Pulsatile Insulin Release Research Articles

Subcellular Compartmentalization of Glucose Mediated Insulin Secretion

Regulation of blood glucose levels depends on the property of beta cells to couple glucose sensing with insulin secretion. This is accomplished by the concentration-dependent flux of glucose through glycolysis and oxidative phosphorylation, generating ATP. The resulting rise in cytosolic ATP/ADP inhibits KATP channels, inducing membrane depolarization and Ca2+ influx, which prompts insulin secretion. Evidence suggests that this coupling of glucose sensing with insulin secretion may be compartmentalized in the submembrane regions of the beta cell. We investigated the subcellular responses of key components involved in this coupling and found mitochondria in the submembrane zone, some tethered to the cytoskeleton near capillaries. Using Fluorescent Lifetime Imaging Microscopy (FLIM), we observed that submembrane mitochondria were the fastest to respond to glucose. In the most glucose-responsive beta cells, glucose triggers rapid, localized submembrane increases in ATP and Ca2+ as synchronized ~4-min oscillations, consistent with pulsatile insulin release after meals. These findings are consistent with the hypothesis that glucose sensing is coupled with insulin secretion in the submembrane zone of beta cells. This zonal adaptation would enhance both the speed and energy efficiency of beta cell responses to glucose, as only a subset of the most accessible mitochondria would be required to trigger insulin secretion.

Periodic insulin stimulation of Akt: Dynamic steady states and robustness

The periodic secretion of insulin is a salient feature of the blood glucose control system in vivo. Insulin levels in the blood exhibit oscillations on multiple time scales – rapid, ultradian, and circadian – and the improved metabolic regulation resulting from pulsatile insulin release has been well established. Although numerous mathematical models investigating the causal mechanisms of insulin oscillations have appeared in the literature, to date there has been comparatively little attention given to the influence of periodic insulin stimulation on downstream components of the insulin signalling pathway.In this paper, we explore the effect of high frequency periodic insulin stimulation on Akt (also known as PKB), a crucial crosstalk node in the insulin signalling pathway that coordinates metabolic and mitogenic processes in the cell. We analyse a mathematical model of Akt translocation to the plasma membrane under both single step insulin perturbations and periodic insulin stimulation with an emphasis on – but not limited to – the physiological range of parameter values.It was shown that the system rapidly attains a robust dynamic steady state entrained to the periodic insulin stimulation. Moreover, the translocation of Akt to the plasma membrane in the model permits a sufficient level of phosphorylation to trigger downstream metabolic regulators. However, the modelling also indicated that further investigation of this activation process is required to determine whether the response of Akt is a key determinant of the enhanced metabolic control observed under periodic insulin stimulation.

Are physiological oscillations physiological?

Despite widespread and striking examples of physiological oscillations, their functional role is often unclear. Even glycolysis, the paradigm example of oscillatory biochemistry, has seen questions about its oscillatory function. Here, we take a systems approach to argue that oscillations play critical physiological roles, such as enabling systems to avoid desensitization, to avoid chronically high and therefore toxic levels of chemicals, and to become more resistant to noise. Oscillation also enables complex physiological systems to reconcile incompatible conditions such as oxidation and reduction, by cycling between them, and to synchronize the oscillations of many small units into one large effect. In pancreatic β-cells, glycolytic oscillations synchronize with calcium and mitochondrial oscillations to drive pulsatile insulin release, critical for liver regulation of glucose. In addition, oscillation can keep biological time, essential for embryonic development in promoting cell diversity and pattern formation. The functional importance of oscillatory processes requires a re-thinking of the traditional doctrine of homeostasis, holding that physiological quantities are maintained at constant equilibrium values, a view that has largely failed in the clinic. A more dynamic approach will initiate a paradigm shift in our view of health and disease. A deeper look into the mechanisms that create, sustain and abolish oscillatory processes requires the language of nonlinear dynamics, well beyond the linearization techniques of equilibrium control theory. Nonlinear dynamics enables us to identify oscillatory ('pacemaking') mechanisms at the cellular, tissue and system levels.

