Mechanisms governing extra- and intra-islet communication in human pancreatic health and disease

Abstract

Pancreatic islets perform homeostatic functions thru complex cell-to-cell communication systems that rely on sensing activity from nearby and distant cells. Diabetes mellitus can result from the perturbation of these communication networks leading to the dysfunction of pancreatic islets which are essential in the regulation of glucose homeostasis. My research's main objective was to elucidate extra- and intra-islet communication mechanisms such as the pancreatic neurovascular architecture, islet purinergic system, and GAD65-mediated autoimmunity to better understand these communication networks and their impact on islet function. To define morphological characteristics of capillaries and nerve fibers in islets and acinar tissue compartments, we analyzed neurovascular assembly across the largest cohort of T1D and normal individuals studied thus far. Then, to determine if islet neurovascular arrangement is altered following β cell loss in T1D, we compared pancreatic tissues from non-diabetic, recent-onset T1D (<10 years duration), and longstanding T1D donors (>10 years duration). Our results indicated that pancreatic capillaries and nerve fibers persist in T1D despite β cell loss, suggesting that α cell secretory changes may be decoupled from neurovascular components. Next, to assess intracellular signaling we developed a live cell microperifusion platform to co-register intra-islet signaling and islet hormone secretion in human pseudoislets (iOrganoids). This integrated microperifusion system gave us the ability to measure pseudoislet insulin and glucagon secretion concurrently and match it with intra-cellular cAMP or Ca2+ dynamics. Next, we assessed how extracellular nucleotides and purinergic signaling mechanisms fine-tune human islet hormone secretion. We defined the cellular distribution of purinergic ecto-enzymes and nucleotide receptors in human islets, we used single-cell RNA sequencing (scRNA-seq) data. We also studied the function of this purinergic niche by assessing extracellular ATP levels which increased concomitantly with insulin secretion after exposure to high glucose and IBMX. Further, we directly tested NTPDase3 loss of function by genetically manipulating human pseudoislets using an shRNA knockdown approach, resulting in a 1.7-fold increase of insulin secretion, mediated thru P2Y1 intra-cellular calcium signaling as assessed by our live cell microperifusion platform. And finally, by utilizing in silico comparisons between bacterial and human β cell GAD65 sequences, we evaluated a potential new mechanism by which bacteria in the gut can induce β cell GAD65 autoimmunity. Overall, my work establishes a greater understanding of a variety of communication systems governing pancreatic function and opens the door for the development of more specific and effective therapeutic targets for diabetes care.