Tissue- and Ligand-Specific Sensing of Gram-Negative Infection in Drosophila by PGRP-LC Isoforms and PGRP-LE

The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. In this study, we used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC and assessed the tissue-specific roles of PGRP-LC isoforms and PGRP-LE in the antibacterial response. Our in vivo studies demonstrate the key role of PGRP-LCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. We also highlight the contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. Our results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. Our in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, our study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila.     

Lactobacillus plantarum promotes Drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing

There is growing evidence that intestinal bacteria are important beneficial partners of their metazoan hosts. Recent observations suggest a strong link between commensal bacteria, host energy metabolism, and metabolic diseases such as diabetes and obesity. As a consequence, the gut microbiota is now considered a "host" factor that influences energy uptake. However, the impact of intestinal bacteria on other systemic physiological parameters still remains unclear. Here, we demonstrate that Drosophila microbiota promotes larval growth upon nutrient scarcity. We reveal that Lactobacillus plantarum, a commensal bacterium of the Drosophila intestine, is sufficient on its own to recapitulate the?natural microbiota growth-promoting effect. L.?plantarum exerts its benefit by acting genetically upstream of the TOR-dependent host nutrient sensing system controlling hormonal growth signaling. Our results indicate that the intestinal microbiota should also be envisaged as a factor that influences the systemic growth of its host.     

Review: Chemosensing of nutrients and non-nutrients in the human and porcine gastrointestinal tract

The gastrointestinal tract (GIT) is an interface between the external and internal milieus that requires continuous monitoring for nutrients or pathogens and toxic chemicals. The study of the physiological/molecular mechanisms, mediating the responses to the monitoring of the GIT contents, has been referred to as chemosensory science. While most of the progress in this area of research has been obtained in laboratory rodents and humans, significant steps forward have also been reported in pigs. The objective of this review was to update the current knowledge on nutrient chemosensing in pigs in light of recent advances in humans and laboratory rodents. A second objective relates to informing the existence of nutrient sensors with their functionality, particularly linked to the gut peptides relevant to the onset/offset of appetite. Several cell types of the intestinal epithelium such as Paneth, goblet, tuft and enteroendocrine cells (EECs) contain subsets of chemosensory receptors also found on the tongue as part of the taste system. In particular, EECs show specific co-expression patterns between nutrient sensors and/or transceptors (transport proteins with sensing functions) and anorexigenic hormones such as cholecystokinin (CCK), peptide tyrosine tyrosine (PYY) or glucagon-like peptide-1 (GLP-1), amongst others. In addition, the administration of bitter compounds has an inhibitory effect on GIT motility and on appetite through GLP-1-, CCK-, ghrelin- and PYY-labelled EECs in the human small intestine and colon. Furthermore, the mammalian chemosensory system is the target of some bacterial metabolites. Recent studies on the human microbiome have discovered that commensal bacteria have developed strategies to stimulate chemosensory receptors and trigger host cellular functions. Finally, the study of gene polymorphisms related to nutrient sensors explains differences in food choices, food intake and appetite between individuals.     

Gastrointestinal chemosensation: chemosensory cells in the alimentary tract

Sensing potentially beneficial or harmful constituents in the luminal content by specialized cells in the gastrointestinal mucosa is an essential prerequisite for governing digestive processes, initiating protective responses and regulating food intake. Until recently, it was poorly understood how the gastrointestinal tract senses and responds to nutrients and non-nutrients in the diet; however, the enormous progress in unraveling the molecular machinery underlying the responsiveness of gustatory cells in the lingual taste buds to these compounds has been an important starting point for studying intestinal chemosensation. Currently, the field of nutrient sensing in the gastrointestinal tract is evolving rapidly and is benefiting from the deorphanization of previously unliganded G-protein-coupled receptors which respond to important nutrients, such as protein degradation products and free fatty acids as well as from the FACS-assisted isolation of distinct cell populations. This review focuses on mechanisms and principles underlying the chemosensory responsiveness of the alimentary tract. It describes the cell types which might potentially contribute to chemosensation within the gut: cells that can operate as specialized sensors and transducers for luminal factors and which communicate information from the gut lumen by releasing paracrine or endocrine acting messenger molecules. Furthermore, it addresses the current knowledge regarding the expression and localization of molecular elements that may be part of the chemosensory machinery which render some of the mucosal cells responsive to constituents of the luminal content, concentrating on candidate receptors and transporters for sensing nutrients.     

