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).

GENE STRUCTURE

cDNA clone length - 902

Bases in 5' UTR - 116

Exons - 2

Bases in 3' UTR - 192

PROTEIN STRUCTURE

Amino Acids - Adipokinetic hormone precursor - 79, Adipokinetic hormone - 8

Structural Domains

A member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of arthropod neuropeptides was identified in the fruitfly Drosophila melanogaster, and its structure was determined by automated Edman degradation and m.s. using fast-atom-bombardment ionization and a tandem hybrid instrument capable of high sensitivity. The sequence of this peptide, which has been called 'DAKH', is pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 (where pGlu is pyroglutamic acid and Trp-NH2 is tryptophan carboxyamide). H.p.l.c. analyses of extracts of the three body segments revealed that more than 80% of the peptide is contained in the thorax. Although DAKH is typical of family members in its general structure and distribution in the animal, it is unique in containing a residue which is charged under physiological conditions (Schaffer, 1990).

Drosophila adipokinetic hormone (DAKH) is an eight amino acid member of a large arthropod neuropeptide family. The gene encoding the peptide precursor has been identified and sequenced providing an inferred precursor structure of 79 amino acids including a 46 amino acid carboxy-terminal fragment of unknown function (Noyes, 1995).

date revised: 10 December 2004

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