Leucokinins: Multifunctional Neuropeptides and Hormones in Insects and Other Invertebrates

Leucokinins (LKs) constitute a neuropeptide family first discovered in a cockroach and later identified in numerous insects and several other invertebrates. The LK receptors are only distantly related to other known receptors. Among insects, there are many examples of species where genes encoding LKs and their receptors are absent. Furthermore, genomics has revealed that LK signaling is lacking in several of the invertebrate phyla and in vertebrates. In insects, the number and complexity of LK-expressing neurons vary, from the simple pattern in the Drosophila larva where the entire CNS has 20 neurons of 3 main types, to cockroaches with about 250 neurons of many different types. Common to all studied insects is the presence or 1–3 pairs of LK-expressing neurosecretory cells in each abdominal neuromere of the ventral nerve cord, that, at least in some insects, regulate secretion in Malpighian tubules. This review summarizes the diverse functional roles of LK signaling in insects, as well as other arthropods and mollusks. These functions include regulation of ion and water homeostasis, feeding, sleep–metabolism interactions, state-dependent memory formation, as well as modulation of gustatory sensitivity and nociception. Other functions are implied by the neuronal distribution of LK, but remain to be investigated.     

Drosophila to Explore Nucleolar Stress

Nucleolar stress occurs when ribosome production or function declines. Nucleolar stress in stem cells or progenitor cells often leads to disease states called ribosomopathies. Drosophila offers a robust system to explore how nucleolar stress causes cell cycle arrest, apoptosis, or autophagy depending on the cell type. We provide an overview of nucleolar stress in Drosophila by depleting nucleolar phosphoprotein of 140 kDa (Nopp140), a ribosome biogenesis factor (RBF) in nucleoli and Cajal bodies (CBs). The depletion of Nopp140 in eye imaginal disc cells generates eye deformities reminiscent of craniofacial deformities associated with the Treacher Collins syndrome (TCS), a human ribosomopathy. We show the activation of c-Jun N-terminal Kinase (JNK) in Drosophila larvae homozygous for a Nopp140 gene deletion. JNK is known to induce the expression of the pro-apoptotic Hid protein and autophagy factors Atg1, Atg18.1, and Atg8a; thus, JNK is a central regulator in Drosophila nucleolar stress. Ribosome abundance declines upon Nopp140 loss, but unusual cytoplasmic granules accumulate that resemble Processing (P) bodies based on marker proteins, Decapping Protein 1 (DCP1) and Maternal expression at 31B (Me31B). Wild type brain neuroblasts (NBs) express copious amounts of endogenous coilin, but coilin levels decline upon nucleolar stress in most NB types relative to the Mushroom body (MB) NBs. MB NBs exhibit resilience against nucleolar stress as they maintain normal coilin, Deadpan, and EdU labeling levels.     

New Insights of SF1 Neurons in Hypothalamic Regulation of Obesity and Diabetes

Despite the substantial role played by the hypothalamus in the regulation of energy balance and glucose homeostasis, the exact mechanisms and neuronal circuits underlying this regulation remain poorly understood. In the last 15 years, investigations using transgenic models, optogenetic, and chemogenetic approaches have revealed that SF1 neurons in the ventromedial hypothalamus are a specific lead in the brain’s ability to sense glucose levels and conduct insulin and leptin signaling in energy expenditure and glucose homeostasis, with minor feeding control. Deletion of hormonal receptors, nutritional sensors, or synaptic receptors in SF1 neurons triggers metabolic alterations mostly appreciated under high-fat feeding, indicating that SF1 neurons are particularly important for metabolic adaptation in the early stages of obesity. Although these studies have provided exciting insight into the implications of hypothalamic SF1 neurons on whole-body energy homeostasis, new questions have arisen from these results. Particularly, the existence of neuronal sub-populations of SF1 neurons and the intricate neurocircuitry linking these neurons with other nuclei and with the periphery. In this review, we address the most relevant studies carried out in SF1 neurons to date, to provide a global view of the central role played by these neurons in the pathogenesis of obesity and diabetes.     