Diverse roles for the Drosophila fructose sensor Gr43a

The detection of nutrients, both in food and within the body, is crucial for the regulation of feeding behavior, growth, and metabolism. While the molecular basis for sensing food chemicals by the taste system has been firmly linked to specific taste receptors, relatively little is known about the molecular nature of the sensors that monitor nutrients internally. Recent reports of taste receptors expressed in other organ systems, foremost in the gastrointestinal tract of mammals and insects, has led to the proposition that some taste receptors may also be used as sensors of internal nutrients. Indeed, we provided direct evidence that the Drosophila gustatory receptor 43a (Gr43a) plays a critical role in sensing internal fructose levels in the fly brain. In addition to the brain and the taste system, Gr43a is also expressed in neurons of the proventricular ganglion and the uterus. Here, we discuss the multiple potential roles of Gr43a in the fly. We also provide evidence that its activation in the brain is likely mediated by the neuropeptide Corazonin. Finally, we posit that Gr43a may represent only a precedent for other taste receptors that sense internal nutrients, not only in flies but, quite possibly, in other animals, including mammals.     

Diverse roles for the Drosophila fructose sensor Gr43a

The detection of nutrients, both in food and within the body, is crucial for the regulation of feeding behavior, growth, and metabolism. While the molecular basis for sensing food chemicals by the taste system has been firmly linked to specific taste receptors, relatively little is known about the molecular nature of the sensors that monitor nutrients internally. Recent reports of taste receptors expressed in other organ systems, foremost in the gastrointestinal tract of mammals and insects, has led to the proposition that some taste receptors may also be used as sensors of internal nutrients. Indeed, we provided direct evidence that the Drosophila gustatory receptor 43a (Gr43a) plays a critical role in sensing internal fructose levels in the fly brain. In addition to the brain and the taste system, Gr43a is also expressed in neurons of the proventricular ganglion and the uterus. Here, we discuss the multiple potential roles of Gr43a in the fly. We also provide evidence that its activation in the brain is likely mediated by the neuropeptide Corazonin. Finally, we posit that Gr43a may represent only a precedent for other taste receptors that sense internal nutrients, not only in flies but, quite possibly, in other animals, including mammals.     

TRP channels in disease

"Transient receptor potential" cation channels (TRP channels) play a unique role as cell sensors, are involved in a plethora of Ca(2+)-mediated cell functions, and play a role as "gate-keepers" in many homeostatic processes such as Ca(2+) and Mg(2+) reabsorption. The variety of functions to which TRP channels contribute and the polymodal character of their activation predict that failures in correct channel gating or permeation will likely contribute to complex pathophysiological mechanisms. Dysfunctions of TRPs cause human diseases but are also involved in a complex manner to contribute and determine the progress of several diseases. Contributions to this special issue discuss channelopathias for which mutations in TRP channels that induce "loss-" or "gain-of-function" of the channel and can be considered "disease-causing" have been identified. The role of TRPs will be further elucidated in complex diseases of the intestinal, renal, urogenital, respiratory, and cardiovascular systems. Finally, the role of TRPs will be discussed in neuronal diseases and neurodegenerative disorders.     

Altered modes of stem cell division drive adaptive intestinal growth

Throughout life, adult organs continually adapt to variable environmental factors. Adaptive mechanisms must fundamentally differ from homeostatic maintenance, but little is known about how physiological factors elicit tissue remodeling. Here, we show that specialized stem cell responses underlie the adaptive resizing of a mature organ. In the adult Drosophila midgut, intestinal stem cells interpret a nutrient cue to "break homeostasis" and drive growth when food is abundant. Activated in part by niche production of insulin, stem cells direct a growth program through two altered modes of behavior: accelerated division rates and predominance of symmetric division fates. Together, these altered modes produce a net increase in total intestinal cells, which is reversed upon withdrawal of food. Thus, tissue renewal programs are not committed to maintain cellular equilibrium; stem cells can remodel organs in response to physiological triggers.     

