Mouse Pancreatic Islets Of Langerhans Research Articles

The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signaling.

The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting β-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet β-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.

Determination of secretory granule maturation times in pancreatic islet β-cells by serial block-face electron microscopy

It is shown how serial block-face electron microscopy (SBEM) of insulin-secreting β-cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the β-cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It was found that 7.2 ± 1.2% (±st. dev.) of the β-cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically ≳100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96 ± 12 h, as previously obtained from pulsed 35S-radiolabeling of cysteine and methionine. Analysis of β-cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically ≲30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135 ± 14 min, and the immature transforming granules for an average time of 130 ± 17 min.

Isolation, Purification, and Culture of Mouse Pancreatic Islets of Langerhans.

Pancreatic islets constitute an important tool for research and clinical applications in the field of diabetes. They are used for transplantation, unraveling new mechanisms in insulin secretion, studying pathophysiological pathways in diseased cells, and pharmacological research aimed at developing improved therapeutic strategies. Therefore, fine-tuning islet isolation protocols remains an important objective for reliable investigations. Here we describe a relatively simple mouse islet isolation protocol that relies on enzymatic digestion using low-activity collagenase and several sedimentation and Percoll gradient steps.

Mitochondrial Networks in Beta Cells of Pancreatic Islet of Langerhans Investigated by Serial Block Face Scanning Electron Microscopy

The process whereby mitochondrial tubules fuse to form extended networks within cells has been associated with metabolic activity, and may provide a defense against generation of reactive oxygen species, whereas fission of such mitochondrial networks has been associated with apoptosis. We have used serial block-face scanning electron microscopy (SBF-SEM) to analyze distributions of mitochondrial network lengths in metabolically active insulin secreting beta cells of mouse pancreatic islets of Langerhans, with the aim of quantifying differences between wild type and diabetic disease models (Pfeifer et al., 2015). This technique provides three-dimensional ultrastructure of beta cells at a spatial resolution of about 10 nm in the x-y plane and 25 nm in the z-direction, from which we have been able to obtain cross sectional areas, volumes and network lengths of mitochondrial tubules in a population of beta cells. Blocks of isolated pancreatic islets were prepared by fixation, staining with heavy atoms, and embedding in Epon-Araldite resin, and were imaged at 1.5-keV primary beam energy with the backscattered electron signal. Our results showed that approximately one-third of the total mitochondrial volume originated from short networks less than 10 µm in length, one-third came from tubules of length 10 to 45 µm, and one-third from networks of length 45 to 150 µm. We conclude that SBF-SEM can provide a quantitative characterization of mitochondrial networks in wild type and disease models of endocrine tissues. This research was funded by the intramural program of NIBIB, NIH.C.R. Pfeifer, A. Shomorony, M.A. Aronova, G. Zhang, T. Cai, H. Xu. A.L. Notkins, R.D. Leapman. Quantitative analysis of mouse pancreatic islet architecture by serial block-face SEM. J Struct Biol, 189 (2015) 44-52.

Isolation of Mouse Pancreatic Islets of Langerhans.

The aim of any pancreatic islet isolation is obtaining pure, viable and functional pancreatic islets, either for in vitro or in vivo purposes. The islets of Langerhans are complex microorgans with the important role of regulating glucose homeostasis. Imbalances in glucose homeostasis lead to diabetes, which is defined by the American Diabetes Association as a "group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both" (American Diabetes Association 2011). Currently, the rising demand of human islets is provoking a shortage of this tissue, limiting research and clinical practice on this field. In this scenario, it is essential to investigate and improve islet isolation procedures in animal models, while keeping in mind the anatomical and functional differences between species. This chapter discusses the main aspects of mouse islet isolation research, highlighting the critical factors and shortcomings to take into account for the selection and/or optimization of a mouse islet isolation protocol.

Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells.

SummaryA combination of two‐dimensional (2D) and three‐dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and β cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a β cell.

ChREBP regulates Pdx-1 and other glucose-sensitive genes in pancreatic β-cells

Carbohydrate responsive element-binding protein (ChREBP) is a transcription factor whose expression and activity are increased in pancreatic β-cells maintained at elevated glucose concentrations. We show here that ChREBP inactivation in clonal pancreatic MIN6 β-cells results in an increase in Pdx-1 expression at low glucose and to a small, but significant, increase in Ins2, GcK and MafA gene expression at high glucose concentrations. Conversely, adenovirus-mediated over-expression of ChREBP in mouse pancreatic islets results in decreases in Pdx-1, MafA, Ins1, Ins2 and GcK mRNA levels. These data suggest that strategies to reduce ChREBP activity might protect against β-cell dysfunction in type 2 diabetes.

