The insulin signaling pathway a century after its discovery: Sexual dimorphism in insulin signaling.

Since practically a century ago, the insulin pathway was discovered in both vertebrates and invertebrates, implying an evolutionarily ancient origin. After a century of research, it is now clear that the insulin signal transduction pathway is a critical, flexible and pleiotropic pathway, evolving into multiple anabolic functions besides glucose homeostasis. It regulates paramount aspects of organismal well-being like growth, longevity, intermediate metabolism, and reproduction. Part of this diversification has been attained by duplications and divergence of both ligands and receptors riding on a common general signal transduction system. One of the aspects that is strikingly different is its usage in reproduction, particularly in male versus female development and fertility within the same species. This review highlights sexual divergence in metabolism and reproductive tract differences, the occurrence of sexually "exaggerated" traits, and sex size differences that are due to the sexes' differential activity/response to the insulin signaling pathway.

Circadian and rhythmic-related behavioral co-morbidities of the diabetic state in Drosophila melanogaster.

Circadian phenomena rule many activities of life on earth. Disruptions in circadian rhythmicity and rhythms have been recognized as a contributing factor for diseased states, for instance metabolic disruptions like diabetes. Diabetes develops as a consequence of faulty insulin pathway signaling, either by lack of insulin production (diabetes type I), or by loss of responsiveness in target tissues (diabetes type 2). In this work we use the model organism Drosophila melanogaster with three different mutant hypomorphic conditions at different levels of the insulin pathway. The insulin pathway is a very evolutionarily conserved pathway. We study these different diabetic conditions as a source of circadian rhythm abnormalities and circadian-related co-morbidities. We do so by studying circadian rhythmicity, activity, sleep and sleep structure, and feeding behavior. Results show that flies with impaired insulin signaling show circadian rhythm and rhythmic-related co-morbidities, especially female flies, as a consequence of the diabetic state. The most extreme disruptions occur in flies with impaired insulin receptor signaling, which stands at the beginning of the insulin pathway, in principle affecting most if not all aspects of this pathway. Our work shows that defective insulin signaling is a source of circadian rhythm and rhythmic related co-morbidities.

The various and shared roles of lncRNAs during development.

lncRNAs, genes transcribed but not translated, longer than 200 nucleotides, are classified as a separate class of nonprotein coding genes. Since their discovery, largely from RNAseq data, a number of pioneer studies have begun to unravel its myriad functions, centered on gene expression regulation, suggesting developmental and evolutionary conservation. Since they do not code for proteins and have no open reading frames, their functional constraints likely differ from that of protein coding genes, or of genes where the majority of the nucleotide sequence is required for function, like tRNAs. This has complicated assessment of both developmental and evolutionary conservation, and the identification of homologs in different species. Here we argue that other characteristics: general synteny and particular chromosomal placement regardless of sequence, sequence micro-motifs, and secondary structure allow for "homologs" to be identified and compared, confirming developmental and evolutionary conservation of lncRNAs. We conclude exemplifying a case in point: that of the evolutionarily conserved lncRNA acal, characterized and required for embryogenesis in Drosophila.

acal is a long non-coding RNA in JNK signaling in epithelial shape changes during drosophila dorsal closure.

Dorsal closure is an epithelial remodeling process taking place during Drosophila embryogenesis. JNK signaling coordinates dorsal closure. We identify and characterize acal as a novel negative dorsal closure regulator. acal represents a new level of JNK regulation. The acal locus codes for a conserved, long, non-coding, nuclear RNA. Long non-coding RNAs are an abundant and diverse class of gene regulators. Mutations in acal are lethal. acal mRNA expression is dynamic and is processed into a collection of 50 to 120 bp fragments. We show that acal lies downstream of raw, a pioneer protein, helping explain part of raw functions, and interacts genetically with Polycomb. acal functions in trans regulating mRNA expression of two genes involved in JNK signaling and dorsal closure: Connector of kinase to AP1 (Cka) and anterior open (aop). Cka is a conserved scaffold protein that brings together JNK and Jun, and aop is a transcription factor. Misregulation of Cka and aop can account for dorsal closure phenotypes in acal mutants.

Regulating cell morphogenesis: the Drosophila Jun N-terminal kinase pathway.

