Readily releasable ß cells with tight Ca2+-exocytosis coupling dictate biphasic glucose-stimulated insulin secretion.

Biphasic glucose-stimulated insulin secretion (GSIS) is essential for blood glucose regulation, but a mechanistic model incorporating the recently identified islet ß cell heterogeneity remains elusive. Here, we show that insulin secretion is spatially and dynamically heterogeneous across the islet. Using a zinc-based fluorophore with spinning-disc confocal microscopy, we reveal that approximately 40% of islet cells, which we call readily releasable ß cells (RRßs), are responsible for 80% of insulin exocytosis events. Although glucose up to 18.2 mM fully mobilized RRßs to release insulin synchronously (first phase), even higher glucose concentrations enhanced the sustained secretion from these cells (second phase). Release-incompetent ß cells show similarities to RRßs in glucose-evoked Ca2+ transients but exhibit Ca2+-exocytosis coupling deficiency. A decreased number of RRßs and their altered secretory ability are associated with impaired GSIS progression in ob/ob mice. Our data reveal functional heterogeneity at the level of exocytosis among ß cells and identify RRßs as a subpopulation of ß cells that make a disproportionally large contribution to biphasic GSIS from mouse islets.

Pancreatic α and ß cells are globally phase-locked.

The Ca2+ modulated pulsatile glucagon and insulin secretions by pancreatic α and ß cells play a crucial role in glucose homeostasis. However, how α and ß cells coordinate to produce various Ca2+ oscillation patterns is still elusive. Using a microfluidic device and transgenic mice, we recorded Ca2+ signals from islet α and ß cells, and observed heterogeneous Ca2+ oscillation patterns intrinsic to each islet. After a brief period of glucose stimulation, α and ß cells' oscillations were globally phase-locked. While the activation of α cells displayed a fixed time delay of ~20 s to that of ß cells, ß cells activated with a tunable period. Moreover, islet α cell number correlated with oscillation frequency. We built a mathematical model of islet Ca2+ oscillation incorporating paracrine interactions, which quantitatively agreed with the experimental data. Our study highlights the importance of cell-cell interaction in generating stable but tunable islet oscillation patterns.

Glucagon Potentiates Insulin Secretion Via ß-Cell GCGR at Physiological Concentrations of Glucose.

Incretin-potentiated glucose-stimulated insulin secretion (GSIS) is critical to maintaining euglycemia, of which GLP-1 receptor (GLP-1R) on ß-cells plays an indispensable role. Recently, α-cell-derived glucagon but not intestine-derived GLP-1 has been proposed as the critical hormone that potentiates GSIS via GLP-1R. However, the function of glucagon receptors (GCGR) on ß-cells remains elusive. Here, using GCGR or GLP-1R antagonists, in combination with glucagon, to treat single ß-cells, α-ß cell clusters and isolated islets, we found that glucagon potentiates insulin secretion via ß-cell GCGR at physiological but not high concentrations of glucose. Furthermore, we transfected primary mouse ß-cells with RAB-ICUE (a genetically encoded cAMP fluorescence indicator) to monitor cAMP level after glucose stimulation and GCGR activation. Using specific inhibitors of different adenylyl cyclase (AC) family members, we revealed that high glucose concentration or GCGR activation independently evoked cAMP elevation via AC5 in ß-cells, thus high glucose stimulation bypassed GCGR in promoting insulin secretion. Additionally, we generated ß-cell-specific GCGR knockout mice which glucose intolerance was more severe when fed a high-fat diet (HFD). We further found that ß-cell GCGR activation promoted GSIS more than GLP-1R in HFD, indicating the critical role of GCGR in maintaining glucose homeostasis during nutrient overload.

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

From Micro to Macro: The Hierarchical Design in a Micropatterned Scaffold for Cell Assembling and Transplantation.

A microwell-patterned membranous scaffold that integrates nano- and microscale topographical characteristics based on polyurethane is fabricated for transplanting syngeneic islets and allogeneic mesenchymal stem cells into diabetic rodents. The scaffold effectively allows for assembling of single cells/microtissues, enables the transplantation of cells with spatial control, and improves the transplant's engraftment efficacy in vivo for treating diabetes.

Overcoming foreign-body reaction through nanotopography: Biocompatibility and immunoisolation properties of a nanofibrous membrane.

Implantable immunoisolation membranes need to possess superior biocompatibility to prohibit the fibrotic deposition that would reduce the nutrient supply and impair the viability/function of the encapsulated cells. Here, electrospun membranes based on thermoplastic polyurethane (TPU) were fabricated to contain microfibers (PU-micro) or nanofibers (PU-nano). The two types of membranes were compared in terms of their interaction with macrophage cells and the host tissues. It was found that the fibrous membranes of different topographies possess distinct material properties: PU-nano caused minimal macrophage responses in vitro and in vivo and induced only mild foreign body reactions compared to PU-micro membranes. A flat macroencapsulation device was fabricated using PU-nano membranes and its immunoisolation function investigated in subcutaneous transplantation models. The nanofibrous device demonstrated the capability to effectively shield the allografts from the immune attack of the host. Nanotopography may confer biocompatibility to materials and nanofibrous materials warrant further study for development of "invisible" immunoisolation devices for cell transplantation.

An optical method to evaluate both mass and functional competence of pancreatic α- and ß-cells.

Imbalanced glucagon and insulin release leads to the onset of type 2 diabetes. To pinpoint the underlying primary driving force, here we have developed a fast, non-biased optical method to measure ratios of pancreatic α- and ß-cell mass and function simultaneously. We firstly label both primary α- and ß-cells with the red fluorescent probe ZinRhodaLactam-1 (ZRL1), and then highlight α-cells by selectively quenching the ZRL1 signal from ß-cells. Based on the signals before and after quenching, we calculate the ratio of the α-cell to ß-cell mass within live islets, which we found matched the results from immunohistochemistry. From the same islets, glucagon and insulin release capability can be concomitantly measured. Thus, we were able to measure the ratio of α-cell to ß-cell mass and their function in wild-type and diabetic Lepr(db)/Lepr(db) (denoted db/db) mice at different ages. We find that the initial glucose intolerance that appears in 10-week-old db/db mice is associated with further expansion of α-cell mass prior to deterioration in functional ß-cell mass. Our method is extendable to studies of islet mass and function in other type 2 diabetes animal models, which shall benefit mechanistic studies of imbalanced hormone secretion during type 2 diabetes progression.