Rapid non-genomic regulation of Ca2+ signals and insulin secretion by PPARα ligands in mouse pancreatic islets of Langerhans

PPAR alpha is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. PPAR alpha is involved in the regulation of in vivo triglyceride levels, presumably through its effects on fatty acid and lipoprotein metabolism. Some nuclear receptors have been involved in rapid effects mediated by non-genomic mechanisms. In this paper, we report the rapid non-genomic effects of PPAR alpha ligands on the intracellular calcium concentration ([Ca2+]i), mitochondrial function, reactive oxygen species (ROS) generation, and secretion of insulin in freshly isolated mouse islets of Langerhans. The hypolipidemic fibrate PPAR alpha agonist WY-14 643 decreased the glucose-induced calcium oscillations in intact islets. This effect was mimicked by the synthetic agonist GW7647 and the endogenous agonist oleylethanolamide. The WY-14 643 action was rapid in onset (5 min) and was still produced in the presence of protein and mRNA synthesis inhibitors, cycloheximide, and actinomycin-d. This suggests that it is independent of gene transcription. In addition, WY-14 623 impaired mitochondrial function, increased ROS formation and decreased insulin release. PPAR alpha is present in beta-cells, mainly in the cytosol and nucleus, with a small subpopulation localized in the plasma membrane. However, the presence of the PPAR alpha ligand effects in mice bearing a disrupted Ppar alpha gene raises the possibility that the rapid effects of the agonists in pancreatic beta-cells are independent of the receptor. We conclude that PPAR alpha agonists produce a decrease in glucose-induced [Ca2+]i signals and insulin secretion in beta-cells through a rapid, non-genomic mechanism.

Anx7 Is Required for Nutritional Control of Gene Expression in Mouse Pancreatic Islets of Langerhans

Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control islets that are principally associated with cell division and DNA repair. The latter genes have not specifically been associated with islet physiology in the past. By contrast, Anx7(+/-) mouse islets exhibit a greatly reduced ability to discriminate genomically between fed and fasted states for all classes of identified genes. Many of the validated genes are specific to islets in comparison to liver tissue examined. Real-time quantitative RT-PCR analysis of islets from Anx7 heterozygous mice and littermate controls revealed remarkable down-regulation in PTEN, Glut-2, PDX-1, IGF-1, and Neuro D1 expression, but not in liver. We conclude that reduced gene dosage in the Anx7(+/-) islet, with concomitant loss of ITPR3 expression and consequent defects in Ca(2+) signaling, may substantially contribute to the mechanism of the loss of genomic discrimination, in vivo, between the fed and fasted states. We believe that the requirement for complete Anx7 gene dosage and IPTR3 expression in islets of Langerhans will prove to be of fundamental importance for understanding the mechanism of nutritional sensing in health and disease.

Synchronous glucose-dependent [Ca 2+] i oscillations in mouse pancreatic islets of Langerhans recorded in vivo

Using microfluorescence in combination with image-analysis techniques we monitored intracellular calcium ([Ca 2+] i) dynamics in mouse islets of Langerhans loaded with fura-2 and recorded in vivo. [Ca 2+] i oscillates in the glycaemias range 5–10 mM, the duration of the oscillations being directly proportional to the blood glucose concentration. The analysis of different areas within the same islet shows that [Ca 2+] i oscillations are synchronous throughout the islet. These results show that in vivo, individual islets of Langerhans behave as a functional syncytium and suggest the existence of secretory pulses of insulin.

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.

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

Pancreatic beta-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 beta-cell activation could be observed, as well as the existence, in a very few cases, of oscillatory non-periodic patterns. Key words beta-cells middle dot Pancreas middle dot Electrophysiology middle dot Oscillations

Amino acid-induced [Ca2+]i oscillations in single mouse pancreatic islets of Langerhans.