Effects of Overexpression of Neurosecretory Protein GL-Precursor Gene on Glucose Homeostasis and Insulin Sensitivity in Mice

A high-fat diet (HFD) quickly induces obesity with insulin resistance and hyperglycemia. We previously reported that a novel hypothalamic small protein, named neurosecretory protein GL (NPGL), stimulates feeding and fat accumulation in mice. However, the effects of NPGL on insulin sensitivity and glucose homeostasis remain unknown. Hence, we subjected NPGL-precursor gene (Npgl)-overexpressing mice to the oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT) under normal chow (NC) and HFD conditions. Npgl overexpression promoted body mass gain and tended to increase food intake of NC-fed mice, whereas it had little effect on HFD-fed mice. The OGTT showed elevated blood glucose and insulin levels in Npgl-overexpressing NC-fed mice 15 min after glucose administration. Both the OGTT and IPITT demonstrated that Npgl overexpression decreased blood glucose levels in HFD-fed mice 60 min after glucose and insulin treatments. Notably, Npgl overexpression increased adipose tissue masses only in NC-fed mice, and it decreased blood glucose and insulin levels in HFD-fed mice at the experimental end point. It also increased the mRNA expression of galanin, one of the feeding and metabolic regulatory neuropeptides, in the hypothalamus of HFD-fed mice. Therefore, NPGL may alleviate HFD-induced hyperglycemia and insulin resistance in mice.     

Glutamate and GABA in Microglia-Neuron Cross-Talk in Alzheimer’s Disease

The physiological balance between excitation and inhibition in the brain is significantly affected in Alzheimer’s disease (AD). Several neuroactive compounds and their signaling pathways through various types of receptors are crucial in brain homeostasis, among them glutamate and γ-aminobutyric acid (GABA). Activation of microglial receptors regulates the immunological response of these cells, which in AD could be neuroprotective or neurotoxic. The novel research approaches revealed the complexity of microglial function, including the interplay with other cells during neuroinflammation and in the AD brain. The purpose of this review is to describe the role of several proteins and multiple receptors on microglia and neurons, and their involvement in a communication network between cells that could lead to different metabolic loops and cell death/survival. Our review is focused on the role of glutamatergic, GABAergic signaling in microglia–neuronal cross-talk in AD and neuroinflammation. Moreover, the significance of AD-related neurotoxic proteins in glutamate/GABA-mediated dialogue between microglia and neurons was analyzed in search of novel targets in neuroprotection, and advanced pharmacological approaches.     

Effects of Indole Glucosinolates on Performance and Sequestration by the Sawfly Athalia rosae and Consequences of Feeding on the Plant Defense System

In this study, the performance and sequestration specificity of the sawfly Athalia rosae L. (Hymenoptera: Tenthredinidae) reared on two wild Brassica oleracea L. (Brassicaceae) populations (KIM and WIN), which express indole glucosinolates (GSs) in very high concentrations, were investigated. The local and systemic plant responses to larval feeding also were analyzed. Indole GSs predominated in KIM plants, whereas aliphatic GSs prevailed in the WIN population, which had several times higher total GS concentrations than KIM plants. Plants of the KIM population had lower specific leaf areas, and higher carbon/nitrogen ratios than WIN plants. The insects reared on WIN plants performed better for most traits than insects reared on the KIM population. The larvae preferentially sequestered aliphatic GSs but when feeding on KIM plants, indole GSs were also concentrated in the hemolymph. In response to feeding by A. rosae larvae, indole GSs were induced locally and systemically, whereas aliphatic GSs were reduced only in systemic leaves of the WIN population. Soluble myrosinase activities were constitutively higher in WIN than in KIM plants, and feeding damage led to a significant reduction of this glucosinolate-degrading enzyme in WIN plants only. The data suggest that the sawfly is well adapted to high concentrations of aliphatic GSs, which are sequestered by the larvae. In contrast, the larvae may be less adapted to plants dominated by indole GSs. Selective induction of indole GSs by the plants in response to larval feeding may be adaptive as accumulation of indole GSs may lower the performance of the larvae.     

Drosophila Solute Carrier 5A5 Regulates Systemic Glucose Homeostasis by Mediating Glucose Absorption in the Midgut

The small intestine is the initial site of glucose absorption and thus represents the first of a continuum of events that modulate normal systemic glucose homeostasis. A better understanding of the regulation of intestinal glucose transporters is therefore pertinent to our efforts in curbing metabolic disorders. Using molecular genetic approaches, we investigated the role of Drosophila Solute Carrier 5A5 (dSLC5A5) in regulating glucose homeostasis by mediating glucose uptake in the fly midgut. By genetically knocking down dSLC5A5 in flies, we found that systemic and circulating glucose and trehalose levels are significantly decreased, which correlates with an attenuation in glucose uptake in the enterocytes. Reciprocally, overexpression of dSLC5A5 significantly increases systemic and circulating glucose and trehalose levels and promotes glucose uptake in the enterocytes. We showed that dSLC5A5 undergoes apical endocytosis in a dynamin-dependent manner, which is essential for glucose uptake in the enterocytes. Furthermore, we showed that the dSLC5A5 level in the midgut is upregulated by glucose and that dSLC5A5 critically directs systemic glucose homeostasis on a high-sugar diet. Together, our studies have uncovered the first Drosophila glucose transporter in the midgut and revealed new mechanisms that regulate glucose transporter levels and activity in the enterocyte apical membrane.     