The Jun-N-terminal Kinase pathway (JNK), known also as stress activated protein kinase pathway (SAPK), is an eukaryotic evolutionarily conserved signaling pathway. From a purported evolutionarily "ancient" function as stress mediator, it evolved in multicellular eukaryotes to permanent roles in development, without leaving its original function. In Drosophila melanogaster, it is required for follicle cell morphogenesis, embryonic dorsal closure, pupal thoracic closure and genital disc rotation closure, all processes with requisite cell shape changes. Besides, it is activated during wound healing and in response to stress (UV irradiation, oxidative stress) where it may signal cell death or proliferation. Despite these varied roles, it has a conserved core of molecules that follow the MAPKKK/MAPKK/MAPK logic of mitogen activated protein kinases pathways. Regulation of the JNK pathway appears majorly negative, with phosphatases, transcription factors and proteins of novel structure "holding back" on JNK activation in different tissues. This particular mode of regulation may hark back to the pathway's origin as stress detector and responder, implying readiness to respond, from which the developmental roles may have evolved as conditions demanding obligate and predicted stress responses (i.e., embryonic dorsal closure viewed as a "wound of development").

Chronic Consumption of Moringa Leaf Powder (Moringa oleifera) Concentration-Dependent Effects in a Drosophila melanogaster Type 2 Diabetes Model.

OBJECTIVE: The metabolic effects of chronic consumption of food laced with different doses of moringa leaf powder (MLP) were assessed using a heteroallelic mutant of the sole insulin receptor gene of Drosophila melanogaster (InR), and the yellow,white (y,w) control stock. METHODS: The MLP composition was partially determined. Both strains were raised in a standard diet (SD) or in a SD supplemented with different MLP doses (0.5, 1.5, 2.5, 4.0, and 5.5%) until 4-5 days of emergence. Afterward, the total carbohydrate, lipid, glucose, and triacylglyceride levels were measured in the flies. Additionally, survival and weight changes were reported. For metabolic tests, female and male virgin flies were evaluated separately. RESULTS: Low MLP supplementation improved carbohydrate and glucose levels in the y,w strain. Additionally, the InR-mutant strain reported lower lipid content when subjected to the same regimes. Survival improved in both strains with low MLP doses, while chronic consumption of high MLP doses resulted in triacylglycerides increase, weight gain, and survival reduction. CONCLUSION: Low doses of MLP supplementation improves some metabolic parameters that affect flies' survival, especially in the y,w strain. Furthermore, the same low doses of MLP treatments also resulted in metabolic improvements in the InR-mutant flies; however, MLP consumption levels should be carefully assessed.Supplemental data for this article is available online at.

High glucose concentrations induce oxidative stress by inhibiting Nrf2 expression in rat Müller retinal cells in vitro.

Diabetic retinopathy (DR) is a complication of diabetes. Several studies have implicated oxidative stress as a fundamental factor in the progression of the disease. The nuclear factor erythroid-2-related factor 2 (Nrf2) is one of the main regulators of redox homeostasis. Glia Müller cells (MC) maintain the structural and functional stability of the retina. The objective of this study was to evaluate the effect of high glucose concentrations on reactive oxygen species (ROS) production and Nrf2 expression levels in rat MC. MC were incubated with normal (NG; 5 mM) or high glucose (HG; 25 mM) for different times. Incubation with HG increased ROS levels from 12 to 48 h but did not affect cell viability. However, exposure to 3 h of HG caused a transient decrease Nrf2 levels. At that time, we also observed a decrease in the mRNA expression of Nrf2 target genes, glutathione levels, and catalase activity, all of which increased significantly beyond initial levels after 48 h of incubation. HG exposure leads to an increase in the p65 subunit of nuclear factor-κB (NF-kB) levels, and its target genes. These results suggest that high glucose concentrations lead to alteration of the redox regulatory capacity of Nrf2 mediated by NF-kB regulation.

Synphilin suppresses α-synuclein neurotoxicity in a Parkinson's disease Drosophila model.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in humans. It affects 1% of the population over 65-years old. Its causes are environmental and genetic. As the world population ages, there is an urgent need for better and more detailed animal models for this kind of disease. In this work we show that the use of transgenic Drosophila is comparable to more complicated and costly animal models such as mice. The Drosophila model behaves very similar to the equivalent transgenic mice model. We show that both Synphilin-1 and α-synuclein are toxic by themselves, but when co-expressed, they suppress their toxicity reciprocally. Importantly, the symptoms induced in the fly can be treated and partially reverted using standard PD pharmacological treatments. This work showcases Drosophila as a detailed and multifaceted model for Parkinson's disease, providing a convenient platform in which to study and find new genetic modifiers of PD. genesis 49:392-402, 2011.

Retinal Nrf2 expression in normal and early streptozotocin-diabetic rats.

