Glucagon‐like peptide‐1 protects synaptic and learning functions from neuroinflammation in rodents

Glucagon‐like peptide‐1 (GLP‐1) is derived from the processing of proglucagon in intestinal L‐cells and releases insulin from pancreatic β‐cells as an incretin. The GLP‐1 receptor has been proposed as a possible therapeutic target for the treatment of Alzheimer's disease, in which neuroinflammation is critical in the pathogenesis. The present study investigates whether GLP‐1 (7–36) amide, an active fragment of GLP‐1, protected against synaptic impairments induced by inflammation‐related injurious agents (lipopolysaccharide [LPS], interleukin‐1β [IL‐1β], and H₂O₂). In the Y‐maze test, LPS (10 μg/mouse, i.c.v) significantly decreased the percentage alternation. Pretreatment with GLP‐1 (7–36) amide (0.09–0.9 nmol/mouse, i.c.v.) prevented an impairment in spontaneous alternation performance. Pretreatment with LPS (10 μg/ml, 2 hr) impaired LTP induction but not paired‐pulse facilitation in the CA1 region of rat hippocampal slices. This impairment was prevented by cotreatment with GLP‐1 (7–36) amide (50 nM). IL‐1β (0.57 nM) or H₂O₂ (50 μM) also impaired LTP induction. This impairment was prevented by GLP‐1 (7–36) amide (50 nM). These results suggest that GLP‐1 (7–36) amide improves the synaptic impairments induced by inflammation‐related injurious agents in the CA1 region of the hippocampus. © 2014 Wiley Periodicals, Inc.     

Glucagon‐like peptide‐1 cleavage product GLP‐1 (9‐36) amide enhances hippocampal long‐term synaptic plasticity in correlation with suppression of Kv4.2 expression and eEF2 phosphorylation

Glucagon‐like peptide‐1 (GLP‐1) is an endogenous gut hormone and a key regulator in maintaining glucose homeostasis by stimulating insulin secretion. Its natural cleavage product GLP‐1 (9‐36), used to be considered a “bio‐inactive” metabolite mainly because of its lack of insulinotropic effects and low affinity for GLP‐1 receptors, possesses unique properties such as anti‐oxidant and cardiovascular protection. Little is known about the role of GLP‐1 (9‐36) in central nervous system. Here we report that chronic, systemic application of GLP‐1 (9‐36) in adult mice facilitated both the induction and maintenance phases of hippocampal long‐term potentiation (LTP), a major form of synaptic plasticity. In contrast, spatial learning and memory, as assessed by the Morris water maze test, was not altered by GLP‐1 (9‐36) administration. At the molecular level, GLP‐1 (9‐36) reduced protein levels of the potassium channel Kv4.2 in hippocampus, which is linked to elevated dendritic membrane excitability. Moreover, GLP‐1(9‐36) treatment inhibited phosphorylation of mRNA translational factor eEF2, which is associated with increased capacity for de novo protein synthesis. Finally, we showed that the LTP‐enhancing effects by GLP‐1 (9‐36) treatment in vivo were blunted by application of exendin(9‐39)amide [EX(9‐39)], the GLP‐1 receptor (GLP‐1R) antagonist, suggesting its role as a GLP‐1R agonist. These findings demonstrate that GLP‐1 (9‐36), which was considered a “bio‐inactive” peptide, clearly exerts physiological effects on neuronal plasticity in the hippocampus, a brain region critical for learning and memory.     

Glucagon‐like peptide‐1 inhibits voltage‐gated potassium currents in mouse nodose ganglion neurons

Background Glucagon‐like peptide‐1 (GLP‐1) is a major hormone known to regulate glucose homeostasis and gut function, and is an important satiety mediator. These actions are at least in part mediated via an action on vagal afferent neurons. However, the mechanism by which GLP‐1 activates vagal afferents remains unknown. We hypothesized that GLP‐1 acts on nodose ganglion neuron voltage‐gated potassium (KV) channels, increasing membrane excitability. Methods Employing perforated patch clamp recordings we examined the effects of GLP‐1 on membrane properties as well as voltage‐gated potassium currents. Extracellular recordings of jejunal afferents were performed to demonstrate the functional relevance of these effects at the nerve terminal. Key Results Glucagon‐like peptide‐1 depolarized a subpopulation of nodose neurons. This membrane depolarization was used to identify neurons containing functional GLP‐1 receptors. In these neurons, GLP‐1 decreased rheobase and broadened the action potential, and increased the number of action potentials elicited at twice rheobase. We identified a GLP‐1 sensitive current whose reversal potential shifted in a depolarizing direction when extracellular potassium was increased. We identified two macroscopic K currents, IA, an inactivating current and IK a sustained current. GLP‐1 caused inhibition of these currents, IK by 45%, P < 0.05 and IA currents by 52%P < 0.01, associated with a hyperpolarizing shift of steady‐state inactivation curves for both currents. In extracellular recordings of jejunal afferents, GLP‐1 increased firing rate, the effect blocked by the K⁺ channel antagonist 4‐AP. Conclusions & Inferences These experiments indicate that GLP‐1 receptor activation results in vagal afferent excitation, due at least in part to inhibition of sustained and inactivating potassium currents. This mechanism may be important in satiety and glucose homeostatic signals arising from the gastrointestinal tract.     

