Inhibiting RHOA Signaling in Mice Increases Glucose Tolerance and Numbers of Enteroendocrine and Other Secretory Cells in the Intestine.

BACKGROUND & AIMS: Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation. METHODS: We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for in vitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose. RESULTS: Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared with mice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucose tolerance compared with administration of saline. CONCLUSIONS: In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of L cells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance.

EBI2 overexpression in mice leads to B1 B-cell expansion and chronic lymphocytic leukemia-like B-cell malignancies.

Human and mouse chronic lymphocytic leukemia (CLL) develops from CD5+ B cells that in mice and macaques are known to define the distinct B1a B-cell lineage. B1a cells are characterized by lack of germinal center (GC) development, and the B1a cell population is increased in mice with reduced GC formation. As a major mediator of follicular B-cell migration, the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) directs B-cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells toward the extrafollicular area, whereas downregulation is essential for GC formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to CLL. Here, we demonstrate that B-cell-targeted expression of human EBI2 (hEBI2) in mice reduces GC-dependent immune responses, reduces total immunoglobulin M (IgM) and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5+ B1a B-cell subset (present as early as 4 days after birth), late-onset lymphoid cancer development, and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B-cell malignancies.

An RNA-seq atlas of mouse brain areas during fasting and diet-induced obesity.

Mammalian energy homeostasis is primarilly regulated by the hypothalamus and hindbrain, with the hippocampus, midbrain nuclei, and other regions implicated by evidence from human genetics studies. To understand how these non-canonical brain regions respond to imbalances in energy homeostasis, we performed two experiments examining the effects of different diets in male C57BL6 mice. In our first study, groups of six pair-housed mice were given access to chow, high-fat diet or fasted for 16 hours. In our subsequent study, two groups of 10 mice were single-housed and given access to chow or fasted for 24 h. We recorded food intake for each cage, the change in body weight for each animal, and collected hypothalamus, hippocampus, superior colliculus, inferior colliculus, frontal cortex, and zona incerta-centric samples. We performed bulk RNA sequencing on 185 samples and validated them by a series of quality control assessments including alignment quality and gene expression profiling. We believe these studies capture the transcriptomic effects of acute fasting and high-fat diet in the rodent brain and provide a valuable reference.

An atlas of GPCRs in dopamine neurons: Identification of the free fatty acid receptor 4 as a regulator of food and water intake.

Midbrain dopaminergic neurons (DANs) are subject to extensive metabotropic regulation, but the repertoire of G protein-coupled receptors (GPCRs) present in these neurons has not been mapped. Here, we isolate DANs from Dat-eGFP mice to generate a GPCR atlas by unbiased qPCR array expression analysis of 377 GPCRs. Combined with data mining of scRNA-seq databases, we identify multiple receptors in DAN subpopulations with 38 of these receptors representing the majority of transcripts. We identify 41 receptors expressed in midbrain DANs but not in non-DAN midbrain cells, including the free fatty acid receptor 4 (FFAR4). Functional expression of FFAR4 is validated by ex vivo Ca2+ imaging, and in vivo experiments support that FFAR4 negatively regulates food and water intake and bodyweight. In addition to providing a critical framework for understanding metabotropic DAN regulation, our data suggest fatty acid sensing by FFAR4 as a mechanism linking high-energy intake to the dopamine-reward pathway.

Endocytic collagen degradation: a novel mechanism involved in protection against liver fibrosis.

Fibrosis of the liver and its end-stage, cirrhosis, represent major health problems worldwide. In these fibrotic conditions, activated fibroblasts and hepatic stellate cells display a net deposition of collagen. This collagen deposition is a major factor leading to liver dysfunction, thus making it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked up-regulation of uPARAP/Endo180 in activated fibroblasts and hepatic stellate cells located close to the collagen deposits. In a hepatic stellate cell line, uPARAP/Endo180 was shown to be active in, and required for, the uptake and intracellular degradation of collagen. To evaluate the functional importance of this collagen receptor in vivo, liver fibrosis was induced in uPARAP/Endo180-deficient mice and littermate wild-type mice by chronic CCl(4) administration. A strong up-regulation of uPARAP/Endo180 was observed in wild-type mice, and a quantitative comparison of collagen deposits in the two groups of mice clearly revealed a fibrosis protective role of uPARAP/Endo180. This effect appeared to directly reflect the activity of the collagen receptor, since no compensatory events were noted when comparing the mRNA expression profiles of the two groups of mice in an array system focused on matrix-degrading components. This function of uPARAP/Endo180 defines a novel role of intracellular collagen turnover in fibrosis protection.

