Partial ablation of adult Drosophilainsulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance

In Drosophila melanogaster (D. melanogaster), neurosecretory insulin-like peptide-producing cells (IPCs), analogous to mammalian pancreatic b cells are involved in glucose homeostasis. Extending those findings, we have developed in the adult fly an oral glucose tolerance test and demonstrated that IPCs indeed are responsible for executing an acute glucose clearance response. To further develop D. melanogaster as a relevant system for studying age-associated metabolic disorders, we set out to determine the impact of adult-specific partial ablation of IPCs (IPC knockdown) on insulin-like peptide (ILP) action, metabolic outcomes and longevity. Interestingly, while IPC knockdown flies are hyperglycemic and glucose intolerant, these flies remain insulin sensitive as measured by peripheral glucose disposal upon insulin injection and serine phosphorylation of a key insulin-signaling molecule, Akt. Significant increases in stored glycogen and triglyceride levels as well as an elevated level of circulating lipid measured in adult IPC knockdown flies suggest profound modulation in energy metabolism. Additional physiological outcomes measured in those flies include increased resistance to starvation and impaired female fecundity. Finally, increased life span and decreased mortality rates measured in IPC knockdown flies demonstrate that it is possible to modulate ILP action in adult flies to achieve life span extension without insulin resistance. Taken together, we have established and validated an invertebrate genetic system to further investigate insulin action, metabolic homeostasis and regulation of aging regulated by adult IPCs.     

Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance

In Drosophila melanogaster (D. melanogaster), neurosecretory insulin-like peptide-producing cells (IPCs), analogous to mammalian pancreatic β cells are involved in glucose homeostasis. Extending those findings, we have developed in the adult fly an oral glucose tolerance test and demonstrated that IPCs indeed are responsible for executing an acute glucose clearance response. To further develop D. melanogaster as a relevant system for studying age-associated metabolic disorders, we set out to determine the impact of adult-specific partial ablation of IPCs (IPC knockdown) on insulin-like peptide (ILP) action, metabolic outcomes and longevity. Interestingly, while IPC knockdown flies are hyperglycemic and glucose intolerant, these flies remain insulin sensitive as measured by peripheral glucose disposal upon insulin injection and serine phosphorylation of a key insulin-signaling molecule, Akt. Significant increases in stored glycogen and triglyceride levels as well as an elevated level of circulating lipid measured in adult IPC knockdown flies suggest profound modulation in energy metabolism. Additional physiological outcomes measured in those flies include increased resistance to starvation and impaired female fecundity. Finally, increased life span and decreased mortality rates measured in IPC knockdown flies demonstrate that it is possible to modulate ILP action in adult flies to achieve life span extension without insulin resistance. Taken together, we have established and validated an invertebrate genetic system to further investigate insulin action, metabolic homeostasis and regulation of aging regulated by adult IPCs.Key words: Drosophila melanogaster, insulin-producing cells (IPCs), drosophila insulin-like peptides (DILPs), type 2 diabetes, oral glucose tolerance test (OGTT), insulin sensitivity, energy metabolism, life span     

Interaction between Sleep and Metabolism in Drosophila with Altered Octopamine Signaling

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.     

Characterization of a Drosophila Alzheimer's disease model: pharmacological rescue of cognitive defects

Transgenic models of Alzheimer's disease (AD) have made significant contributions to our understanding of AD pathogenesis, and are useful tools in the development of potential therapeutics. The fruit fly, Drosophila melanogaster, provides a genetically tractable, powerful system to study the biochemical, genetic, environmental, and behavioral aspects of complex human diseases, including AD. In an effort to model AD, we over-expressed human APP and BACE genes in the Drosophila central nervous system. Biochemical, neuroanatomical, and behavioral analyses indicate that these flies exhibit aspects of clinical AD neuropathology and symptomology. These include the generation of Aβ(40) and Aβ(42), the presence of amyloid aggregates, dramatic neuroanatomical changes, defects in motor reflex behavior, and defects in memory. In addition, these flies exhibit external morphological abnormalities. Treatment with a γ-secretase inhibitor suppressed these phenotypes. Further, all of these phenotypes are present within the first few days of adult fly life. Taken together these data demonstrate that this transgenic AD model can serve as a powerful tool for the identification of AD therapeutic interventions.     

Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.     