Fasting and postprandial concentrations of GLP-1 in intestinal lymph and portal plasma: evidence for selective release of GLP-1 in the lymph system

Glucagon like peptide 1 (GLP-1) is an intestinal hormone that plays an important role in glucose metabolism. GLP-1 is released from mucosal L cells following nutrient ingestion and contributes to the incretin effect, with the enhancement of insulin secretion occurring with enteral compared with intravenous glucose administration. The mechanisms linking nutrient absorption and GLP-1 secretion are unknown, and studies addressing this topic, particularly in small animal models, have been hampered by the relatively low concentrations of GLP-1 in the circulation. We hypothesized that GLP-1 levels would be higher in samples of intestinal lymph compared with plasma and could provide a novel system in which to study meal-induced hormone secretion. We addressed this hypothesis in conscious rats with indwelling catheters in the portal vein and distal intestinal lymph duct. These animals had plasma and lymph sampled before and for 240 min after instillation of a liquid meal in the gastrointestinal tract. Lymph contained detectable concentrations of glucose, insulin, and GLP-1 that were reliably measured using our assays. Before and after the Ensure feeding, plasma insulin levels were approximately two times as high in portal plasma as intestinal lymph. In marked contrast, GLP-1 levels were five to six times higher in lymph relative to portal plasma following nutrient administration. This relative difference in GLP-1 levels was even greater when lymph was compared with peripheral plasma and dramatically exceeded the ratio of lymph to plasma peptide tyrosine-tyrosine concentrations. This is the first observation of a gastrointestinal hormone being disproportionately transported in lymph. The remarkable levels of GLP-1 in intestinal lymph demonstrate the potential for lymphatic sampling as a more sensitive means of studying the secretory physiology of this hormone in vivo. In addition, these data raise the possibility that intestinal lymph may serve as a specialized signaling conduit for regulatory peptides secreted by gastrointestinal endocrine cells.     

Nitrate transceptor(s) in plants

The availability of mineral nutrients in the soil dramatically fluctuates in both time and space. In order to optimize their nutrition, plants need efficient sensing systems that rapidly signal the local external concentrations of the individual nutrients. Until recently, the most upstream actors of the nutrient signalling pathways, i.e. the sensors/receptors that perceive the extracellular nutrients, were unknown. In Arabidopsis, increasing evidence suggests that, for nitrate, the main nitrogen source for most plant species, a major sensor is the NRT1.1 nitrate transporter, also contributing to nitrate uptake by the roots. Membrane proteins that fulfil a dual nutrient transport/signalling function have been described in yeast and animals, and are called 'transceptors'. This review aims to illustrate the nutrient transceptor concept in plants by presenting the current evidence indicating that NRT1.1 is a representative of this class of protein. The various facets, as well as the mechanisms of nitrate sensing by NRT1.1 are considered, and the possible occurrence of other nitrate transceptors is discussed.     

Cross-Species Comparison of Genes Related to Nutrient Sensing Mechanisms Expressed along the Intestine

Introduction

Intestinal chemosensory receptors and transporters are able to detect food-derived molecules and are involved in the modulation of gut hormone release. Gut hormones play an important role in the regulation of food intake and the control of gastrointestinal functioning. This mechanism is often referred to as “nutrient sensing”. Knowledge of the distribution of chemosensors along the intestinal tract is important to gain insight in nutrient detection and sensing, both pivotal processes for the regulation of food intake. However, most knowledge is derived from rodents, whereas studies in man and pig are limited, and cross-species comparisons are lacking.

Aim

To characterize and compare intestinal expression patterns of genes related to nutrient sensing in mice, pigs and humans.

Methods

Mucosal biopsy samples taken at six locations in human intestine (n = 40) were analyzed by qPCR. Intestinal scrapings from 14 locations in pigs (n = 6) and from 10 locations in mice (n = 4) were analyzed by qPCR and microarray, respectively. The gene expression of glucagon, cholecystokinin, peptide YY, glucagon-like peptide-1 receptor, taste receptor T1R3, sodium/glucose cotransporter, peptide transporter-1, GPR120, taste receptor T1R1, GPR119 and GPR93 was investigated. Partial least squares (PLS) modeling was used to compare the intestinal expression pattern between the three species.

Results and conclusion

The studied genes were found to display specific expression patterns along the intestinal tract. PLS analysis showed a high similarity between human, pig and mouse in the expression of genes related to nutrient sensing in the distal ileum, and between human and pig in the colon. The gene expression pattern was most deviating between the species in the proximal intestine. Our results give new insights in interspecies similarities and provide new leads for translational research and models aiming to modulate food intake processes in man.     

