Modulation of Metabolic Hormone Signaling via a Circadian Hormone and Biogenic Amine in Drosophila melanogaster.

In insects, adipokinetic hormone is the primary hormone responsible for the mobilization of stored energy. While a growing body of evidence has solidified the role of adipokinetic hormone (AKH) in modulating the physiological and behavioral responses to metabolic stress, little is known about the upstream endocrine circuit that directly regulates AKH release. We evaluated the AKH-producing cell (APC) transcriptome to identify potential regulatory elements controlling APC activity and found that a number of receptors showed consistent expression levels, including all known dopamine receptors and the pigment dispersing factor receptor (PDFR). We tested the consequences of targeted genetic knockdown and found that APC limited expression of RNAi elements corresponding to each dopamine receptor and caused a significant reduction in survival under starvation. In contrast, PDFR knockdown significantly extended lifespan under starvation, whereas expression of a tethered PDF in APCs resulted in significantly shorter lifespans. These manipulations caused various changes in locomotor activity under starvation. We used live-cell imaging to evaluate the acute effects of the ligands for these receptors on APC activation. Dopamine application led to a transient increase in intracellular calcium in a trehalose-dependent manner. Furthermore, coapplication of dopamine and ecdysone led to a complete loss of this response, suggesting that these two hormones act antagonistically. We also found that PDF application led to an increase in cAMP in APCs and that this response was dependent on expression of the PDFR in APCs. Together, these results suggest a complex circuit in which multiple hormones act on APCs to modulate metabolic state.

Regulation of Metabolism by an Ensemble of Different Ion Channel Types: Excitation-Secretion Coupling Mechanisms of Adipokinetic Hormone Producing Cells in Drosophila.

Adipokinetic Hormone (AKH) is the primary insect hormone that mobilizes stored energy and is functional equivalent to mammalian glucagon. While most studies have focused on exploring the functional roles of AKH, relatively little is known about how AKH secretion is regulated. We assessed the AKH cell transcriptome and mined the data set for specific insight into the identities of different ion channels expressed in this cell lineage. We found reliable expression of multiple ion channel genes with multiple members for each ionic species. Specifically, we found significant signals for 39 of the either known or suspected ion channel genes within the Drosophila genome. We next performed a targeted RNAi screen aimed to identify the functional contribution of these different ion channels that may participate in excitation-secretion coupling in AKH producing cells (APCs). We assessed starvation survival, because changes in AKH signaling have previously been shown to impact starvation sensitivity. Genetic knockdown of three genes (Ca-Beta, Sur, and sei), in AKH producing cells caused highly significant changes (P < 0.001) in both male and female lifespan, and knockdown of six other genes (Shaw, cac, Ih, NaCP60E, stj, and TASK6) caused significant changes (P < 0.05) in only female lifespan. Specifically, the genetic knockdown of Ca-Beta and Sur led to increases in starvation lifespan, whereas the knockdown of sei decreased starvation survivorship. Focusing on these three strongest candidates from the behavioral screen, we assessed other AKH-dependent phenotypes. The AKH hormone is required for starvation-induced hyperactivity, and we found that these three ion channel gene knockdowns changed activity profiles and further suggest a modulatory role of these channels in AKH release. We eliminated the possibility that these genetic elements caused AKH cell lethality, and using independent methods, we verified expression of these genes in AKH cells. Collectively, these results suggest a model of AKH-cell excitability and establish an experimental framework for evaluating intrinsic mechanisms of AKH release.

Behavioral Aversion to AITC Requires Both Painless and dTRPA1 in Drosophila.

There has been disagreement over the functional roles of the painless gene product in the detection and subsequent behavioral aversion to the active ingredient in wasabi, allyl isothiocyanate (AITC). Originally, painless was reported to eliminate the behavioral aversion to AITC, although subsequent reports suggested that another trpA homolog, dTRPA1, was responsible for AITC aversion. We re-evaluated the role of the painless gene in the detection of AITC, employing several different behavioral assays. Using the proboscis extension reflex (PER) assay, we observed that AITC did not reduce PER frequencies in painless or dTRPA1 mutants but did in wild-type genotypes. Quantification of food intake showed a significant decline in food consumption in the presence of AITC in wild-type, but not painless mutants. We adapted an oviposition choice assay and found wild-type oviposit on substrates lacking AITC, in contrast to painless and dTRPA1 mutants. Lastly, tracking individual flies relative to a point source of AITC, showed a consistent clustering of wild-type animals away from the point source, which was absent in painless mutants. We evaluated expression patterns of both dTRPA1 and painless, which showed expression in distinct central and peripheral populations. We identified the transmitter phenotypes of subsets of painless and dTRPA1 neurons and found similar neuropeptides as those expressed by mammalian trpA expressing neurons. Using a calcium reporter, we observed AITC-evoked responses in both painless and dTRPA1 expressing neurons. Collectively, these results reaffirm the necessity of painless in nociceptive behaviors and suggest experiments to further resolve the molecular basis of aversion.

Connecting nutrient sensing and the endocrine control of metabolic allocation in insects.

It is clear that specific hormones control an organism's energy use and regulate the differential allocations of energy to activity, growth and maintenance of specific tissues, and reproduction. Appropriate metabolic allocations require an assessment of the nutrient state of the animal, and nutrient sensing must be tied to appropriate signals in order to coordinate the repertoire of behaviors and physiologies accompanying a particular metabolic investment. Here, we review the known and speculated connections between nutrient sensing and the endocrine control of energy allocation in insects. Insects, being speciose and diverse in life history strategies, offer a unique perspective into the general architecture of the signaling mechanisms of energetic allocation and also into unique elements that correlate with specific life histories.

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. Here, we report 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 life span 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 that AMPK promotes AKH secretion. We observed reduced secretion 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 is highly conserved and that AMPK plays a prominent role in these networks.