Visually controlled pulsatile release of insulin from chitosan poly-acrylic acid nanobubbles triggered by focused ultrasound

Insulin therapy is the most effective way to control the blood glucose value of diabetic patients. The most effective administration route for insulin is subcutaneous injection because bioavailability for non-injection administration is low and unstable. However, patients often need a multiple daily insulin injection regimen to control basal and postprandial blood glucose, which causes various complications. Controlled pulsatile drug release technology using ultrasound as an external stimulus source is a very promising method to avoid multiple injections of insulin. However, most of the drug-loaded microbubbles used for ultrasound-mediated treatment have a short half-life, which limits their use in controlled pulsatile drug release. More importantly, how to control insulin release is still a challenge. In this paper, chitosan poly-acrylic acid nanobubbles as drug carriers of insulin were prepared to achieve a visually controlled pulsatile release of insulin triggered by focused ultrasound. The experimental results in vivo demonstrated that nanobubbles were stable enough to achieve long-term visualization for 7 days after intramuscular injection in rats. Under the guidance of ultrasound imaging, it is visible to find the position and observe the gray values change of nanobubbles. Thus, when triggered by focused ultrasound, the amount of insulin could be accurately pulsatile released from nanobubbles. In vivo experiments in rats showed that the visually controlled pulsatile release of insulin could be achieved for a long time, up to 3 consecutive days. The blood glucose level could be repeatedly reduced by focused ultrasound irradiation with just one injection. Our research provided a promising way for visually controlled pulsatile release of insulin, which would significantly reduce the injection frequency of insulin.

1748-P: Assessing the Presence of Delta-Cell–Mediated Electrical Coupling in the Islet

Somatostatin-producing delta cells play an important role in the regulation of paracrine cross talk in the islet through inhibition of insulin and glucagon secretion by beta and alpha cells, respectively. However, recent studies have suggested the presence of electrical coupling via gap junctions between delta and beta cells. Electrical coupling mediated cross-talk would support the correlation between pulsatile insulin and somatostatin release and reveal a structural source of cell-cell communication within the islet. To test whether beta and delta cells are gap junction coupled, we measured the permeability of cell to cell connections and the relative dynamics of Ca2+. Islets of Sst-Cre;Rosa-LSL-tdTomato mice were used which show delta-cell specific labelling. They were then loaded with the cationic dye rhodamine-123 at 5mM glucose to measure performance of fluorescence recovery after photobleaching to assess gap junction coupling of delta cells and non-delta cells. Calcium dynamics were assessed using calcium dye Fluo-4 upon stimulating the islet from low glucose (2mM or 5 mM) to high glucose(11mM). No significant differences between recovery rate were observed when comparing delta cells and beta cells (p>0.05), which indicates some level of gap junction permeability among delta cells exists. The calcium activity of a population of delta cells was similar to those of neighboring beta cells, indicating a functional connection exists between these cell types. Furthermore, first-responder beta cells showed a preferential proximity to delta cells suggesting an impact of delta cells on functional sub-populations of nearby beta-cells. These results suggest a possible feedback system exists in the islet that is governed by electrical coupling of delta cells, which is important for both the function of beta cells and insulin secretion, and may also impact secretion of other islet hormones. Disclosure C.H.Levitt: None. R.K.Benninger: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (P30DK116073, R01DK102950, R01DK106412)

Synchronisation of glycolytic activity in yeast cells.

Glycolysis is the central metabolic pathway of almost every cell and organism. Under appropriate conditions, glycolytic oscillations may occur in individual cells as well as in entire cell populations or tissues. In many biological systems, glycolytic oscillations drive coherent oscillations of other metabolites, for instance in cardiomyocytes near anorexia, or in pancreas where they lead to a pulsatile release of insulin. Oscillations at the population or tissue level require the cells to synchronize their metabolism. We review the progress achieved instudying a model organism for glycolytic oscillations, namely yeast. Oscillations may occur on the level of individual cells as well as on the level of the cell population. In yeast, the cell-to-cell interaction is realized by diffusion-mediated intercellular communication via a messenger molecule. The present mini-review focuses on the synchronisation of glycolytic oscillations in yeast. Synchronisation is a quorum-sensing phenomenon because the collective oscillatory behaviour of a yeast cell population ceases when the cell density falls below a threshold. We review the question, under which conditions individual cells in a sparse population continue or cease to oscillate. Furthermore, we provide an overview of the pathway leading to the onset of synchronized oscillations. We also address the effects of spatial inhomogeneities (e.g., the formation of spatial clusters) on the collective dynamics, and also review the emergence of travelling waves of glycolytic activity. Finally, we briefly review the approaches used in numerical modelling of synchronized cell populations.