Regulation of adult stem cell behavior by nutrient signaling

Adult stem cells play an essential role throughout life, maintaining tissue and organ function by providing a reservoir of cells for homeostasis and repair. Maintenance and activity of adult stem cells have been the focus of numerous studies that have revealed stem cell-intrinsic factors and signals from the local microenvironment that regulate stem cell behavior. A growing body of work has provided evidence that circulating, systemic factors also contribute to the regulation of stem cell behavior in numerous tissues. We have demonstrated that Drosophila male germline stem cells (GSCs) and intestinal stem cells (ISCs) respond to changes in nutrient availability, specifically amino acids. Furthermore, we have shown that insulin signaling plays an important role in mediating the effects of changes in nutritional conditions. Notably, insulin signaling is cell-autonomously required within male GSCs for maintenance. Here we discuss our data regarding the effects and mechanisms by which changes in systemic nutritional conditions may influence the maintenance and activity of adult stem cells via insulin signaling.Key words: Drosophila, stem cells, nutrition, insulin, niche     

Functions and roles of the extracellular Ca2+-sensing receptor in the gastrointestinal tract

The gastrointestinal tract is vital to food digestion and nutrient absorption as well as normal salt and water homeostasis. Studies over the last several years have shown that the Ca2+-sensing receptor is expressed along the entire gastrointestinal tract. The potential roles for the receptor in gastrointestinal biology are now only beginning to be elucidated and much work remains. Well-studied physiological effects include regulation of gastric acid secretion and modulation of fluid transport in the colon. It remains to be determined if the Ca2+-sensing receptor is involved in calcium handling by the gastrointestinal tract. The ability of organic nutrient receptor agonists/allosteric modifiers, such as polyamines and L-amino acids, to activate the Ca2+-sensing receptor suggest potential roles in signalling nutrient availability to gastric and intestinal epithelial cells. In addition, polyamines are crucial for normal cell proliferation and differentiation required to sustain the rapid turnover of gastrointestinal epithelial cells and the Ca2+-sensing receptor may be involved in this function. Activation of the colonic Ca2+-sensing receptor can abrogate cyclic nucleotide-mediated fluid secretion suggesting a role for the receptor in modifying secretory diarrheas like cholera. Finally, the Ca2+-sensing receptor has been suggested to provide a mechanism for the effect of calcium intake in reducing the risk of colon cancer.     

In developing Drosophila neurones the production of gamma-amino butyric acid is tightly regulated downstream of glutamate decarboxylase translation and can be influenced by calcium

The presented work pioneers the embryonic Drosophila CNS for studies of the developmental regulation and function of gamma-amino butyric acid (GABA). We describe for the first time the developmental pattern of GABA in Drosophila and address underlying regulatory mechanisms. Surprisingly, and in contrast to vertebrates, detectable levels of GABA occur late during Drosophila neurogenesis, after essential neuronal proliferation and growth have taken place and synaptogenesis has been initiated. This timeline is almost unchanged when the GABA synthetase glutamate decarboxylase (GAD) is strongly misexpressed throughout the nervous system suggesting a tight post-translational regulation of GABA expression. We confirmed such GABA control mechanisms in an independent model system, i.e. primary Drosophila cell cultures raised in elevated [K+]. The data suggest that, in both systems, GABA suppression occurs via control of GAD activity. Using developing embryos and cell cultures as parallel assay systems for pharmacological and genetic studies we show that the negative regulation of GAD can be overridden by drugs known to elevate intracellular free [Ca2+]. Our results provide the basis for investigations of genetic mechanisms underlying the observed phenomenon, and we discuss the potential implications of this work for Drosophila neurogenesis but also for a general understanding of GAD regulation.     

Differential expression of heat shock proteins in Drosophila embryonic cells following metal ion exposure

Drosophila embryonic cells were exposed to a number of metal ions that have been previously reported to act as teratogens in mammalian systems, including some known to induce heat shock (stress) proteins in a variety of model systems. This study examined the effects of these ions both on differentiation of muscles and neurons and on the induction of heat shock proteins. Metals such as arsenate, cadmium, and mercury all inhibited neuron and/or muscle differentiation in Drosophila embryonic cultures, while they also induced the entire set of heat shock proteins. Two metal ions, nickel and zinc, were shown to induce only the 22- and 23-K proteins, a pattern similar to that seen in "classical" teratogens reported previously. None of the metals tested induced only the 26- and 27-K proteins. These results suggest that there exist different regulatory mechanisms responsible for the heat shock response.     

