The pancreatic islets of Langerhans are multicellular micro-organs integral to maintaining glucose homeostasis through secretion of the hormone insulin. β-cells within the islet exist as a highly coupled electrical network which coordinates electrical activity and insulin release at high glucose, but leads to global suppression at basal glucose. Despite its importance, how network dynamics generate this emergent binary on/off behavior remains to be elucidated. Previous work has suggested that a small threshold of quiescent cells is able to suppress the entire network. By modeling the islet as a Boolean network, we predicted a phase-transition between globally active and inactive states would emerge near this threshold number of cells, indicative of critical behavior. This was tested using islets with an inducible-expression mutation which renders defined numbers of cells electrically inactive, together with pharmacological modulation of electrical activity. This was combined with real-time imaging of intracellular free-calcium activity [Ca2+]i and measurement of physiological parameters in mice. As the number of inexcitable cells was increased beyond ∼15%, a phase-transition in islet activity occurred, switching from globally active wild-type behavior to global quiescence. This phase-transition was also seen in insulin secretion and blood glucose, indicating physiological impact. This behavior was reproduced in a multicellular dynamical model suggesting critical behavior in the islet may obey general properties of coupled heterogeneous networks. This study represents the first detailed explanation for how the islet facilitates inhibitory activity in spite of a heterogeneous cell population, as well as the role this plays in diabetes and its reversal. We further explain how islets utilize this critical behavior to leverage cellular heterogeneity and coordinate a robust insulin response with high dynamic range. These findings also give new insight into emergent multicellular dynamics in general which are applicable to many coupled physiological systems, specifically where inhibitory dynamics result from coupled networks.

Myelin membrane, which ensheaths axons, has an unusually high amount of cholesterol. Cholesterol influences membrane fluidity and assembles lipid-rich microdomains within membranes, and some studies have shown that cholesterol is important for myelination. How cholesterol influences the development and differentiation of oligodendrocytes, glial cells that make myelin, is not known nor is clear whether isoprenoids, which also are products of the cholesterol biosynthetic pathway, contribute to myelination. Through a forward genetic screen in zebrafish we discovered that mutation of hmgcs1, which encodes an enzyme necessary for isoprenoid and cholesterol synthesis, causes oligodendrocyte progenitor cells (OPCs) to migrate past their target axons and to fail to express myelin genes. Drawing on a combination of pharmacological inhibitor and rescue experiments, we provide evidence that isoprenoids and protein prenylation, but not cholesterol, are required in OPCs to halt their migration at target axons. On the other hand, cholesterol, but not isoprenoids, is necessary both for axon ensheathment and myelin gene expression. Our data reveal that different products of the cholesterol biosynthetic pathway have distinct roles in oligodendrocyte development and that they together help to coordinate directed migration, axon wrapping, and gene expression.

Key pointsr Gap junctions provide electrical coupling that is critical to the function of pancreatic islets.Disruptions to connexin36 (Cx36) have been suggested to occur in diabetes.r No accurate and non-invasive technique has yet been established to quantify changes in Cx36 gap junction coupling in the intact islet.r This study developed fluorescence recovery after photobleaching (FRAP) as a non-invasive technique for quantifying Cx36 gap junction coupling in living islets.r The study identified treatments that modulate gap junction coupling, confirmed that the cellular distribution of coupling throughout the islet is highly heterogeneous and confirmed that α cells and β cells do not form functional Cx36 gap junctions.r This technique will enable future studies to examine the regulation of Cx36 gap junction coupling and its disruption in diabetes, and to uncover potential novel therapeutic targets associated with gap junction coupling. AbstractThe pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose-and cAMP-dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

Diabetes is caused by dysfunction to β-cells in the islets of Langerhans, disrupting insulin secretion and glucose homeostasis. Gap junction-mediated electrical coupling between β-cells in the islet plays a major role in coordinating a pulsatile secretory response at elevated glucose and suppressing insulin secretion at basal glucose. Previously, we demonstrated that a critical number of inexcitable cells can rapidly suppress the overall islet response, as a result of gap junction coupling. This was demonstrated in a murine model of Neonatal Diabetes Mellitus (NDM) involving expression of ATP-insensitive KATP channels, and by a multi-cellular computational model of islet electrical activity. Here we examined the mechanisms by which gap junction coupling contributes to islet dysfunction in NDM. We first verified the computational model against [Ca2+] and insulin secretion measurements in islets expressing ATP-insensitive KATP channels under different levels of gap junction coupling. We then applied this model to predict how different KATP channel mutations found in NDM suppress [Ca2+], and the role of gap junction coupling in this suppression. We further extended the model to account for stochastic noise and insulin secretion dynamics. We found experimentally and in the islet model that reductions in gap junction coupling allow progressively greater glucose-stimulated [Ca2+] and insulin secretion following expression of ATP-insensitive KATP channels. The model demonstrated good correspondence between suppression of [Ca2+] and clinical presentation of different NDM mutations. Significant recoveries in [Ca2+] and insulin secretion were predicted for many mutations upon reductions in gap junction coupling, where stochastic noise played a significant role in the recoveries. These findings provide new understanding how the islet functions as a multicellular system and for the role of gap junction channels in exacerbating the effects of decreased cellular excitability. They further suggest novel therapeutic options for NDM and other monogenic forms of diabetes.

Mutations to the ATP-sensitive K+ channel (KATP channel) that reduce the sensitivity of ATP inhibition cause neonatal diabetes mellitus via suppression of β-cell glucose-stimulated free calcium activity ([Ca2+]i) and insulin secretion. Connexin-36 (Cx36) gap junctions also regulate islet electrical activity; upon knockout of Cx36, β-cells show [Ca2+]i elevations at basal glucose. We hypothesized that in the presence of overactive ATP-insensitive KATP channels, a reduction in Cx36 would allow elevations in glucose-stimulated [Ca2+]i and insulin secretion to improve glucose homeostasis. To test this, we introduced a genetic knockout of Cx36 into mice that express ATP-insensitive KATP channels and measured glucose homeostasis and islet metabolic, electrical, and insulin secretion responses. In the normal presence of Cx36, after expression of ATP-insensitive KATP channels, blood glucose levels rapidly rose to >500 mg/dL. Islets from these mice showed reduced glucose-stimulated [Ca2+]i and no insulin secretion. In mice lacking Cx36 after expression of ATP-insensitive KATP channels, normal glucose levels were maintained. Islets from these mice had near-normal glucose-stimulated [Ca2+]i and insulin secretion. We therefore demonstrate a novel mechanism by which islet function can be recovered in a monogenic model of diabetes. A reduction of gap junction coupling allows sufficient glucose-stimulated [Ca2+]i and insulin secretion to prevent the emergence of diabetes.