Heléne Dansk

Glucose Generates Coincident Insulin and Somatostatin Pulses and Antisynchronous Glucagon Pulses from Human Pancreatic Islets

The kinetics of insulin, glucagon and somatostatin release was studied in human pancreatic islets. Batches of 10-15 islets were perifused and the hormones measured with RIA in 30-sec fractions. Increase of glucose from 3 to 20 mm resulted in a brief pulse of glucagon coinciding with suppression of basal insulin and somatostatin release. There was a subsequent drop of glucagon release concomitant with the appearance of a pronounced pulse of insulin and a slightly delayed pulse of somatostatin. Continued exposure to 20 mm glucose generated pulsatile release of the three hormones with 7- to 8-min periods accounting for 60-70% of the secreted amounts. Glucose caused pronounced stimulation of average insulin and somatostatin release. However, the nadirs between the glucagon pulses were lower than the secretion at 3 mm glucose, resulting in 18% suppression of average release. The repetitive glucagon pulses were antisynchronous to coincident pulses of insulin and somatostatin. The resulting greater than 20-fold variations of the insulin to glucagon ratio might be essential for minute-to-minute regulation of the hepatic glucose production.

Nitric oxide induces synchronous Ca2+ transients in pancreatic beta cells lacking contact.

Aims To evaluate the role of nitric oxide (NO) in the coordination of the Ca 2+ signals generating pulsatile insulin release in pancreatic &bgr; cells isolated from ob/ob mice. Methodology Using ratiometric fura-2 technique for recording glucose-induced cytoplasmic Ca 2+ transients, it was possible to demonstrate a synchronization of &bgr; cells lacking contact. Results The frequency of the transients increased 10-fold in the presence of 20 n M glucagon. Additional increase in frequency with maintenance of synchronization was observed when the &bgr; cells were exposed to 100 &mgr;M of the NO donors sodium nitroprusside and hydroxylamine. Bolus additions of 0.1–10 &mgr;M gaseous NO resulted in prompt appearance of cytoplasmic Ca 2+ transients. An activator of soluble guanylate cyclase (mesoporphyrin) increased the frequency of the transients, and inhibition of this enzyme with 1H-(1,2,4) oxadiazolo [4,3-a] quinoxalin-1-one had the opposite effect. Conclusion The results support the idea that nitrergic nerves generate &bgr;-cell transients of Ca 2+ synchronizing the activity of the numerous islets in the pancreas.

Signaling Underlying Pulsatile Insulin Secretion

Regular oscillations of the circulating insulin concentrations were discovered in the monkey [28] and subsequently found in normal human subjects [50]. The characteristic insulin pattern is deteriorated in patients with type 2 diabetes [49] as well as in their close relatives [6 11. Studies in non-diabetic subjects with suppressed endogenous insulin secretion and diabetic patients have indicated that less insulin is required to maintain normoglycaemia if the hormone is infused in a pulsatile manner compared to a constant rate [12, 57, 59, 63, 641. This difference is probably explained by higher expression of insulin receptors, when insulin is delivered in pulses [27]. It is easy to envision a scenario for the development of type 2 diabetes in which deteriorated oscillations leads to insulin resistance with a compensating hypersecretion of the hormone. In susceptible individuals the increased insulin demand may eventually exhaust the pancreatic p-cells with resulting development of overt diabetes. What is then the origin of the regular insulin oscillations? One possibility is that they result from a negative feedback loop between the liver and the pancreatic pcell [50]. However, later studies have indicated that the oscillations occur independent of changes in plasma glucose, reflecting a pacemaker function in the pancreas [49, 581. This conclusion is consistent with measurements of secretion from the isolated perfused dog pancreas [76]. Another fundamental aspect is the frequency of the insulin oscillations. Whereas the early studies on humans and monkeys indicated a periodicity of 10-15 min [28, 501, measurements in the dog showed 4-8 min. The latter estimate is similar to the periodicity observed from the perfused dog pancreas [75] and that based on blood sampling from the portal vein of dogs [66]. The portal insulin oscillations are very prominent, indicating that pulsatile secretion accounts for 70% of total secretion (Fig. 1). In the periphery the oscillations are less pronounced due to recirculation and the fact that the liver extracts almost 50% of the portal hormone [ 131. The report of a lower frequency of the insulin oscillations in the peripheral blood may simply reflect difficulties in detecting insulin peaks due to a low signal-to-noise ratio [66]. Indeed, measurements on portal blood from patients with liver cirrhosis indicated a periodicity of 4.1-6.5 min [77]. Mechanisms underlying pulsatile insulin release will now be discussed at different levels of integration, starting with isolated pancreatic p-cells.