Molecular consequences of peripheral Influenza A infection on cell populations in the murine hypothalamus.

Infection with Influenza A virus (IAV) causes the well-known symptoms of the flu, including fever, loss of appetite, and excessive sleepiness. These responses, mediated by the brain, will normally disappear once the virus is cleared from the system, but a severe respiratory virus infection may cause long-lasting neurological disturbances. These include encephalitis lethargica and narcolepsy. The mechanisms behind such long lasting changes are unknown. The hypothalamus is a central regulator of the homeostatic response during a viral challenge. To gain insight into the neuronal and non-neuronal molecular changes during an IAV infection, we intranasally infected mice with an H1N1 virus and extracted the brain at different time points. Using single-nucleus RNA sequencing (snRNA-seq) of the hypothalamus, we identify transcriptional effects in all identified cell populations. The snRNA-seq data showed the most pronounced transcriptional response at 3 days past infection, with a strong downregulation of genes across all cell types. General immune processes were mainly impacted in microglia, the brain resident immune cells, where we found increased numbers of cells expressing pro-inflammatory gene networks. In addition, we found that most neuronal cell populations downregulated genes contributing to the energy homeostasis in mitochondria and protein translation in the cytosol, indicating potential reduced cellular and neuronal activity. This might be a preventive mechanism in neuronal cells to avoid intracellular viral replication and attack by phagocytosing cells. The change of microglia gene activity suggest that this is complemented by a shift in microglia activity to provide increased surveillance of their surroundings.

When you are ill, your behaviour changes. You sleep more, eat less and are less likely to go out and be active. This behavioural change is called the 'sickness response' and is believed to help the immune system fight infection. An area of the brain called the hypothalamus helps to regulate sleep and appetite. Previous research has shown that when humans are ill, the immune system sends signals to the hypothalamus, likely initiating the sickness response. However, it was not clear which brain cells in the hypothalamus are involved in the response and how long after infection the brain returns to its normal state. To better understand the sickness response, Lemcke et al. infected mice with influenza then extracted and analysed brain tissue at different timepoints. The experiments showed that the major changes to gene expression in the hypothalamus early during an influenza infection are not happening in neurons ­ the cells in the brain that transmit electrical signals and usually control behaviour. Instead, it is cells called glia ­ which provide support and immune protection to the neurons ­ that change during infection. The findings suggest that these cells prepare to protect the neurons from influenza should the virus enter the brain. Lemcke et al. also found that the brain takes a long time to go back to normal after an influenza infection. In infected mice, molecular changes in brain cells could be detected even after the influenza infection had been cleared from the respiratory system. In the future, these findings may help to explain why some people take longer than others to fully recover from viral infections such as influenza and aid development of medications that speed up recovery.

Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism.

GPR39, a constitutively active 7TM receptor important for glucose-induced insulin secretion and maturation of pancreatic ß-cell function, is up-regulated in adipose tissue on abstinence from food and chemically induced diabetes. In the present study, we investigated the effect of GPR39 deficiency on body weight and adipocyte metabolism. GPR39-deficient mice were subjected to a high-fat diet and body composition, glucose tolerance, insulin secretion, food intake, and energy expenditure were evaluated. The cell biology of adipocyte metabolism was studied on both mRNA and protein levels. A significant increase in body weight corresponding to a 2-fold selective increase in fat mass was observed in GPR39-deficient mice fed a high-fat diet as compared with wild-type littermate controls fed the same diet. The GPR39-deficient animals had similar food intake but displayed almost eliminated diet-induced thermogenesis, measured by the oxygen consumption rate (Vo(2)) on change from normal to high-fat diet. Analysis of the adipose tissue for lipolytic enzymes demonstrated decreased level of phosphorylated hormone-sensitive lipase (HSL) and a decreased level of adipose triglyceride lipase (ATGL) by 35 and 60%, respectively, after food withdrawal in the GPR39-deficient mice. Extracellular signal-regulated kinases (ERK1/2), a signaling pathway known to be important for lipolysis, was decreased by 56% in the GPR39-deficient mice. GPR39 deficiency is associated with increased fat accumulation on a high-fat diet, conceivably due to decreased energy expenditure and adipocyte lipolytic activity.

