Drosophila as a Rapid Screening Model to Evaluate the Hypoglycemic Effects of Dipeptidyl Peptidase 4 (DPP4) Inhibitors: High Evolutionary Conservation of DPP4.

Dipeptidyl peptidase 4 (DPP4) inhibitors, commonly known as gliptins, have been an integral part of the treatment of type 2 diabetes mellitus (T2DM) for several years. Despite their remarkable efficacy in lowering glucose levels and their compatibility with other hypoglycemic drugs, recent studies have revealed adverse effects, prompting the search for improved drugs within this category, which has required the use of animal models to verify the hypoglycemic effects of these compounds. Currently, in many countries the use of mammals is being significantly restricted, as well as cost prohibitive, and alternative in vivo approaches have been encouraged. In this sense, Drosophila has emerged as a promising alternative for several compelling reasons: it is cost-effective, offers high experimental throughput, is genetically manipulable, and allows the assessment of multigenerational effects, among other advantages. In this study, we present evidence that diprotin A, a DPP4 inhibitor, effectively reduces glucose levels in Drosophila hemolymph. This discovery underscores the potential of Drosophila as an initial screening tool for novel compounds directed against DPP4 enzymatic activity.

The Statin Target Hmgcr Regulates Energy Metabolism and Food Intake through Central Mechanisms.

The statin drug target, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), is strongly linked to body mass index (BMI), yet how HMGCR influences BMI is not understood. In mammals, studies of peripheral HMGCR have not clearly identified a role in BMI maintenance and, despite considerable central nervous system expression, a function for central HMGCR has not been determined. Similar to mammals, Hmgcr is highly expressed in the Drosophila melanogaster brain. Therefore, genetic and pharmacological studies were performed to identify how central Hmgcr regulates Drosophila energy metabolism and feeding behavior. We found that inhibiting Hmgcr, in insulin-producing cells of the Drosophila pars intercerebralis (PI), the fly hypothalamic equivalent, significantly reduces the expression of insulin-like peptides, severely decreasing insulin signaling. In fact, reducing Hmgcr expression throughout development causes decreased body size, increased lipid storage, hyperglycemia, and hyperphagia. Furthermore, the Hmgcr induced hyperphagia phenotype requires a conserved insulin-regulated α-glucosidase, target of brain insulin (tobi). In rats and mice, acute inhibition of hypothalamic Hmgcr activity stimulates food intake. This study presents evidence of how central Hmgcr regulation of metabolism and food intake could influence BMI.

The Statin Target HMG-Coenzyme a Reductase (Hmgcr) Regulates Sleep Homeostasis in Drosophila.

Statins, HMG Coenzyme A Reductase (HMGCR) inhibitors, are a first-line therapy, used to reduce hypercholesterolemia and the risk for cardiovascular events. While sleep disturbances are recognized as a side-effect of statin treatment, the impact of statins on sleep is under debate. Using Drosophila, we discovered a novel role for Hmgcr in sleep modulation. Loss of pan-neuronal Hmgcr expression affects fly sleep behavior, causing a decrease in sleep latency and an increase in sleep episode duration. We localized the pars intercerebralis (PI), equivalent to the mammalian hypothalamus, as the region within the fly brain requiring Hmgcr activity for proper sleep maintenance. Lack of Hmgcr expression in the PI insulin-producing cells recapitulates the sleep effects of pan-neuronal Hmgcr knockdown. Conversely, loss of Hmgcr in a different PI subpopulation, the corticotropin releasing factor (CRF) homologue-expressing neurons (DH44 neurons), increases sleep latency and decreases sleep duration. The requirement for Hmgcr activity in different neurons signifies its importance in sleep regulation. Interestingly, loss of Hmgcr in the PI does not affect circadian rhythm, suggesting that Hmgcr regulates sleep by pathways distinct from the circadian clock. Taken together, these findings suggest that Hmgcr activity in the PI is essential for proper sleep homeostasis in flies.

Dibutyl phthalate disrupts conserved circadian rhythm in Drosophila and human cells.

