Synaptic Inputs to the Mouse Dorsal Vagal Complex and Its Resident Preproglucagon Neurons.

Stress responses are coordinated by widespread neural circuits. Homeostatic and psychogenic stressors activate preproglucagon (PPG) neurons in the caudal nucleus of the solitary tract (cNTS) that produce glucagon-like peptide-1; published work in rodents indicates that these neurons play a crucial role in stress responses. While the axonal targets of PPG neurons are well established, their afferent inputs are unknown. Here we use retrograde tracing with cholera toxin subunit b to show that the cNTS in male and female mice receives axonal inputs similar to those reported in rats. Monosynaptic and polysynaptic inputs specific to cNTS PPG neurons were revealed using Cre-conditional pseudorabies and rabies viruses. The most prominent sources of PPG monosynaptic input include the lateral (LH) and paraventricular (PVN) nuclei of the hypothalamus, parasubthalamic nucleus, lateral division of the central amygdala, and Barrington's nucleus (Bar). Additionally, PPG neurons receive monosynaptic vagal sensory input from the nodose ganglia and spinal sensory input from the dorsal horn. Sources of polysynaptic input to cNTS PPG neurons include the hippocampal formation, paraventricular thalamus, and prefrontal cortex. Finally, cNTS-projecting neurons within PVN, LH, and Bar express the activation marker cFOS in mice after restraint stress, identifying them as potential sources of neurogenic stress-induced recruitment of PPG neurons. In summary, cNTS PPG neurons in mice receive widespread monosynaptic and polysynaptic input from brain regions implicated in coordinating behavioral and physiological stress responses, as well as from vagal and spinal sensory neurons. Thus, PPG neurons are optimally positioned to integrate signals of homeostatic and psychogenic stress.SIGNIFICANCE STATEMENT Recent research has indicated a crucial role for glucagon-like peptide-1-producing preproglucagon (PPG) neurons in regulating both appetite and behavioral and autonomic responses to acute stress. Intriguingly, the central glucagon-like peptide-1 system defined in rodents is conserved in humans, highlighting the translational importance of understanding its anatomical organization. Findings reported here indicate that PPG neurons receive significant monosynaptic and polysynaptic input from brain regions implicated in autonomic and behavioral responses to stress, as well as direct input from vagal and spinal sensory neurons. Improved understanding of the neural pathways underlying the recruitment of PPG neurons may facilitate the development of novel therapies for the treatment of stress-related disorders.

Diversification of the functions of proglucagon and glucagon receptor genes in fish.

The teleost fish-specific genome duplication gave rise to a great number of species inhabiting diverse environments with different access to nutrients and life histories. This event produced duplicated gcg genes, gcga and gcgb, for proglucagon-derived peptides, glucagon and GLP-1 and duplicated gcgr receptor genes, gcgra and gcgrb, which play key roles connecting the consumption of nutrients with glucose metabolism. We conducted a systematic survey of the genomes from 28 species of fish (24 bony (Superclass Osteichthyes), 1 lobe-finned (Class Sarcoperygii), 1 cartilaginous (Superclass Chondrichthyes), and 2 jawless (Superclass Agnatha)) and find that almost all surveyed ray-finned fish contain gcga and gcgb genes with different coding potential and duplicated gcgr genes, gcgra and gcgrb that form two separate clades in the phylogenetic tree consistent with the accepted species phylogeny. All gcgb genes encoded only glucagon and GLP-1 and gcga genes encoded glucagon, GLP-1, and GLP-2, indicating that gcga was subfunctionalized to produce GLP-2. We find a single glp2r, but no glp1r suggesting that duplicated gcgrb was neofunctionalized to bind GLP-1, as demonstrated for the zebrafish gcgrb (Oren et al., 2016). In functional experiments with zebrafish gcgrb and GLP-1 from diverse fish we find that anglerfish GLP-1a, encoded by gcga, is less biologically active than the gcgb anglerfish GLP-1b paralog. But some other fish (zebrafish, salmon, and catfish) gcga GLP-1a display similar biological activities, indicating that the regulation of glucose metabolism by GLP-1 in ray-finned fish is species-specific. Searches of genomes in cartilaginous fish identified a proglucagon gene that encodes a novel GLP-3 peptide in addition to glucagon, GLP-1, and GLP-2, as well as a single gcgr, glp2r, and a new glucagon receptor-like receptor whose identity still needs to be confirmed. The sequence of the shark GLP-1 contained an N-terminal mammalian-like extension that in mammals undergoes a proteolytic cleavage to release biologically active GLP-1. Our results indicate that early in vertebrate evolution diverse regulatory mechanisms emerged for the control of glucose metabolism by proglucagon-derived peptides and their receptors and that in ray-finned fish they included subfunctionalization and neofunctionalization of these genes.