Thermodynamic Optimality of Glycolytic Oscillations.

Temporal order in living matters reflects the self-organizing nature of dynamical processes driven out of thermodynamic equilibrium. Because of functional reasons, the period of a biochemical oscillation must be tuned to a specific value with precision; however, according to the thermodynamic uncertainty relation (TUR), the precision of the oscillatory period is constrained by the thermodynamic cost of generating it. After reviewing the basics of chemical oscillations using the Brusselator as a model system, we study the glycolytic oscillation generated by octameric phosphofructokinase (PFK), which is known to display a period of several minutes. By exploring the phase space of glycolytic oscillations, we find that the glycolytic oscillation under the cellular condition is realized in a cost-effective manner. Specifically, over the biologically relevant range of parameter values of glycolysis and octameric PFK, the entropy production from the glycolytic oscillation is minimal when the oscillation period is (5-10) min. Furthermore, the glycolytic oscillation is found at work near the phase boundary of limit cycles, suggesting that a moderate increase of glucose injection rate leads to the loss of oscillatory dynamics, which is reminiscent of the loss of pulsatile insulin release resulting from elevated blood glucose level.

Reducing Glucokinase Activity Restores Endogenous Pulsatility and Enhances Insulin Secretion in Islets From db/db Mice.

An early sign of islet failure in type 2 diabetes (T2D) is the loss of normal patterns of pulsatile insulin release. Disruptions in pulsatility are associated with a left shift in glucose sensing that can cause excessive insulin release in low glucose (relative hyperinsulinemia, a hallmark of early T2D) and β-cell exhaustion, leading to inadequate insulin release during hyperglycemia. Our hypothesis was that reducing excessive glucokinase activity in diabetic islets would improve their function. Isolated mouse islets were exposed to glucose and varying concentrations of the glucokinase inhibitor d-mannoheptulose (MH) to examine changes in intracellular calcium ([Ca2+]i) and insulin secretion. Acutely exposing islets from control CD-1 mice to MH in high glucose (20 mM) dose dependently reduced the size of [Ca2+]i oscillations detected by fura-2 acetoxymethyl. Glucokinase activation in low glucose (3 mM) had the opposite effect. We then treated islets from male and female db/db mice (age, 4 to 8 weeks) and heterozygous controls overnight with 0 to 10 mM MH to determine that 1 mM MH produced optimal oscillations. We then used 1 mM MH overnight to measure [Ca2+]i and insulin simultaneously in db/db islets. MH restored oscillations and increased insulin secretion. Insulin secretion rates correlated with MH-induced increases in amplitude of [Ca2+]i oscillations (R2 = 0.57, P < 0.01, n = 10) but not with mean [Ca2+]i levels in islets (R2 = 0.05, not significant). Our findings show that correcting glucose sensing can restore proper pulsatility to diabetic islets and improved pulsatility correlates with enhanced insulin secretion.

Somatostatin promotes glucose generation of Ca2+oscillations in pancreatic islets both in the absence and presence of tolbutamide

Many cellular processes, including pulsatile release of insulin, are triggered by increase of cytoplasmic Ca2+. This study examines how somatostatin affects glucose generation of cytoplasmic Ca2+ oscillations in mouse islets in absence and presence of tolbutamide blockade of the KATP channels. Ca2+ was measured with dual wavelength microflurometry in isolated islets loaded with the indicator Fura-2. Rise of glucose from 3 to 20 mM evoked introductory lowering of Ca2+ prolonged by activation of somatostatin receptors. During continued superfusion exposure to somatostatin triggered oscillations mediated by periodic increase from the basal level (absence of tolbutamide) or by periodic interruption of an elevated level (presence of tolbutamide). In the latter situation the oscillations were transformed into sustained elevation by activation of muscarinic receptors (acetylcholine) or increase of cyclic AMP (IBMX, 8-bromo-cyclic AMP, forskolin). The observed effect of cyclic AMP raises the question whether high proportions of the glucagon-producing α-cells promote steady-state elevation of Ca2+. In support for this idea somatostatin was found to trigger glucose-induced Ca2+ oscillations essentially in small islets that contain very few α-cells. The results indicate that somatostatin promotes glucose generation of Ca2+oscillations with similar characteristics both in the absence and presence of functional KATP channels.