People are constantly exposed to phthalates, due to their common use in the production of plastics, pharmaceuticals, cosmetics and skin care products. The ability of phthalates to disrupt endocrine signaling, leading to developmental, reproductive and metabolic defects, has been studied, yet how phthalates interfere with these biological functions is still unclear. To uncover DBP interacting molecular pathways, we raised Drosophila melanogaster on food containing dibutyl phthalate (DBP) at various concentrations. Whole transcriptome analysis of adult Drosophila reveals that DBP exposure throughout development disrupts the expression of genes central to circadian rhythm regulation, including increased expression of vrille (vri, human NFIL3), timeless (tim, human TIMELESS) and period (per, human PER3), with decreased expression of Pigment-dispersing factor (Pdf). DBP exposure also alters the expression of the evolutionarily conserved nuclear receptor Hormone receptor-like in 38 (Hr38, human NR4A2), which is known to regulate Pdf expression. Furthermore, behavioral assays determined that exposing Drosophila to DBP throughout development modifies the circadian rhythm of adults. Although DBP inhibits the expression of signaling systems regulating vision, including Rh5 and Rh6, two light-sensing G-protein coupled receptors involved in the daily resetting of circadian rhythm, it does not influence eye development. Circadian rhythm genes are well conserved from flies to humans; therefore, we tested the effect of DBP exposure on human breast cells (MCF10A) and demonstrate that, similar to the fruit fly model, this exposure disrupts circadian rhythm (BMAL1 expression) at doses that promote the proliferation and migration ability of MCF10A cells. Our results are the first to provide comprehensive evidence that DBP interferes with circadian rhythm in both adult Drosophila and human cells, which may help to explain the broad physiological action of phthalates.

Impact of high-fat diet on lifespan, metabolism, fecundity and behavioral senescence in Drosophila.

Excess consumption of high-fat diet (HFD) is likely to result in obesity and increases the predisposition to associated health disorders. Drosophila melanogaster has emerged as an important model to study the effects of HFD on metabolism, gut function, behavior, and ageing. In this study, we investigated the effects of HFD on physiology and behavior of female flies at different time-points over several weeks. We found that HFD decreases lifespan, and also with age leads to accelerated decline of climbing ability in both virgins and mated flies. In virgins HFD also increased sleep fragmentation with age. Furthermore, long-term exposure to HFD results in elevated adipokinetic hormone (AKH) transcript levels and an enlarged crop with increased lipid stores. We detected no long-term effects of HFD on body mass, or levels of triacylglycerides (TAG), glycogen or glucose, although fecundity was diminished. However, one week of HFD resulted in decreased body mass and elevated TAG levels in mated flies. Finally, we investigated the role of AKH in regulating effects of HFD during aging. Both with normal diet (ND) and HFD, Akh mutant flies displayed increased longevity compared to control flies. However, both mutants and controls showed shortened lifespan on HFD compared to ND. In flies exposed to ND, fecundity is decreased in Akh mutants compared to controls after one week, but increased after three weeks. However, HFD leads to a similar decrease in fecundity in both genotypes after both exposure times. Thus, long-term exposure to HFD increases AKH signaling, impairs lifespan and fecundity and augments age-related behavioral senescence.

Drosophila Insulin-Like Peptide 8 (DILP8) in Ovarian Follicle Cells Regulates Ovulation and Metabolism.