Interleukin‐6 in the central amygdala is bioactive and co‐localised with glucagon‐like peptide‐1 receptor

Neuronal circuits involving the central amygdala (CeA) are gaining prominence as important centres for regulation of metabolic functions. As a part of the subcortical food motivation circuitry, CeA is associated with food motivation and hunger. We have previously shown that interleukin (IL)‐6 can act as a downstream mediator of the metabolic effects of glucagon‐like peptide‐1 (GLP‐1) receptor (R) stimulation in the brain, although the sites of these effects are largely unknown. In the present study, we used the newly generated and validated RedIL6 reporter mouse strain to investigate the presence of IL‐6 in the CeA, as well as possible interactions between IL‐6 and GLP‐1 in this nucleus. IL‐6 was present in the CeA, mostly in cells in the medial and lateral parts of this structure, and a majority of IL‐6‐containing cells also co‐expressed GLP‐1R. Triple staining showed GLP‐1 containing fibres co‐staining with synaptophysin close to or overlapping with IL‐6 containing cells. GLP‐1R stimulation enhanced IL‐6 mRNA levels. IL‐6 receptor‐alpha (IL‐6Rα) was found to a large part in neuronal CeA cells. Using electrophysiology, we determined that cells with neuronal properties in the CeA could be rapidly stimulated by IL‐6 administration in vitro. Moreover, microinjections of IL‐6 into the CeA could slightly reduce food intake in vivo in overnight fasted rats. In conclusion, IL‐6 containing cells in the CeA express GLP‐1R, are close to GLP‐1‐containing synapses, and demonstrate increased IL‐6 mRNA in response to GLP‐1R agonist treatment. IL‐6, in turn, exerts biological effects in the CeA, possibly via IL‐6Rα present in this nucleus.     

Glucagon‐Like Peptide‐1 Receptor Agonist Administration Suppresses Both Water and Saline Intake in Rats

Glucagon‐like peptide‐1 (GLP‐1) plays an important role in energy homeostasis. Injections of GLP‐1 receptor (GLP‐1R) agonists suppress food intake, and endogenous GLP‐1 is released when nutrients enter the gut. There is also growing evidence that the GLP‐1 system is involved in the regulation of body fluid homeostasis. GLP‐1R agonists suppress water intake independent of their effects on food intake. It is unknown, however, whether this suppressive effect of GLP‐1R agonists extends to saline intake. Accordingly, we tested the effect of the GLP‐1R agonists liraglutide (0.05 μg) and exendin‐4 (0.05 μg) on water and saline intake, as stimulated either by angiotensin II (AngII) or by water deprivation with partial rehydration (WD‐PR). Each agonist suppressed AngII‐induced water intake; however, only exendin‐4 suppressed saline intake. WD‐PR‐induced water and saline intakes were both attenuated by each agonist. Analysis of drinking microstructure after WD‐PR found a reliable effect of the agonists on burst number. Furthermore, exendin‐4 conditioned a robust taste avoidance to saccharine; however, there was no similar effect of liraglutide. To evaluate the relevance of the conditioned taste avoidance, we tested whether inducing visceral malaise by injection of lithium chloride (LiCl) suppressed fluid intake. Injection of LiCl did not suppress water or saline intakes. Overall, these results indicate that the fluid intake suppression by GLP‐1R activation is not selective to water intake, is a function of post‐ingestive feedback, and is not secondary to visceral malaise.     

