Role of cholinergic neurons in the motor effects of glucagon-like peptide-2 in mouse colon.

Glucagon-like peptide-2 (GLP-2) reduces mouse gastric tone and small intestine transit, but its action on large intestine motility is still unknown. The purposes of the present study were 1) to examine the influence of GLP-2 on spontaneous mechanical activity and on neurally evoked responses, by recording intraluminal pressure from mouse isolated colonic segments; 2) to characterize GLP-2 mechanism of action; and 3) to determine the distribution of GLP-2 receptor (GLP-2R) in the mouse colonic muscle coat by immunohistochemistry. Exogenous GLP-2 (0.1\u2013 300 nM) induced a concentration-dependent reduction of the spontaneous mechanical activity, which was abolished by the desensitization of GLP-2 receptor or by tetrodotoxin, a voltage-dependent Na+-channel blocker. GLP-2 inhibitory effect was not affected by Nomega-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor), apamin (a blocker of small conductance Ca2+-dependent K+ channels), or [Lys1,Pro2,5,Arg3,4,Tyr6]VIP7\u201328 (a VIP receptor antagonist), but it was prevented by atropine or pertussis toxin (PTX), a Gi/o protein inhibitor. Proximal colon responses to electrical field stimulation were characterized by nitrergic relaxation, which was followed by cholinergic contraction. GLP-2 reduced only the cholinergic evoked contractions. This effect was almost abolished by GLP-2 receptor desensitization or PTX. GLP-2 failed to affect the contractile responses to exogenous carbachol. GLP-2R immunoreactivity (IR) was detected only in the neuronal cells of both plexuses of the colonic muscle coat. More than 50% of myenteric GLP-2R-IR neurons shared the choline acetyltransferase IR. In conclusion, the activation of GLP-2R located on cholinergic neurons may modulate negatively the colonic spontaneous and electrically evoked contractions through inhibition of acetylcholine release. The effect is mediated by Gi protein

GLP-2 receptor expression in excitatory and inhibitory enteric neurons and its role in mouse duodenum contractility.

Background. Glucagon-like peptide 2 (GLP-2), a nutrient-responsive hormone, exerts various actions in the gastrointestinal tract that are mediated by a G-protein coupled receptor called GLP-2R. A little information is available on GLP-2R expression in enteric neurons and nothing on the interstitial cells of Cajal (ICC). Methods. We investigated presence and distribution of the GLP-2R in the mouse duodenum by immunohistochemistry and the potential motor effects of GLP-2 on the spontaneous and neurally evoked mechanical activity. Key Results. The GLP-2R was expressed by the myenteric and submucosal neurons. Labelling was also present in nerve varicosities within the circular muscular layer and at the deep muscular plexus (DMP). No immunoreactive nerve fiber was seen within the longitudinal muscle layer. The GLP-2R-positive neurons were either excitatory (SP- and choline-acetyltransferase-positive) or inhibitory (vasoactive intestinal polypeptide and nNOS-positive). The ICC, both at the myenteric plexus and at theDMP,never expressed GLP-2R but, especially those at the DMP, were surrounded by GLP-2R-positive nerve varicosities co-expressing either excitatory or inhibitory neurotransmitters. Quantitative analysis demonstrated a consistent prevalence of GLP-2R on the excitatory pathways. In agreement, the functional results showed that the administration of GLP-2 in vitro caused decrease of the spontaneous contractions mediated by nitric oxide release and reduction of the evoked cholinergic contractions. Conclusions & Inferences. The present findings indicate that the GLP-2R is expressed by inhibitory and excitatory neurons, the GLP-2 inhibits the muscle contractility likely decreasing cholinergic neurotransmission and increasing nitric oxide production, and this effect is possibly mediated by the ICC-DMP recruitment

A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy

The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence nuclear and eosin Y staining that enabled us to obtain hematoxylin and eosin pseudo-coloring comparable with the gold standard H&E analysis. The computational reconstructions obtained with 3D optical imaging can be analyzed by a pathologist without any specific training in volumetric microscopy, paving the way for new biomedical applications in clinical pathology

Tisochrysis lutea F&M-M36 Mitigates Risk Factors of Metabolic Syndrome and Promotes Visceral Fat Browning through β3-Adrenergic Receptor/UCP1 Signaling

Pre-metabolic syndrome (pre-MetS) may represent the best transition phase to start treatments aimed at reducing cardiometabolic risk factors of MetS. In this study, we investigated the effects of the marine microalga Tisochrysis lutea F&M-M36 (T. lutea) on cardiometabolic components of pre-MetS and its underlying mechanisms. Rats were fed a standard (5% fat) or a high-fat diet (20% fat) supplemented or not with 5% of T. lutea or fenofibrate (100 mg/Kg) for 3 months. Like fenofibrate, T. lutea decreased blood triglycerides (p < 0.01) and glucose levels (p < 0.01), increased fecal lipid excretion (p < 0.05) and adiponectin (p < 0.001) without affecting weight gain. Unlike fenofibrate, T. lutea did not increase liver weight and steatosis, reduced renal fat (p < 0.05), diastolic (p < 0.05) and mean arterial pressure (p < 0.05). In visceral adipose tissue (VAT), T. lutea, but not fenofibrate, increased the β3-adrenergic receptor (β3ADR) (p < 0.05) and Uncoupling protein 1 (UCP-1) (p < 0.001) while both induced glucagon-like peptide-1 receptor (GLP1R) protein expression (p < 0.001) and decreased interleukin (IL)-6 and IL-1β gene expression (p < 0.05). Pathway analysis on VAT whole-gene expression profiles showed that T. lutea up-regulated energy-metabolism-related genes and down-regulated inflammatory and autophagy pathways. The multitarget activity of T. lutea suggests that this microalga could be useful in mitigating risk factors of MetS