In Drosophila melanogaster eight insulin-like peptides (DILP1-8) are encoded on separate genes. These DILPs are characterized by unique spatial and temporal expression patterns during the lifecycle. Whereas, functions of several of the DILPs have been extensively investigated at different developmental stages, the role of DILP8 signaling is primarily known from larvae and pupae where it couples organ growth and developmental transitions. In adult female flies, a study showed that a specific set of neurons that express the DILP8 receptor, Lgr3, is involved in regulation of reproductive behavior. Here, we further investigated the expression of dilp8/DILP8 and Lgr3 in adult female flies and the functional role of DILP8 signaling. The only site where we found both dilp8 expression and DILP8 immunolabeling was in follicle cells around mature eggs. Lgr3 expression was detected in numerous neurons in the brain and ventral nerve cord, a small set of peripheral neurons innervating the abdominal heart, as well as in a set of follicle cells close to the oviduct. Ovulation was affected in dilp8 mutants as well as after dilp8-RNAi using dilp8 and follicle cell Gal4 drivers. More eggs were retained in the ovaries and fewer were laid, indicating that DILP8 is important for ovulation. Our data suggest that DILP8 signals locally to Lgr3 expressing follicle cells as well as systemically to Lgr3 expressing efferent neurons in abdominal ganglia that innervate oviduct muscle. Thus, DILP8 may act at two targets to regulate ovulation: follicle cell rupture and oviduct contractions. Furthermore, we could show that manipulations of dilp8 expression affect starvation resistance suggesting effects on metabolism. Possibly this reflects a feedback signaling between ovaries and the CNS that ensures nutrients for ovary development. In summary, it seems that DILP8 signaling in regulation of reproduction is an ancient function, conserved in relaxin signaling in mammals.

Regulatory Roles of Drosophila Insulin-Like Peptide 1 (DILP1) in Metabolism Differ in Pupal and Adult Stages.

The insulin/IGF-signaling pathway is central in control of nutrient-dependent growth during development, and in adult physiology and longevity. Eight insulin-like peptides (DILP1-8) have been identified in Drosophila, and several of these are known to regulate growth, metabolism, reproduction, stress responses, and lifespan. However, the functional role of DILP1 is far from understood. Previous work has shown that dilp1/DILP1 is transiently expressed mainly during the pupal stage and the first days of adult life. Here, we study the role of dilp1 in the pupa, as well as in the first week of adult life, and make some comparisons to dilp6 that displays a similar pupal expression profile, but is expressed in fat body rather than brain neurosecretory cells. We show that mutation of dilp1 diminishes organismal weight during pupal development, whereas overexpression increases it, similar to dilp6 manipulations. No growth effects of dilp1 or dilp6 manipulations were detected during larval development. We next show that dilp1 and dilp6 increase metabolic rate in the late pupa and promote lipids as the primary source of catabolic energy. Effects of dilp1 manipulations can also be seen in the adult fly. In newly eclosed female flies, survival during starvation is strongly diminished in dilp1 mutants, but not in dilp2 and dilp1/dilp2 mutants, whereas in older flies, only the double mutants display reduced starvation resistance. Starvation resistance is not affected in male dilp1 mutant flies, suggesting a sex dimorphism in dilp1 function. Overexpression of dilp1 also decreases survival during starvation in female flies and increases egg laying and decreases egg to pupal viability. In conclusion, dilp1 and dilp6 overexpression promotes metabolism and growth of adult tissues during the pupal stage, likely by utilization of stored lipids. Some of the effects of the dilp1 manipulations may carry over from the pupa to affect physiology in young adults, but our data also suggest that dilp1 signaling is important in metabolism and stress resistance in the adult stage.

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

Insulin/IGF signaling (IIS) regulates essential processes including development, metabolism, and aging. The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. Many reports show that longevity is increased by manipulations that decrease DILP2 levels. It has been shown that dilp1 is expressed primarily in pupal stages, but also during adult reproductive diapause. Here, we find that dilp1 is also highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Here, we study dilp1 and dilp2 single and double mutants to describe epistatic and synergistic 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. On the other hand, dilp1 and dilp2 redundantly or synergistically interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. These interactions do not correlate with patterns for how dilp1 and dilp2 affect longevity and AKH. Thus, 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. Our data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro-longevity insulin-like peptide.

Modulation of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin.