Insights on glicentin, a promising peptide of the proglucagon family.

Glicentin is a proglucagon-derived peptide mainly produced in the L-intestinal cells. While the roles of other members of the proglucagon family including glucagon-like peptide 1, glucagon-like peptide 2 and oxyntomodulin has been well studied, the functions and variation of glicentin in human are not fully understood. Experimental and clinical studies have highlighted its role in both intestinal physiology and glucose metabolism, pointing to its potential interest in a wide range of pathological states including gastrointestinal and metabolic disorders. Due to its structure presenting many similarities with the other proglucagon-derived peptides, its measurement is technically challenging. The recent commercialization of specific detection methods has offered new opportunities to go further in the understanding of glicentin physiology. Here we summarize the current knowledge on glicentin biogenesis and physiological roles. In the limelight of clinical studies investigating glicentin variation in human, we discuss future directions for potential applications in clinical practice.

Preproglucagon neurons in the hindbrain have IL-6 receptor-α and show Ca2+ influx in response to IL-6.

Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure, and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analog of the proglucagon-derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA expression of the cytokine interleukin-6 (IL-6) in the hypothalamus and hindbrain and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1-producing PPG neurons in the nucleus of the solitary tract (NTS) in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca(2+) concentration in neurons capable of expressing PPG. We also show that the Ca(2+) increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNP:ase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca(2+) to the cytosol from the extracellular space.

Endogenous glucagon-like peptide-1 reduces drinking behavior and is differentially engaged by water and food intakes in rats.

Glucagon-like peptide-1 (GLP-1) is produced in the ileum and the nucleus of the solitary tract. It is well known that GLP-1 controls food intake, but there is a growing literature indicating that GLP-1 also is involved in fluid intake. It is not known, however, if the observed effects are pharmacological or if endogenous GLP-1 and its receptor contribute to physiological fluid intake control. Accordingly, we blocked endogenous GLP-1 by application of a receptor antagonist and measured subsequent drinking. Furthermore, we measured changes in GLP-1-associated gene expression after water intake, and compared the effects of fluid intake to those caused by food intake. Rats injected with the antagonist exendin-9 (Ex-9) drank more fluid in response to either subcutaneous hypertonic saline or water deprivation with partial rehydration than did vehicle-treated rats. Analysis of licking behavior showed that Ex-9 increased fluid intake by increasing the number of licking bursts, without having an effect on the number of licks per burst, suggesting that endogenous GLP-1 suppresses fluid intake by influencing satiety. Subsequent experiments showed that water intake had a selective effect on central GLP-1-related gene expression, unlike food intake, which affected both central and peripheral GLP-1. Although water and food intakes both affected central GLP-1-relevant gene expression, there were notable differences in the timing of the effect. These results show a novel role of the endogenous GLP-1 system in fluid intake, and indicate that elements of the GLP-1 system can be engaged separately by different forms of ingestive behavior.

Short communication: expression of peptide YY, proglucagon, neuropeptide Y receptor Y2, and glucagon-like peptide-1 receptor in bovine peripheral tissues.

The role of distal gut signals in control of feed intake and metabolism in cattle has received scant attention. Peptide YY (PYY) and glucagon-like peptide-1, which are secreted from enteroendocrine cells of the distal gut in monogastrics have several functions, including regulation of energy balance. However, little is known of the tissue expression of these peptides and their receptors in cattle. The aim of the current study was to characterize the tissue distribution of PYY, neuropeptide Y receptor Y2 (Y2), proglucagon (GCG), and glucagon-like peptide-1 receptor (GLP1R) in various peripheral tissues of cattle. Four male 7-wk-old dairy calves were euthanized and 16 peripheral tissues were collected. Conventional PCR and quantitative real-time PCR were performed to confirm tissue expression and quantify the transcript abundance in various tissues. The results of conventional PCR revealed that mRNA for both PYY and Y2 was detectable in the rumen, abomasum, duodenum, jejunum, ileum, and colon but not in other tissues. Quantitative real-time PCR data demonstrated that PYY mRNA was 2- to 3-fold greater in the pancreas, kidney, and heart relative to the liver. By conventional PCR, GCG mRNA was detected in the abomasum, duodenum, jejunum, ileum, and colon and GLP1R mRNA was expressed in all gut segments, pancreas, spleen, and kidney. Quantitative real-time PCR data demonstrated that, relative to transcript abundance in the liver, GCG mRNA was 4- to 40-fold higher from abomasum to colon, and GLP1R mRNA was 50- to 300-fold higher from the rumen to colon, 14-fold greater in the pancreas, 18-fold higher in the spleen, and 166-fold greater in the kidney. The tissue distribution of PYY, GCG, and their receptors observed in the current study is, in general, consistent with expression patterns in monogastrics. The predominant expression of PYY, Y2, and GCG in the gut, and the presence of GLP1R in multiple peripheral tissues suggest a role for PYY in controlling gut functions and for GLP-1 in regulating multiple physiological functions in cattle.