Synchronization of pancreatic β-cell rhythmicity after glucagon induction of Ca2+ transients

Pancreatic beta-cells are biological oscillators requiring a coupling force for the synchronization of the cytoplasmic Ca(2+) oscillations responsible for pulsatile insulin release. Testing the idea that transients, superimposed on the oscillations, are important for this synchronization, the concentration of cytoplasmic Ca(2+) ([Ca(2+)](i)) was measured with ratiometric fura-2 technique in single beta-cells and small aggregates prepared from islets isolated from ob/ob-mice. Image analyses revealed asynchronous [Ca(2+)](i) oscillations in adjacent beta-cells lacking physical contact. The addition of glucagon stimulated the firing of [Ca(2+)](i) transients, which appeared in synchrony in adjacent beta-cells. Moreover, the presence of glucagon promoted synchronization of the [Ca(2+)](i) oscillations in beta-cells separated by a distance <100 microm but not in those >200 microm apart. The results support the proposal that the repolarizing effect of [Ca(2+)](i) transients provides a coupling force for co-ordinating the pulses of insulin release generated by pancreatic beta-cells.

The neurotransmitter ATP triggers Ca2+ responses promoting coordination of pancreatic islet oscillations.

Objectives Pulsatile insulin release into the portal vein is critically dependent on entrainment of the islets in the pancreas into a common oscillatory phase. Because the pulses reflect periodic variations of the cytoplasmic Ca2+ concentration ([Ca2+]i), we studied whether the neurotransmitters adenosine triphosphate (ATP) and acetylcholine promote synchronization of [Ca2+]i oscillations between islets lacking contact. Methods Medium-sized and small mouse islets and cell aggregates were used for measuring [Ca2+]i with the indicator fura-2. Results Exposure to acetylcholine resulted in an initial [Ca2+]i peak followed by disappearance of the [Ca2+]i oscillations induced by 11-mmol/L glucose. The effect of ATP was often restricted to an elusive [Ca2+]i peak. The incidence of distinct [Ca2+]i responses to ATP increased under conditions (accelerated superfusion, small islets, or cell aggregates) intended to counteract purinoceptor desensitization owing to intercellular accumulation of ATP. Attempts to imitate neural activity by brief (15 seconds) exposure to ATP or acetylcholine resulted in temporary synchronization of the glucose-induced [Ca2+]i oscillations between islets lacking contact. Conclusions The data support the idea that purinergic signaling has a key role for coordinating the oscillatory activity of the islets in the pancreas, reinforcing previous arguments for the involvement of nonadrenergic, noncholinergic neurons.

Stretch activation of Ca2+ transients in pancreatic β cells by mobilization of intracellular stores