Astrocytic Vesicle Mobility in Health and Disease

Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5′-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.     

Functional Inactivation of Drosophila GCK Orthologs Causes Genomic Instability and Oxidative Stress in a Fly Model of MODY-2

Maturity-onset diabetes of the young (MODY) type 2 is caused by heterozygous inactivating mutations in the gene encoding glucokinase (GCK), a pivotal enzyme for glucose homeostasis. In the pancreas GCK regulates insulin secretion, while in the liver it promotes glucose utilization and storage. We showed that silencing the Drosophila GCK orthologs Hex-A and Hex-C results in a MODY-2-like hyperglycemia. Targeted knock-down revealed that Hex-A is expressed in insulin producing cells (IPCs) whereas Hex-C is specifically expressed in the fat body. We showed that Hex-A is essential for insulin secretion and it is required for Hex-C expression. Reduced levels of either Hex-A or Hex-C resulted in chromosome aberrations (CABs), together with an increased production of advanced glycation end-products (AGEs) and reactive oxygen species (ROS). This result suggests that CABs, in GCK depleted cells, are likely due to hyperglycemia, which produces oxidative stress through AGE metabolism. In agreement with this hypothesis, treating GCK-depleted larvae with the antioxidant vitamin B6 rescued CABs, whereas the treatment with a B6 inhibitor enhanced genomic instability. Although MODY-2 rarely produces complications, our data revealed the possibility that MODY-2 impacts genome integrity.     

Adult Frass Provides a Pheromone Signature for <Emphasis Type="Italic">Drosophila</Emphasis> Feeding and Aggregation

Adult Drosophila melanogaster locate food resources by using distinct olfactory cues that often are associated with the fermentation of fruit. However, in addition to being an odorous food source and providing a possible site for oviposition, fermenting fruit also provides a physical substrate upon which flies can attract and court a potential mate. In this study, we demonstrate that Drosophila adults are able to recruit additional flies to a food source by covering the exposed surface area with fecal spots, and that this recruitment is mediated via olfactory receptors (Ors). Analyses of the deposited frass material demonstrates that frass contains several previously studied pheromone components, such as methyl laurate (ML), methyl myristate (MM), methyl palmitate (MP), and 11-cis-vaccenyl acetate (cVA), in addition to several cuticular hydrocarbons (CHCs) that are known to be behaviorally active. Moreover, this study also demonstrates that adult feeding is increased in the presence of frass, although it appears that Ors are less likely to mediate this phenomenon. In summary, the frass deposited by the fly onto the fruit provides both pheromone and CHC cues that lead to increased feeding and aggregation in Drosophila. This research is the first step in examining Drosophila frass as an important chemical signature that provides information about both the sex and the species of the fly that generated the fecal spots.     

Head-to-tail cyclization of a heptapeptide eliminates its cytotoxicity and significantly increases its inhibition effect on amyloid β-protein fibrillation and cytotoxicity

Amyloid-β (Aβ) protein aggregation is the main hallmark of Alzheimer’s disease (AD). Inhibition of Aβ fibrillation is thus a promising therapeutic approach to the prevention and treatment of AD. Recently, we designed a heptapeptide inhibitor, LVFFARK (LK7). LK7 shows a promising inhibitory capability on Aβ fibrillation, but is prone to self-assembling and displays high cytotoxicity, which would hinder its practical application. Herein, we modified LK7 by a head-to-tail cyclization and obtained a cyclic LK7 (cLK7). cLK7 exhibits a different self-assembly behavior from LK7, and has higher stability against proteolysis than LK7 and little cytotoxicity to SH-SY5Y cells. Thermodynamic analysis revealed that both LK7 and cLK7 could bind to Aβ₄₀ by electrostatic interactions, hydrogen bonding and hydrophobic interactions, but the binding affinity of cLK7 for Aβ₄₀ (K_D = 4.96 µmol/L) is six times higher than that of LK7 (K_D = 32.2 µmol/L). The strong binding enables cLK7 to stabilize the secondary structure of Aβ₄₀ and potently inhibit its nucleation, fibrillation and cytotoxicity at extensive concentration range, whereas LK7 could only moderately inhibit Aβ₄₀ fibrillation and cytotoxicity at low concentrations. The findings indicate that the peptide cyclization is a promising approach to enhance the performance of peptide-based amyloid inhibitors.     