The brainstem preproglucagon system in a non-human primate (Macaca mulatta).

The nucleus of the solitary tract (NTS) contains a small population of neurons expressing preproglucagon. In these neurons preproglucagon is processed to the glucagon-like-peptides 1 and 2 (GLP-1 and GLP-2) and oxyntomodulin. Whereas the neuroanatomy of these neurons is well characterized in rodents the location and projection of preproglucagon neurons have never been described in primates. The purpose of the present study was to characterize the location of preproglucagon neurons and their projections in the non-human primate using radioactive in situ hybridization and immunohistochemistry. In situ hybridization revealed preproglucagon mRNA expressing neurons in the caudal nucleus of the solitary tract extending laterally through the intermediate reticular nucleus into the A1 area. Using an antibody raised against rat GLP-2, GLP-2-immunoreactive (-ir) cell bodies were found in the same areas as the preproglucagon mRNA. Only very few GLP-2-ir nerve fibers were observed in the caudal brainstem and mostly in the same areas as the GLP-2-ir cell bodies. The most prominent GLP-2-ir terminal fields were detected in the hypothalamus and rostrally in the bed nucleus of the stria terminalis complex. In the hypothalamus, GLP-2-ir fibers arborized extensively in the paraventricular nucleus of the hypothalamus (PVN), the dorsomedial hypothalamic nucleus (DMH) and the arcuate nucleus (Arc), the latter containing the densest fiber-plexus. The findings indicate that the brainstem preproglucagon neuronal system is highly conserved between rat and non-human primate with the exception of a much denser innervation of the mediobasal hypothalamus in the primate brain.

Leptin directly depolarizes preproglucagon neurons in the nucleus tractus solitarius: electrical properties of glucagon-like Peptide 1 neurons.

OBJECTIVE: Glucagon-like peptide (GLP)-1 inhibits food intake, acting both in the periphery and within the central nervous system. It is unclear if gut-derived GLP-1 can enter the brain, or whether GLP-1 from preproglucagon (PPG) cells in the lower brainstem is required to activate central GLP-1 receptors. Brainstem PPG neurons, however, have been poorly characterized, due to the difficulties in identifying these cells while viable. This study provides data on the electrical properties of brainstem PPG cells and their regulation by orexigenic and anorexigenic peptides. RESEARCH DESIGN AND METHODS: Transgenic mice expressing Venus under control of the PPG promoter were used to identify PPG neurons in vitro in brainstem slice preparations for electrophysiological recordings. RESULTS The majority of PPG neurons were spontaneously active. Further electrical and molecular characterization revealed that GLP-1 receptor activation had no pre- or postsynaptic effect and that PPG neurons lack GLP-1 receptors. Similarly, they were unresponsive to PYY and ghrelin. In contrast, leptin rapidly and reversibly depolarized these neurons. Responses to electrical stimulation of the solitary tract suggest that PPG cells are mostly second-order neurons, receiving direct input from vagal afferent fibers. Both evoked and spontaneous excitatory postsynaptic currents were predominantly glutamatergic. CONCLUSIONS: The study introduces PPG-promoter-Venus transgenic mice as a viable and important tool to study brainstem PPG cells. PPG neuron activity is directly modulated by leptin but was unaffected by other satiety or hunger peptides. Direct synaptic input from the solitary tract suggests that peripheral signals (including GLP-1) could modulate PPG cells via vagal afferents.

Resolution of type 2 diabetes following gastric bypass surgery: involvement of gut-derived glucagon and glucagonotropic signalling?