LPS counter regulates RNA expression of extracellular proteases and their inhibitors in murine macrophages.

Besides their evident importance in host defense, macrophages have been shown to play a detrimental role in different pathological conditions, including chronic inflammation, atherosclerosis, and cancer. Regardless of the exact situation, macrophage activation and migration are intimately connected to extracellular matrix degradation. This process is accomplished by multiple proteolytic enzymes, including serine proteases and members of the matrix metalloproteinase family. In this study, we have utilized qPCR arrays to simultaneously analyze the temporal expression pattern of a range of genes involved in extracellular matrix metabolism in the mouse derived-macrophage cell line RAW 264.7 following stimulation with LPS. Our results revealed that LPS induces the expression of matrix metalloproteinases while at the same time decreased the expression of matrix metalloproteinase inhibitors. The opposite scenario was found for the genes encoding serine proteases, which were downregulated while their inhibitors were upregulated. In addition, intergenic comparison of the expression levels of related proteases revealed large differences in their basal expression level. These data highlight the complexity of the gene expression regulation implicated in macrophage-dependent matrix degradation and furthermore emphasize the value of qPCR array techniques for the investigation of the complex regulation of the matrix degradome.

Single-Cell Mapping of GLP-1 and GIP Receptor Expression in the Dorsal Vagal Complex.

The dorsal vagal complex (DVC) in the hindbrain, composed of the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus, plays a critical role in modulating satiety. The incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) act directly in the brain to modulate feeding, and receptors for both are expressed in the DVC. Given the impressive clinical responses to pharmacologic manipulation of incretin signaling, understanding the central mechanisms by which incretins alter metabolism and energy balance is of critical importance. Here, we review recent single-cell approaches used to detect molecular signatures of GLP-1 and GIP receptor-expressing cells in the DVC. In addition, we discuss how current advancements in single-cell transcriptomics, epigenetics, spatial transcriptomics, and circuit mapping techniques have the potential to further characterize incretin receptor circuits in the hindbrain.

Adhesion receptor ADGRG2/GPR64 is in the GI-tract selectively expressed in mature intestinal tuft cells.

OBJECTIVE: GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract. METHODS: Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNA-seq analysis. RESULTS: Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells. CONCLUSIONS: GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells.

A genetic map of the mouse dorsal vagal complex and its role in obesity.

The brainstem dorsal vagal complex (DVC) is known to regulate energy balance and is the target of appetite-suppressing hormones, such as glucagon-like peptide 1 (GLP-1). Here we provide a comprehensive genetic map of the DVC and identify neuronal populations that control feeding. Combining bulk and single-nucleus gene expression and chromatin profiling of DVC cells, we reveal 25 neuronal populations with unique transcriptional and chromatin accessibility landscapes and peptide receptor expression profiles. GLP-1 receptor (GLP-1R) agonist administration induces gene expression alterations specific to two distinct sets of Glp1r neurons-one population in the area postrema and one in the nucleus of the solitary tract that also expresses calcitonin receptor (Calcr). Transcripts and regions of accessible chromatin near obesity-associated genetic variants are enriched in the area postrema and the nucleus of the solitary tract neurons that express Glp1r and/or Calcr, and activating several of these neuronal populations decreases feeding in rodents. Thus, DVC neuronal populations associated with obesity predisposition suppress feeding and may represent therapeutic targets for obesity.

