Time and amplitude regulation of pulsatile insulin secretion by triggering and amplifying pathways in mouse islets.

Glucose-induced insulin secretion is pulsatile. We investigated how the triggering pathway (rise in beta-cell [Ca(2+)](i)) and amplifying pathway (greater Ca(2+) efficacy on exocytosis) influence this pulsatility. Repetitive [Ca(2+)](i) pulses were imposed by high K(+)+ diazoxide in single mouse islets. Insulin secretion (measured simultaneously) tightly followed [Ca(2+)](i) changes. Lengthening [Ca(2+)](i) pulses increased the duration but not the amplitude of insulin pulses. Increasing glucose (5-20 mmol/l) augmented the amplitude of insulin pulses without changing that of [Ca(2+)](i) pulses. Larger [Ca(2+)](i) pulses augmented the amplitude of insulin pulses at high, but not low glucose. In conclusion, the amplification pathway ensures amplitude modulation of insulin pulses whose time modulation is achieved by the triggering pathway

Imaging a target of Ca2+ signalling: Dense core granule exocytosis viewed by total internal reflection fluorescence microscopy

Ca2+ ions are the most ubiquitous second messenger found in all cells, and play a significant role in controlling regulated secretion from neurons, endocrine, neuroendocrine and exocrine cells. Here, we describe microscopic techniques to image regulated secretion, a target of Ca2+ signalling. The first of these, total internal reflection fluorescence (TIRF), is well suited for optical sectioning at cell–substrate regions with an unusually thin region of fluorescence excitation (<150 nm). It is thus particularly useful for studies of regulated hormone secretion. A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope. We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained. A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included

Imaging a target of Ca(2+) signalling: Dense core granule exocytosis viewed by total internal reflection fluorescence microscopy.

Ca(2+) ions are the most ubiquitous second messenger found in all cells, and play a significant role in controlling regulated secretion from neurons, endocrine, neuroendocrine and exocrine cells. Here, we describe microscopic techniques to image regulated secretion, a target of Ca(2+) signalling. The first of these, total internal reflection fluorescence (TIRF), is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation (<150nm). It is thus particularly useful for studies of regulated hormone secretion. A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope. We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained. A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included

Disorganization of cytoplasmic Ca(2+) oscillations and pulsatile insulin secretion in islets from ob/ obmice.

AIMS/HYPOTHESIS: In normal mouse islets, glucose induces synchronous cytoplasmic [Ca(2+)](i) oscillations in beta cells and pulses of insulin secretion. We investigated whether this fine regulation of islet function is preserved in hyperglycaemic and hyperinsulinaemic ob/ obmice. METHODS: Intact islets from ob/ ob mice and their lean littermates were used after overnight culture for measurement of [Ca(2+)](i) and insulin secretion. RESULTS: We observed three types of [Ca(2+)](i) responses during stimulation by 9 to 12 mmol/l of glucose: sustained increase, rapid oscillations and slow (or mixed) oscillations. They occurred in 8, 18 and 74% of lean islets and 9, 0 and 91% of ob/ ob islets, respectively. Subtle desynchronisation of [Ca(2+)](i) oscillations between regions occurred in 11% of lean islets. In ob/ ob islets, desynchronisation was frequent (66-82% depending on conditions) and prominent: oscillations were out of phase in different regions because of distinct periods and shapes. Only small ob/ ob islets were well synchronised, but sizes of synchronised lean and desynchronised ob/ ob islets were markedly overlapped. The occurrence of desynchronisation in clusters of 5 to 50 islet cells from ob/ obmice and not from lean mice further indicates that islet hypertrophy is not the only causal factor. In both types of islets, synchronous [Ca(2+)](i) oscillations were accompanied by oscillations of insulin secretion. In poorly synchronised ob/ ob islets, secretion was irregular but followed the pattern of the global [Ca(2+)](i) changes. CONCLUSIONS/INTERPRETATION: The regularity of glucose-induced [Ca(2+)](i) oscillations is disrupted in islets from ob/ ob mice and this desynchronisation perturbs the pulsatility of insulin secretion. A similar mechanism could contribute to the irregularity of insulin oscillations in Type II (non-insulin-dependent) diabetes mellitus

Dual mechanism of the potentiation by glucose of insulin secretion induced by arginine and tolbutamide in mouse islets