Efficacy of the glucagon‐like peptide‐1 agonist exenatide in the treatment of short bowel syndrome

Background Short bowel syndrome (SBS) is a serious clinical disorder characterized by diarrhea and nutritional deprivation. Glucagon‐like peptide‐1 (GLP‐1) is a key hormone, produced by L‐cells in the ileum, that regulates proximal gut transit. When extensive ileal resection occurrs, as in SBS, GLP‐1 levels may be deficient. In this study, we test whether the use of GLP‐1 agonist exenatide can improve the nutritional state and intestinal symptoms of patients with SBS. Methods Five consecutive patients with SBS based on ≤90 cm of small bowel and clinical evidence of nutritional deprivation were selected. Baseline SBS symptoms, demographic and laboratory data were obtained. Antroduodenal manometry was performed on each subject. Each patient was then started on exenatide and over the following month, the baseline parameters were repeated. Key Results The subjects consisted of four males and one female, aged 46–69 years. At baseline, all had severe diarrhea that ranged from 6 to 15 bowel movements per day, often occurring within minutes of eating. After exenatide, all five patients had immediate improvement in bowel frequency and form; bowel movements were no longer meal‐related. Total parenteral nutrition was stopped successfully in three patients. Antroduodenal manometry revealed continuous low amplitude gastric contractions during fasting which completely normalized with exenatide. Conclusions & Inferences Exenatide is a novel and safe treatment option for SBS. It produced substantial improvement in the bowel habits, nutritional status and quality of life of SBS patients. Successful treatment with exenatide may significantly reduce the need for parenteral nutrition and small bowel transplant.     

DEP domain‐containing mTOR‐interacting protein in the rat brain: Distribution of expression and potential implication

DEP domain–containing mTOR‐interacting protein (DEPTOR) has been recently discovered as an endogenous regulator of the mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2. mTORC1 is present in the brain, and there is growing evidence that its dysregulation contributes to several brain alterations. This suggests the involvement of mTOR signaling and its modulators in neurobiological controls. Here, we characterized and mapped the expression of DEPTOR in the rat brain. We show that DEPTOR was widely expressed from the forebrain to the hindbrain, including the hippocampus, the mediobasal hypothalamus, and the circumventricular organs (CVOs). In the hippocampus, DEPTOR protein and Deptor mRNA were highly expressed in the dendate gyrus and CA3 field. In the CVOs, DEPTOR was expressed in the subfornical organ, the median eminence, and the area postrema. In the mediobasal hypothalamus, DEPTOR was expressed in neurons of the ventromedial nucleus (VMH) and colocalized with proopiomelanocortin (POMC) in the arcuate nucleus (ARC). The hypothalamic distribution suggested a role for DEPTOR in energy balance. Supporting this possibility, we observed that Deptor hypothalamic expression was modulated by the nutritional status in a context of diet‐induced and genetic obesity; food deprivation increased Deptor mRNA in both the ARC and VMH of obese rats. In conclusion, the present results illustrate the presence of DEPTOR in the rat brain and suggest a role for DEPTOR in the hypothalamic regulation of energy balance, which further supports the role of mTOR in energy homeostasis. J. Comp. Neurol. 523:93–107, 2015. © 2014 Wiley Periodicals, Inc.     

Activation of Nesfatin‐1‐Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin‐1 in Rats

Peripheral anorectic hormones, such as glucagon‐like peptide (GLP)‐1, cholecystokinin (CCK)‐8 and leptin, suppress food intake. The newly‐identified anorectic neuropeptide, nesfatin‐1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP‐1 and CCK‐8 and co‐administrations of GLP‐1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin‐1‐immunoreactive (‐IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP‐1 (100 μg/kg) caused significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK‐8 (50 μg/kg) resulted in marked increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP‐1 (33 μg/kg) or leptin. However, co‐administration of GLP‐1 (33 μg/kg) and leptin resulted in significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP‐1, CCK‐8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin‐1. These results indicate that nesfatin‐1‐expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP‐1 and leptin in addition to CCK‐8, and also suppress food intake in rats.     