Transcriptomic analysis links diverse hypothalamic cell types to fibroblast growth factor 1-induced sustained diabetes remission.

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.

The Molecular Diversity of Vagal Afferents Revealed.

Using single-cell RNA sequencing (RNA-seq), Kupari and coworkers (Cell Rep., 2019) have generated a long sought-after molecular atlas of vagal afferents in the mouse. Vagal afferents were found to be organized into 24 subtypes, revealing a level of diversity that was not previously recognized.

RhoA in tyrosine hydroxylase neurones regulates food intake and body weight via altered sensitivity to peripheral hormones.

Dopamine-producing tyrosine hydroxylase (TH) neurones in the hypothalamic arcuate nucleus (ARC) have recently been shown to be involved in ghrelin signalling and body weight homeostasis. In the present study, we investigate the role of the intracellular regulator RhoA in hypothalamic TH neurones in response to peripheral hormones. Diet-induced obesity was found to be associated with increased phosphorylation of TH in ARC, indicating obesity-associated increased activity of ARC TH neurones. Mice in which RhoA was specifically knocked out in TH neurones (TH-RhoA-/- mice) were more sensitive to the orexigenic effect of peripherally administered ghrelin and displayed an abolished response to the anorexigenic hormone leptin. When TH-RhoA-/- mice were challenged with a high-fat high-sucrose (HFHS) diet, they became hyperphagic and gained more body weight and fat mass compared to wild-type control mice. Importantly, lack of RhoA prevented development of ghrelin resistance, which is normally observed in wild-type mice after long-term HFHS diet feeding. Patch-clamp electrophysiological analysis demonstrated increased ghrelin-induced excitability of TH neurones in lean TH-RhoA-/- mice compared to lean littermate control animals. Additionally, increased expression of the orexigenic hypothalamic neuropeptides agouti-related peptide and neuropeptide Y was observed in TH-RhoA-/- mice. Overall, our data indicate that TH neurones in ARC are important for the regulation of body weight homeostasis and that RhoA is both a central effector in these neurones and important for the development of obesity-induced ghrelin resistance. The obese phenotype of TH-RhoA-/- mice may be a result of increased sensitivity to ghrelin and decreased sensitivity to leptin, resulting in increased food intake.

The aromatic amino acid sensor GPR142 controls metabolism through balanced regulation of pancreatic and gut hormones.

OBJECTIVES: GPR142, which is highly expressed in pancreatic islets, has recently been deorphanized as a receptor for aromatic amino acids; however, its physiological role and pharmacological potential is unclear. METHODS AND RESULTS: We find that GPR142 is expressed not only in ß- but also in α-cells of the islets as well as in enteroendocrine cells, and we confirm that GPR142 is a highly selective sensor of essential aromatic amino acids, in particular Trp and oligopeptides with N-terminal Trp. GPR142 knock-out mice displayed a very limited metabolic phenotype but demonstrated that L-Trp induced secretion of pancreatic and gut hormones is mediated through GPR142 but that the receptor is not required for protein-induced hormone secretion. A synthetic GPR142 agonist stimulated insulin and glucagon as well as GIP, CCK, and GLP-1 secretion. In particular, GIP secretion was sensitive to oral administration of the GPR142 agonist an effect which in contrast to the other hormones was blocked by protein load. Oral administration of the GPR142 agonist increased [3H]-2-deoxyglucose uptake in muscle and fat depots mediated through insulin action while it lowered liver glycogen conceivably mediated through glucagon, and, consequently, it did not lower total blood glucose. Nevertheless, acute administration of the GPR142 agonist strongly improved oral glucose tolerance in both lean and obese mice as well as Zucker fatty rat. Six weeks in-feed chronic treatment with the GPR142 agonist did not affect body weight in DIO mice, but increased energy expenditure and carbohydrate utilization, lowered basal glucose, and improved insulin sensitivity. CONCLUSIONS: GPR142 functions as a sensor of aromatic amino acids, controlling GIP but also CCK and GLP-1 as well as insulin and glucagon in the pancreas. GPR142 agonists could have novel interesting potential in modifying metabolism through a balanced action of gut hormones as well as both insulin and glucagon.

