Endocrine cells in human pancreatic islets express cholinergic markers. (a) Z-stack of confocal images of a mouse pancreatic section showing an islet immunostained for vesicular acetylcholine transporter (vAChT, green) and glucagon (red). Intense vAChT staining is present in nerve fibers and fiber varicosities in the islet but not in islet cells. (b) Z-stack of confocal images of a human pancreatic section showing strong vAChT immunostaining in islet cells. Merge of glucagon and vAChT immunostaining appears yellow. (c) Preincubation with control peptide abolishes vAChT staining in human islets. Scale bar = 50 µm (a–c). (d) Western blotting analyses of lysates from four separate human islet preparations (HI1–4) and human pancreatic exocrine tissue (HP), with mouse brain (MB) as a positive control. Specific bands were seen in human islet lysates for vAChT (~70 kDa; upper), for choline acetyltransferase (ChAT; ~63 kDA and ~68 kDa; middle), and for choline transporter 1 (ChT1; ~68 kDa; lower). Notice the reduced expression of these cholinergic markers in exocrine tissue. A molecular marker was run in parallel (second lane). (e–g) vAChT (e), ChAT (f), and ChT1 (g) mRNA expression in brain (B, n = 4), human islets (I, n = 12), and human pancreas (P, n = 3). (h) vAChT mRNA levels were associated with ChAT mRNA levels (r² = 0.57; slope significantly different from 0, P < 0.01).

Isolated human islets secrete acetylcholine (ACh) in response to alpha cell-specific stimuli. (a) Photomicrograph of an ACh biosensor (colorized green) apposed to an isolated human islet to monitor ACh secretion evoked by stimulation of islet cells. Responses in the biosensor were recorded by loading biosensors with fura-2 and imaging cytoplasmic [Ca²⁺]. Scale bar, 50 µm. (b) In the absence of human islets, biosensors responded to direct application of ACh (10 µM), but not to kainate (100 µM), KCl (25 mM), or a change in glucose concentration. Responses to ACh were inhibited by the muscarinic antagonist atropine (5 µM). (c) Trace shows stimulus-induced secretion of ACh from endocrine cells in a human islet, measured with an ACh biosensor positioned against the islet as in a. Kainate (100 µM) and a decrease in glucose concentration (return from 16 mM to 3 mM) evoked responses in the biosensor. Biosensor responses were blocked by atropine 5 µM). Bars denote drug applications. (d), Summary of data from experiments such as those shown in c. Bars show means ± SEM for ACh biosensor signals in response to stimulation of islets. KCl (25 mM) depolarization (n = 8 experiments), kainate (100 µM, n = 11) and decreases in glucose concentration (from 16 mM to 3 mM, n = 4) induced ACh secretion. Biosensor responses were blocked by atropine (5 µM). (e) ACh release was stimulated by lowering the glucose concentration from 11 mM to 3 mM or by depolarizing with KCl (25 mM), as determined with a fluorescent enzymatic assay (see Methods; n = 6 islet preparations; ANOVA followed by multiple comparison, *P < 0.05).

Human alpha cells express vAChT and ChAT. (a) Confocal images of human pancreatic sections showing that vAChT immunostaining (green) colocalized with glucagon immunostaining (red, left) in many human alpha cells, with somatostatin in some delta cells (right), but not with insulin immunostaining in beta cells (middle). Colocalization appears yellow. (b) Quantification shows the percentage of vAChT immunostained cells also labeled for glucagon, insulin, or somatostatin (n = 3 human pancreata). Percentages do not add exactly to 100% because analyses were performed on different sections. (c) At high magnification, glucagon (red) and vAChT immunostaining (green) appear localized to different regions in alpha cells. Shown are three optical planes through an alpha cell. (d) Fluorescence colocalization in alpha and beta cells showing strong colocalization of insulin and C-peptide in beta cells and lack of colocalization of DAPI and glucagon in alpha cells. The degree of glucagon and vAChT Colocalization was significantly lower than that of C-peptide and insulin (ANOVA followed by multiple comparison, *P < 0.05). Shown are scatter plots of pixel intensities (PI) in the specified channels (left) and the respective thresholded Pearson’s correlation coefficients (right, n = 12 cells). (e) ChAT immunostaining (green, left) was present in glucagon-labeled alpha cells (red, middle). Colocalization appears yellow (merge, right). (f) High magnification confocal image of an alpha cell stained for glucagon (red) and ChAT (green). Scale bars, 50 µm (a, e) and 5 µm (c, f).

Endogenously released ACh amplifies glucose-induced insulin secretion in human islets. (a) ACh (black trace) and the muscarinic agonist oxotremorine (green trace), but not nicotine (blue trace), elicited concentration-dependent insulin release from human islets. Bars denote stimulus application. Representative traces of n = 3 islet preparations. (b) Summary of data from experiments similar to those shown in a, but conducted in the presence of low (3 mM; black symbols) and high (11 mM; red symbols) glucose (n = 3 preparations). (c) The acetylcholinesterase inhibitor physostigmine (30 µM) increased insulin secretion at 3 mM glucose (n = 5 human islet preparations). (d–f) Physostigmine-induced increases in insulin secretion were significantly inhibited by the vAChT blocker vesamicol (10 µM, d), by the M3 receptor antagonist J-104129 (50 nM, e), or when the experiment was performed at a higher glucose concentration (11 mM, f; Student’s t-test, P < 0.05). (g) Insulin secretion induced by repeatedly raising glucose from 3 mM to 11 mM was increased in the presence of physostigmine (30 µM; black symbols) and reduced in the presence of J-104129 (50 nM; red symbols; representative traces of four experiments). A control experiment with untreated islets was run in parallel (grey symbols). Bar denotes drug application. 11G indicates 15 min of elevated glucose (11 mM). Islets were stimulated four times with glucose followed by 25 mM KCl. (h) Summary of data from experiments such as those shown in c. J-104129 significantly reduced glucose-induced insulin secretion (red bars; n = 4 preparations), whereas the cholinesterase inhibitor physostigmine (30 µM) increased insulin secretion (black bars; n = 5 preparations). Responses are expressed as percentage of the respective insulin response of control islets (100%, grey dashed line). One sample t-tests were used to compare the actual mean to a theoretical mean of 100% (control; *P < 0.05).