1. The effects of amino acids on cytosolic free calcium concentration ([Ca2+]i) were measured, using fura-2 fluorescence imaging, in mouse pancreatic islets of Langerhans. 2. Slow [Ca2+]i oscillations appeared when isolated islets were incubated with a solution containing a mixture of amino acids and glucose at concentrations found in the plasma of fed animals. 3. In the presence of 11 mM glucose, alanine (5 mM) and arginine (10 mM) induced a transient rise in [Ca2+]i followed by an oscillatory pattern, while leucine (3 mM) and isoleucine (10 mM) triggered the appearance of slow [Ca2+]i oscillations. 4. Also in the presence of glucose (11 mM), tolbutamide (10 microM) increased the duration of the glucose-induced [Ca2+]i oscillations. While tolbutamide (10 microM) did not modify the leucine-induced slow oscillatory pattern, addition of diazoxide (10 microM) resulted in the gradual appearance of [Ca2+]i oscillations which resembled the glucose-induced fast oscillations. 5. Like stimulatory glucose concentrations (11 mM), glyceraldehyde (10 mM) induced fast oscillations of [Ca2+]i. 6. Fluoroacetate (2 mM) transformed leucine-induced slow [Ca2]i oscillations into fast [Ca2+]i oscillations. Iodoacetate (1 mM) completely inhibited any oscillatory pattern. 7. It is suggested that mitochondrially generated signals, derived from amino acid oxidative metabolism, acting in conjunction with glucose-signalled messengers, are very effective at closing ATP-dependent K+ channels (KATP+). 8. We propose that metabolic regulation of KATP+ channels is one of the mechanisms underlying the modulation of the oscillatory [Ca2+]i response to nutrient secretagogues.

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

The effect of alpha-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 beta-cell electrical activity plays a role in the origin of slow [Ca2+]i oscillations.

Metabolic regulation of intracellular calcium concentration in mouse pancreatic islets of Langerhans

Intracellular Ca2+ concentration ([Ca2+]i) handling during K(+)-induced Ca2+ loads was studied in single islets of Langerhans. K(+)-induced depolarization caused a rapid and transient rise in [Ca2+]i. After K+ removal [Ca2+]i declined with a time course usually fitted by the sum of two exponential functions. Partial Na+ removal increased the resting [Ca2+]i level, indicating the existence of a Na+/Ca2+ exchange, but only slightly impaired the recovery from Ca2+ loads. Metabolic poisoning with CN- increased the resting Ca2+ level and slowed down the recovery from Ca2+ loads. Removal of external Na+ in islets poisoned with CN- strongly inhibited Ca2+ removal mechanisms. An increase in the glucose concentration from 0 to 16 mM (in the presence of diazoxide) resulted in a decrease in the resting [Ca2+]i and an acceleration of [Ca2+]i recovery from K+ loads. These results suggest that the main mechanism responsible for Ca2+ homeostasis is dependent on metabolic energy and that such energy can be provided by glucose metabolism.

Fluorescence digital image analysis of glucose-induced [Ca2+]i oscillations in mouse pancreatic islets of Langerhans.

At intermediate glucose concentrations, [Ca2+]i (intracellular calcium) measured in single islets of Langerhans undergo oscillations that are caused by glucose-induced bursting of electrical activity. Using digital video imaging of fura-2--loaded islets, we have analyzed the spatial distribution of [Ca2+]i in response to the natural secretagogue glucose and the KATP channel blocker tolbutamide. When the glucose level is increased, [Ca2+]i first increases and then starts to oscillate with a synchronous pattern through the islet. The synchrony is maintained even during nonrhythmic oscillatory patterns. In the presence of tolbutamide, [Ca2+]i increases in all the islet regions, suggesting that the calcium signal is derived mainly from the beta-cell population. These results demonstrate that the islets behave as a functional syncytium in response to stimulatory glucose levels, canceling out heterogeneities at the single cell level.

Fluorescence digital image analysis of glucose-induced [Ca2+]i oscillations in mouse pancreatic islets of Langerhans

Fluorescence digital image analysis of glucose-induced [Ca2+]i oscillations in mouse pancreatic islets of Langerhans

Glucose-induced oscillatory changes in extracellular ionized potassium concentration in mouse islets of Langerhans

