Our long-term goal is to enable diabetes treatments through precise control of pancreatic nerve activity. We will create a detailed and systemic view of defined pancreatic nerves, their gene expression profiles, function and the effects of high fat diet (HFD) that are necessary to achieve this. Our pilot data already suggest HFD causes rapid disruption of pancreatic nerve structure and their ability to control insulin release in response to blood glucose. Our proposal will create new insights into pancreatic nerves' control of blood glucose by bringing an unprecedented level of precision and specificity by combining 3d imaging, RNAseq and neuromodulation tools in which we have unique and deep experience. Without this knowledge, the likelihood of successfully targeting neural pathways to control blood glucose in diabetes will remain remote. The overall objective of this proposal, which is the next step toward attaining new avenues to treat diabetes, is to understand the effects of HFD on the structure, transcriptome and function of pancreatic parasympathetic and sympathetic efferent nerves. Our central hypothesis is that HFD increases islet sympathetic innervation and reduces islet parasympathetic innervation leading to insufficient insulin to maintain normal glucose. The rationale that underlies the proposed research is that pancreatic islets are highly innervated by sympathetic and parasympathetic nerves and HFD disrupts pancreatic nerve structure, gene expression and function. However, we do not know if the structural changes from HFD are uniform, which endocrine cells are affected or the time course. We do not have a comprehensive gene expression profile of neurons innervating the pancreas or the molecular pathways disrupted by HFD. In addition, we do not know the precise functions of pancreatic parasympathetic and sympathetic nerves or how HFD affects these roles. These represent major gaps in our understanding. To test our central hypothesis and attain the overall objective, we will a) determine the effects of HFD on the 3D structure of islet sympathetic and parasympathetic efferent nerves, b) determine the effects of HFD on gene expression in pancreatic sympathetic and parasympathetic efferent nerves b) determine the effects of HFD on the function of islet sympathetic and parasympathetic nerves to regulate islet hormone release. To do so, we will use 3D imaging of cleared pancreata to determine the effects of low (10%) or HFD (45%) on pancreatic parasympathetic and sympathetic nerve structure and their relationship with beta, alpha and delta cells in fed and fasted mice. We will use single cell RNAseq to identify the effects of HFD on gene expression and the molecular pathways disrupted by HFD in pancreatic parasympathetic and sympathetic nerves. We will use highly target neuromodulation to determine the effects of HFD on pancreatic sympathetic and parasympathetic nerve function. The proposed studies will provide a comprehensive understanding of the unique biology of pancreatic sympathetic and parasympathetic innervation to form a crucial foundation for future studies identifying critical periods, reversibility and therapeutic targets to prevent and treat type 2 diabetes.