Certain types of bariatric surgical procedures have proved not only to be effective with regard to treating obesity, but they also seem to be associated with endocrine changes which independently of weight loss give rise to remission of type 2 diabetes. Currently, it is speculated that surgical re-routing of nutrients triggers changes in the release of gastrointestine-derived hormones, which in turn cause amelioration of the diabetic state. The 'hindgut hypothesis' states that surgical re-routing of nutrients to the distal part of the small intestine results in increased secretion and concomitant glucose-lowering effects of glucagon-like peptide-1, whereas the 'foregut hypothesis' emphasises that surgical bypass of the foregut prevents the release of a hitherto unidentified nutrient-induced diabetogenic signal in susceptible individuals. Recent studies have shown that in patients with type 2 diabetes, glucagon is differentially secreted in response to oral and i.v. glucose, respectively, with lack of suppression (and initial net secretion) during oral glucose administration and a perfectly normal suppression during isoglycaemic i.v. glucose administration. These findings could point towards a role for glucagon or gut-derived glucagonotropic signalling as putative diabetogenic signals of the foregut hypothesis. In the present paper the hypotheses describing the glucose-lowering mechanisms of bariatric surgical procedures sharing the common feature of a bypass of the duodenum and the proximal jejunum are outlined and a possible role for glucagon in these is proposed.

Physiology of incretins (GIP and GLP-1) and abnormalities in type 2 diabetes.

Incretin hormones are defined as intestinal hormones released in response to nutrient ingestion, which potentiate the glucose-induced insulin response. In humans, the incretin effect is mainly caused by two peptide hormones, glucose-dependent insulin releasing polypeptide (GIP), and glucagon-like peptide-1 (GLP-1). GIP is secreted by K cells from the upper small intestine while GLP-1 is mainly produced in the enteroendocrine L cells located in the distal intestine. Their effect is mediated through their binding with specific receptors, though part of their biological action may also involve neural modulation. GIP and GLP-1 are both rapidly degraded into inactive metabolites by the enzyme dipeptidyl-peptidase-IV (DPP-IV). In addition to its effects on insulin secretion, GLP-1 exerts other significant actions, including stimulation of insulin biosynthesis, inhibition of glucagon secretion, inhibition of gastric emptying and acid secretion, reduction of food intake, and trophic effects on the pancreas. As the insulinotropic action of GLP-1 is preserved in type 2 diabetic patients, this peptide was likely to be developed as a therapeutic agent for this disease.

The alpha cell expresses glucagon-like peptide-2 receptors and glucagon-like peptide-2 stimulates glucagon secretion from the rat pancreas.

AIMS/HYPOTHESIS: Glucagon-like peptide-2 (GLP-2) is a gut hormone regulating intestinal growth and nutrient absorption. Recently, GLP-2 has been reported to stimulate glucagon secretion in healthy humans. We sought to clarify the mechanism and physiological significance of this endocrine effect. MATERIALS AND METHODS: The expression of the GLP-2 receptor gene, Glpr2, and the localisation of the protein were evaluated by real-time PCR on cDNA from isolated rat islets and by immunohistochemistry in rat and human pancreas. The glucagon, insulin and somatostatin responses to 0.1, 1 and 10 nmol/l GLP-2 and to GLP-1 and GLP-2 given simultaneously were studied in the isolated perfused rat pancreas. RESULTS: Expression of Glp2r transcript was confirmed by PCR. In both human and rat pancreas, GLP-2r immunoreactivity was colocalised with proglucagon. GLP-2 at 10 nmol/l increased glucagon secretion significantly from a pre-infusion level of 0.314 +/- 0.07 to 0.508 +/- 0.09 pmol/min (p < 0.0005), whereas lower GLP-2 concentrations were ineffective. Neither insulin nor somatostatin output was influenced. During simultaneous administration of GLP-1 and GLP-2, net glucagon release was no longer reduced by 0.1, 1 or 10 nmol/l GLP-1, which, when given alone, inhibited glucagon secretion by 25.0 +/- 9.9, 46.2 +/- 4.8, and 44.1 +/- 2.9%, respectively. CONCLUSIONS/INTERPRETATION: Our results suggest that GLP-2 stimulates glucagon secretion through GLP-2r present on the alpha cell in rats. In the presence of GLP-2, the glucagonostatic effect of GLP-1, normally co-secreted with GLP-2, is markedly inhibited. Based on our analogous immunohistochemical findings in human pancreas, this mechanism also applies in all likelihood to humans. However, further in vivo studies are required to assess the physiological significance of the glucagonotropic action of GLP-2 in humans.

The role of gut hormones in glucose homeostasis.

The gastrointestinal tract has a crucial role in the control of energy homeostasis through its role in the digestion, absorption, and assimilation of ingested nutrients. Furthermore, signals from the gastrointestinal tract are important regulators of gut motility and satiety, both of which have implications for the long-term control of body weight. Among the specialized cell types in the gastrointestinal mucosa, enteroendocrine cells have important roles in regulating energy intake and glucose homeostasis through their actions on peripheral target organs, including the endocrine pancreas. This article reviews the biological actions of gut hormones regulating glucose homeostasis, with an emphasis on mechanisms of action and the emerging therapeutic roles of gut hormones for the treatment of type 2 diabetes mellitus.