Glucose induces insulin secretion (IS) and also potentiates the insulin-releasing action of secretagogues such as arginine and sulfonylureas. This potentiating effect is known to be impaired in type 2 diabetic patients, but its cellular mechanisms are unclear. IS and cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse islets during perifusion with 3-15 mmol/l glucose (G3-G15, respectively) and pulse or stepwise stimulation with 1-10 mmol/l arginine or 5-250 micromol/l tolbutamide. In G3, arginine induced small increases in [Ca(2+)](i) but no IS. G7 alone only slightly increased [Ca(2+)](i) and IS but markedly potentiated arginine effects on [Ca(2+)](i), which resulted in significant IS (already at 1 mmol/l). For each arginine concentration, both responses further increased at G10 and G15, but the relative change was distinctly larger for IS than [Ca(2+)](i). At all glucose concentrations, tolbutamide dose dependently increased [Ca(2+)](i) and IS with thresholds of 25 micromol/l for [Ca(2+)](i) and 100 micromol/l for IS at G3 and of 5 micromol/l for both at G7 and above. Between G7 and G15, the effect of tolbutamide on [Ca(2+)](i) increased only slightly, whereas that on IS was strongly potentiated. The linear relationship between IS and [Ca(2+)](i) at increasing arginine or tolbutamide concentrations became steeper as the glucose concentration was raised. Thus glucose augmented more the effect of each agent on IS than that on [Ca(2+)](i). In conclusion, glucose potentiation of arginine- or tolbutamide-induced IS involves increases in both the rise of [Ca(2+)](i) and the action of Ca(2+) on exocytosis. This dual mechanism must be borne in mind to interpret the alterations of the potentiating action of glucose in type 2 diabetic patients

Oscillations of insulin secretion can be triggered by imposed oscillations of cytoplasmic Ca2+ or metabolism in normal mouse islets

Glucose-induced insulin secretion depends on an acceleration of glucose metabolism, requires a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i), and is modulated by activation of protein kinases in beta-cells. Normal mouse islets were used to determine whether oscillations of these three signals are able and necessary to trigger oscillations of insulin secretion. The approach was to minimize or abolish spontaneous oscillations and to compare the impact of forced oscillations of each signal on insulin secretion. In a control medium, repetitive increases in the glucose concentration triggered oscillations in metabolism [NAD(P)H fluorescence], [Ca2+]i (fura-PE3 method), and insulin secretion. In the presence of diazoxide, metabolic oscillations persisted, but [Ca2+]i and insulin oscillations were abolished. When the islets were depolarized with high K+ with or without diazoxide, [Ca2+]i was elevated, and insulin secretion was stimulated. Forced metabolic oscillations transiently decreased or did not affect [Ca2+]i and potentiated insulin secretion with oscillations of small amplitude. These oscillations of secretion followed metabolic oscillations only when [Ca2+]i did not change. When [Ca2+]i fluctuated, these changes prevailed over those of metabolism for timing secretion. Repetitive depolarizations with high K+ in the presence of stable glucose (10 mmol/l) induced synchronous pulses of [Ca2+]i and insulin secretion with only small oscillations of metabolism. Continuous stimulation of protein kinase A (PKA) and protein kinase C (PKC) did not dissociate the [Ca2+]i and insulin pulses from the high K+ pulses. However, the amplitude of the insulin pulses was consistently increased, whereas that of the [Ca2+]i pulses was either increased (PKA) or decreased (PKC). In conclusion, metabolic oscillations can induce oscillations of insulin secretion independently of but with a lesser effectiveness than [Ca2+]i oscillations. Although oscillations in metabolism may cyclically influence secretion through an ATP-sensitive K+ channel (K+-ATP channel)-independent pathway, their regulatory effects are characterized by a hysteresis that makes them unlikely drivers of fast oscillations, unless they also involve [Ca2+]i changes through the K+-ATP channel-dependent pathway

Dual mechanism of the potentiation by glucose of insulin secretion induced by arginine and tolbutamide in mouse islets.

Glucose induces insulin secretion (IS) and also potentiates the insulin-releasing action of secretagogues such as arginine and sulfonylureas. This potentiating effect is known to be impaired in type 2 diabetic patients, but its cellular mechanisms are unclear. IS and cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse islets during perifusion with 3-15 mmol/l glucose (G3-G15, respectively) and pulse or stepwise stimulation with 1-10 mmol/l arginine or 5-250 micromol/l tolbutamide. In G3, arginine induced small increases in [Ca(2+)](i) but no IS. G7 alone only slightly increased [Ca(2+)](i) and IS but markedly potentiated arginine effects on [Ca(2+)](i), which resulted in significant IS (already at 1 mmol/l). For each arginine concentration, both responses further increased at G10 and G15, but the relative change was distinctly larger for IS than [Ca(2+)](i). At all glucose concentrations, tolbutamide dose dependently increased [Ca(2+)](i) and IS with thresholds of 25 micromol/l for [Ca(2+)](i) and 100 micromol/l for IS at G3 and of 5 micromol/l for both at G7 and above. Between G7 and G15, the effect of tolbutamide on [Ca(2+)](i) increased only slightly, whereas that on IS was strongly potentiated. The linear relationship between IS and [Ca(2+)](i) at increasing arginine or tolbutamide concentrations became steeper as the glucose concentration was raised. Thus glucose augmented more the effect of each agent on IS than that on [Ca(2+)](i). In conclusion, glucose potentiation of arginine- or tolbutamide-induced IS involves increases in both the rise of [Ca(2+)](i) and the action of Ca(2+) on exocytosis. This dual mechanism must be borne in mind to interpret the alterations of the potentiating action of glucose in type 2 diabetic patients

