ß-cell intrinsic dynamics rather than gap junction structure dictates subpopulations in the islet functional network.

Diabetes is caused by the inability of electrically coupled, functionally heterogeneous ß-cells within the pancreatic islet to provide adequate insulin secretion. Functional networks have been used to represent synchronized oscillatory [Ca2+] dynamics and to study ß-cell subpopulations, which play an important role in driving islet function. The mechanism by which highly synchronized ß-cell subpopulations drive islet function is unclear. We used experimental and computational techniques to investigate the relationship between functional networks, structural (gap junction) networks, and intrinsic ß-cell dynamics in slow and fast oscillating islets. Highly synchronized subpopulations in the functional network were differentiated by intrinsic dynamics, including metabolic activity and KATP channel conductance, more than structural coupling. Consistent with this, intrinsic dynamics were more predictive of high synchronization in the islet functional network as compared to high levels of structural coupling. Finally, dysfunction of gap junctions, which can occur in diabetes, caused decreases in the efficiency and clustering of the functional network. These results indicate that intrinsic dynamics rather than structure drive connections in the functional network and highly synchronized subpopulations, but gap junctions are still essential for overall network efficiency. These findings deepen our interpretation of functional networks and the formation of functional subpopulations in dynamic tissues such as the islet.

Restoring connexin-36 function in diabetogenic environments precludes mouse and human islet dysfunction.

The secretion of insulin from ß-cells in the islet of Langerhans is governed by a series of metabolic and electrical events, which can fail during the progression of type 2 diabetes (T2D). ß-cells are electrically coupled via connexin-36 (Cx36) gap junction channels, which coordinates the pulsatile dynamics of [Ca2+ ] and insulin release across the islet. Factors such as pro-inflammatory cytokines and free fatty acids disrupt gap junction coupling under in vitro conditions. Here we test whether gap junction coupling and coordinated [Ca2+ ] dynamics are disrupted in T2D, and whether recovery of gap junction coupling can recover islet function. We examine islets from donors with T2D, from db/db mice, and islets treated with pro-inflammatory cytokines (TNF-α, IL-1ß, IFN-É£) or free fatty acids (palmitate). We modulate gap junction coupling using Cx36 over-expression or pharmacological activation via modafinil. We also develop a peptide mimetic (S293) of the c-terminal regulatory site of Cx36 designed to compete against its phosphorylation. Cx36 gap junction permeability and [Ca2+ ] dynamics were disrupted in islets from both human donors with T2D and db/db mice, and in islets treated with pro-inflammatory cytokines or palmitate. Cx36 over-expression, modafinil treatment and S293 peptide all enhanced Cx36 gap junction coupling and protected against declines in coordinated [Ca2+ ] dynamics. Cx36 over-expression and S293 peptide also reduced apoptosis induced by pro-inflammatory cytokines. Critically, S293 peptide rescued gap junction coupling and [Ca2+ ] dynamics in islets from both db/db mice and a sub-set of T2D donors. Thus, recovering or enhancing Cx36 gap junction coupling can improve islet function in diabetes. KEY POINTS: Connexin-36 (Cx36) gap junction permeability and associated coordination of [Ca2+ ] dynamics is diminished in human type 2 diabetes (T2D) and mouse models of T2D. Enhancing Cx36 gap junction permeability protects against disruptions to the coordination of [Ca2+ ] dynamics. A novel peptide mimetic of the Cx36 c-terminal regulatory region protects against declines in Cx36 gap junction permeability. Pharmacological elevation in Cx36 or Cx36 peptide mimetic recovers [Ca2+ ] dynamics and glucose-stimulated insulin secretion in human T2D and mouse models of T2D.

Functional architecture of pancreatic islets identifies a population of first responder cells that drive the first-phase calcium response.

Insulin-secreting ß-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine "first responder" cells, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified "hub" and "leader" cells. We used islets isolated from Mip-CreER; Rosa-Stop-Lox-Stop-GCamP6s mice (ß-GCamP6s) that show ß-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of ß-cells in the islet was spatially organized, dependent on the cell's distance to the first responder cell, and consistent over time up to approximately 24 h. When first responder cells were laser ablated, the first-phase [Ca2+] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder ß-cell state, critical for the islet first-phase response to glucose.

ENTPD3 Marks Mature Stem Cell-Derived ß-Cells Formed by Self-Aggregation In Vitro.

Stem cell-derived ß-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult ß-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human ß-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient ß cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among ß cells, yet testing this in vivo in the intact pancreas is challenging. Robo ßKO mice, in which the genes Robo1 and Robo2 are deleted selectively in ß cells, provide a unique model of altered islet spatial architecture without loss of ß cell differentiation or islet damage from diabetes. Combining Robo ßKO mice with intravital microscopy, we show here that Robo ßKO islets have reduced synchronized intra-islet Ca2+ oscillations among ß cells in vivo. We provide evidence that this loss is not due to a ß cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

Small subpopulations of ß-cells do not drive islet oscillatory [Ca2+] dynamics via gap junction communication.

