Juan Vicente Sanchez-Andres

Junctional communication of pancreatic beta cells contributes to the control of insulin secretion and glucose tolerance.

Proper insulin secretion requires the coordinated functioning of the numerous beta cells that form pancreatic islets. This coordination depends on a network of communication mechanisms whereby beta cells interact with extracellular signals and adjacent cells via connexin channels. To assess whether connexin-dependent communication plays a role in vivo, we have developed transgenic mice in which connexin 32 (Cx32), one of the vertebrate connexins found in the pancreas, is expressed in beta cells. We show that the altered beta-cell coupling that results from this expression causes reduced insulin secretion in response to physiologically relevant concentrations of glucose and abnormal tolerance to the sugar. These alterations were observed in spite of normal numbers of islets, increased insulin content, and preserved secretory response to glucose by individual beta cells. Moreover, glucose-stimulated islets showed improved electrical synchronization of these cells and increased cytosolic levels of Ca(2+). The results show that connexins contribute to the control of beta cells in vivo and that their excess is detrimental for insulin secretion.

In vivo synchronous membrane potential oscillations in mouse pancreatic beta-cells: lack of co-ordination between islets.

1. The properties of the oscillations in electrical activity of different beta‐cells within the same islet of Langerhans, and of different islets within the same pancreas, recorded in vivo, are described. 2. Simultaneous recordings of two cells within the same islet showed that the oscillations were synchronous. A rapid increase in blood glucose led to the simultaneous appearance of a transitory phase of continuous electrical activity in both cells. These results indicate that under physiological conditions, the islets operate as a functional syncytium. 3. Simultaneous recordings of cells from two different islets within the same pancreas showed that the oscillations in the electrical activity were not synchronous, which suggests that each islet is a functionally independent unit. Rapid changes in blood glucose led to the appearance of a transitory phase of increased electrical activity in both islets, although of different duration. These results suggest that the endocrine pancreas lacks a pacemaker driving the electrical activity of all the islets. 4. The comparison of the degree of activation of different islets, simultaneously recorded at different glucose concentrations, indicated that all the islets had a similar sensitivity to glucose. Furthermore, when the glucose concentration was increased, the electrical activity in both islets increased in parallel, suggesting that the amount of insulin released due to the increase in glycaemia was produced by the simultaneous response of all the islets and not by the recruitment of islets with different sensitivities to glucose. 5. Our results predict that the synchronous electrical activity of all the cells within an islet will result in widespread intracellular calcium oscillations and pulsatile insulin secretion. The periodicity of the pulses of insulin secretion in different islets is suggested to be of slightly different length and asynchronous.

Oscillation of gap junction electrical coupling in the mouse pancreatic islets of Langerhans.

1. Pancreatic beta‐cells oscillate synchronously when grouped in islets. Coupling seems essential to maintain this oscillatory behaviour, as isolated cells are unable to oscillate. This allows the islet to be used as a model system for studying the role of coupling in the generation of oscillatory patterns. 2. Pairs of beta‐cells were intracellularly recorded in islets. beta‐Cells oscillated synchronously. Propagated voltage deflections were observed as a function of glucose concentration and of the distance between the recording electrodes. Space constants were smaller in the silent than in the active phases, suggesting a higher intercellular connection in the active phases. 3. Coupling coefficients and estimated coupling conductances were larger in the active than in the silent phases. 4. Coupling coefficients and coupling conductances changed dynamically and in phase with the membrane potential oscillations, pointing to an active modulation of the gap junctions. 5. We hypothesize a role for coupling in the generation of the oscillatory events, providing different levels of permeability dependent on the state of conductance during the oscillatory phases.

