The neurotransmitter acetylcholine plays a major role in regulating insulin secretion. In several species parasympathetic innervation provides cholinergic input to beta cells, but recent studies show that the glucagon secreting alpha cell is a major source of acetylcholine in human islets. The existence of this novel source of acetylcholine in the islet implies that insulin secretion and glucose metabolism can be regulated by local cholinergic mechanisms that remain unknown. The long-term goal of this research program is to understand the contribution of cholinergic signaling to human islet biology in health and disease. The objective of this application is to determine how acetylcholine is secreted, how it is degraded, and how it impacts beta cell biology, using a combination of innovative in vitro and in vivo approaches. The central hypothesis is that paracrine cholinergic input from the alpha cell influences human beta cell function. In our model, acetylcholine is released independently of glucagon and activates beta cell muscarinic receptors, stimulating signaling cascades that promote insulin secretion and glucose homeostasis. Cholinesterases produced by beta cells shape the duration and magnitude of cholinergic signaling. The rationale for the proposed research is that the results will contribute a missing, fundamental element to basic knowledge, without which islet biology cannot be understood. The proposed research is therefore relevant to the mission of the NIH that pertains to the pursuit of fundamental knowledge about the nature and behavior of living systems. Guided by strong preliminary data, our central hypothesis will be tested by pursuing three specific aims: 1) Identify the mechanisms of acetylcholine release from human alpha cells; 2) Determine the location and role of acetylcholinesterase in alpha-beta cell communication; and 3) Determine the impact of cholinergic signaling on human beta cell function. Under the first aim, we will visualize secretion of acetylcholine and glucagon using optical indicators of exocytosis (pHluorins) and measure acetylcholine and glucagon release from human islets in real time using biosensor cells. Under the second aim, we will study the expression of cholinesterases in human islets using biochemical assays, RT-PCR, and immunohistochemistry, and test FDA-approved cholinesterase inhibitors for their ability to increase insulin secretion. Under the third aim, we will stimulate alpha cells and measure beta cell responses with novel probes for signaling molecules. To investigate long-term effects of islet cholinergic signaling in vivo we will use a humanized mouse model in which human islets are transplanted into the mouse eye. We will inhibit acetylcholine secretion, acetylcholine breakdown, and muscarinic receptors in intraocular human islet grafts and measure the effects on human insulin plasma levels and glycemia in the recipient mouse. The proposed research is significant because it is expected to make a strong and lasting impact on our understanding of the role of cholinergic signaling in human islet biology. Ultimately, such knowledge has the potential to impact the way diabetes is treated.