L.A. Blackshaw

Mechanisms of activation of mouse and human enteroendocrine cells by nutrients

Objective Inhibition of food intake and glucose homeostasis are both promoted when nutrients stimulate enteroendocrine cells (EEC) to release gut hormones. Several specific nutrient receptors may be located on EEC that respond to dietary sugars, amino acids and fatty acids. Bypass surgery for obesity and type II diabetes works by shunting nutrients to the distal gut, where it increases activation of nutrient receptors and mediator release, but cellular mechanisms of activation are largely unknown. We determined which nutrient receptors are expressed in which gut regions and in which cells in mouse and human, how they are associated with different types of EEC, how they are activated leading to hormone and 5-HT release. Design and results mRNA expression of 17 nutrient receptors and EEC mediators was assessed by quantitative PCR and found throughout mouse and human gut epithelium. Many species similarities emerged, in particular the dense expression of several receptors in the distal gut. Immunolabelling showed specific colocalisation of receptors with EEC mediators PYY and GLP-1 (L-cells) or 5-HT (enterochromaffin cells). We exposed isolated proximal colonic mucosa to specific nutrients, which recruited signalling pathways within specific EEC extracellular receptor-regulated kinase (p-ERK) and calmodulin kinase II (pCAMKII), as shown by subsequent immunolabelling, and activated release of these mediators. Aromatic amino acids activated both pathways in mouse, but in humans they induced only pCAMKII, which was colocalised mainly with 5-HT expression. Activation was pertussis toxin-sensitive. Fatty acid (C12) potently activated p-ERK in human in all EEC types and evoked potent release of all three mediators. Conclusions Specific nutrient receptors associate with distinct activation pathways within EEC. These may provide discrete, complementary pharmacological targets for intervention in obesity and type II diabetes.

TRPM8 on mucosal sensory nerves regulates colitogenic responses by innate immune cells via CGRP

TRPM8 is the molecular sensor for cold; however, the physiological role of TRPM8+ neurons at mucosal surfaces is unclear. Here we evaluated the distribution and peptidergic properties of TRPM8+ fibers in naive and inflamed colons, as well as their role in mucosal inflammation. We found that Trpm8−/− mice were hypersusceptible to dextran sodium sulfate (DSS)-induced colitis, and that Trpm8−/− CD11c+ DCs (dendritic cells) showed hyperinflammatory responses to toll-like receptor (TLR) stimulation. This was phenocopied in calcitonin gene–related peptide (CGRP) receptor-deficient mice, but not in substance P receptor-deficient mice, suggesting a functional link between TRPM8 and CGRP. The DSS phenotype of CGRP receptor-deficient mice could be adoptively transferred to wild-type (WT) mice, suggesting that CGRP suppresses the colitogenic activity of bone marrow–derived cells. TRPM8+ mucosal fibers expressed CGRP in human and mouse colon. Furthermore, neuronal CGRP contents were increased in colons from naive and DSS-treated Trpm8−/− mice, suggesting deficient CGRP release in the absence of TRPM8 triggering. Finally, treatment of Trpm8−/− mice with CGRP reversed their hyperinflammatory phenotype. These results suggest that TRPM8 signaling in mucosal sensory neurons is indispensable for the regulation of innate inflammatory responses via the neuropeptide CGRP.

Modulation of murine gastric vagal afferent mechanosensitivity by neuropeptide W.

Neuropeptide W (NPW) is an endogenous ligand for the receptors GPR7 and GPR8 and is involved in central regulation of energy homeostasis. NPW in the periphery is found in gastric gastrin (G) cells. In the stomach, energy intake is influenced by vagal afferent signals, so we aimed to determine the effect of NPW on mechanosensitive gastric vagal afferents under different feeding conditions.

Peripheral KV7 channels regulate visceral sensory function in mouse and human colon

Background Chronic visceral pain is a defining symptom of many gastrointestinal disorders. The KV7 family (KV7.1–KV7.5) of voltage-gated potassium channels mediates the M current that regulates excitability in peripheral sensory nociceptors and central pain pathways. Here, we use a combination of immunohistochemistry, gut-nerve electrophysiological recordings in both mouse and human tissues, and single-cell qualitative real-time polymerase chain reaction of gut-projecting sensory neurons, to investigate the contribution of peripheral KV7 channels to visceral nociception. Results Immunohistochemical staining of mouse colon revealed labelling of KV7 subtypes (KV7.3 and KV7.5) with CGRP around intrinsic enteric neurons of the myenteric plexuses and within extrinsic sensory fibres along mesenteric blood vessels. Treatment with the KV7 opener retigabine almost completely abolished visceral afferent firing evoked by the algogen bradykinin, in agreement with significant co-expression of mRNA transcripts by single-cell qualitative real-time polymerase chain reaction for KCNQ subtypes and the B2 bradykinin receptor in retrogradely labelled extrinsic sensory neurons from the colon. Retigabine also attenuated responses to mechanical stimulation of the bowel following noxious distension (0–80 mmHg) in a concentration-dependent manner, whereas the KV7 blocker XE991 potentiated such responses. In human bowel tissues, KV7.3 and KV7.5 were expressed in neuronal varicosities co-labelled with synaptophysin and CGRP, and retigabine inhibited bradykinin-induced afferent activation in afferent recordings from human colon. Conclusions We show that KV7 channels contribute to the sensitivity of visceral sensory neurons to noxious chemical and mechanical stimuli in both mouse and human gut tissues. As such, peripherally restricted KV7 openers may represent a viable therapeutic modality for the treatment of gastrointestinal pathologies.

Effects of Obesity and Gastric Bypass Surgery on Nutrient Sensors, Endocrine Cells, and Mucosal Innervation of the Mouse Colon

Background: Nutrient-sensing receptors located on enteroendocrine (EEC) cells modulate appetite via detection of luminal contents. Colonic ‘tasting’ of luminal contents may influence changes to appetite observed in obesity and after weight loss induced by bariatric surgery. We assessed the effects of obesity and gastric bypass-induced weight loss on expression of nutrient-sensing G-protein coupled receptors (GPCRs), EEC and enterochromaffin (EC) cells and mucosal innervation. Methods: qPCR and immunohistochemistry were used to study colonic tissue from (a) chow-fed/lean, (b) high-fat fed/obese, (c) Roux-en-Y gastric bypass surgery (RYGB), and (d) calorie restriction-induced weight loss mice. Results: Expression of GPR41, GPR43, GPR40, GPR120, GPR84, GPR119, GPR93 and T1R3 was increased in obese mice. Obesity-induced overexpression of GPR41, 40, 84, and 119 further increased after RYGB whereas GPR120 and T1R3 decreased. RYGB increased TGR5 expression. L-cells, but not EC cells, were increased after RYGB. No differences in mucosal innervation by protein gene product (PGP) 9.5 and GLP-1R-positive nerve fibers were observed. Stimulation of colonic mucosa with GPR41, GPR40, GPR85, GPR119, and TGR5 agonists increased cell activation marker expression. Conclusions: Several nutrient-sensing receptors induced activation of colonic EEC. Profound adaptive changes to the expression of these receptors occur in response to diet and weight loss induced by RYGB or calorie restriction.