Regulation of Smad activity

Finding that peripodial cells in wing and eye imaginal discs are essential for the growth and patterning of the separate layer of disc cells now opens the study of interacting cell layers to the powerful developmental genetic techniques with which the Drosophila system is blessed. We can anticipate that future work will identify how such interactions contribute to patterning and how the mechanisms and processes that are involved are conserved in vertebrates. We can also look forward to contributions that this work will make to understand-ing the role of interconnecting cell extensions in such signaling processes. In this minireview, we have noted numerous types of signaling cells in which cellular extensions have been observed. At present, neither the functional nor structural relationship of these related structures is known. It is certainly tempting to suggest that these structures are conduits for signals or that they function as sensors. There is, as yet, no direct experimental evidence for such roles.     

Lipid-induced peroxidation in the intestine is involved in glucose homeostasis imbalance in mice

Background

Daily variations in lipid concentrations in both gut lumen and blood are detected by specific sensors located in the gastrointestinal tract and in specialized central areas. Deregulation of the lipid sensors could be partly involved in the dysfunction of glucose homeostasis. The study aimed at comparing the effect of Medialipid (ML) overload on insulin secretion and sensitivity when administered either through the intestine or the carotid artery in mice.

Methodology/Principal Findings

An indwelling intragastric or intracarotid catheter was installed in mice and ML or an isocaloric solution was infused over 24 hours. Glucose and insulin tolerance and vagus nerve activity were assessed. Some mice were treated daily for one week with the anti-lipid peroxidation agent aminoguanidine prior to the infusions and tests. The intestinal but not the intracarotid infusion of ML led to glucose and insulin intolerance when compared with controls. The intestinal ML overload induced lipid accumulation and increased lipid peroxidation as assessed by increased malondialdehyde production within both jejunum and duodenum. These effects were associated with the concomitant deregulation of vagus nerve. Administration of aminoguanidine protected against the effects of lipid overload and normalized glucose homeostasis and vagus nerve activity.

Conclusions/Significance

Lipid overload within the intestine led to deregulation of gastrointestinal lipid sensing that in turn impaired glucose homeostasis through changes in autonomic nervous system activity.     

Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway

In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence we examined the role of TOR pathway genes in regulating lifespan by using Drosophila. We show that inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2 (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. We find that overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction.     

Effects of luminal thymol on epithelial transport in human and rat colon

Gut lumen is continually exposed to a great variety of agents, including noxious compounds. Chemical receptors that detect the luminal environment are thought to play an important role as sensors and to modulate gastrointestinal functions. Recently, it has been reported that odorant receptors (ORs) are expressed in the small intestinal mucosa and that odorants stimulate serotonin secretion. However, ion transport in the responses to odorants has rarely been discussed, particularly in relation to the large intestine. In the present study, we examined the effects of the OR ligand thymol on ion transport in human and rat colonic epithelia using an Ussing chamber. In the mucosal-submucosal preparations, the mucosal addition of thymol evoked anion secretion concentration dependently. In addition, dextran (4 kDa) permeability was enhanced by the mucosal treatment with thymol. The response to thymol was not affected by tetrodotoxin (TTX) or piroxicam treatments in human or rat colon. Thymol-evoked electrogenic anion secretion was abolished under Ca(2+)-free conditions or mucosal treatment with transient receptor potential (TRP) A1 blocker (HC-030031). Pretreatment of thymol did not affect electrical field stimulation-evoked anion secretion but significantly attenuated short-chain fatty acid-evoked secretion in a concentration-dependent manner. OR1G1 and TRPA1 expression was investigated in isolated colonic mucosa by RT-PCR. The present results provide evidence that the OR ligand thymol modulates epithelial permeability and electrogenic anion secretion in human and rat colon. The anion secretion by luminal thymol is most likely mediated by direct activation of TRPA1 channel. We suggest that the sensing and responding to odorants in the colon also plays a role in maintaining intestinal homeostasis.