Ultrafast glucose-responsive, high loading capacity erythrocyte to self-regulate the release of insulin

Insulin (INS) delivery system that can mimic normal insulin secretion to maintain the blood glucose level (BGL) in the normal range is an ideal treatment for diabetes. However, most of the existing closed-loop INS delivery systems respond slowly to the changes in BGL, resulting in a time lag between the abnormal BGL and the release of INS, which is not suitable for practical application. In this study, glucose oxidase (GOx)-modified erythrocytes are used as INS carriers (GOx-INS-ER) that can rapidly self-regulate the release of INS upon the changes in BGL. In this system, glucose can be broken down into gluconic acid and hydrogen peroxide by GOx-INS-ER, and the latter will rupture the erythrocyte membrane to release INS within minutes. A pulsatile release of INS can be achieved upon the changes in the glucose concentration. This GOx-INS-ER enables diabetic rats to overcome hyperglycemia within 1 h, and a single injection of this GOx-INS-ER into the STZ-induced diabetic rats can maintain the BGL in the normal range up to 9 days. Statement of SignificanceDiabetes mellitus has been a major public health threatener with global prevalence. Although, glucose-responsive carriers that can release insulin (INS) in a closed loop have been explored greatly in recent years, their sluggish glucose-responsive property and low INS-loading content greatly restrict their practical application [ACS Nano, 2013, 7, 4194].In this work, we reported INS-loaded erythrocytes featuring ultrafast glucose-responsive property and high INS loading content, which could release INS in a closed loop. These GOX-INS-ERs could respond to the changes in glucose level within several minutes and self-regulate the release of INS for a long time. Single injection of GOX-INS-ER can overcome hyperglycemia in diabetic mice within 1 h and maintain the baseline level of glucose up to 9 days. We think our method may provide a robust way to potentiate diabetes treatment.

A mathematical model of the impact of insulin secretion dynamics on selective hepatic insulin resistance

Physiological insulin secretion exhibits various temporal patterns, the dysregulation of which is involved in diabetes development. We analyzed the impact of first-phase and pulsatile insulin release on glucose and lipid control with various hepatic insulin signaling networks. The mathematical model suggests that atypical protein kinase C (aPKC) undergoes a bistable switch-on and switch-off, under the control of insulin receptor substrate 2 (IRS2). The activation of IRS1 and IRS2 is temporally separated due to the inhibition of IRS1 by aPKC. The model further shows that the timing of aPKC switch-off is delayed by reduced first-phase insulin and reduced amplitude of insulin pulses. Based on these findings, we propose a sequential model of postprandial hepatic control of glucose and lipid by insulin, according to which delayed aPKC switch-off contributes to selective hepatic insulin resistance, which is a long-standing paradox in the field.

A Ratiometric Sensor for Imaging Insulin Secretion in Single β Cells

SummaryDespite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 β cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in β cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse β cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated β cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.

Ultrasound-triggered noninvasive regulation of blood glucose levels using microgels integrated with insulin nanocapsules

Diabetes is a serious public health problem affecting 422 million people worldwide. Traditional diabetes management often requires multiple daily insulin injections, associated with pain and inadequate glycemia control. Herein, we have developed an ultrasound-triggered insulin delivery system capable of pulsatile insulin release that can provide both long-term sustained and fast on-demand responses. In this system, insulin-loaded poly(lactic-co-glycolic acid) (PLGA) nanocapsules are encapsulated within chitosan microgels. The encapsulated insulin in nanocapsules can passively diffuse from the nanoparticle but remain restricted within the microgel. Upon ultrasound treatment, the stored insulin in microgels can be rapidly released to regulate blood glucose levels. In a chemically-induced type 1 diabetic mouse model, we demonstrated that this system, when activated by 30 s ultrasound administration, could effectively achieve glycemic control for up to one week in a noninvasive, localized, and pulsatile manner.