Localization of Neurotrophin Specific Trk Receptors in Mechanosensory Systems of Killifish (Nothobranchius guentheri)

Neurotrophins (NTs) and their signal-transducing Trk receptors play a crucial role in the development and maintenance of specific neuronal subpopulations in nervous and sensory systems. NTs are supposed to regulate two sensory systems in fish, the inner ear and the lateral line system (LLS). The latter is one of the major mechanosensory systems in fish. Considering that annual fishes of the genus Nothobranchius, with their short life expectancy, have become a suitable model for aging studies and that the occurrence and distribution of neurotrophin Trk receptors have never been investigated in the inner ear and LLS of killifish (Nothobranchius guentheri), our study aimed to investigate the localization of neurotrophin-specific Trk receptors in mechanosensory systems of N. guentheri. For histological and immunohistochemical analysis, adult specimens of N. guentheri were processed using antibodies against Trk receptors and S100 protein. An intense immunoreaction for TrkA and TrkC was found in the sensory cells of the inner ear as well as in the hair cells of LLS. Moreover, also the neurons localized in the acoustic ganglia displayed a specific immunoreaction for all Trk receptors (TrkA, B, and C) analyzed. Taken together, our results demonstrate, for the first time, that neurotrophins and their specific receptors could play a pivotal role in the biology of the sensory cells of the inner ear and LLS of N. guentheri and might also be involved in the hair cells regeneration process in normal and aged conditions.     

Feeding Deterrents and Sensitivity Suppressors for Pieris rapae Larvae in Wheat Germ Diet

Rejection of wheat germ diet by cabbage-reared Pieris rapae larvae is explained by the fact that the diet contains strong feeding deterrents to the larvae. These deterrents were present mainly in hexane and butanol (BuOH) fractions of diet extracts, but the post-BuOH water fraction also showed some deterrent activity. Although minor diet components such as sorbic acid had a weak deterrent effect, wheat germ was primarily responsible for the activity. Seven compounds isolated from the BuOH extract of wheat germ were deterrent to the larvae. UV spectra suggested that most of these compounds are apigenin-based flavones. Fractionation of the hexane extracts showed that most of the active compounds were methanol-soluble, and HPLC of the MeOH fractions revealed the presence of active compounds that were absent in the BuOH extract of wheat germ, as well as other compounds that were common to both fractions. Acceptance of nasturtium by P. rapae larvae reared on wheat germ diet is explained by a type of “cross habituation” of the larvae to feeding deterrents. When larvae were reared on cabbage leaves treated with chemical fractions from wheat germ diet, they readily accepted nasturtium, which is normally refused by cabbage-reared P. rapae larvae, due to the presence of strong feeding deterrents in the plant. A high percentage of diet-reared larvae also fed on Erysimum cheiranthoides and Iberis amara, which are normally rejected. The chemical constituents encountered in wheat germ can almost completely account for the larval acceptance of nasturtium. The suitability of wheat germ diet for rearing phytophagous insects for behavioral assays must be questioned.     

20-Hydroxyecdysone Deters Oviposition and Larval Feeding in the European Grapevine Moth, Lobesia botrana

European grapevine moth females (Lobesia botrana, Lepidoptera Tortricidae) select an oviposition site by tasting the host plant surface and then gluing a single egg on berries from grapes or from several other host plant species. In doing so, females should avoid ovipositing on plants that are detrimental to their progeny. Do they sense the same deterrent compounds as larvae, despite the fact that they do not have access to the same compartments of the plants? We tested this hypothesis with 20-hydroxyecdysone (20E), purified from Leuzea carthamoides. Phytoecdysteroids are usually found inside plant tissues and accessible to larvae in an aqueous phase, while adults would access them only through the epicuticular wax. We first confirmed that larvae avoid feeding on 20E and that they taste 20E with their lateral sensilla styloconica, at a threshold of 10^?6 M. Then, we tested whether adult females avoid ovipositing on glass spheres sprayed with 20E. When given a choice, females avoided laying eggs on a treated surface, at a threshold of 8 ng/cm². In addition, they deposited significantly fewer eggs in the presence of 20E. Presuming that legs play an important role in assessing the oviposition substrate, we assessed the sensitivity of their taste receptors. In females, 14 taste sensilla are located on the ventral side of the last tarsus of the prothoracic leg. One group of these sensilla house one neuron that is sensitive to 20E, with a detection threshold of about 10^?7 M. The same molecule is thus sensed both in larvae and adults of L. botrana where it respectively inhibits feeding and oviposition.     

