BK channels mediate a novel ionic mechanism that regulates glucose-dependent electrical activity and insulin secretion in mouse pancreatic β-cells

BK channels are large unitary conductance K + channels cooperatively activated by intracellular calcium and membrane depolarisation. We show that BK channels regulate electrical activity in β-cells of mouse pancreatic islets exposed to elevated glucose. In 11.1 m m glucose, the non-peptidyl BK channel blocker paxilline increased the height of β-cell action potentials (APs) by 21 mV without affecting burst- or silent-period durations. In isolated β-cells, paxilline increased AP height by 16 mV without affecting resting membrane potential. In voltage clamp, paxilline blocked a transient component of outward current activated by a short depolarisation, which accounted for at least 90% of the initial outward K + current. This BK current ( I BK ) was blocked by the Ca 2+ channel blockers Cd 2+ (200 μ m ) or nimodipine (1 μ m ), and potentiated by FPL-64176 (1 μ m ). I BK was also 56% blocked by the BK channel blocker iberiotoxin (100 n m ). I BK activated more than 10-fold faster than the delayed rectifier I Kv over the physiological voltage range, and partially inactivated. An AP-like command revealed that I BK activated and deactivated faster than I Kv and accounted for 86% of peak I K , explaining why I BK block increased AP height. A higher amplitude AP-like command, patterned on an AP recorded in 11.1 m m glucose plus paxilline, activated 4-fold more I Kv and significantly increased Ca 2+ entry. Paxilline increased insulin secretion in islets exposed to 11.1 m m glucose by 67%, but did not affect basal secretion in 2.8 m m glucose. These data suggest a modified model of β-cell AP generation where I BK and I Kv coordinate the AP repolarisation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79246/1/jphysiol.2009.184341.pd

The Phantom Burster Model for Pancreatic β-Cells

Abstract Pancreatic β-cells exhibit bursting oscillations with a wide range of periods. Whereas periods in isolated cells are generally either a few seconds or a few minutes, in intact islets of Langerhans they are intermediate (10–60 s). We develop a mathematical model for β-cell electrical activity capable of generating this wide range of bursting oscillations. Unlike previous models, bursting is driven by the interaction of two slow processes, one with a relatively small time constant (1–5 s) and the other with a much larger time constant (1–2 min). Bursting on the intermediate time scale is generated without need for a slow process having an intermediate time constant, hence phantom bursting. The model suggests that isolated cells exhibiting a fast pattern may nonetheless possess slower processes that can be brought out by injecting suitable exogenous currents. Guided by this, we devise an experimental protocol using the dynamic clamp technique that reliably elicits islet-like, medium period oscillations from isolated cells. Finally, we show that strong electrical coupling between a fast burster and a slow burster can produce synchronized medium bursting, suggesting that islets may be composed of cells that are intrinsically either fast or slow, with few or none that are intrinsically medium

Pharmacological Properties and Functional Role of Kslow Current in Mouse Pancreatic β-Cells: SK Channels Contribute to Kslow Tail Current and Modulate Insulin Secretion

The pharmacological properties of slow Ca2+-activated K+ current (Kslow) were investigated in mouse pancreatic β-cells and islets to understand how Kslow contributes to the control of islet bursting, [Ca2+]i oscillations, and insulin secretion. Kslow was insensitive to apamin or the KATP channel inhibitor tolbutamide, but UCL 1684, a potent and selective nonpeptide SK channel blocker reduced the amplitude of Kslow tail current in voltage-clamped mouse β-cells. Kslow was also selectively and reversibly inhibited by the class III antiarrythmic agent azimilide (AZ). In isolated β-cells or islets, pharmacologic inhibition of Kslow by UCL 1684 or AZ depolarized β-cell silent phase potential, increased action potential firing, raised [Ca2+]i, and enhanced glucose-dependent insulin secretion. AZ inhibition of Kslow also supported mediation by SK, rather than cardiac-like slow delayed rectifier channels since bath application of AZ to HEK 293 cells expressing SK3 cDNA reduced SK current. Further, AZ-sensitive Kslow current was extant in β-cells from KCNQ1 or KCNE1 null mice lacking cardiac slow delayed rectifier currents. These results strongly support a functional role for SK channel-mediated Kslow current in β-cells, and suggest that drugs that target SK channels may represent a new approach for increasing glucose-dependent insulin secretion. The apamin insensitivity of β-cell SK current suggests that β-cells express a unique SK splice variant or a novel heteromultimer consisting of different SK subunits