Diabetic retinopathy is the most common cause of vision loss among diabetic patients. Although hyperglycemia produces retinal oxidative stress in long-standing diabetes, the pathogenesis mechanism is unknown. The Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a central role in cell responses against oxidative damage. We used adult Long Evans rats where diabetes was induced by streptozotocin. Normal and treated rats were sacrificed at 7, 20, and 45 days after streptozotocin injection. We analyzed Nrf2 and Keap1 expression in retinal homogenates, cytoplasmic, and nuclear retinal fractions. Normal retina showed Nrf2 expression in all retina nuclear layers. We found a transitory decrease of Nrf2 mRNA and protein expression at 7 and 20 days after the streptozotocin injection that recovered later on: moreover, the protein level increased after 45 days. Keap1 immunoprecipitation revealed similar levels as Nrf2 in normal and diabetic rat retinas, indicating that the diabetic condition did not lead to dissociation of the Keap1-Nrf2 complex. Indeed, glutathione levels and superoxide dismutase activity were not altered in the treated rat retinas. These results do not support oxidative stress in the retina shortly after diabetes induction.

Diet Composition Differentially Affects Insulin Pathway Compromised and Control Flies.

The insulin pathway is an anabolic pathway that controls, amongst other things, glucose homeostasis. It is an evolutionarily conserved pathway. Disruptions in insulin pathway functions can lead to diabetic states. Diabetes, a very common occurrence in modern life, afflicts a significant portion of the population of developed and developing countries worldwide. Yet, few studies have addressed the evolution of diabetic states on a long-term basis. Here, we cultured three different insulin pathway signaling compromised flies (heteroallelic mutant combinations, akin to diabetes mellitus type II) and wild type control flies, for the extent of one generation in different isocaloric diets fed at libitum, with or without extra methionine added. All fly stocks have a homogenized genetic background. We measured weight, total lipid, and carbohydrate content of adults at two different time points, and survival of adults reared in some of the different diets. Results show that, despite the fact that all diet regimes allow survival of at least a fraction of flies to adulthood, life histories are significantly different. Higher protein content diets promote better survival compared to higher percentage lipid and carbohydrate diets, and added methionine promotes survival in moderately reduced protein content diets. In mutants, survival is significantly reduced, and added methionine generally has an effect, albeit a more modest one. Our results highlight the value of higher percentage protein diets, and differences in effects in "healthy" versus "diabetic" states. They also show that added methionine, proposed as a "sensor" for protein content in food for flies, leads to differential effects depending on the adequacy of the diet regime.

Drosophila bioassays are very sensitive methods to assess tarantula species venoms.

INTRODUCTION: Assaying venom toxicity in a suitable model system is often tricky, since normally the amount of venom is in short supply, and the assay subjects, i.e., typically mice, require large amounts. There is also no guarantee that the effects observed in the bioassay reflect the true nature of the venom's intended effects, as the animals used for assessment might not be the prey items to which the venom has evolved. METHODS: We harvested tarantula venoms from the Indian Poecilotheria regalis and the Mexican Bonnetina papalutlensis using light anesthesia and electrical stimulation. We follow the definition of venom as stated in (Nelsen et al., 2014). The recovered venom was lyophilized and reconstituted in sterile saline solution for injections. Drosophila melanogaster third instar larvae and adult flies were injected with 4.6 nanoliters of different concentrations of the venoms into the sixth abdominal segment, and scored for survival and development to adulthood. RESULTS: The injected venoms were very effective in provoking lethality of injected larvae and adults, with an LD50 of 1-5 nanomoles protein /gram wet weight. Comparison with other toxicity bioassays, i.e., mice and crickets -using the same P. regalis venom- renders the Drosophila bioassays three orders of magnitude more sensitive. The P. regalis and B. papalutlensis venoms have similar LD50. DISCUSSION: These bioassays use a small amount of venom compared to other bioassays, allowing characterization with far fewer starting material. As it uses insects, phylogenetically close to the intended prey victims, it also points to the efficiency of the tarantula venom for its preferred prey items, and thus, links as well to the tarantulas' ecology.

Fos metamorphoses: Lessons from mutants in model organisms.