GPR39 splice variants versus antisense gene LYPD1: expression and regulation in gastrointestinal tract, endocrine pancreas, liver, and white adipose tissue.

G protein-coupled receptor 39 (GPR39) is a constitutively active, orphan member of the ghrelin receptor family that is activated by zinc ions. GPR39 is here described to be expressed in a full-length, biologically active seven-transmembrane form, GPR39-1a, as well as in a truncated splice variant five-transmembrane form, GPR39-1b. The 3' exon of the GPR39 gene overlaps with an antisense gene called LYPD1 (Ly-6/PLAUR domain containing 1). Quantitative RT-PCR analysis demonstrated that GPR39-1a is expressed selectively throughout the gastrointestinal tract, including the liver and pancreas as well as in the kidney and adipose tissue, whereas the truncated GPR39-1b form has a more broad expression pattern, including the central nervous system but with highest expression in the stomach and small intestine. In contrast, the LYPD1 antisense gene is highly expressed throughout the central nervous system as characterized with both quantitative RT-PCR and in situ hybridization analysis. A functional analysis of the GPR39 promoter region identified sites for the hepatocyte nuclear factors 1alpha and 4alpha (HNF-1alpha and -4alpha) and specificity protein 1 (SP1) transcription factors as being important for the expression of GPR39. In vivo experiments in rats demonstrated that GPR39 is up-regulated in adipose tissue during fasting and in response to streptozotocin treatment, although its expression is kept constant in the liver from the same animals. GPR39-1a was expressed in white but not brown adipose tissue and was down-regulated during adipocyte differentiation of fibroblasts. It is concluded that the transcriptional control mechanism, the tissue expression pattern, and in vivo response to physiological stimuli all indicate that the GPR39 receptor very likely is of importance for the function of a number of metabolic organs, including the liver, gastrointestinal tract, pancreas, and adipose tissue.

Enterochromaffin 5-HT cells - A major target for GLP-1 and gut microbial metabolites.

OBJECTIVES: 5-HT storing enterochromaffin (EC) cells are believed to respond to nutrient and gut microbial components, and 5-HT receptor-expressing afferent vagal neurons have been described to be the major sensors of nutrients in the GI-tract. However, the molecular mechanism through which EC cells sense nutrients and gut microbiota is still unclear. METHODS AND RESULTS: TPH1, the 5-HT generating enzyme, and chromogranin A, an acidic protein responsible for secretory granule storage of 5-HT, were highly enriched in FACS-purified EC cells from both small intestine and colon using a 5-HT antibody-based method. Surprisingly, EC cells from the small intestine did not express GPCR sensors for lipid and protein metabolites, such as FFAR1, GPR119, GPBAR1 (TGR5), CaSR, and GPR142, in contrast to the neighboring GLP-1 storing enteroendocrine cell. However, the GLP-1 receptor was particularly highly expressed and enriched in EC cells as judged both by qPCR and by immunohistochemistry using a receptor antibody. GLP-1 receptor agonists robustly stimulated 5-HT secretion from intestinal preparations using both HPLC and a specific amperometric method. Colonic EC cells expressed many different types of known and potential GPCR sensors of microbial metabolites including three receptors for SCFAs, i.e. FFAR2, OLF78, and OLF558 and receptors for aromatic acids, GPR35; secondary bile acids GPBAR1; and acyl-amides and lactate, GPR132. CONCLUSION: Nutrient metabolites apparently do not stimulate EC cells of the small intestine directly but through a paracrine mechanism involving GLP-1 secreted from neighboring enteroendocrine cells. In contrast, colonic EC cells are able to sense a multitude of different metabolites generated by the gut microbiota as well as gut hormones, including GLP-1.

Profiling of G protein-coupled receptors in vagal afferents reveals novel gut-to-brain sensing mechanisms.