Glucose Metabolism, Islet Architecture, and Genetic Homogeneity in Imprinting of [Ca2+]i and Insulin Rhythms in Mouse Islets

We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca2+]i) that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed “islet imprinting.” We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J) as well as outbred mouse strains (Swiss-Webster; CD1). Second, imprinting was observed in NAD(P)H oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca2+]i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a KATP-channel opener that blocks [Ca2+]i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca2+]i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca2+]i oscillations. Lastly, to test whether the imprinted [Ca2+]i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca2+]i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon

Mice Deficient in GEM GTPase Show Abnormal Glucose Homeostasis Due to Defects in Beta-Cell Calcium Handling

Glucose-stimulated insulin secretion from beta-cells is a tightly regulated process that requires calcium flux to trigger exocytosis of insulin-containing vesicles. Regulation of calcium handling in beta-cells remains incompletely understood. Gem, a member of the RGK (Rad/Gem/Kir) family regulates calcium channel handling in other cell types, and Gem over-expression inhibits insulin release in insulin-secreting Min6 cells. The aim of this study was to explore the role of Gem in insulin secretion. We hypothesised that Gem may regulate insulin secretion and thus affect glucose tolerance in vivo

Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase Modulates Oscillations of Pancreatic Islet Metabolism

Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. It has been proposed that a positive feedback circuit exists within the glycolytic pathway, the autocatalytic activation of phosphofructokinase-1 (PFK1), which endows pancreatic beta-cells with the ability to generate oscillations in metabolism. Flux through PFK1 is controlled by the bifunctional enzyme PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) in two ways: via (1) production/degradation of fructose-2,6-bisphosphate (Fru2,6-BP), a potent allosteric activator of PFK1, as well as (2) direct activation of glucokinase due to a protein-protein interaction. In this study, we used a combination of live-cell imaging and mathematical modeling to examine the effects of inducibly-expressed PFK2/FBPase2 mutants on glucose-induced Ca2+ pulsatility in mouse islets. Irrespective of the ability to bind glucokinase, mutants of PFK2/FBPase2 that increased the kinase:phosphatase ratio reduced the period and amplitude of Ca2+ oscillations. Mutants which reduced the kinase:phosphatase ratio had the opposite effect. These results indicate that the main effect of the bifunctional enzyme on islet pulsatility is due to Fru2,6-BP alteration of the threshold for autocatalytic activation of PFK1 by Fru1,6-BP. Using computational models based on PFK1-generated islet oscillations, we then illustrated how moderate elevation of Fru-2,6-BP can increase the frequency of glycolytic oscillations while reducing their amplitude, with sufficiently high activation resulting in termination of slow oscillations. The concordance we observed between PFK2/FBPase2-induced modulation of islet oscillations and the models of PFK1-driven oscillations furthermore suggests that metabolic oscillations, like those found in yeast and skeletal muscle, are shaped early in glycolysis

Pulsatile insulin secretion, impaired glucose tolerance and type 2 diabetes

Type 2 diabetes (T2DM) results when increases in beta cell function and/or mass cannot compensate for rising insulin resistance. Numerous studies have documented the longitudinal changes in metabolism that occur during the development of glucose intolerance and lead to T2DM. However, the role of changes in insulin secretion, both amount and temporal pattern has been understudied. Most of the insulin secreted from pancreatic beta cells of the pancreas is released in a pulsatile pattern, which is disrupted in T2DM. Here we review the evidence that changes in beta cell pulsatility occur during the progression from glucose intolerance to T2DM in humans, and contribute significantly to the etiology of the disease. We review the evidence that insulin pulsatility improves the efficacy of secreted insulin on its targets, particularly hepatic glucose production, but also examine evidence that pulsatility alters or is altered by changes in peripheral glucose uptake. Finally, we summarize our current understanding of the biophysical mechanisms responsible for oscillatory insulin secretion. Understanding how insulin pulsatility contributes to normal glucose homeostasis and is altered in metabolic disease states may help improve the treatment of T2DM