Xenin-25 increases cytosolic free calcium levels and acetylcholine release from a subset of myenteric neurons

Abstract

Xenin-25 (Xen) is a 25 amino acid neurotensin-related peptide reportedly produced with glucose-dependent insulinotropic polypeptide (GIP) by a subset of K cells in the proximal gut. We previously showed exogenously administered Xen, with GIP but not alone, increases insulin secretion in humans and mice. In mice, this effect is indirectly mediated via a central nervous system-independent cholinergic relay in the periphery. Xen also delays gastric emptying, reduces food intake, induces gall bladder contractions, and increases gut motility and secretion from the exocrine pancreas, suggesting that some effects of Xen could be mediated by myenteric neurons (MENs). To determine whether Xen activates these neurons, MENs were isolated from guinea pig proximal small intestines. Cells expressed neuronal markers and exhibited typical neuron-like morphology with extensive outgrowths emanating from cell bodies. Cytosolic free Ca(2+) levels ([Ca(2+)](i)) were measured using Fura-2. ATP/UTP, KCl, and forskolin increased [Ca(2+)](i) in 99.6%, 92%, and 23% of the MENs imaged, respectively, indicating that they are functional and activated by nucleotide receptor signaling, direct depolarization, and cAMP. [Ca(2+)](i) increased in only 12.7% of MENs treated with Xen. This rise was blocked by pretreatment with EGTA, diazoxide, SR48692, and neurotensin. Thus the Xen-mediated increase in [Ca(2+)](i) involves influx of extracellular Ca(2+) and activation of neurotensin receptor-1 (NTSR1). Xen also increased acetylcholine release from MENs. Amylin, produced by β-and enteroendocrine cells, delays gastric emptying and increased [Ca(2+)](i) almost exclusively in Xen-responsive MENs. Immunohistochemistry demonstrated NTSR1 expression in human duodenal MENs. Thus myenteric rather than central neurons could mediate some effects of Xen and amylin.

Metabolic responses to xenin-25 are altered in humans with Roux-en-Y gastric bypass surgery

“…This result also indicates that Xen does not act directly on intestinal L cells to regulate GLP-1 release. Because effects of Xen are mediated by neurotensin receptor-1 on neurons [8,41–43], we hypothesize that a Xen-sensitive pathway, mediated by enteric neurons, that inhibits GLP-1 secretion is severed during the RYGB procedure itself. The loss of this inhibitory pathway could play an important role in the exaggerated postprandial GLP-1 release after RYGB.…”

Xenin-25 delays gastric emptying and reduces postprandial glucose levels in humans with and without Type 2 diabetes

“…Paraffin-embedded blocks of human stomach were obtained from our Department of Pathology. Sections were deparaffinized, subjected to antigen retrieval with EDTA (pH 8), blocked with CAS-Block (Invitrogen, Frederick, MD), and incubated with primary antibodies overnight at 4°C as previously described (52,57). After washing, bound primary antibodies were detected following incubation for 45 min at room temperature with the appropriate conjugated secondary antibodies.…”

“…Genetic and pharmacological data indicate that effects of Xen are mediated by neurotensin receptor-1 [NTSR1 (19,24,35,57)]. In animals, Xen delays gastric emptying (25), reduces food intake (2,10,27), increases gut motility (17), augments gall bladder contractions (23), and enhances secretion from the exocrine pancreas (16).…”

Inter-organ communication and regulation of beta cell function

“…In vitro studies on xenin-25 and GIP treatment of mouse islets indicate that xenin-25 does not act directly on beta cells. Rather, xenin-25 acts by stimulating cholinergic signalling to beta cells via non-ganglionic circuitry that, to date, remains to be characterised (14). …”