Slow [Ca2+]i Oscillations Induced by Ketoisocaproate in Single Mouse Pancreatic Islets

The effect of α-ketoisocaproate (KIC), the first catabolic metabolite of the amino acid leucine, on [Ca2+]i, insulin release, and membrane potential was measured in mouse pancreatic islets of Langerhans. Stimulatory concentrations of KIC (2.5–10 mmol/l) caused slow oscillations of [Ca2+]i and cyclic variations of the membrane potential. Slow [Ca2+]i oscillations depended on extracellular calcium. Simultaneous measurements of [Ca2+]i and insulin release resolved pulsatile insulin secretion that paralleled slow [Ca2+]i oscillations. Whereas 11 mmol/l glucose induced a significant increase in cAMP, KIC was unable to modify it. Glucagon (10 nmol/l), which significantly increased cAMP in mouse islets, also increased the frequency of glucose-induced fast [Ca2+]i oscillations. However, neither glucagon (10 nmol/l) nor dibutyryl cAMP (1 mmol/l) was able to change the slow oscillation pattern into a fast pattern. Imaging of Ca2+ showed that KIC-induced slow oscillations were synchronic throughout the whole islet. It is suggested that β-cell electrical activity plays a role in the origin of slow [Ca2+]i oscillations.

The electrical activity of mouse pancreatic beta-cells recorded in vivo shows glucose-dependent oscillations.

1. The characteristics of the electrical activity of beta‐cells from islets of Langerhans recorded in vivo are described. For blood glucose concentrations from 4 to 11 mM, the electrical activity of pancreatic beta‐cells is oscillatory, with alternating depolarized and hyperpolarized phases. During the depolarized phases, action potentials are triggered. 2. The main effect of increasing glucose concentration consists of an increase in the duration of the depolarized phase. The relationship between blood glucose concentration and the percentage of time in the depolarized phase can be described by a sigmoidal function with half‐activation at 6.8 mM glucose. The equivalent value obtained in parallel experiments in vitro is 13.3 mM, a significant rightward shift in the activation curve that suggests a role for other neural or humoral factors in determining the islet sensitivity to glucose. 3. The injection of glucose into the bloodstream produces a transitory phase of continuous electrical activity that is recorded within seconds after the change and that leads to a decrease of the glycaemia to the prestimulatory value. 4. The results demonstrate that under physiological conditions the electrical response of beta‐cells to glucose consists of membrane potential oscillations, validating previous data obtained with isolated preparations. Furthermore, the electrical response occurs at lower levels of glycaemia than those predicted from recordings in isolated preparations and is maximal within the physiological range of blood glucose.

Regulation of pancreatic β-cell electrical activity and insulin release by physiological amino acid concentrations

Abstract The mutual enhancement of insulin release by glucose and amino acids is not clearly understood. In this study, the effects on electrical activity and insulin release of a mixture of amino acids and glucose at concentrations found in fed (aaFD) and fasted (aaFT) animals were determined using freshly isolated mouse islets. Islets perifused with aaFD mixture showed an oscillatory pattern of electrical activity at lower glucose concentrations (5 mmol/l) than in islets perifused with the aaFT mixture and with glucose (G) alone (10 mmol/l). The concentration/response curve for the fraction of time spent by the membrane potential in the active phase in aaFD-stimulated islets was found to be significantly shifted to the left and had a smaller slope than that for glucose-stimulated islets. Insulin release followed the same pattern. This resulted in a concentration/response curve for glucose that was closer to that recorded ”in vivo”. We have also found that four amino acids (leucine, isoleucine, alanine and arginine) are largely responsible for the observed effects and that there is a non-linear enhancement of insulin release as a consequence of the combined effect of amino acids and glucose. This effect was more pronounced in the second phase of insulin release and was dependent on intracellular Ca2+. These findings indicate that amino acids account for most of the leftward shift in the concentration/response curve for glucose and that a reduction in the threshold for the glucose-induced oscillatory electrical activity response and in the generation of Ca2+ spikes accounts for the triggering of insulin release at lower glucose concentrations. Nevertheless, the effects on insulin release at high glucose concentrations cannot be explained solely by the increase in glucose-induced electrical activity.

