Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes. Pancreatic islets derived from stem cells are benchmarked against primary cells.Peer reviewe

Regulation of docking and priming in pancreatic α- and β-cells

The secretion of islet hormones from endocrine cells of the pancreas plays vital roles in maintaining glucose homeostasis. Dysfunction of these cells leads to diabetes, a devastating metabolic disorder affecting millions worldwide, but underlying mechanisms remain poorly understood. In hyperglycemic conditions, β-cells secrete insulin, whereas α-cells secrete an increased amount of glucagon in hypoglycemic conditions. Both insulin and glucagon are stored in secretory granules preceding their release by regulated exocytosis. This process involves several steps, including tethering, docking, priming, and finally, a fusion of the granules with the plasma membrane. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins and phosphoinositides (PIs) drive pancreatic hormone exocytosis and secretion, which follows a biphasic time course. Biphasic secretion is thought to reflect the vastly different release probabilities of individual granules, but direct evidence for this is still lacking.  Therefore, this thesis investigates exocytosis in the two main pancreatic cell types with a particular focus on preceding steps docking and priming, to identify rate-limiting steps in health and type-2 diabetes (T2D). Our data indicated that granule docking is critical for sustained secretion in α- and β-cells. Glucagon granule exocytosis had a U-shaped sensitivity to glucose in both healthy and T2D α-cells. However, T2D α-cells exhibited a marginal decrease in exocytosis, as well as docking, and they were markedly insensitive to somatostatin and insulin. T2D β-cells reduced exocytosis dramatically, and docking was compromised and no longer responsive to glucose, which correlated with reduced insulin secretion and elevated donor HbA1c. These results were further strengthened by the finding that expression of a group of genes that are involved explicitly in granule docking was reduced (by RNAseq of islets from over 200 human donors), and overexpression of the corresponding proteins increased granule docking in human β-cells. We further aimed to study the basis for the recruitment of these proteins to the docking site. Here we tested the hypothesis that highly charged lipids mainly PIs act as a hotspot to interact with SNARE proteins that initiate docking. We showed the homogenous distribution of all PIs markers in the plasma membrane, with no PIs microdomains at the exocytotic site during granule docking. However, rapid and local PI(4,5)P2 signaling at fusion sites was crucial for stabilizing fusion pore by binding to proteins related to the release site. These results suggested a role of PI(4,5)P2 in priming and fusion regulation rather than docking. Overall, this work gives new insights into the mechanisms underlying pancreatic hormone secretion in both healthy and diabetic conditions

Regulation of docking and priming in pancreatic α- and β-cells

The secretion of islet hormones from endocrine cells of the pancreas plays vital roles in maintaining glucose homeostasis. Dysfunction of these cells leads to diabetes, a devastating metabolic disorder affecting millions worldwide, but underlying mechanisms remain poorly understood. In hyperglycemic conditions, β-cells secrete insulin, whereas α-cells secrete an increased amount of glucagon in hypoglycemic conditions. Both insulin and glucagon are stored in secretory granules preceding their release by regulated exocytosis. This process involves several steps, including tethering, docking, priming, and finally, a fusion of the granules with the plasma membrane. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins and phosphoinositides (PIs) drive pancreatic hormone exocytosis and secretion, which follows a biphasic time course. Biphasic secretion is thought to reflect the vastly different release probabilities of individual granules, but direct evidence for this is still lacking.  Therefore, this thesis investigates exocytosis in the two main pancreatic cell types with a particular focus on preceding steps docking and priming, to identify rate-limiting steps in health and type-2 diabetes (T2D). Our data indicated that granule docking is critical for sustained secretion in α- and β-cells. Glucagon granule exocytosis had a U-shaped sensitivity to glucose in both healthy and T2D α-cells. However, T2D α-cells exhibited a marginal decrease in exocytosis, as well as docking, and they were markedly insensitive to somatostatin and insulin. T2D β-cells reduced exocytosis dramatically, and docking was compromised and no longer responsive to glucose, which correlated with reduced insulin secretion and elevated donor HbA1c. These results were further strengthened by the finding that expression of a group of genes that are involved explicitly in granule docking was reduced (by RNAseq of islets from over 200 human donors), and overexpression of the corresponding proteins increased granule docking in human β-cells. We further aimed to study the basis for the recruitment of these proteins to the docking site. Here we tested the hypothesis that highly charged lipids mainly PIs act as a hotspot to interact with SNARE proteins that initiate docking. We showed the homogenous distribution of all PIs markers in the plasma membrane, with no PIs microdomains at the exocytotic site during granule docking. However, rapid and local PI(4,5)P2 signaling at fusion sites was crucial for stabilizing fusion pore by binding to proteins related to the release site. These results suggested a role of PI(4,5)P2 in priming and fusion regulation rather than docking. Overall, this work gives new insights into the mechanisms underlying pancreatic hormone secretion in both healthy and diabetic conditions

