Hormonal responses to cholinergic input are different in humans with and without type 2 diabetes mellitus

<div><p>Peripheral muscarinic acetylcholine receptors regulate insulin and glucagon release in rodents but their importance for similar roles in humans is unclear. Bethanechol, an acetylcholine analogue that does not cross the blood-brain barrier, was used to examine the role of peripheral muscarinic signaling on glucose homeostasis in humans with normal glucose tolerance (NGT; n = 10), impaired glucose tolerance (IGT; n = 11), and type 2 diabetes mellitus (T2DM; n = 9). Subjects received four liquid meal tolerance tests, each with a different dose of oral bethanechol (0, 50, 100, or 150 mg) given 60 min before a meal containing acetaminophen. Plasma pancreatic polypeptide (PP), glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), glucose, glucagon, C-peptide, and acetaminophen concentrations were measured. Insulin secretion rates (ISRs) were calculated from C-peptide levels. Acetaminophen and PP concentrations were surrogate markers for gastric emptying and cholinergic input to islets. The 150 mg dose of bethanechol increased the PP response 2-fold only in the IGT group, amplified GLP-1 release in the IGT and T2DM groups, and augmented the GIP response only in the NGT group. However, bethanechol did not alter ISRs or plasma glucose, glucagon, or acetaminophen concentrations in any group. Prior studies showed infusion of xenin-25, an intestinal peptide, delays gastric emptying and reduces GLP-1 release but not ISRs when normalized to plasma glucose levels. Analysis of archived plasma samples from this study showed xenin-25 amplified postprandial PP responses ~4-fold in subjects with NGT, IGT, and T2DM. Thus, increasing postprandial cholinergic input to islets augments insulin secretion in mice but not humans.</p><p><b><i>Trial Registration</i>:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01434901?term=NCT01434901&rank=1" target="_blank">NCT01434901</a></p></div

Activation of Serum Response Factor in the Depolarization Induction of Egr-1 Transcription in Pancreatic Islet β-Cells

The results of the current studies define the major elements whereby glucose metabolism in islet β-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2–20 mm) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that β-cell depolarization and Ca2+ influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca2+/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal −198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca2+-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser103-phosphorylated SRF in nuclear extracts, indicated that the SRE·SRF complexes contribute to the Ca2+-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells

Targeted Ablation of Glucose-dependent Insulinotropic Polypeptide-producing Cells in Transgenic Mice Reduces Obesity and Insulin Resistance Induced by a High Fat Diet*

The K cell is a specific sub-type of enteroendocrine cell located in the proximal small intestine that produces glucose-dependent insulinotropic polypeptide (GIP), xenin, and potentially other unknown hormones. Because GIP promotes weight gain and insulin resistance, reducing hormone release from K cells could lead to weight loss and increased insulin sensitivity. However, the consequences of coordinately reducing circulating levels of all K cell-derived hormones are unknown. To reduce the number of functioning K cells, regulatory elements from the rat GIP promoter/gene were used to express an attenuated diphtheria toxin A chain in transgenic mice. K cell number, GIP transcripts, and plasma GIP levels were profoundly reduced in the GIP/DT transgenic mice. Other enteroendocrine cell types were not ablated. Food intake, body weight, and blood glucose levels in response to insulin or intraperitoneal glucose were similar in control and GIP/DT mice fed standard chow. In contrast to single or double incretin receptor knock-out mice, the incretin response was absent in GIP/DT animals suggesting K cells produce GIP plus an additional incretin hormone. Following high fat feeding for 21-35 weeks, the incretin response was partially restored in GIP/DT mice. Transgenic versus wild-type mice demonstrated significantly reduced body weight (25%), plasma leptin levels (77%), and daily food intake (16%) plus enhanced energy expenditure (10%) and insulin sensitivity. Regardless of diet, long term glucose homeostasis was not grossly perturbed in the transgenic animals. In conclusion, studies using GIP/DT mice demonstrate an important role for K cells in the regulation of body weight and insulin sensitivity