Oscillatory patterns of electrical activity in mouse pancreatic islets of Langerhans recorded in vivo

Pancreatic β-cells secrete insulin as a function of blood glucose concentration. One of the key steps in stimulus-secretion coupling is the depolarisation of the membrane and the appearance of bursts of calcium action potentials. Recently, the characteristics and glucose dependence of the oscillations in electrical activity in vivo have been described. The experiments described here were designed to determine the temporal evolution of such electrical activity when no experimental changes in the glycaemia are imposed. The absolute duration of the active and silent phases has been analysed and compared with the values obtained in vitro. We have found that in vivo, at glycaemia ranging from 6.0 to 7.5 mM, the electrical activity of the islets of Langerhans is permanently oscillatory, the mean duration of the depolarisation phase being 28 s. In general, the oscillatory pattern remains very constant for relatively long (up to 60 min) periods of time. In some experiments, slow or transitory changes in the degree of β-cell activation could be observed, as well as the existence, in a very few cases, of oscillatory non-periodic patterns.

Evidence that muscarinic potentiation of insulin release is initiated by an early transient calcium entry

The increased insulin release induced by carbamoylcholine (CbCh) in pancreatic islets requires the presence of extracellular Ca2+. Intracellular recordings demonstrate that CbCh produces a transient increase in Ca2+ channel activity lasting from 30 to 60 s. Thereafter activity decreased to levels lower than in controls. When extracellular Ca2+ was present during this initial period, the stimulatory effects of CbCh were not different from those in which Ca2+ was present throughout. These experiments suggest that during muscarinic potentiation of insulin release extracellular calcium is only needed in the first minute.

Bursting as a source for predictability in biological neural network activity

Abstract The role of bursting as a unit of neural information has received considerable support in the recent years. Experimental evidence shows that in many different neural systems, e.g. visual cortex or hippocampus, bursting is essential for coding and processing. We have recently demonstrated (Menendez de la Prida et al., 1996) the spontaneous presence of bursts in in vitro hippocampal slices from newborn animals, providing a good system to investigate bursting dynamics in physiological conditions. Here we analyze the interspike intervals (ISIs) of five intracellularly recorded cells from immature hippocampal networks. First, we test the time series against Poisson processes, typical of pure random behavior, using the Kolmogorov-Smirnov test. Only 2 5 records strongly deviate from Poisson process. Nonlinear diction tests are then applied to compare original series with its Gaussian-scaled random phase surrogates and signs of short time predictability are observed ( 1 5 ). This predictability is originated by the intrinsic structure of bursts, in an otherwise purely random process, and can be removed completely by eliminating the bursts from the original time series. Here we introduce this method of eliminating bursts to get insight into the nonlinear dynamics of firing. Also the interburst intervals are indistinguishable from pure noise. The analysis of unstable periodicities within the bursts in the original ISIs shows that signs of nonlinearities can be statistically differentiated from their surrogate realizations (Pierson-Moss method). We discuss the computational implication of these results.

Effects of calcium buffering on glucose‐induced insulin release in mouse pancreatic islets: an approximation to the calcium sensor

1 The properties of the calcium sensor for glucose‐induced insulin secretion have been studied using cell‐permeant Ca2+ buffers with distinct kinetics and affinities. In addition, submembrane cytosolic Ca2+ distribution has been modelled after trains of glucose‐induced action potential‐like depolarizations. 2 Slow Ca2+ buffers (around 1 mmol l−1 intracellular concentration) with different affinities (EGTA and Calcium Orange‐5N) did not significantly affect glucose‐induced insulin release. Modelling showed no effect on cytosolic Ca2+ concentrations at the outermost shell (0.05 μm), their effects being observed in the innermost shells dependent on Ca2+ affinity. 3 In contrast, fast Ca2+ buffers (around 1 mmol l−1 intracellular concentration) with different affinities (BAPTA and Calcium Green‐5N) caused a 50% inhibition of early insulin response and completely blocked the late phase of glucose‐induced insulin response, their simulations showing a decrease of [Ca2+]i at both the inner and outermost shells. 4 These data are consistent with the existence in pancreatic β‐cells of a higher affinity Ca2+ sensor than that proposed for neurons. Moreover, these data are consistent with the proposed existence of two distinct pools of granules: (i) ‘primed’ vesicles, colocalized with Ca2+ channels and responsible of the first phase of insulin release; and (ii) ‘reserved pool’ vesicles, not colocalized and responsible for the second phase.