Insulin granule biogenesis and exocytosis

Insulin is produced by pancreatic beta-cells, and once released to the blood, the hormone stimulates glucose uptake and suppresses glucose production. Defects in both the availability and action of insulin lead to elevated plasma glucose levels and are major hallmarks of type-2 diabetes. Insulin is stored in secretory granules that form at the trans-Golgi network. The granules undergo extensive modifications en route to their release sites at the plasma membrane, including changes in both protein and lipid composition of the granule membrane and lumen. In parallel, the insulin molecules also undergo extensive modifications that render the hormone biologically active. In this review, we summarize current understanding of insulin secretory granule biogenesis, maturation, transport, docking, priming and eventual fusion with the plasma membrane. We discuss how different pools of granules form and how these pools contribute to insulin secretion under different conditions. We also highlight the role of the beta-cell in the development of type-2 diabetes and discuss how dysregulation of one or several steps in the insulin granule life cycle may contribute to disease development or progression

Local PI(4,5)P2 signaling inhibits fusion pore expansion during exocytosis

Summary: Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live-cell total internal reflection fluorescence (TIRF) microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. Knockdown of phosphatidylinositol-phosphate-5-kinase-1c or optogenetic recruitment of 5-phosphatase reduces PI(4,5)P2 transients and accelerates fusion pore expansion, suggesting that acute PI(4,5)P2 synthesis is involved. Thus, local phospholipid signaling inhibits fusion pore expansion and peptide release through an unconventional endocytic mechanism

Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes.

Glucagon is released from pancreatic α-cells to activate pathways that raise blood glucose. Its secretion is regulated by α-cell-intrinsic glucose sensing and paracrine control through insulin and somatostatin. To understand the inadequately high glucagon levels that contribute to hyperglycemia in type-2 diabetes (T2D), we analyzed granule behavior, exocytosis and membrane excitability in α-cells of 68 non-diabetic and 21 T2D human donors. We report that exocytosis is moderately reduced in α-cells of T2D donors, without changes in voltage-dependent ion currents or granule trafficking. Dispersed α-cells have a non-physiological V-shaped dose response to glucose, with maximal exocytosis at hyperglycemia. Within intact islets, hyperglycemia instead inhibits α-cell exocytosis, but not in T2D or when paracrine inhibition by insulin or somatostatin is blocked. Surface expression of somatostatin-receptor-2 is reduced in T2D, suggesting a mechanism for the observed somatostatin resistance. Thus, elevated glucagon in human T2D may reflect α-cell insensitivity to paracrine inhibition at hyperglycemia

Syntaxin clusters at secretory granules in a munc18-bound conformation

Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, stx1 clusters at the granule docking site, together with the S/M protein munc18. Here we determined features of stx1 that contribute to its clustering at granules. In live insulin-secreting cells, stx1 and stx3 (but not stx4 or stx11) accumulated at docked granules, and stx1 (but not stx4) rescued docking in cells expressing botulinum neurotoxin-C. Using a series of stx1 deletion mutants and stx1/4 chimeras, we found that all four helical domains (Ha, Hb, Hc, SNARE) and the short N-terminal peptide contribute to recruitment to granules. However, only the Hc domain confers specificity, and it must be derived from stx1 for recruitment to occur. Point mutations in the Hc or the N-terminal peptide designed to interfere with binding to munc18-1 prevent stx1 from clustering at granules, and a mutant munc18 deficient in binding to stx1 does not cluster at granules. We conclude that stx1 is recruited to the docking site in a munc18-1-bound conformation, providing a rationale for the requirement for both proteins for granule docking

Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes

Glucose-stimulated insulin secretion is biphasic, with a rapid first phase and a slowly developing sustained second phase; both are disturbed in type 2 diabetes (T2D). Biphasic secretion results from vastly different release probabilities of individual insulin granules, but the morphological and molecular basis for this is unclear. Here, we show that human insulin secretion and exocytosis critically depend on the availability of membrane-docked granules and that T2D is associated with a strong reduction in granule docking. Glucose accelerated granule docking, and this effect was absent in T2D. Newly docked granules only slowly acquired release competence; this was regulated by major signaling pathways, but not glucose. Gene expression analysis indicated that key proteins involved in granule docking are downregulated in T2D, and overexpression of these proteins increased granule docking. The findings establish granule docking as an important glucose-dependent step in human insulin secretion that is dysregulated in T2D. Insulin secretion is disturbed in type 2 diabetes (T2D). Gandasi et al. show that insulin granule docking to the plasma membrane is necessary for exocytosis and sustained insulin secretion and that this process is dysregulated in T2D

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