The effects of age on radiation resistance and oxidative stress in adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a well-established model organism for genetic studies of development and aging. We examined the effects of lethal ionizing radiation on male and female adult Drosophila of different ages, using doses of radiation from 200 to 1500 Gy. Fifty percent lethality 2 days postirradiation (LD(50/2)) in wild-type 1-day-old adult fruit flies was approximately 1238 Gy for males and 1339 Gy for females. We observed a significant age-dependent decline in the radiation resistance of both males and females. Radiation damage is postulated to occur by the generation of oxygen radicals. An age-related decline in the ability of flies to resist an agent that induces oxygen radicals, paraquat, was observed when comparing 10- and 20-day adults. Female flies are more resistant to paraquat than male flies. Oxidative stress mediated by paraquat was additive with sublethal exposures to radiation in young adults. Therefore, the ability to repair the damage caused by oxygen radicals seems to decline with the age of the flies. Because Drosophila adults are largely post-mitotic, our data suggest that adult Drosophila melanogaster can serve as an excellent model to study the factors responsible for radiation resistance in post-mitotic tissue and age-dependent changes in this resistance.     

Drosophila melanogaster as a model to study drug addiction

Animal studies have been instrumental in providing knowledge about the molecular and neural mechanisms underlying drug addiction. Recently, the fruit fly Drosophila melanogaster has become a valuable system to model not only the acute stimulating and sedating effects of drugs but also their more complex rewarding properties. In this review, we describe the advantages of using the fly to study drug-related behavior, provide a brief overview of the behavioral assays used, and review the molecular mechanisms and neural circuits underlying drug-induced behavior in flies. Many of these mechanisms have been validated in mammals, suggesting that the fly is a useful model to understand the mechanisms underlying addiction.     

果蝇在肿瘤学研究中的优势及应用前景

果蝇作为研究人类疾病的模式生物, 与哺乳动物不仅在基本的生物学、生理学和神经系统机能等方面比较相似, 而且果蝇有其作为模式生物的独特优势。近年来的研究表明, 果蝇和人类在肿瘤发生信号通路等方面的保守性很高, 而且果蝇具有很强的遗传学可操作性, 是肿瘤学研究有效的模型之一, 可用于研究人类肿瘤发生、发展、转移等分子机制。文章综述了果蝇在肿瘤学研究中的优势、已建立的用于研究特定癌症的果蝇模型, 并对其在未来肿瘤学的研究方向进行展望, 以期为国内肿瘤学研究和抗肿瘤药物的研发提供参考。     

Using experimental evolution to study the physiological mechanisms of desiccation resistance in Drosophila melanogaster

Data from populations undergoing experimental evolution can be used to make comparisons between physiologically differentiated populations and to determine evolutionary trajectories. Comparisons of long-established laboratory populations of Drosophila melanogaster that are strongly differentiated with respect to desiccation resistance are used to test alternative hypotheses concerning the mechanisms that fruit flies use to survive bouts of extreme desiccation. This comparative study supports the hypothesis that, in at least one case, D. melanogaster can evolve increased resistance to desiccation by decreasing water loss rates and by increasing bulk water content but not by increasing metabolic water content or dehydration tolerance. While glycogen was involved in water storage, its primary role was in water binding, not the production of metabolic water. Measurement of the trajectories of these component mechanisms during selection for desiccation resistance is used to demonstrate that water loss rate quickly plateaus in response to selection, while water content continues to improve. This disparity reveals the value of studying evolutionary trajectories and the need for longer-term selection studies in evolutionary physiology.     

Searching for sleep mutants of Drosophila melanogaster

The functions of sleep are still unknown, but are probably related to cellular and molecular aspects of neural function. To better understand the benefits that sleep may bring at the cellular level, recent studies have employed Drosophila melanogaster as a model system and shown that fruit flies share the fundamental features of mammalian sleep. As in mammals, sleep in Drosophila is characterized by increased arousal threshold and by changes in brain electrical activity. Fly sleep is homeostatically regulated independent of the circadian clock, is modulated by stimulants and hypnotics, and is affected by age. Also, fly sleep is associated with changes in brain gene expression similar to those observed in mammals. While Drosophila neurobiology is sufficiently complex to permit meaningful generalizations to mammals and humans, Drosophila genetics is simple enough to allow a rapid mutagenesis screening. An ongoing mutagenesis study has screened approximately 5000 mutant Drosophila lines and found that sleep amount, sleep pattern, and the homeostatic regulation of sleep are highly conserved phenotypes in flies. So far, this study has identified 10 short sleeper lines and 4 lines that show no sleep rebound after sleep deprivation. Ultimately, the characterization of these lines should help identifying crucial cellular pathways involved in the regulatory mechanisms of sleep and its functional consequences.     

Insulin Stimulates Translocation of Human GLUT4 to the Membrane in Fat Bodies of Transgenic Drosophila melanogaster

The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in ‘diabetic’ flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin-signaling pathways in Drosophila, demonstrating the utility of TIRFM of tagged sugar transporters to monitor signaling pathways in insects.     