How Kiss-and-Run Can Make Us Sick: SOX4 Puts a Break on the Pore.

Insulin is released by regulated exocytosis of secretory granules. At the heart of this process is a tight complex of three SNARE proteins (SNAP25 and syntaxin in the plasma membrane and synaptobrevin in the vesicle membrane) that forms during exocytosis (1). The complex pulls the two opposing membranes close enough to enable formation of a narrow fusion pore (∼1.5 nm), a proteolipidic structure that behaves somewhat similar to an ion channel (2). Owing to its small size, the pore essentially acts as a molecular sieve that allows passage of small molecules but prevents release of the larger peptide hormones (3,4) (Fig. 1 A ). Once formed, the fusion pore can then either expand irreversibly (“full fusion,” required for insulin secretion) or revert to the closed state (“kiss-and-run”) (5). The sieve effect is of particular interest in β-cells as insulin granules contain many peptides, nucleotides, and amino acids that are cosecreted with insulin and have their own signaling functions (6). Purines, in particular ATP, stimulate both insulin secretion and β-cell survival via P2X and P2Y receptors (7), and the resulting autocrine and paracrine feedback may be involved in synchronizing pulsatile insulin release (8). Glutamate stimulates and GABA inhibits glucagon release …

Submembrane ATP and Ca2+ kinetics in α-cells: unexpected signaling for glucagon secretion.

Cytoplasmic ATP and Ca2+ are implicated in current models of glucose’s control of glucagon and insulin secretion from pancreatic α- and β-cells, respectively, but little is known about ATP and its relation to Ca2+ in α-cells. We therefore expressed the fluorescent ATP biosensor Perceval in mouse pancreatic islets and loaded them with a Ca2+ indicator. With total internal reflection fluorescence microscopy, we recorded subplasma membrane concentrations of Ca2+ and ATP ([Ca2+]pm; [ATP]pm) in superficial α- and β-cells of intact islets and related signaling to glucagon and insulin secretion by immunoassay. Consistent with ATP’s controlling glucagon and insulin secretion during hypo- and hyperglycemia, respectively, the dose-response relationship for glucose-induced [ATP]pm generation was left shifted in α-cells compared to β-cells. Both cell types showed [Ca2+]pm and [ATP]pm oscillations in opposite phase, probably reflecting energy-consuming Ca2+ transport. Although pulsatile insulin and glucagon release are in opposite phase, [Ca2+]pm synchronized in the same phase between α- and β-cells. This paradox can be explained by the overriding of Ca2+ stimulation by paracrine inhibition, because somatostatin receptor blockade potently stimulated glucagon release with little effect on Ca2+. The data indicate that an α-cell-intrinsic mechanism controls glucagon in hypoglycemia and that paracrine factors shape pulsatile secretion in hyperglycemia.—Li, J., Yu, Q., Ahooghalandari, P., Gribble, F. M., Reimann, F., Tengholm, A., Gylfe, E. Submembrane ATP and Ca2+ kinetics in α-cells: unexpected signaling for glucagon secretion.

Neurotransmitter control of islet hormone pulsatility.

Pulsatile secretion is an inherent property of hormone-releasing pancreatic islet cells. This secretory pattern is physiologically important and compromised in diabetes. Neurotransmitters released from islet cells may shape the pulses in auto/paracrine feedback loops. Within islets, glucose-stimulated β-cells couple via gap junctions to generate synchronized insulin pulses. In contrast, α- and δ-cells lack gap junctions, and glucagon release from islets stimulated by lack of glucose is non-pulsatile. Increasing glucose concentrations gradually inhibit glucagon secretion by α-cell-intrinsic mechanism/s. Further glucose elevation will stimulate pulsatile insulin release and co-secretion of neurotransmitters. Excitatory ATP may synchronize β-cells with δ-cells to generate coinciding pulses of insulin and somatostatin. Inhibitory neurotransmitters from β- and δ-cells can then generate antiphase pulses of glucagon release. Neurotransmitters released from intrapancreatic ganglia are required to synchronize β-cells between islets to coordinate insulin pulsatility from the entire pancreas, whereas paracrine intra-islet effects still suffice to explain coordinated pulsatile release of glucagon and somatostatin. The present review discusses how neurotransmitters contribute to the pulsatility at different levels of integration.