Liquid membrane [K+]-sensitive microelectrodes (1-2 micron tip diameter) were used to measure the extracellular ionized potassium concentration in mouse pancreatic islets of Langerhans. With the tip of the microelectrode at the surface of the islet, the time course of the [K+]-sensitive electrode potential changes in response to the application of rapid changes in [K+]o (from 1.25 to 5 mM), could be reproduced by the equation for K+-diffusion through a 100-micron-thick unstirred layer around the islet (diffusion coefficient for K+ at 27 degrees C, DK,o, taken as 1.83 X 10(-5) cm2/s). The time to reach 63% of the steady-state electrode response with the tip in the chamber at the surface of the islet was from 5 to 6 s. When the tip of the [K+]-sensitive electrode was placed in the islet tissue, the time for the response to reach 63% of the steady-state level increased. The time course of the [K+]-sensitive electrode response could be reproduced using the same diffusion model assuming that K+ diffusion into the islet tissue takes place in a tortuous intercellular path with an apparent diffusion coefficient, DK,I, about half of DK,o, in series with the unstirred layer around the islet. In the absence of glucose the potassium concentration in the extracellular space, [K+]I, was found to be higher than the concentration in the external modified Krebs solution, [K+]o. The difference in concentration [K+]I - [K+]o was greater when [K+]o was smaller than 2 mM. In the presence of glucose (between 11 and 16 mM), under steady-state conditions, small oscillatory changes in the [K+], (1.48 +/- 0.94 mM) were detected. Simultaneous recording of membrane potential from one B-cell and [K+], in the same islet indicated that the potassium concentration increased during the active phase of the bursts of electrical activity. Maximum concentration in the intercellular was reached near the end of the active phase of the bursts. We propose that the space between islet cells constitutes a restricted diffusion system where potassium accumulates during the transient activation of potassium channels.

Mouse pancreatic beta-cells: tetraethylammonium blockage of the potassium permeability increase induced by depolarization.

1. Membrane potentials and input resistance were measured in beta-cells from mouse pancreatic islets of Langerhans in the presence or absence of D-glucose. 2. Tetraethylammonium (TEA) (a specific blocker of the K permeability increase induced by shifts in membrane potential from negative to positive values) was externally applied and its effects on potentials and input resistance evaluated. 3. In the absence of glucose, addition of TEA up to 20 mM to the perifusion medium did not affect the resting potential and the input resistance, the selectivity ratio PK/PNa (calculated from the constant field equation) remaining unchanged at about 30. 4. The characteristic response of the beta-cell membrane potential, in the presence of glucose, is a fluctuation between a silent phase at about -50 mV and an active phase at about -40 mV giving rise to a train of spikes. TEA abolishes this pattern and very much reduces the graded response of spike frequency normally seen with different concentrations of glucose. 5. Addition of glucose in the presence of up to 20 mM-TEA induces an increase in membrane resistance of about 4.10(7) omega. 6. TEA lowers the glucose level required to trigger the electrical activity from about 5.6 to about 4.6 mM. 7. TEA blocks the repolarization phase of action potentials induced by the addition of glucose or by depolarizing intracellular current injection. 8. In the presence of 11.1 mM-glucose and 20 mM-TEA the action potentials frequently crossed the zero line, the membrane potential reaching up to 25 mV during the peak of the spikes.

Potassium permeability activated by intracellular calcium ion concentration in the pancreatic beta-cell.

1. Membrane potentials and input resistance were measured in beta-cells from mouse pancreatic islets of Langerhans in a study designed to assess the role of a K permeability specifically blocked by quinine or quinidine and activated by intracellular calcium ion concentration ([Ca2+])i-activated PK). 2. Addition of 100 microM-quinine to the perifusion medium resulted in a 10--30 mV depolarization of the membrane and an increase in the input resistance of ca. 4.10(7) omega. 3. In the absence of glucose, 100 microM-quinine induced electrical activity. 4. In the presence of glucose, 100 microM-quinine abolished the burst pattern of electrical activity and very much reduced the graded response of spike frequency normally seen with different concentrations of glucose. 5. Addition of mitochondrial inhibitors, KCN, NaN3, DNP, CCCP, FCCP, to the perifusion medium containing glucose rapidly hyperpolarized the beta-cell membrane, inducing a concomitant decrease in input resistance. 6. In the presence of glucose, these mitochondrial inhibitors reversibly blocked electrical activity; upon removal of the inhibitor, recovery of electrical activity followed a biphasic pattern. 7. The effects of mitochondrial inhibitors were partially reversed by 100 microM-quinine. 8. It is proposed that the membrane potential of the beta-cell in the absence of glucose is predominantly controlled by the [Ca2+]i-activated PK. It is further suggested that this permeability to K controls the level for glucose stimulation and leads to the generation of the burst pattern.