Palmitoylation of Prolactin-Releasing Peptide Increased Affinity for and Activation of the GPR10, NPFF-R2 and NPFF-R1 Receptors: In Vitro Study

The anorexigenic neuropeptide prolactin-releasing peptide (PrRP) is involved in the regulation of food intake and energy expenditure. Lipidization of PrRP stabilizes the peptide, facilitates central effect after peripheral administration and increases its affinity for its receptor, GPR10, and for the neuropeptide FF (NPFF) receptor NPFF-R2. The two most potent palmitoylated analogs with anorectic effects in mice, palm¹¹-PrRP31 and palm-PrRP31, were studied in vitro to determine their agonist/antagonist properties and mechanism of action on GPR10, NPFF-R2 and other potential off-target receptors related to energy homeostasis. Palmitoylation of both PrRP31 analogs increased the binding properties of PrRP31 to anorexigenic receptors GPR10 and NPFF-R2 and resulted in a high affinity for another NPFF receptor, NPFF-R1. Moreover, in CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1, palm¹¹-PrRP and palm-PrRP significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and cAMP-responsive element-binding protein (CREB). Palm¹¹-PrRP31, unlike palm-PrRP31, did not activate either c-Jun N-terminal kinase (JNK), p38, c-Jun, c-Fos or CREB pathways in cells expressing NPFF-1R. Palm-PrRP31 also has higher binding affinities for off-target receptors, namely, the ghrelin, opioid (KOR, MOR, DOR and OPR-L1) and neuropeptide Y (Y₁, Y₂ and Y₅) receptors. Palm¹¹-PrRP31 exhibited fewer off-target activities; therefore, it has a higher potential to be used as an anti-obesity drug with anorectic effects.     

JNK Signaling in Drosophila Aging and Longevity

The evolutionarily conserved c-Jun N-terminal kinase (JNK) signaling pathway is a critical genetic determinant in the control of longevity. In response to extrinsic and intrinsic stresses, JNK signaling is activated to protect cells from stress damage and promote survival. In Drosophila, global JNK upregulation can delay aging and extend lifespan, whereas tissue/organ-specific manipulation of JNK signaling impacts lifespan in a context-dependent manner. In this review, focusing on several tissues/organs that are highly associated with age-related diseases—including metabolic organs (intestine and fat body), neurons, and muscles—we summarize the distinct effects of tissue/organ-specific JNK signaling on aging and lifespan. We also highlight recent progress in elucidating the molecular mechanisms underlying the tissue-specific effects of JNK activity. Together, these studies highlight an important and comprehensive role for JNK signaling in the regulation of longevity in Drosophila.     

Genetic Deletion of Rheb1 in the Brain Reduces Food Intake and Causes Hypoglycemia with Altered Peripheral Metabolism

Excessive food/energy intake is linked to obesity and metabolic disorders, such as diabetes. The hypothalamus in the brain plays a critical role in the control of food intake and peripheral metabolism. The signaling pathways in hypothalamic neurons that regulate food intake and peripheral metabolism need to be better understood for developing pharmacological interventions to manage eating behavior and obesity. Mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a master regulator of cellular metabolism in different cell types. Pharmacological manipulations of mTOR complex 1 (mTORC1) activity in hypothalamic neurons alter food intake and body weight. Our previous study identified Rheb1 (Ras homolog enriched in brain 1) as an essential activator of mTORC1 activity in the brain. Here we examine whether central Rheb1 regulates food intake and peripheral metabolism through mTORC1 signaling. We find that genetic deletion of Rheb1 in the brain causes a reduction in mTORC1 activity and impairs normal food intake. As a result, Rheb1 knockout mice exhibit hypoglycemia and increased lipid mobilization in adipose tissue and ketogenesis in the liver. Our work highlights the importance of central Rheb1 signaling in euglycemia and energy homeostasis in animals.