On the coherent behavior of pancreatic beta cell clusters

Beta cells in pancreas represent an example of coupled biological oscillators which via communication pathways, are able to synchronize their electrical activity, giving rise to pulsatile insulin release. In this work we numerically analyze scale free self-similarity features of membrane voltage signal power density spectrum, through a stochastic dynamical model for beta cells in the islets of Langerhans fine tuned on mouse experimental data. Adopting the algebraic approach of coherent state formalism, we show how coherent molecular domains can arise from proper functional conditions leading to a parallelism with "phase transition" phenomena of field theory.

Thermoresponsive biodegradable PEG‐PCL‐PEG based injectable hydrogel for pulsatile insulin delivery

An injectable biodegradable hydrogel was prepared for temperature-responsive pulsatile release of insulin. Triblock copolymer of poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) was prepared by ring opening bulk copolymerization and characterized using FT-IR, (1) HNMR, and gel permeation chromatography. Aqueous solution of PECE formed an injectable hydrogel, which was solution at room temperature and transformed into gel at 37°C. The temperature-responsive sol-gel transition and crystallinity of PECE hydrogel was studied and compared with pluronic, a well-studied nonbiodegradable injectable hydrogel. In vitro release study revealed that insulin release profile of PECE was similar to pluronic, and its viscosity was 1/30(th) of pluronic sol at 10,000 s(-1) shear rate. Release behavior of insulin from PECE hydrogels followed Fickian diffusion of first order. Insulin retained its secondary structure after release as confirmed by circular dichroism spectrum. A threefold increase in Fickian diffusion coefficient was evidenced when temperature was increased from 34 to 40°C because of crystalline melting of PCL part of PECE. Pulsatile release of insulin showed a correlation coefficient of 0.90 with the change of temperature.

P2Y1receptor‐dependent diacylglycerol signaling microdomains in β cells promote insulin secretion

Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic β cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 ± 0.4 s) but high-amplitude elevations (up to 109 ± 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y₁, but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.

The In Vivo β-to-β-Cell Chat Room: Connexin Connections Matter

Proper glycemic control requires that numerous β-cells are coordinated to rapidly, but temporarily, secrete insulin after meals. This coordination integrates hundreds of β-cells within each islet into a functionally homogeneous unit. This homogeneity occurs despite the intrinsic heterogeneity of individual β-cells (1). Fine-tuning of this coordination is further required because of the cyclic oscillations that ionic, metabolic, and effector events undergo within each islet, as well as among the many islets of a pancreas. Together, these events ensure normal pulsatility of insulin secretion (2,3). Many mechanisms have evolved over time to achieve this coordination (1–4). One such mechanism is cell-to-cell coupling, the process by which contiguous cells exchange cytosolic molecules (Fig. 1) via channels made of connexin (Cx) proteins. These proteins concentrate at gap junction domains of the cell membrane (4). FIG. 1. In control islets, Cx36 channels allow for the exchange of electrotonic currents and cytosolic molecules between coupled β-cells. Islets null for Cx36 no longer feature this coupling, which causes the loss of the intercellular synchronization of glucose-induced Ca2+ transients and of pulsatile insulin release, as well as an extended off-response of β-cells. These alterations are associated with a decrease in the expression of the Ins genes and in both first and second phases of GSIS, which result in impaired glucose tolerance of the Cx36-null mice. It is still unclear (?) whether loss of Cx36 also sufficiently raises the basal secretion of insulin to alter the circulating levels of the hormone. Whether similar changes also take place after the temporary uncoupling of β-cells, resulting from the closure of Cx36 channels, is still largely undetermined (nd). Many aspects of β-cell function also remain to be investigated in …