The islets of Langerhans exist as multicellular networks that regulate blood glucose levels. The majority of cells in the islet are excitable, insulin-producing ß-cells that are electrically coupled via gap junction channels. ß-cells are known to display heterogeneous functionality. However, due to gap junction coupling, ß-cells show coordinated [Ca2+] oscillations when stimulated with glucose, and global quiescence when unstimulated. Small subpopulations of highly functional ß-cells have been suggested to control [Ca2+] dynamics across the islet. When these populations were targeted by optogenetic silencing or photoablation, [Ca2+] dynamics across the islet were largely disrupted. In this study, we investigated the theoretical basis of these experiments and how small populations can disproportionality control islet [Ca2+] dynamics. Using a multicellular islet model, we generated normal, skewed or bimodal distributions of ß-cell heterogeneity. We examined how islet [Ca2+] dynamics were disrupted when cells were targeted via hyperpolarization or populations were removed; to mimic optogenetic silencing or photoablation, respectively. Targeted cell populations were chosen based on characteristics linked to functional subpopulation, including metabolic rate of glucose oxidation or [Ca2+] oscillation frequency. Islets were susceptible to marked suppression of [Ca2+] when ~10% of cells with high metabolic activity were hyperpolarized; where hyperpolarizing cells with normal metabolic activity had little effect. However, when highly metabolic cells were removed from the model, [Ca2+] oscillations remained. Similarly, when ~10% of cells with either the highest frequency or earliest elevations in [Ca2+] were removed from the islet, the [Ca2+] oscillation frequency remained largely unchanged. Overall, these results indicate small populations of ß-cells with either increased metabolic activity or increased frequency are unable to disproportionately control islet-wide [Ca2+] via gap junction coupling. Therefore, we need to reconsider the physiological basis for such small ß-cell populations or the mechanism by which they may be acting to control normal islet function.

Caloric restriction recovers impaired ß-cell-ß-cell gap junction coupling, calcium oscillation coordination, and insulin secretion in prediabetic mice.

Caloric restriction can decrease the incidence of metabolic diseases, such as obesity and Type 2 diabetes mellitus. The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis are not fully understood. Intercellular communication within the islets of Langerhans, mediated by Connexin36 (Cx36) gap junctions, regulates insulin secretion dynamics and glucose homeostasis. The goal of this study was to determine whether caloric restriction can protect against decreases in Cx36 gap junction coupling and altered islet function induced in models of obesity and prediabetes. C57BL6 mice were fed with a high-fat diet (HFD), showing indications of prediabetes after 2 mo, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to 1 mo of 40% caloric restriction (2 g/day of HFD). Mice under 40% caloric restriction showed reversal in weight gain and recovered insulin sensitivity, fasting glucose, and insulin levels. In islets of mice fed the HFD, caloric restriction protected against obesity-induced decreases in gap junction coupling and preserved glucose-stimulated calcium signaling, including Ca2+ oscillation coordination and oscillation amplitude. Caloric restriction also promoted a slight increase in glucose metabolism, as measured by increased NAD(P)H autofluorescence, as well as recovering glucose-stimulated insulin secretion. We conclude that declines in Cx36 gap junction coupling that occur in obesity can be completely recovered by caloric restriction and obesity reversal, improving Ca2+ dynamics and insulin secretion regulation. This suggests a critical role for caloric restriction in the context of obesity to prevent islet dysfunction.

How Heterogeneity in Glucokinase and Gap-Junction Coupling Determines the Islet [Ca2+] Response.

Understanding how cell subpopulations in a tissue impact overall system function is challenging. There is extensive heterogeneity among insulin-secreting ß-cells within islets of Langerhans, including their insulin secretory response and gene expression profile, and this heterogeneity can be altered in diabetes. Several studies have identified variations in nutrient sensing between ß-cells, including glucokinase (GK) levels, mitochondrial function, or expression of genes important for glucose metabolism. Subpopulations of ß-cells with defined electrical properties can disproportionately influence islet-wide free-calcium activity ([Ca2+]) and insulin secretion via gap-junction electrical coupling. However, it is poorly understood how subpopulations of ß-cells with altered glucose metabolism may impact islet function. To address this, we utilized a multicellular computational model of the islet in which a population of cells deficient in GK activity and glucose metabolism was imposed on the islet or in which ß-cells were heterogeneous in glucose metabolism and GK kinetics were altered. This included simulating GK gene (GCK) mutations that cause monogenic diabetes. We combined these approaches with experimental models in which gck was genetically deleted in a population of cells or GK was pharmacologically inhibited. In each case, we modulated gap-junction electrical coupling. Both the simulated islet and the experimental system required 30-50% of the cells to have near-normal glucose metabolism, fewer than cells with normal KATP conductance. Below this number, the islet lacked any glucose-stimulated [Ca2+] elevations. In the absence of electrical coupling, the change in [Ca2+] was more gradual. As such, electrical coupling allows a large minority of cells with normal glucose metabolism to promote glucose-stimulated [Ca2+]. If insufficient numbers of cells are present, which we predict can be caused by a subset of GCK mutations that cause monogenic diabetes, electrical coupling exacerbates [Ca2+] suppression. This demonstrates precisely how metabolically heterogeneous ß-cell populations interact to impact islet function.