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

Cholinergic signaling mediates the effects of xenin-25 on secretion of pancreatic polypeptide but not insulin or glucagon in humans with impaired glucose tolerance.

We previously demonstrated that infusion of an intestinal peptide called xenin-25 (Xen) amplifies the effects of glucose-dependent insulinotropic polypeptide (GIP) on insulin secretion rates (ISRs) and plasma glucagon levels in humans. However, these effects of Xen, but not GIP, were blunted in humans with type 2 diabetes. Thus, Xen rather than GIP signaling to islets fails early during development of type 2 diabetes. The current crossover study determines if cholinergic signaling relays the effects of Xen on insulin and glucagon release in humans as in mice. Fasted subjects with impaired glucose tolerance were studied. On eight separate occasions, each person underwent a single graded glucose infusion- two each with infusion of albumin, Xen, GIP, and GIP plus Xen. Each infusate was administered ± atropine. Heart rate and plasma glucose, insulin, C-peptide, glucagon, and pancreatic polypeptide (PP) levels were measured. ISRs were calculated from C-peptide levels. All peptides profoundly increased PP responses. From 0 to 40 min, peptide(s) infusions had little effect on plasma glucose concentrations. However, GIP, but not Xen, rapidly and transiently increased ISRs and glucagon levels. Both responses were further amplified when Xen was co-administered with GIP. From 40 to 240 min, glucose levels and ISRs continually increased while glucagon concentrations declined, regardless of infusate. Atropine increased resting heart rate and blocked all PP responses but did not affect ISRs or plasma glucagon levels during any of the peptide infusions. Thus, cholinergic signaling mediates the effects of Xen on insulin and glucagon release in mice but not humans

Baseline Demographic and Clinical Characteristics for Bethanechol and Xenin-25 Studies.

<p>Baseline Demographic and Clinical Characteristics for Bethanechol and Xenin-25 Studies.</p

Bethanechol differentially affects GIP and GLP-1 responses in humans with NGT, IGT, and T2DM.

<p>Selected data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156852#pone.0156852.g003" target="_blank">Fig 3</a> (-15 to 30 min) are expanded to emphasize differential GLP-1 (panels A-C) and GIP (panels D-F) responses to the 150 mg dose of bethanechol.</p

Bethanechol differentially affects PP, GLP-1, and GIP responses in humans with NGT, IGT, and T2DM.

<p>Subjects with NGT, IGT, and T2DM were administered separate meal tolerance tests with placebo (blue dots) or bethanechol at a dose of 50 mg (green squares), 100 mg (yellow triangles), or 150 mg (inverted red triangles). Plasma levels of PP (Panels A-C), intact GLP-1 (Panels D-F), and total GIP (Panels G-I) were measured at the indicated times before and after meal ingestion. Values represent group means ± SEMs for subjects with NGT (Panels A, D, G), IGT (Panels B, E, H), and T2DM (Panels C, F, I). The number of subjects receiving the 0, 50, 100, and 150 mg dose of bethanechol is indicated for each group. Differences in subject number within each group are because several subjects did not receive the 150 mg dose. GLP-1 and GIP levels were only measured in samples from individual subjects receiving both the 0 mg and 150 mg doses of bethanechol. <i>P</i> values for the bethanechol effect (B) and for bethanechol-time interaction (B*T) are indicated in each panel. Statistically significant <i>P</i> values for individual time points are shown if the bethanechol or bethanechol-time interaction was significant.</p

Xen amplifies the effects of GIP on ISRs.

<p>Graded glucose infusions (GGIs) with the saline control (Panels A-C) or atropine infusion (Panels D-F) were conducted. Plasma glucose (Panels A and D) and ISRs (Panels B and E) are shown for the indicated times before and during GGIs. The glucose infusion rate (GIR) at each 40 minute step is shown in white. Note that plasma glucose levels increase progressively even though glucose was administered in a step-wise fashion. Data from panels A and B are re-graphed in panel C whereas data from panels D and E are re-graphed in panel F. Symbols and error bars are eliminated in panels C and F for clarity. The rapid and transient increases in ISR in response to GIP and GIP plus Xen from 0–40 minutes (Panels B and E) are reflected by the initial spikes in ISRs (Panels C and F) that occur in the absence of a significant increase in plasma glucose levels. Values represent group means ± SEM.</p

Atropine inhibits pancreatic polypeptide release.

<p><u>Panels A and B</u>. Pancreatic polypeptide (PP) levels were measured at the indicated times before and during graded glucose infusions (GGIs) in the presence of the indicated peptide(s) and with infusion of saline (<u>Panel A</u>) or atropine (<u>Panel B</u>). Total (<u>Panel C</u>) and incremental <u>(Panel D</u>) AUCs from 0–240 minutes were calculated from data in panels A and B. Incremental and total AUCs were determined for each individual and values represent group means ± SEM. Significance was determined using the mixed effects model. <i>p</i> values for each peptide versus albumin alone are indicated within charts in Panels C and D. <i>p</i> values for the effects of atropine for each peptide infusate (or albumin control) are indicated below Panels C and D. The <i>p</i> values for infusate effects were calculated for all 8 treatments. Note that atropine infusion completely blocks all pancreatic polypeptide responses.</p

The graded glucose infusion protocol.

<p>The glucose infusion rate was increased in a step wise fashion every 40 minutes starting at time zero as shown in red. The primed-constant infusion of each peptide(s) was started at time zero and is shown in yellow. Note that GIP and Xen were each infused at the same rate when administered together. The primed-constant infusion of atropine (or saline) was initiated 30 minutes before the start of the graded glucose infusion as shown in blue. All infusions were terminated 240 minutes after the glucose infusion was initiated.</p

Bethanechol has no effect on glucose homeostasis in humans with NGT, IGT, and T2DM.

<p>Plasma glucose (panels A-C), glucagon (panels G-I), and ACM (panels J-L) levels and insulin secretion rates (panels D-F) were determined during meal tolerances as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156852#pone.0156852.g003" target="_blank">Fig 3</a>. Values represent group means ± SEMs for subjects with NGT (Panels A, D, G, J), IGT (Panels B, E, H, K), and T2DM (Panels C, F, I, L). Symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156852#pone.0156852.g004" target="_blank">Fig 4</a>. <i>P</i> values for bethanechol and for bethanechol-time interaction are indicated in each panel. Data for the 100 mg dose of bethanechol for one subject with T2DM was excluded from the analysis because baseline values were 4.5 standard deviations from the mean.</p

Bethanechol increases the PP iAUC only in humans with IGT.

<p>Incremental AUCs were calculated for each individual at the indicated dose of bethanechol from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156852#pone.0156852.g003" target="_blank">Fig 3</a>. Group means ± SEM are shown. <i>P</i> values were determined using the mixed effects model. Statistics are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156852#pone.0156852.g004" target="_blank">Fig 4</a>.</p