Introduction Nonadrenergic, noncholinergic neurons have been proposed to synchronize pulsatile insulin release from the islets in the pancreas by triggering transient increases of the cytoplasmic Ca2+ concentration ([Ca2+]i) in &bgr;-cells via an inositol trisphoshate-dependent mechanism. Aims To test whether pancreatic &bgr;-cells respond to stretch activation with similar types of transients and whether these Ca2+ signals propagate to other &bgr;-cells in the presence and absence of cell contacts. Methodology Single cells and small aggregates were prepared from &bgr;-cell–rich islets from ob/ob mice. After 2–5 days of culture, [Ca2+]i was measured with digital imaging and the indicator fura-2 during superfusion with a medium containing 20 mmol/L glucose and 50 &mgr;mol/L methoxyverapamil. Membrane stretch was induced by osmotic swelling or focal touch stimulation. Results Lowering the medium osmolarity with 100–102 mOSM/L by removal of sucrose or by dilution resulted in a 2–3-fold increase in the number of transients during an initial 5-minute period. Sucrose omission was stimulatory also after isosmolar replacement with readily penetrating urea. The intracellular Ca2+-ATPase inhibitor thapsigargin suppressed both the spontaneously occurring transients and those initiated by volume expansion. Touch stimuli induced [Ca2+]i transients, which rapidly propagated to cells within the same aggregate or lacking contact. Conclusion The observations support the idea that &bgr;-cells both receive and regenerate extracellular signals triggering [Ca2+]i transients. Touch stimulation is a useful tool for investigating the propagation of [Ca2+]i signals between pancreatic &bgr;-cells lacking physical contact.

Activation of alpha adrenergic and muscarinic receptors modifies early glucose suppression of cytoplasmic Ca2+ in pancreatic β-cells

Elevation of glucose induces transient inhibition of insulin release by lowering cytoplasmic Ca(2+) ([Ca(2+)]i) below baseline in pancreatic β-cells. The period of [Ca(2+)]i decrease (phase 0) coincides with increased glucagon release and is therefore the starting point for antisynchronous pulses of insulin and glucagon. We now examine if activation of adrenergic α2A and muscarinic M3 receptors affects the initial [Ca(2+)]i response to increase of glucose from 3 to 20mM in β-cells situated in mouse islets. In the absence of receptor stimulation the elevation of glucose lowered [Ca(2+)]i during 90-120 s followed by rise due to opening of voltage-dependent Ca(2+) channels. The period of [Ca(2+)]i decrease was prolonged by activation of the α2A adrenergic receptors (1 μM epinephrine or 100 nM clonidine) and shortened by stimulation of the muscarinic M3 receptors (0.1 μM acetylcholine). The latter effect was mimicked by the Na/K pump inhibitor ouabain (10-100 μM). The results indicate that prolonged initial decrease (phase 0) is followed by slow [Ca(2+)]i rise and shorter decrease followed by fast rise. It is concluded that the period of initial decrease of [Ca(2+)]i regulates the subsequent β-cell response to glucose.

Sulfonylurea blockade of KATP channels unmasks a distinct type of glucose-induced Ca2+ decrease in pancreatic β-cells

Objectives This study aimed to explore how sulfonylurea blockade of KATP channels affects the early Ca2+ signals for glucose generation of insulin release. Methods Cytoplasmic Ca2+ was measured with ratiometric microfluorometry in isolated mouse islets loaded with Fura-PE3. Results After sulfonylurea blockade of the KATP channels (50 &mgr;M-1 mM tolbutamide or 1 &mgr;M-1 mM gliclazide), increase of glucose from 3 to 20 mM resulted in suppression of elevated Ca2+ during a 3- to 5-minute period. The Ca2+ decrease was shorter after inhibition of the Na/K pump with ouabain (10 and 100 &mgr;M) but prolonged when the &agr;2A adrenoceptors were activated with clonidine (1 and 10 nM) or epinephrine (10 nM). Inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase pump with 10 &mgr;M cyclopiazonic acid counteracted the action of 10 nM clonidine, making the Ca2+ decrease shorter than in controls. Extended superfusion of islets with a medium containing 20 mM glucose and 1 mM tolbutamide sometimes resulted in delayed appearance of Ca2+ oscillations mediated by periodic interruption of elevated Ca2+. Conclusions Increase of glucose generates prompt suppression of cytoplasmic Ca2+ in &bgr;-cells lacking functional KATP channels. Activation of &agr;2A adrenoceptors markedly prolongs the period of glucose-induced Ca2+ decrease, an effect counteracted by cyclopiazonic acid.

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.