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages ^1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) ³. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis ^4,5. Here, we have modified a previously described procedure for insulin injection ⁶ and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly''s ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.Download video file.^{(37M, mov)}     

共生表皮葡萄球菌促进果蝇胚后发育

【背景】动物体定殖有多种共生微生物,这些共生微生物严重影响着宿主生理和病理,日益成为研究热点之一。【目的】分离与鉴定黑腹果蝇共生菌,探究表皮葡萄球菌对黑腹果蝇的发育影响和潜在作用机制。【方法】用CEM培养基(Carotenoid expression medium)从果蝇肠道内分离细菌,通过16SrRNA基因序列比对鉴定菌株;以发育时间和幼虫表面积检测果蝇的发育时期和生长速率;利用实时定量PCR检测果蝇促前胸腺激素与胰岛素通路的激活。【结果】从果蝇体内分离到的菌株为表皮葡萄球菌,该菌可以有效定殖于果蝇的肠道。表皮葡萄球菌通过提高果蝇生长速率而显著促进其发育。在分子水平上,表皮葡萄球菌激活PTTH和胰岛素信号以刺激宿主的生长发育。【结论】表皮葡萄球菌是果蝇的一种共生菌,可以通过调控PTTH和胰岛素信号而刺激果蝇生长发育。     

Alcohol consumption as self-medication against blood-borne parasites in the fruit fly

Plants and fungi often produce toxic secondary metabolites that limit their consumption, but herbivores and fungivores that evolve resistance gain access to these resources and can also gain protection against nonresistant predators and parasites. Given that Drosophila melanogaster fruit fly larvae consume yeasts growing on rotting fruit and have evolved resistance to fermentation products, we decided to test whether alcohol protects flies from one of their common natural parasites, endoparasitoid wasps. Here, we show that exposure to ethanol reduces wasp oviposition into fruit fly larvae. Furthermore, if infected, ethanol consumption by fruit fly larvae causes increased death of wasp larvae growing in the hemocoel and increased fly survival without need of the stereotypical antiwasp immune response. This multifaceted protection afforded to fly larvae by ethanol is significantly more effective against a generalist wasp than a wasp that specializes on D. melanogaster. Finally, fly larvae seek out ethanol-containing food when infected, indicating that they use alcohol as an antiwasp medicine. Although the high resistance of D. melanogaster may make it uniquely suited to exploit curative properties of alcohol, it is possible that alcohol consumption may have similar protective effects in other organisms.     

Sterols as dietary markers for Drosophila melanogaster

During cold acclimation fruit flies switch their feeding from yeast to plant food, however there are no robust molecular markers to monitor this in the wild. Drosophila melanogaster is a sterol auxotroph and relies on dietary sterols to produce lipid membranes, lipoproteins and molting hormones. We employed shotgun lipidomics to quantify eight major food sterols in total lipid extracts of heads and genital tracts of adult male and female flies. We found that their sterol composition is dynamic and reflective of fly diet in an organ-specific manner. Season-dependent changes observed in the organs of wild-living flies suggested that the molar ratio between yeast (ergosterol, zymosterol) and plant (sitosterol, stigmasterol) sterols is a quantifiable, generic and unequivocal marker of their feeding behavior suitable for ecological and environmental population-based studies. The enrichment of phytosterols over yeast sterols in wild-living flies at low temperatures is consistent with switching from yeast to plant diet and corroborates the concomitantly increased unsaturation of their membrane lipids.     

Quality of two aphid species (Rhopalosiphum padi and Sitobion avenae) as food for the generalist predator Tachyporus hypnorum (Col., Staphylinidae)

Abstract:  The quality of two aphid species ( Rhopalosiphum padi and Sitobion avenae ) as food for the staphylinid generalist predator Tachyporus hypnorum was investigated. Fruit flies ( Drosophila melanogaster ) were used as prey for comparison. T. hypnorum has been reported to prey on aphids, to consume large quantities and to show a high preference for aphids. This study showed that compared with fruit flies aphids are not high-quality prey for neither adult females nor larvae of T. hypnorum . Larvae were less able to utilize aphids than adult females. Reproduction was affected by both aphid species; a diet of S. avenae reduced fecundity, and a diet of R. padi reduced egg hatching success and prolonged the time spent in the egg stage compared with a fruit fly diet. For the T. hypnorum larvae pure diets of both S. avenae and R. padi resulted in high larval mortality. Overall ranking of the three prey types based on several fitness parameters differed between larvae and adults. The adult fitness parameters gave rather conflicting rankings of all three prey types though the two aphid species were of approximately the same overall value, whereas the larval fitness parameters gave identical rankings: D. melanogaster  >  S. avenae  >  R. padi . In the larval stage survival was greatly reduced by both aphid diets, while this was not the case for the adult females.     

