR. Although a number of studies have demonstrated that AKH is a regulator of metabolism, the current findings uncover a key role of AKH in governing nutrient-specific feeding decisions. It is becoming clear that the APCs integrate many signals that affect AKH release, and these signals may therefore also affect food choice. The APCs therefore seem to function as a signal-integration hub, similar to the IPCs, which receive many different inputs to control insulin production and release. AstC, Bursicon and NPF from the gut control AKH expression and secretion, indicating that multiple signals, even from the same organ, converge on the APCs. These signals presumably convey different aspects of nutritional status and may act with different dynamics to regulate AKH production and/or release, or even in a redundant manner to regulate AKH signalling. Likewise, many signals released from the fat body convey similar and seemingly redundant nutritional information to the IPCs (Malita, 2022).

Recent work has also revealed a sex-specific role of AKH, with lower activity in females underlying differences in male and female metabolism. Consistent with this notion, the current results indicate that in mated females the midgut NPF system inhibits AKH signalling, suppressing intake of sugar-rich food. Furthermore, it was recently shown that in mated females, midgut-derived AstC acts in a sex-specific manner through AKH to coordinate metabolism and food intake under nutritional stress. The current work shows that NPF also works sex-specifically to sustain physiological requirements in mated females by signalling from the gut to control AKH, suggesting that the gut-AKH axis occupies a central link in the hormonal relays underlying sex-specific regulation of physiology. A recent report showed that female germline cells modulate sugar appetite, but this effect is not induced by mating and does not affect yeast feeding as this study has found for gut NPF and AKH, suggesting that it is an independent mechanism (Malita, 2022).

How nutrient signals from the gut modulate feeding is key to understanding how nutritional needs are translated into specific feeding actions to maintain balance. This study has identified a homeostatic circuit triggered by gut-derived NPF that limits sugar consumption. Similar mechanisms for sugar-induced satiety that promote protein consumption may also enable mammals to balance their intake of different nutrients with their metabolic needs. Explaining how nutrient-responsive gut hormones such as NPF affect dietary choice is important to better understand hunger and cravings for specific nutrients that may ultimately lead to obesity (Malita, 2022).

Dual lipolytic control of body fat storage and mobilization in Drosophila

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

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

In order to examine the effect of AKHR signaling on fat storage and mobilization in vivo, two different P element-insertion mutants were used, 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).

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

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

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

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

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

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

REGULATION

Promoter Structure

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The neuropeptide Allatostatin A regulates metabolism and feeding decisions in Drosophila

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

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

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

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

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

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

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

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

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

Protein Interactions

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

DEVELOPMENTAL BIOLOGY

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

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

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

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

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

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

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

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

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

Adipokinetic hormone (AKH), energy budget and their effect on feeding and gustatory processes of foraging honey bees

The adipokinetic hormone (AKH) of insects is considered an equivalent of the mammalian hormone glucagon as it induces fast mobilization of carbohydrates and lipids from the fat body upon starvation. Yet, in foraging honey bees, which lack fat body storage for carbohydrates, it was suggested that AKH may have lost its original function. This study manipulated the energy budget of bee foragers to determine the effect of AKH on appetitive responses. As AKH participates in a cascade leading to acceptance of unpalatable substances in starved Drosophila, its effect on foragers presented with sucrose solution spiked with salicin was also assessed. Starved and partially-fed bees were topically exposed with different doses of AKH to determine if this hormone modifies food ingestion and sucrose responsiveness. A significant effect of the energy budget (i.e. starved vs. partially-fed) was found on the decision to ingest or respond to both pure sucrose solution and sucrose solution spiked with salicin, but no effect of AKH per se. These results are consistent with a loss of function of AKH in honey bee foragers, in accordance with a social life that implies storing energy resources in the hive, in amounts that exceed individual needs (de Brito, 2021).

AKH in non-insect arthropods

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

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

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

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

Physiological effects of AKH in hormones

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Search PubMed for articles about Drosophila Adipokinetic hormone

Al-Anzi, B., Sapin, V., Waters, C., Zinn, K., Wyman, R. J. and Benzer, S. (2009). Obesity-blocking neurons in Drosophila. Neuron 63(3): 329-341. PubMed ID: 19679073

Arrese, E. L. and Wells, M. A. (1997). Adipokinetic hormone-induced lipolysis in the fat body of an insect, Manduca sexta: synthesis of sn-1,2-diacylglycerols. J. Lipid Res. 38(1): 68-76. 9034201

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

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

Braco, J. T., et al. (2012). Energy-dependent modulation of glucagon-like signaling in Drosophila via the AMP-activated protein kinase. Genetics [Epub ahead of print]. PubMed Citation: 22798489

Bradfield, J. Y., Lee, Y. H. and Keeley, L. L. (1991). Cytochrome P450 family 4 in a cockroach: molecular cloning and regulation by regulation by hypertrehalosemic hormone. Proc. Natl. Acad. Sci. 88(10): 4558-62.

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 Citation: 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

de Brito Sanchez, G., Exposito Munoz, A., Chen, L., Huang, W., Su, S. and Giurfa, M. (2021). Adipokinetic hormone (AKH), energy budget and their effect on feeding and gustatory processes of foraging honey bees. Sci Rep 11(1): 18311. PubMed ID: 34526585

Döring, F., Wischmeyer, E., Kuhnlein, R. P., Jäckle, H. and Karschin, A. (2002). Inwardly rectifying K+ (Kir) channels in Drosophila. A crucial role of cellular milieu factors Kir channel function. J. Biol. Chem. 277: 25554-25561. PubMed citation: 11964404

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

Garfias, A., Rodriguez-Sosa, L. and Arechiga, H. (1995). Modulation of crayfish retinal function by red pigment concentrating hormone J. Exp. Biol. 198: 1447-54. 9319346

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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 Citation: 9731220

Hentze, J. L., Carlsson, M. A., Kondo, S., Nassel, D. R. and Rewitz, K. F. (2015). The neuropeptide Allatostatin A regulates metabolism and feeding decisions in Drosophila. Sci Rep 5: 11680. PubMed ID: 26123697

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., et al. (2005). Ablation of AKH-producing neuroendocrine cells decreases trehalose levels and induces behavioral changes in Drosophila. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288(2): R531-8. 15374818

Jaffe, H., et al. (1989). Primary structure of two neuropeptide hormones with adipokinetic and hypotrehalosemic activity isolated from the corpora cardiaca of horse flies (Diptera). Proc. Natl. Acad. Sci. 86: 8161-8164. 2813385

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

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

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

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date revised: 12 December 2023

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