Calcium signaling in pancreatic beta-cells in health and in Type 2 diabetes

International audienc

Control mechanisms of the oscillations of insulin secretion in vitro and in vivo

The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free Ca(2+) ([Ca(2+)](c)) in beta-cells play a key role in the control of insulin secretion, and because oscillations of these two factors have been observed in single isolated islets and beta-cells, pulsatile insulin secretion could theoretically result from [Ca(2+)](c) or metabolism oscillations. We could not detect metabolic oscillations independent from [Ca(2+)](c) changes in beta-cells, and imposed metabolic oscillations were poorly effective in inducing oscillations of secretion when [Ca(2+)](c) was kept stable, which suggests that metabolic oscillations are not the direct regulator of the oscillations of secretion. By contrast, tight temporal and quantitative correlations between the changes in [Ca(2+)](c) and insulin release strongly suggest that [Ca(2+)](c) oscillations are the direct drivers of insulin secretion oscillations. Metabolism may play a dual role, inducing [Ca(2+)](c) oscillations (via changes in ATP-sensitive K(+) channel activity and membrane potential) and amplifying the secretory response by increasing the efficiency of Ca(2+) on exocytosis. The mechanisms underlying the oscillations of insulin secretion by the isolated pancreas and those observed in vivo remain elusive. It is not known how the functioning of distinct islets is synchronized, and the possible role of intrapancreatic ganglia in this synchronization requires confirmation. That pulsatile insulin secretion is beneficial in vivo, by preventing insulin resistance, is suggested by the greater hypoglycemic effect of exogenous insulin when it is infused in a pulsatile rather than continuous manner. The observation that type 2 diabetic patients have impaired pulsatile insulin secretion has prompted the suggestion that such dysregulation contributes to the disease and justifies the efforts toward understanding of the mechanism underlying the pulsatility of insulin secretion both in vitro and in vivo

Do oscillations of insulin secretion occur in the absence of cytoplasmic Ca2+ oscillations in beta-cells?

That oscillations of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in beta-cells induce oscillations of insulin secretion is not disputed, but whether metabolism-driven oscillations of secretion can occur in the absence of [Ca(2+)](i) oscillations is still debated. Because this possibility is based partly on the results of experiments using islets from aged, hyperglycemic, hyperinsulinemic ob/ob mice, we compared [Ca(2+)](i) and insulin secretion patterns of single islets from 4- and 10-month-old, normal NMRI mice to those of islets from 7- and 10-month-old ob/ob mice (Swedish colony) and their lean littermates. The responses were subjected to cluster analysis to identify significant peaks. Control experiments without islets and with a constant insulin concentration were run to detect false peaks. Both ob/ob and NMRI islets displayed large synchronous oscillations of [Ca(2+)](i) and insulin secretion in response to repetitive depolarizations with 30 mmol/l K(+) in the presence of 0.1 mmol/l diazoxide and 12 mmol/l glucose. Continuous depolarization with high K(+) steadily elevated [Ca(2+)](i) in all types of islets, with no significant oscillation, and caused a biphasic insulin response. In islets from young (4-month-old) NMRI mice and 7-month-old lean mice, the insulin profile did not show significant peaks when [Ca(2+)](i) was stable. In contrast, two or more peaks were detected over 20 min in the response of most ob/ob islets. Similar insulin peaks appeared in the insulin response of 10-month-old lean and NMRI mice. However, the size of the insulin peaks detected in the presence of stable [Ca(2+)](i) was small, so that no more than 10-13% of total insulin secretion occurred in a pulsatile manner. In conclusion, insulin secretion does not oscillate when [Ca(2+)](i) is stably elevated in beta-cells from young normal mice. Some oscillations are observed in aged mice and are seen more often in ob/ob islets. These fluctuations of the insulin secretion rate at stably elevated [Ca(2+)](i), however, are small compared with the large oscillations induced by [Ca(2+)](i) oscillations in beta-cells