SLOB, a SLOWPOKE channel binding protein, regulates insulin pathway signaling and metabolism in Drosophila

There is ample evidence that ion channel modulation by accessory proteins within a macromolecular complex can regulate channel activity and thereby impact neuronal excitability. However, the downstream consequences of ion channel modulation remain largely undetermined. The Drosophila melanogaster large conductance calcium-activated potassium channel SLOWPOKE (SLO) undergoes modulation via its binding partner SLO-binding protein (SLOB). Regulation of SLO by SLOB influences the voltage dependence of SLO activation and modulates synaptic transmission. SLO and SLOB are expressed especially prominently in median neurosecretory cells (mNSCs) in the pars intercerebralis (PI) region of the brain; these cells also express and secrete Drosophila insulin like peptides (dILPs). Previously, we found that flies lacking SLOB exhibit increased resistance to starvation, and we reasoned that SLOB may regulate aspects of insulin signaling and metabolism. Here we investigate the role of SLOB in metabolism and find that slob null flies exhibit changes in energy storage and insulin pathway signaling. In addition, slob null flies have decreased levels of dilp3 and increased levels of takeout, a gene known to be involved in feeding and metabolism. Targeted expression of SLOB to mNSCs rescues these alterations in gene expression, as well as the metabolic phenotypes. Analysis of fly lines mutant for both slob and slo indicate that the effect of SLOB on metabolism and gene expression is via SLO. We propose that modulation of SLO by SLOB regulates neurotransmission in mNSCs, influencing downstream insulin pathway signaling and metabolism.     

High sugar-induced insulin resistance in Drosophila relies on the lipocalin Neural Lazarillo

In multicellular organisms, insulin/IGF signaling (IIS) plays a central role in matching energy needs with uptake and storage, participating in functions as diverse as metabolic homeostasis, growth, reproduction and ageing. In mammals, this pleiotropy of action relies in part on a dichotomy of action of insulin, IGF-I and their respective membrane-bound receptors. In organisms with simpler IIS, this functional separation is questionable. In Drosophila IIS consists of several insulin-like peptides called Dilps, activating a unique membrane receptor and its downstream signaling cascade. During larval development, IIS is involved in metabolic homeostasis and growth. We have used feeding conditions (high sugar diet, HSD) that induce an important change in metabolic homeostasis to monitor possible effects on growth. Unexpectedly we observed that HSD-fed animals exhibited severe growth inhibition as a consequence of peripheral Dilp resistance. Dilp-resistant animals present several metabolic disorders similar to those observed in type II diabetes (T2D) patients. By exploring the molecular mechanisms involved in Drosophila Dilp resistance, we found a major role for the lipocalin Neural Lazarillo (NLaz), a target of JNK signaling. NLaz expression is strongly increased upon HSD and animals heterozygous for an NLaz null mutation are fully protected from HSD-induced Dilp resistance. NLaz is a secreted protein homologous to the Retinol-Binding Protein 4 involved in the onset of T2D in human and mice. These results indicate that insulin resistance shares common molecular mechanisms in flies and human and that Drosophila could emerge as a powerful genetic system to study some aspects of this complex syndrome.     

Effect of plant oils on quality parameters of Bactrocera tryoni (Froggatt) reared on liquid larval diet

Liquid larval diets have been developed for several tephritid fruit flies including Queensland fruit fly, Bactrocera tryoni (Frogatt) (Q‐fly). In liquid diets, wheat germ oil (WGO) is usually added to improve performance in some quality parameters of reared flies, especially flight ability. However, for some flies, other plant oils may be more readily available, cheaper or produce flies of superior performance. In the present study, four alternative types of plant oils – rice bran, canola, vegetable, and sesame – were incorporated into a fruit fly liquid larval diet to replace the currently used wheat germ oil and their efficacy on the quality parameters of reared Q‐fly was compared to diets containing wheat germ oil or no oil. The quality parameters included: total pupal yield (N), pupal recovery (%), larval duration (days), pupal weight (mg), adult emergence (%), adult fliers (%), rate of fliers (%), sex ratio (%), F₁ egg/female/day and egg hatching (%). There were significant differences among treatments in performance of Q‐fly. Vegetable oil appeared better in terms of total pupal yield, percentage of pupal recovery, percentage of adult emergence, percentage of fliers, mean egg/female/day and % F₁ egg hatch compared with other oil treatments, especially from that of WGO treated diet. The result suggests that WGO can be substituted with rice bran and vegetable oil to improve the liquid larval diet for rearing of B. tryoni, with vegetable oil being the best replacement.