The Fos oncogene gene family is evolutionarily conserved throughout Eukarya. Fos proteins characteristically have a leucine zipper and a basic region with a helix-turn-helix motif that binds DNA. In vertebrates, there are several Fos homologs. They can homo- or hetero-dimerize via the leucine zipper domain. Fos homologs coupled with other transcription factors, like Jun oncoproteins, constitute the Activator Protein 1 (AP-1) complex. From its original inception as an oncogene, the subsequent finding that they act as transcription factors binding DNA sequences known as TRE, to the realization that they are activated in many different scenarios, and to loss-of-function analysis, the Fos proteins have traversed a multifarious path in development and physiology. They are instrumental in 'immediate early genes' responses, and activated by a seemingly myriad assemblage of different stimuli. Yet, the majority of these studies were basically gain-of-function studies, since it was thought that Fos genes would be cell lethal. Loss-of-function mutations in vertebrates were recovered later, and were not cell lethal. In fact, c-fos null mutations are viable with developmental defects (osteopetrosis and myeloid lineage abnormalities). It was then hypothesized that vertebrate genomes exhibit partial redundancy, explaining the 'mild' phenotypes, and complicating assessment of complete loss-of-function phenotypes. Due to its promiscuous activation, fos genes (especially c-fos) are now commonly used as markers for cellular responses to stimuli. fos homologs high sequence conservation (including Drosophila) is advantageous as it allows critical assessment of fos genes functions in this genetic model. Drosophila melanogaster contains only one fos homolog, the gene kayak. kayak mutations are lethal, and allow study of all the processes where fos is required. The kayak locus encodes several different isoforms, and is a pleiotropic gene variously required for development involving cell shape changes. In general, fos genes seem to primarily activate programs involved in cellular architectural rearrangements and cell shape changes.

aaquetzalli is required for epithelial cell polarity and neural tissue formation in Drosophila.

Morphogenetic movements during embryogenesis require dynamic changes in epithelial cell polarity and cytoskeletal reorganization. Such changes involve, among others, rearrangements of cell-cell contacts and protein traffic. In Drosophila melanogaster, neuroblast delamination during early neurogenesis is a well-characterized process requiring a polarized neuroepithelium, regulated by the Notch signaling pathway. Maintenance of epithelial cell polarity ensues proper Notch pathway activation during neurogenesis. We characterize here aaquetzalli (aqz), a gene whose mutations affect cell polarity and nervous system specification. The aqz locus encodes a protein that harbors a domain with significant homology to a proline-rich conserved domain of nuclear receptor co-activators. aqz expression occurs at all stages of the fly life cycle, and is dynamic. aqz mutants are lethal, showing a disruption of cell polarity during embryonic ventral neuroepithelium differentiation resulting in loss of epithelial integrity and mislocalization of membrane proteins (shown by mislocalization of Crumbs, DE-Cadherin, and Delta). As a consequence, aqz mutant embryos with compromised apical-basal cell polarity develop spotty changes of neuronal and epithelial numbers of cells.

Drosophila melanogaster as a Model for Diabetes Type 2 Progression.

Drosophila melanogaster has been used as a very versatile and potent model in the past few years for studies in metabolism and metabolic disorders, including diabetes types 1 and 2. Drosophila insulin signaling, despite having seven insulin-like peptides with partially redundant functions, is very similar to the human insulin pathway and has served to study many different aspects of diabetes and the diabetic state. Yet, very few studies have addressed the chronic nature of diabetes, key for understanding the full-blown disease, which most studies normally explore. One of the advantages of having Drosophila mutant viable combinations at different levels of the insulin pathway, with significantly reduced insulin pathway signaling, is that the abnormal metabolic state can be studied from the onset of the life cycle and followed throughout. In this review, we look at the chronic nature of impaired insulin signaling. We also compare these results to the results gleaned from vertebrate model studies.

piragua encodes a zinc finger protein required for development in Drosophila.

We isolated and characterized embryonic lethal mutations in piragua (prg). The prg locus encodes a protein with an amino terminus Zinc Finger-Associated-Domain (ZAD) and nine C2H2 zinc fingers (ZF). prg mRNA and protein expression during embryogenesis is dynamic with widespread maternal contribution, and subsequent expression in epithelial precursors. About a quarter of prg mutant embryos do not develop cuticle, and from those that do a small fraction have cuticular defects. Roughly half of prg mutants die during embryogenesis. prg mutants have an extended phenocritical period encompassing embryogenesis and first instar larval stage, since the other half of prg mutants die as first or second instar larvae. During dorsal closure, time-lapse high-resolution imaging shows defects arising out of sluggishness in closure, resolving at times in failures of closure. prg is expressed in imaginal discs, and is required for imaginal development. prg was identified in imaginal tissue in a cell super competition screen, together with other genes, like flower. We find that flower mutations are also embryonic lethal with a similar phenocritical period and strong embryonic mutant phenotypes (head involution defects, primarily). The two loci interact genetically in the embryo, as they increase embryonic mortality to close to 90% with the same embryonic phenotypes (dorsal closure and head involution defects, plus lack of cuticle). Mutant prg clones generated in developing dorsal thorax and eye imaginal tissue have strong developmental defects (lack of bristles and ommatidial malformations). prg is required in several developmental morphogenetic processes.