Behavior and physiology are orchestrated by neuropeptides acting as central neuromodulators and circulating hormones. An outstanding question is how these neuropeptides function to coordinate complex and competing behaviors. In Drosophila, the neuropeptide leucokinin (LK) modulates diverse functions, but mechanisms underlying these complex interactions remain poorly understood. As a first step towards understanding these mechanisms, we delineated LK circuitry that governs various aspects of post-feeding physiology and behavior. We found that impaired LK signaling in Lk and Lk receptor (Lkr) mutants affects diverse but coordinated processes, including regulation of stress, water homeostasis, feeding, locomotor activity, and metabolic rate. Next, we sought to define the populations of LK neurons that contribute to the different aspects of this physiology. We find that the calcium activity in abdominal ganglia LK neurons (ABLKs), but not in the two sets of brain neurons, increases specifically following water consumption, suggesting that ABLKs regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of Lkr expression, mined a brain single-cell transcriptome dataset for genes coexpressed with Lkr, and identified synaptic partners of LK neurons. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and chemosensory cells, correlates well with regulatory roles detected in the Lk and Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) and transcripts in IPCs and increased starvation resistance. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons have different targets, but modulate the establishment of post-prandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating feeding-related neuroendocrine regulation of vital behavior and physiology.

Behavioral Senescence and Aging-Related Changes in Motor Neurons and Brain Neuromodulator Levels Are Ameliorated by Lifespan-Extending Reproductive Dormancy in Drosophila.

The lifespan of Drosophilamelanogaster can be extended substantially by inducing reproductive dormancy (also known as diapause) by lowered temperature and short days. This increase of longevity is accompanied by lowered metabolism and increased stress tolerance. We ask here whether behavioral senescence is ameliorated during adult dormancy. To study this we kept flies for seven or more weeks in normal rearing conditions or in diapause conditions and compared to 1-week-old flies in different behavioral assays of sleep, negative geotaxis and exploratory walking. We found that the senescence of geotaxis and locomotor behavior seen under normal rearing conditions was negligible in flies kept in dormancy. The normal senescence of rhythmic activity and sleep patterns during the daytime was also reduced by adult dormancy. Investigating the morphology of specific neuromuscular junctions (NMJs), we found that changes normally seen with aging do not take place in dormant flies. To monitor age-associated changes in neuronal circuits regulating activity rhythms, sleep and walking behavior we applied antisera to tyrosine hydroxylase (TH), serotonin and several neuropeptides to examine changes in expression levels and neuron morphology. In most neuron types the levels of stored neuromodulators decreased during normal aging, but not in diapause treated flies. No signs of neurodegeneration were seen in either condition. Our data suggest that age-related changes in motor neurons could be the cause of part of the behavioral senescence and that this is ameliorated by reproductive diapause. Earlier studies established a link between age-associated decreases in neuromodulator levels and behavioral decline that could be rescued by overexpression of neuromodulator. Thus, it is likely that the retained levels of neuromodulators in dormant flies alleviate behavioral senescence.

Drosophila insulin-like peptide 1 (DILP1) is transiently expressed during non-feeding stages and reproductive dormancy.

The insulin/insulin-like growth factor signaling pathway is evolutionarily conserved in animals, and is part of nutrient-sensing mechanisms that control growth, metabolism, reproduction, stress responses, and lifespan. In Drosophila, eight insulin-like peptides (DILP1-8) are known, six of which have been investigated in some detail, whereas expression and functions of DILP1 and DILP4 remain enigmatic. Here we demonstrate that dilp1/DILP1 is transiently expressed in brain insulin producing cells (IPCs) from early pupa until a few days of adult life. However, in adult female flies where diapause is triggered by low temperature and short days, within a time window 0-10h post-eclosion, the dilp1/DILP1 expression remains high for at least 9 weeks. The dilp1 mRNA level is increased in dilp2, 3, 5 and dilp6 mutant flies, indicating feedback regulation. Furthermore, the DILP1 expression in IPCs is regulated by short neuropeptide F, juvenile hormone and presence of larval adipocytes. Male dilp1 mutant flies display increased lifespan and reduced starvation resistance, whereas in female dilp1 mutants oviposition is reduced. Thus, DILP1 is expressed in non-feeding stages and in diapausing flies, is under feedback regulation and appears to play sex-specific functional roles.