OBJECTIVES: G protein-coupled receptors (GPCRs) act as transmembrane molecular sensors of neurotransmitters, hormones, nutrients, and metabolites. Because unmyelinated vagal afferents richly innervate the gastrointestinal mucosa, gut-derived molecules may directly modulate the activity of vagal afferents through GPCRs. However, the types of GPCRs expressed in vagal afferents are largely unknown. Here, we determined the expression profile of all GPCRs expressed in vagal afferents of the mouse, with a special emphasis on those innervating the gastrointestinal tract. METHODS: Using a combination of high-throughput quantitative PCR, RNA sequencing, and in situ hybridization, we systematically quantified GPCRs expressed in vagal unmyelinated Nav1.8-expressing afferents. RESULTS: GPCRs for gut hormones that were the most enriched in Nav1.8-expressing vagal unmyelinated afferents included NTSR1, NPY2R, CCK1R, and to a lesser extent, GLP1R, but not GHSR and GIPR. Interestingly, both GLP1R and NPY2R were coexpressed with CCK1R. In contrast, NTSR1 was coexpressed with GPR65, a marker preferentially enriched in intestinal mucosal afferents. Only few microbiome-derived metabolite sensors such as GPR35 and, to a lesser extent, GPR119 and CaSR were identified in the Nav1.8-expressing vagal afferents. GPCRs involved in lipid sensing and inflammation (e.g. CB1R, CYSLTR2, PTGER4), and neurotransmitters signaling (CHRM4, DRD2, CRHR2) were also highly enriched in Nav1.8-expressing neurons. Finally, we identified 21 orphan GPCRs with unknown functions in vagal afferents. CONCLUSION: Overall, this study provides a comprehensive description of GPCR-dependent sensing mechanisms in vagal afferents, including novel coexpression patterns, and conceivably coaction of key receptors for gut-derived molecules involved in gut-brain communication.

GPR39 signaling is stimulated by zinc ions but not by obestatin.

GPR39 is an orphan member of the ghrelin receptor family that recently was suggested to be the receptor for obestatin, a peptide derived from the ghrelin precursor. Here, we compare the effect of obestatin to the effect of Zn(2+) on signal transduction and study the effect of obestatin on food intake. Although Zn(2+) stimulated inositol phosphate turnover, cAMP production, arrestin mobilization, as well as cAMP response element-dependent and serum response element-dependent transcriptional activity in GPR39-expressing cells as opposed to mock-transfected cells, no reproducible effect was obtained with obestatin in the GPR39-expressing cells. Moreover, no specific binding of obestatin could be detected in two different types of GPR39-expressing cells using three different radioiodinated forms of obestatin. By quantitative PCR analysis, GPR39 expression was readily detected in peripheral organs such as duodenum and kidney but not in the pituitary and hypothalamus, i.e. presumed central target organs for obestatin. Obestatin had no significant and reproducible effect on acute food intake in either freely fed or fasted lean mice. It is concluded that GPR39 is probably not the obestatin receptor. In contrast, the potency and efficacy of Zn(2+) in respect of activating signaling indicates that this metal ion could be a physiologically relevant agonist or modulator of GPR39.

Model-Based Discovery of Synthetic Agonists for the Zn2+-Sensing G-Protein-Coupled Receptor 39 (GPR39) Reveals Novel Biological Functions.

The G-protein-coupled receptor 39 (GPR39) is a G-protein-coupled receptor activated by Zn2+. We used a homology model-based approach to identify small-molecule pharmacological tool compounds for the receptor. The method focused on a putative binding site in GPR39 for synthetic ligands and knowledge of ligand binding to other receptors with similar binding pockets to select iterative series of minilibraries. These libraries were cherry-picked from all commercially available synthetic compounds. A total of only 520 compounds were tested in vitro, making this method broadly applicable for tool compound development. The compounds of the initial library were inactive when tested alone, but lead compounds were identified using Zn2+ as an allosteric enhancer. Highly selective, highly potent Zn2+-independent GPR39 agonists were found in subsequent minilibraries. These agonists identified GPR39 as a novel regulator of gastric somatostatin secretion.