Development and diabetes on the fly.

We review the use of a model organism to study the effects of a slow course, degenerative disease: namely, diabetes mellitus. Development and aging are biological phenomena entailing reproduction, growth, and differentiation, and then decline and progressive loss of functionality leading ultimately to failure and death. It occurs at all biological levels of organization, from molecular interactions to organismal well being and homeostasis. Yet very few models capable of addressing the different levels of complexity in these chronic, developmental phenomena are available to study, and model organisms are an exception and a welcome opportunity for these approaches. Genetic model organisms, like the common fruit fly, Drosophila melanogaster, offer the possibility of studying the panoply of life processes in normal and diseased states like diabetes mellitus, from a plethora of different perspectives. These long-term aspects are now beginning to be characterized.

Drosophila chem mutations disrupt epithelial polarity in Drosophila embryos.

Drosophila embryogenesis has proven to be an extremely powerful system for developmental gene discovery and characterization. We isolated five new EMS-induced alleles that do not complement the l(3R)5G83 lethal line isolated in the Nüsslein-Volhard and Wieschaus screens. We have named this locus chem. Lethality of the new alleles as homozygous zygotic mutants is not completely penetrant, and they have an extended phenocritical period. Like the original allele, a fraction of mutant embryos die with cuticular defects, notably head involution and dorsal closure defects. Embryonic defects are much more extreme in germline clones, where the majority of mutant embryos die during embryogenesis and do not form cuticle, implying a strong chem maternal contribution. chem mutations genetically interact with mutations in cytoskeletal genes (arm) and with mutations in the epithelial polarity genes coracle, crumbs, and yurt. chem mutants dorsal open defects are similar to those present in yurt mutants, and, likewise, they have epithelial polarity defects. chem1 and chem3 mutations suppress yurt3 , and chem3 mutants suppress crumbs1 mutations. In contrast, chem1 and coracle2 mutations enhance each other. Compared to controls, in chem mutants in embryonic lateral epithelia Crumbs expression is mislocalized and reduced, Coracle is increased and mislocalized basally at embryonic stages 13-14, then reduced at stage 16. Arm expression has a similar pattern but levels are reduced.

Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death.

The identification of pathways necessary for retinal pigment epithelium (RPE) function is fundamental to uncover therapies for blindness. Prolactin (PRL) receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19) human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2) expression, which inhibits the TRPM2-mediated intracellular Ca(2+) rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr(-/-)) mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.

Prolactins are natural inhibitors of angiogenesis in the retina.

PURPOSE: Disruption of the normally antiangiogenic environment of the retina leads to aberrant angiogenesis, the major cause of vision loss throughout the world. Prolactin (PRL), the hormone originally associated with milk production, can be proteolytically processed to 16K-PRL, a 16 kDa N-terminal PRL fragment with potent antiangiogenic and vasoconstrictive actions. This study was conducted to determine whether 16K-PRL is found naturally in the retina and plays a role in angiogenesis and vasodilation. METHODS: Expression of PRL mRNA in rat retina was determined by RT-PCR and in situ hybridization. Western blot was used to examine the expression of PRL and derived fragments in retinal homogenates. The role of PRL and 16K-PRL in the retina was studied by intravitreal injection of either antibodies against PRL or small interfering RNAs (siRNA), to suppress expression of retinal PRL mRNA. RESULTS: Rat retina expressed PRL mRNA in the outer nuclear, outer plexiform, inner nuclear, and ganglion cell layers. Both full-length PRL and N-terminal 16K-PRL were detected in retinal homogenates by polyclonal and monoclonal antibodies. The intravitreal injection of antibodies able to neutralize the actions of 16K-PRL increased the number of retinal blood vessels and capillary area by threefold. Furthermore, siRNA-mediated inhibition of PRL mRNA increased retinal neovascularization threefold and resulted in a significant increase in vasodilation. CONCLUSIONS: These results demonstrate that PRL is synthesized and cleaved to antiangiogenic 16K-PRL by retinal tissue and that these molecules play a key role in preventing angiogenesis in the healthy retina.