Peripheral motor action of glucagon‐like peptide‐1 through enteric neuronal receptors

Background Glucagon‐like peptide‐1 (GLP‐1) is a proglucagon‐derived peptide expressed in the enteroendocrine‐L cells of small and large intestine and released in response to meal ingestion. Glucagon‐like peptide‐1 exerts inhibitory effects on gastrointestinal motility through vagal afferents and central nervous mechanisms; however, no data is available about a direct influence on the gastrointestinal wall. Our aim was to investigate the effects of GLP‐1 on the spontaneous and evoked mechanical activity of mouse duodenum and colon and to identify the presence and distribution of GLP‐1 receptors (GLP‐1R) in the muscle coat. Methods Organ bath recording technique and immunohistochemistry were used. Key Results Glucagon‐like peptide‐1 (up to the concentration of 1 μmol L^?1) failed to affect spontaneous mechanical activity. It caused concentration‐dependent reduction of the electrically evoked cholinergic contractions in circular smooth muscle of both intestinal segments, without affecting the longitudinal muscle responses. Glucagon‐like peptide‐1 inhibitory effect was significantly antagonized by exendin (9–39), an antagonist of GLP‐1R. In both intestinal preparations, GLP‐1 effect was not affected by guanethidine, a blocker of adrenergic neurotransmission, but it was significantly reduced by N_ω‐nitro‐l‐arginine methyl ester, inhibitor of nitric oxide (NO) synthase. Glucagon‐like peptide‐1 failed to affect the contractions evoked by exogenous carbachol. Immunohistochemistry demonstrated GLP‐1R expression in the enteric neurons. Furthermore, 27% of GLP‐1R immunoreactive (IR) neurons in the duodenum and 79% of GLP‐1R‐IR neurons in the colon, co‐expressed nNOS. Conclusions & Inferences The present results suggest that GLP‐1 is able to act in the enteric nervous system by decreasing the excitatory cholinergic neurotransmission through presynaptic GLP‐1Rs, which modulate NO release.     

Oxidized low‐density lipoprotein (oxLDL)‐induced cell death in dorsal root gangion cell cultures depends not on the lectin‐like oxLDL receptor‐1 but on the toll‐like receptor‐4

DRG cells have been found to undergo apoptosis and necrosis after oxidized low‐density lipoprotein (oxLDL) stimulation in vitro. However, the mechanism of oxLDL‐induced DRG cell death is unclear. For this reason, we studied the expression of two potential oxLDL receptors: lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) and toll‐like receptor‐4 (TLR4) in dorsal root ganglion (DRG) cell cultures from postnatal rats. Cells were cultivated with and without oxLDL. In oxLDL‐treated DRG cell cultures, the increase of cleaved caspase‐3 protein was observed as a sign of enhanced apoptosis. Untreated and oxLDL‐treated DRG cell cultures expressed LOX‐1 and TLR4 at similar levels. The LOX‐1 expression remained unchanged after receptor blockade. However, the inhibition of LOX‐1 caused a significant increase of cleaved caspase‐3 and a decrease of TLR4 levels. The TLR4‐inhibited DRG cell cultures lacked changes in LOX‐1 expression for all experimental groups. The inhibition of TLR4 caused activation of jun N‐terminal kinase (JNK) and a significant decrease of cleaved caspase‐3 but did not change the TLR4 level. We conclude that LOX‐1 and TLR4 are expressed in cultivated rat DRG cells and that the oxLDL‐induced cell death in DRG cell cultures does not depend on the LOX‐1 but on the TLR4. © 2009 Wiley‐Liss, Inc.     

Distribution of the SynDIG4/proline‐rich transmembrane protein 1 in rat brain

The modulation of AMPA receptor (AMPAR) content at synapses is thought to be an underlying molecular mechanism of memory and learning. AMPAR content at synapses is highly plastic and is regulated by numerous AMPAR accessory transmembrane proteins such as TARPs, cornichons, and CKAMPs. SynDIG (synapse differentiation‐induced gene) defines a family of four genes (SynDIG1–4) expressed in distinct and overlapping patterns in the brain. SynDIG1 was previously identified as a novel transmembrane AMPAR‐associated protein that regulates synaptic strength. The related protein SynDIG4 [also known as Prrt1 (proline‐rich transmembrane protein 1)] has recently been identified as a component of AMPAR complexes. In this study, we show that SynDIG1 and SynDIG4 have distinct yet overlapping patterns of expression in the central nervous system, with SynDIG4 having especially prominent expression in the hippocampus and particularly within CA1. In contrast to SynDIG1 and other traditional AMPAR auxiliary subunits, SynDIG4 is de‐enriched at the postsynaptic density and colocalizes with extrasynaptic GluA1 puncta in primary dissociated neuron culture. These results indicate that, although SynDIG4 shares sequence similarity with SynDIG1, it might act through a unique mechanism as an auxiliary factor for extrasynaptic GluA1‐containing AMPARs. J. Comp. Neurol. 524:2266–2280, 2016. © 2015 Wiley Periodicals, Inc.